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    Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl. Gregory Mark A,Phang Tzu L,Neviani Paolo,Alvarez-Calderon Francesca,Eide Christopher A,O'Hare Thomas,Zaberezhnyy Vadym,Williams Richard T,Druker Brian J,Perrotti Danilo,Degregori James Cancer cell Although Bcr-Abl kinase inhibitors have proven effective in the treatment of chronic myeloid leukemia (CML), they generally fail to eradicate Bcr-Abl(+) leukemia cells. To identify genes whose inhibition sensitizes Bcr-Abl(+) leukemias to killing by Bcr-Abl inhibitors, we performed an RNAi-based synthetic lethal screen with imatinib mesylate in CML cells. This screen identified numerous components of a Wnt/Ca(2+)/NFAT signaling pathway. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production, and enhanced sensitivity to Bcr-Abl inhibition. Furthermore, NFAT inhibition with cyclosporin A facilitated leukemia cell elimination by the Bcr-Abl inhibitor dasatinib and markedly improved survival in a mouse model of Bcr-Abl(+) acute lymphoblastic leukemia (ALL). Targeting this pathway in combination with Bcr-Abl inhibition could improve treatment of Bcr-Abl(+) leukemias. 10.1016/j.ccr.2010.04.025
    Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3. Yount Jacob S,Moltedo Bruno,Yang Yu-Ying,Charron Guillaume,Moran Thomas M,López Carolina B,Hang Howard C Nature chemical biology Identification of immune effectors and the post-translational modifications that control their activity is essential for dissecting mechanisms of immunity. Here we demonstrate that the antiviral activity of interferon-induced transmembrane protein 3 (IFITM3) is post-translationally regulated by S-palmitoylation. Large-scale profiling of palmitoylated proteins in a dendritic cell line using a chemical reporter strategy revealed over 150 lipid-modified proteins with diverse cellular functions, including innate immunity. We discovered that S-palmitoylation of IFITM3 on membrane-proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. The sites of S-palmitoylation are highly conserved among the IFITM family of proteins in vertebrates, which suggests that S-palmitoylation of these immune effectors may be an ancient post-translational modification that is crucial for host resistance to viral infections. The S-palmitoylation and clustering of IFITM3 will be important for elucidating its mechanism of action and for the design of antiviral therapeutics. 10.1038/nchembio.405
    Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model. Velin Dominique,Favre Laurent,Bernasconi Eric,Bachmann Daniel,Pythoud Catherine,Saiji Essia,Bouzourene Hanifa,Michetti Pierre Gastroenterology BACKGROUND & AIMS:Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS:In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS:Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS:IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa. 10.1053/j.gastro.2009.02.077
    Identification of malignant cells in multiple myeloma bone marrow with immunoglobulin VH gene probes by fluorescent in situ hybridization and flow cytometry. Cao J,Vescio R A,Hong C H,Kim A,Lichtenstein A K,Berenson J R The Journal of clinical investigation Because it has been difficult to identify and separate malignant cells in human lymphoid malignancies, we have developed a flow cytometry-based fluorescent in situ hybridization (FISH) technique using immunoglobulin (Ig) heavy chain variable region (VH) gene probes. After obtaining the specific VH gene sequence expressed by the multiple myeloma IM-9 cell line and the malignant cells in five multiple myeloma patients, sense and antisense biotinylated single-stranded RNA probes were prepared by transcription from the malignant clone's VH DNA sequences. The cells from the IM-9 cell line and from the mononuclear bone marrow cells of multiple myeloma patients were fixed, hybridized with the above biotinylated RNA probes, incubated with streptavidin-phycoerythrin, and analyzed by FACS analysis. The myeloma cells stained positive with their own specific antisense VH biotinylated RNa probes, whereas sense and irrelevant antisense biotinylated probes demonstrated only background staining. Dilutional concentrations of the IM-9 cell line with normal bone marrow cells were also accurately quantitated by this procedure. The application of this technique will allow a more accurate assessment of tumor burden in patients with multiple myeloma and should permit an accurate method of tumor cell purification for clinical as well as biological studies. Furthermore, this technological advance should be equally effective at identifying specific VH gene-expressing cells in other lymphoid malignancies, as well as in nonmalignant B cell disorders. 10.1172/JCI117805
    Suppression of tumour-specific CD4⁺ T cells by regulatory T cells is associated with progression of human colorectal cancer. Betts Gareth,Jones Emma,Junaid Syed,El-Shanawany Tariq,Scurr Martin,Mizen Paul,Kumar Mayur,Jones Sion,Rees Brian,Williams Geraint,Gallimore Awen,Godkin Andrew Gut BACKGROUND:There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4(+)Foxp3(+) regulatory T cells (Tregs) has also been documented. OBJECTIVE:To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression. METHODS:A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4(+) T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4. RESULTS:Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4(+) T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4(+) T cell responses. CONCLUSION:These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4(+) T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention. 10.1136/gutjnl-2011-300970
    Differential effects of unfolded protein response pathways on axon injury-induced death of retinal ganglion cells. Hu Yang,Park Kevin K,Yang Liu,Wei Xin,Yang Qiang,Cho Kin-Sang,Thielen Peter,Lee Ann-Hwee,Cartoni Romain,Glimcher Laurie H,Chen Dong Feng,He Zhigang Neuron Loss of retinal ganglion cells (RGCs) accounts for visual function deficits after optic nerve injury, but how axonal insults lead to neuronal death remains elusive. By using an optic nerve crush model that results in the death of the majority of RGCs, we demonstrate that axotomy induces differential activation of distinct pathways of the unfolded protein response in axotomized RGCs. Optic nerve injury provokes a sustained CCAAT/enhancer binding homologous protein (CHOP) upregulation, and deletion of CHOP promotes RGC survival. In contrast, IRE/XBP-1 is only transiently activated, and forced XBP-1 activation dramatically protects RGCs from axon injury-induced death. Importantly, such differential activations of CHOP and XBP-1 and their distinct effects on neuronal cell death are also observed in RGCs with other types of axonal insults, such as vincristine treatment and intraocular pressure elevation, suggesting a new protective strategy for neurodegeneration associated with axonal damage. 10.1016/j.neuron.2011.11.026
    Weakness, fatigue, and an abnormal white blood cell count. Abboud Ramzi,Sham Ronald JAMA 10.1001/jama.2014.2440
    Glucocorticoid receptor in stromal cells is essential for glucocorticoid-mediated suppression of inflammation in arthritis. Koenen Mascha,Culemann Stephan,Vettorazzi Sabine,Caratti Giorgio,Frappart Lucien,Baum Wolfgang,Krönke Gerhard,Baschant Ulrike,Tuckermann Jan P Annals of the rheumatic diseases BACKGROUND:Glucocorticoid (GC) therapy is frequently used to treat rheumatoid arthritis due to potent anti-inflammatory actions of GCs. Direct actions of GCs on immune cells were suggested to suppress inflammation. OBJECTIVES:Define the role of the glucocorticoid receptor (GR) in stromal cells for suppression of inflammatory arthritis. METHODS:Bone marrow chimeric mice lacking the GR in the hematopoietic or stromal compartment, respectively, and mice with impaired GR dimerisation (GR) were analysed for their response to dexamethasone (DEX, 1 mg/kg) treatment in serum transfer-induced arthritis (STIA). Joint swelling, cell infiltration (histology), cytokines, cell composition (flow cytometry) and gene expression were analysed and RNASeq of wild type and GR primary murine fibroblast-like synoviocytes (FLS) was performed. RESULTS:GR deficiency in immune cells did not impair GC-mediated suppression of STIA. In contrast, mice with GR-deficient or GR dimerisation-impaired stromal cells were resistant to GC treatment, despite efficient suppression of cytokines. Intriguingly, in mice with impaired GR function in the stromal compartment, GCs failed to stimulate non-classical, non-activated macrophages (Ly6C, MHCII) and associated anti-inflammatory markers CD163, CD36, AnxA1, MerTK and Axl. Mice with GR deficiency in FLS were partially resistant to GC-induced suppression of STIA. Accordingly, RNASeq analysis of DEX-treated GR FLS revealed a distinct gene signature indicating enhanced activity and a failure to reduce macrophage inflammatory protein (Mip)-1α and Mip-1β. CONCLUSION:We report a novel anti-inflammatory mechanism of GC action that involves GR dimerisation-dependent gene regulation in non-immune stromal cells, presumably FLS. FLS control non-classical, anti-inflammatory polarisation of macrophages that contributes to suppression of inflammation in arthritis. 10.1136/annrheumdis-2017-212762
    Expression of Free Fatty Acid Receptor 2 by Dendritic Cells Prevents Their Expression of Interleukin 27 and Is Required for Maintenance of Mucosal Barrier and Immune Response Against Colorectal Tumors in Mice. Lavoie Sydney,Chun Eunyoung,Bae Sena,Brennan Caitlin A,Gallini Comeau Carey Ann,Lang Jessica K,Michaud Monia,Hoveyda Hamid R,Fraser Graeme L,Fuller Miles H,Layden Brian T,Glickman Jonathan N,Garrett Wendy S Gastroenterology BACKGROUND & AIMS:Intestinal microbes and their metabolites affect the development of colorectal cancer (CRC). Short-chain fatty acids are metabolites generated by intestinal microbes from dietary fiber. We investigated the mechanisms by which free fatty acid receptor 2 (FFAR2), a receptor for short-chain fatty acids that can affect the composition of the intestinal microbiome, contributes to the pathogenesis of CRC. METHODS:We performed studies with Apc mice, ApcFfar2 mice, mice with conditional disruption of Ffar2 in dendritic cells (DCs) (Ffar2CD11c-Cre mice), ApcFfar2CD11c-Cre mice, and Ffar2 mice (controls); some mice were given dextran sodium sulfate to induce colitis, with or without a FFAR2 agonist or an antibody against interleukin 27 (IL27). Colon and tumor tissues were analyzed by histology, quantitative polymerase chain reaction, and 16S ribosomal RNA gene sequencing; lamina propria and mesenteric lymph node tissues were analyzed by RNA sequencing and flow cytometry. Intestinal permeability was measured after gavage with fluorescently labeled dextran. We collected data on colorectal tumors from The Cancer Genome Atlas. RESULTS:ApcFfar2 mice developed significantly more spontaneous colon tumors than Apc mice and had increased gut permeability before tumor development, associated with reduced expression of E-cadherin. Colon tumors from ApcFfar2 mice had a higher number of bacteria than tumors from Apc mice, as well as higher frequencies of CD39CD8 T cells and exhausted or dying T cells. DCs from ApcFfar2 mice had an altered state of activation, increased death, and higher production of IL27. Administration of an antibody against IL27 reduced the numbers of colon tumors in Apc mice with colitis. Frequencies of CD39CD8 T cells and IL27 DCs were increased in colon lamina propria from Ffar2CD11c-Cre mice with colitis compared with control mice or mice without colitis. ApcFfar2CD11c-Cre mice developed even more tumors than ApcFfar2 mice, and their tumors had even higher numbers of IL27 DCs. Apc mice with colitis given the FFAR2 agonist developed fewer colon tumors, with fewer IL27 DCs, than mice not given the agonist. DCs incubated with the FFAR2 agonist no longer had gene expression patterns associated with activation or IL27 production. CONCLUSIONS:Loss of FFAR2 promotes colon tumorigenesis in mice by reducing gut barrier integrity, increasing tumor bacterial load, promoting exhaustion of CD8 T cells, and overactivating DCs, leading to their death. Antibodies against IL27 and an FFAR2 agonist reduce tumorigenesis in mice and might be developed for the treatment of CRC. 10.1053/j.gastro.2019.12.027
    LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate. Gao Yajing,Yan Yan,Tripathi Sushil,Pentinmikko Nalle,Amaral Ana,Päivinen Pekka,Domènech-Moreno Eva,Andersson Simon,Wong Iris P L,Clevers Hans,Katajisto Pekka,Mäkelä Tomi P Gastroenterology BACKGROUND & AIMS:In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. METHODS:We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1 mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1 mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. RESULTS:Some intestinal crypts from Lkb1 mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1 mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. CONCLUSIONS:LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs. 10.1053/j.gastro.2019.12.033
    HoxA3 is an apical regulator of haemogenic endothelium. Iacovino Michelina,Chong Diana,Szatmari Istvan,Hartweck Lynn,Rux Danielle,Caprioli Arianna,Cleaver Ondine,Kyba Michael Nature cell biology During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells. 10.1038/ncb2137
    Widespread B cell perturbations in HIV-1 infection afflict naive and marginal zone B cells. Liechti Thomas,Kadelka Claus,Braun Dominique L,Kuster Herbert,Böni Jürg,Robbiani Melissa,Günthard Huldrych F,Trkola Alexandra The Journal of experimental medicine Perturbations in B cells are a hallmark of HIV-1 infection. This is signified by increased numbers of exhausted CD21 memory B cells, driven by continuous antigen-specific and bystander activation. Using high-dimensional flow cytometry, we demonstrate that this exhausted phenotype is also prevalent among peripheral antigen-inexperienced naive and marginal zone (MZ) B cells in acute and chronic HIV-1 infection. A substantial fraction of naive and MZ B cells exhibit down-regulated CD21 levels and diminished response to B cell receptor (BCR)-dependent stimulation. Compared with CD21 subsets, the CD21 naive and MZ B cells differ in the expression of chemokine receptors and activation markers. Effective antiretroviral treatment normalizes peripheral naive and MZ B cell populations. Our results emphasize a more widely spread impairment of B cells in HIV-1 infection than previously appreciated, including antigen-inexperienced cells. This highlights the importance of monitoring functional capacities of naive B cells in HIV-1 infection, as exhausted CD21 naive B cells may severely impair induction of novel B cell responses. 10.1084/jem.20181124
    Szilard's dream. Balaban Nathalie Q Nature methods 10.1038/nmeth0905-648
    A microfluidic chemostat for experiments with bacterial and yeast cells. Groisman Alex,Lobo Caroline,Cho HoJung,Campbell J Kyle,Dufour Yann S,Stevens Ann M,Levchenko Andre Nature methods Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer. 10.1038/nmeth784
    Ibrutinib plus Venetoclax for the Treatment of Mantle-Cell Lymphoma. Tam Constantine S,Anderson Mary Ann,Pott Christiane,Agarwal Rishu,Handunnetti Sasanka,Hicks Rodney J,Burbury Kate,Turner Gillian,Di Iulio Juliana,Bressel Mathias,Westerman David,Lade Stephen,Dreyling Martin,Dawson Sarah-Jane,Dawson Mark A,Seymour John F,Roberts Andrew W The New England journal of medicine BACKGROUND:Both the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax are active as monotherapy in the treatment of mantle-cell lymphoma. Complete response rates of 21% have been observed for each agent when administered as long-term continuous therapy. Preclinical models predict synergy in combination. METHODS:We conducted a single-group, phase 2 study of daily oral ibrutinib and venetoclax in patients, as compared with historical controls. Patients commenced ibrutinib monotherapy at a dose of 560 mg per day. After 4 weeks, venetoclax was added in stepwise, weekly increasing doses to 400 mg per day. Both drugs were continued until progression or an unacceptable level of adverse events. The primary end point was the rate of complete response at week 16. Minimal residual disease (MRD) was assessed by flow cytometry in bone marrow and by allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) in blood. RESULTS:The study included 24 patients with relapsed or refractory mantle-cell lymphoma (23 patients) or previously untreated mantle-cell lymphoma (1 patient). Patients were 47 to 81 years of age, and the number of previous treatments ranged from none to six. Half the patients had aberrations of TP53, and 75% had a high-risk prognostic score. The complete response rate according to computed tomography at week 16 was 42%, which was higher than the historical result of 9% at this time point with ibrutinib monotherapy (P<0.001). The rate of complete response as assessed by positron-emission tomography was 62% at week 16 and 71% overall. MRD clearance was confirmed by flow cytometry in 67% of the patients and by ASO-PCR in 38%. In a time-to-event analysis, 78% of the patients with a response were estimated to have an ongoing response at 15 months. The tumor lysis syndrome occurred in 2 patients. Common side effects were generally low grade and included diarrhea (in 83% of the patients), fatigue (in 75%), and nausea or vomiting (in 71%). CONCLUSIONS:In this study involving historical controls, dual targeting of BTK and BCL2 with ibrutinib and venetoclax was consistent with improved outcomes in patients with mantle-cell lymphoma who had been predicted to have poor outcomes with current therapy. (Funded by Janssen and others; AIM ClinicalTrials.gov number, NCT02471391 .). 10.1056/NEJMoa1715519
    Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. Keating Michael J,O'Brien Susan,Albitar Maher,Lerner Susan,Plunkett William,Giles Francis,Andreeff Michael,Cortes Jorge,Faderl Stefan,Thomas Deborah,Koller Charles,Wierda William,Detry Michelle A,Lynn Alice,Kantarjian Hagop Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Fludarabine and cyclophosphamide (FC), which are active in treatment of chronic lymphocytic leukemia (CLL), are synergistic with the monoclonal antibody rituximab in vitro in lymphoma cell lines. A chemoimmunotherapy program consisting of fludarabine, cyclophosphamide, and rituximab (FCR) was developed with the goal of increasing the complete remission (CR) rate in previously untreated CLL patients to >/= 50%. PATIENTS AND METHODS:We conducted a single-arm study of FCR as initial therapy in 224 patients with progressive or advanced CLL. Flow cytometry was used to measure residual disease. Results and safety were compared with a previous regimen using FC. RESULTS:The median age was 58 years; 75 patients (33%) had Rai stage III to IV disease. The CR rate was 70% (95% CI, 63% to 76%), the nodular partial remission rate was 10%, and the partial remission rate was 15%, for an overall response rate of 95% (95% CI, 92% to 98%). Two thirds of patients evaluated with flow cytometry had less than 1% CD5- and CD19-coexpressing cells in bone marrow after therapy. Grade 3 to 4 neutropenia occurred during 52% of courses; major and minor infections were seen in 2.6% and 10% of courses, respectively. One third of the 224 patients had >/= one episode of infection, and 10% had a fever of unknown origin. CONCLUSION:FCR produced a high CR rate in previously untreated CLL. Most patients had no detectable disease on flow cytometry at the end of therapy. Time to treatment failure analysis showed that 69% of patients were projected to be failure free at 4 years (95% CI, 57% to 81%). 10.1200/JCO.2005.12.051
    Specific peripheral B cell tolerance defects in patients with multiple sclerosis. Kinnunen Tuure,Chamberlain Nicolas,Morbach Henner,Cantaert Tineke,Lynch Megan,Preston-Hurlburt Paula,Herold Kevan C,Hafler David A,O'Connor Kevin C,Meffre Eric The Journal of clinical investigation Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. B cells have recently emerged as major contributors to disease pathogenesis, but the mechanisms responsible for the loss of B cell tolerance in patients with MS are largely unknown. In healthy individuals, developing autoreactive B cells are removed from the repertoire at 2 tolerance checkpoints during early B cell development. Both of these central and peripheral B cell tolerance checkpoints are defective in patients with rheumatoid arthritis (RA) and type 1 diabetes (T1D). Here, we found that only the peripheral, but not the central, B cell tolerance checkpoint is defective in patients with MS. We show that this specific defect is accompanied by increased activation and homeostatic proliferation of mature naive B cells. Interestingly, all of these MS features parallel defects observed in FOXP3-deficient IPEX patients, who harbor nonfunctional Tregs. We demonstrate that in contrast to patients with RA or T1D, bone marrow central B cell selection in MS appears normal in most patients. In contrast, patients with MS suffer from a specific peripheral B cell tolerance defect that is potentially attributable to impaired Treg function and that leads to the accumulation of autoreactive B cell clones in their blood. 10.1172/JCI68775
    Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing NCRNA disease. Ghiasvand Noor M,Rudolph Dellaney D,Mashayekhi Mohammad,Brzezinski Joseph A,Goldman Daniel,Glaser Tom Nature neuroscience Individuals with nonsyndromic congenital retinal nonattachment (NCRNA) are totally blind from birth. The disease afflicts ∼1% of Kurdish people living in a group of neighboring villages in North Khorasan, Iran. We found that NCRNA is caused by a 6,523-bp deletion that spans a remote cis regulatory element 20 kb upstream from ATOH7 (Math5), a bHLH transcription factor gene that is required for retinal ganglion cell (RGC) and optic nerve development. In humans, the absence of RGCs stimulates massive neovascular growth of fetal blood vessels in the vitreous and early retinal detachment. The remote ATOH7 element appears to act as a secondary or 'shadow' transcriptional enhancer. It has minimal sequence similarity to the primary enhancer, which is close to the ATOH7 promoter, but drives transgene expression with an identical spatiotemporal pattern in the mouse retina. The human transgene also functions appropriately in zebrafish, reflecting deep evolutionary conservation. These dual enhancers may reinforce ATOH7 expression during early critical stages of eye development when retinal neurogenesis is initiated. 10.1038/nn.2798
    Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks. Kuhar Ryan,Gwiazda Kamila S,Humbert Olivier,Mandt Tyler,Pangallo Joey,Brault Michelle,Khan Iram,Maizels Nancy,Rawlings David J,Scharenberg Andrew M,Certo Michael T Nucleic acids research The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks. 10.1093/nar/gkt872
    RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation. Collins Amélie,Hewitt Susannah L,Chaumeil Julie,Sellars Maclean,Micsinai Mariann,Allinne Jeanne,Parisi Fabio,Nora Elphège P,Bolland Dan J,Corcoran Anne E,Kluger Yuval,Bosselut Remy,Ellmeier Wilfried,Chong Mark M W,Littman Dan R,Skok Jane A Immunity T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells. 10.1016/j.immuni.2011.03.004
    Autocrine transforming growth factor-β1 promotes in vivo Th17 cell differentiation. Gutcher Ilona,Donkor Moses K,Ma Qian,Rudensky Alexander Y,Flavell Richard A,Li Ming O Immunity TGF-β1 is a regulatory cytokine that has an important role in controlling T cell differentiation. T cell-produced TGF-β1 acts on T cells to promote Th17 cell differentiation and the development of experimental autoimmune encephalomyelitis (EAE). However, the exact TGF-β1-producing T cell subset required for Th17 cell generation and its cellular mechanism of action remain unknown. Here we showed that deletion of the Tgfb1 gene from activated T cells and Treg cells, but not Treg cells alone, abrogated Th17 cell differentiation, resulting in almost complete protection from EAE. Furthermore, differentiation of T cells both in vitro and in vivo demonstrated that TGF-β1 was highly expressed by Th17 cells and acted in a predominantly autocrine manner to maintain Th17 cells in vivo. These findings reveal an essential role for activated T cell-produced TGF-β1 in promoting the differentiation of Th17 cells and controlling inflammatory diseases. 10.1016/j.immuni.2011.03.005
    Peritoneal Level of CD206 Associates With Mortality and an Inflammatory Macrophage Phenotype in Patients With Decompensated Cirrhosis and Spontaneous Bacterial Peritonitis. Stengel Sven,Quickert Stefanie,Lutz Philipp,Ibidapo-Obe Oluwatomi,Steube Arndt,Köse-Vogel Nilay,Yarbakht Melina,Reuken Philipp A,Busch Martin,Brandt Annette,Bergheim Ina,Deshmukh Sachin D,Stallmach Andreas,Bruns Tony Gastroenterology BACKGROUND & AIMS:Peritoneal macrophages (PMs) regulate inflammation and control bacterial infections in patients with decompensated cirrhosis. We aimed to characterize PMs and associate their activation with outcomes of patients with spontaneous bacterial peritonitis (SBP). METHODS:We isolated PMs from ascites samples of 66 patients with decompensated cirrhosis (19 with SBP) and analyzed them by flow cytometry, quantitative real-time polymerase chain reaction, functional analysis, and RNA microarrays. We used ascites samples of a separate cohort of 111 patients with decompensated cirrhosis (67 with SBP) and quantified the soluble form of the mannose receptor (CD206) and tumor necrosis factor by enzyme-linked immunosorbent assay (test cohort). We performed logistic regression analysis to identify factors associated with 90-day mortality. We validated our findings using data from 71 patients with cirrhosis and SBP. Data from 14 patients undergoing peritoneal dialysis for end-stage renal disease but without cirrhosis were included as controls. RESULTS:We used surface levels of CD206 to identify subsets of large PMs (LPM) and small PMs (SPM), which differed in granularity and maturation markers, in ascites samples from patients with cirrhosis. LPMs vs SPMs from patients with cirrhosis had different transcriptomes; we identified more than 4000 genes that were differentially regulated in LPMs vs SPMs, including those that regulate the cycle, metabolism, self-renewal, and immune cell signaling. LPMs had an inflammatory phenotype, were less susceptible to tolerance induction, and released more tumor necrosis factor than SPMs. LPMs from patients with cirrhosis produced more inflammatory cytokines than LPMs from controls. Activation of PMs by Toll-like receptor agonists and live bacteria altered levels of CD206 on the surface of LPMs and release of soluble CD206. Analysis of serial ascites fluid from patients with SBP revealed loss of LPMs in the early phase of SBP, but levels increased after treatment. In the test and validation cohorts, patients with SBP and higher concentrations of soluble CD206 in ascites fluid (>0.53 mg/L) were less likely to survive for 90 days than those with lower levels. CONCLUSIONS:Surface level of CD206 can be used to identify mature, resident, inflammatory PMs in patients with cirrhosis. Soluble CD206 is released from activated LPMs and increased concentrations in patients with cirrhosis and SBP indicate reduced odds of surviving for 90 days. 10.1053/j.gastro.2020.01.029
    Dysregulated neutrophil responses and neutrophil extracellular trap formation and degradation in PAPA syndrome. Mistry Pragnesh,Carmona-Rivera Carmelo,Ombrello Amanda K,Hoffmann Patrycja,Seto Nickie L,Jones Anne,Stone Deborah L,Naz Faiza,Carlucci Philip,Dell'Orso Stefania,Gutierrez-Cruz Gustavo,Sun Hong-Wei,Kastner Daniel L,Aksentijevich Ivona,Kaplan Mariana J Annals of the rheumatic diseases OBJECTIVES:Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is characterised by flares of sterile arthritis with neutrophil infiltrate and the overproduction of interleukin (IL)-1β. The purpose of this study was to elucidate the potential role of neutrophil subsets and neutrophil extracellular traps (NET) in the pathogenesis of PAPA. METHODS:Neutrophils and low-density granulocytes (LDG) were quantified by flow cytometry. Circulating NETs were measured by ELISA and PAPA serum was tested for the ability to degrade NETs. The capacity of NETs from PAPA neutrophils to activate macrophages was assessed. Skin biopsies were analysed for NETs and neutrophil gene signatures. RESULTS:Circulating LDGs are elevated in PAPA subjects. PAPA neutrophils and LDGs display enhanced NET formation compared with control neutrophils. PAPA sera exhibit impaired NET degradation and this is corrected with exogenous DNase1. Recombinant human IL-1β induces NET formation in PAPA neutrophils but not healthy control neutrophils. NET formation in healthy control neutrophils is induced by PAPA serum and this effect is inhibited by the IL-1 receptor antagonist, anakinra. NETs from PAPA neutrophils and LDGs stimulate IL-6 release in healthy control macrophages. NETs are detected in skin biopsies of patients with PAPA syndrome in association with increased tissue IL-1β, IL-8 and IL-17. Furthermore, LDG gene signatures are detected in PAPA skin. CONCLUSIONS:PAPA syndrome is characterised by an imbalance of NET formation and degradation that may enhance the half-life of these structures in vivo, promoting inflammation. Anakinra ameliorates NET formation in PAPA and this finding supports a role for IL-1 signalling in exacerbated neutrophil responses in this disease. The study also highlights other inflammatory pathways potentially pathogenic in PAPA, including IL-17 and IL-6, and these results may help guide new therapeutic approaches in this severe and often treatment-refractory condition. 10.1136/annrheumdis-2018-213746
    High Serum Levels of Cholesterol Increase Antitumor Functions of Nature Killer Cells and Reduce Growth of Liver Tumors in Mice. Qin Wen-Hao,Yang Zhi-Shi,Li Mian,Chen Yao,Zhao Xiao-Fang,Qin Ying-Yi,Song Jia-Qi,Wang Bi-Bo,Yuan Bo,Cui Xiu-Liang,Shen Feng,He Jia,Bi Yu-Fang,Ning Guang,Fu Jing,Wang Hong-Yang Gastroenterology BACKGROUND AND AIMS:The relationship between serum cholesterol level and development of hepatocellular carcinoma (HCC) remains unclear. We investigated the effects of serum cholesterol level on development of liver tumors in mice. METHODS:We performed studies with C57BL/6J mice, mice with disruption of the low-density lipoprotein receptor gene (Ldlrmice), and mice with conditional deletion of nature killer (NK) cells (NK mice). Some C57BL/6J and NK mice were given injections of diethylinitrosamine to induce liver tumor formation. Mice were placed on a normal diet (ND) or high-cholesterol diet (HCD) to induce high serum levels of cholesterol. We also studied mice with homozygous disruption of ApoE (ApoE mice), which spontaneously develop high serum cholesterol. C57BL/6J and NK mice on the ND or HCD were implanted with Hep1-6 (mouse hepatoma) cells and growth of xenograft tumors and lung metastases were monitored. Blood samples were collected from mice and analyzed by biochemistry and flow cytometry; liver and tumor tissues were collected and analyzed by histology, immunohistochemistry, and RNA-sequencing analysis. NK cells were isolated from mice and analyzed for cholesterol content, lipid raft formation, immune signaling, and changes in functions. We obtained matched tumor tissues and blood samples from 30 patients with HCC and blood samples from 40 healthy volunteers; levels of cholesterol and cytotoxicity of NK cells were measured. RESULTS:C57BL/6J mice on HCD and ApoE mice with high serum levels of cholesterol developed fewer and smaller liver tumors and lung metastases after diethylinitrosamine injection or implantation of Hep1-6 cells than mice on ND. Liver tumors from HCD-fed mice and ApoE mice had increased numbers of NK cells compared to tumors from ND-fed mice. NK mice or mice with antibody-based depletion for NK cells showed similar tumor number and size in ND and HCD groups after diethylinitrosamine injection or implantation of Hep1-6 cells. NK cells isolated from C57BL/6J mice fed with HCD had increased expression of NK cell-activating receptors (natural cytotoxicity triggering receptor 1 and natural killer group 2, member D), markers of effector function (granzyme B and perforin), and cytokines and chemokines compared with NK cells from mice on ND; these NK cells also had enhanced cytotoxic activity against mouse hepatoma cells, accumulated cholesterol, increased lipid raft formation, and immune signaling activation. NK cells isolated from HCD-fed Ldlr mice did not have increased cholesterol content or cytotoxic activity against mouse hepatoma cells compared with ND-fed Ldlr mice. Serum levels of cholesterol correlated with number and activity of NK cells isolated from human HCCs. CONCLUSIONS:Mice with increased serum levels of cholesterol due to an HCD or genetic disruption of ApoE develop fewer and smaller tumors after injection of hepatoma cells or a chemical carcinogen. We found cholesterol to accumulate in NK cells and activate their effector functions against hepatoma cells. Strategies to increase cholesterol uptake by NK cells can be developed for treatment of HCC. 10.1053/j.gastro.2020.01.028
    Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells. Peng Dunfa,Belkhiri Abbes,Hu Tianling,Chaturvedi Rupesh,Asim Mohammad,Wilson Keith T,Zaika Alexander,El-Rifai Wael Gut OBJECTIVE:Exposure of the oesophageal mucosa to gastric acid and bile acids leads to the accumulation of reactive oxygen species (ROS), a known risk factor for Barrett's oesophagus and progression to oesophageal adenocarcinoma (OAC). This study investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage. DESIGN:Using in-vitro cell models, experiments were performed that included glutathione peroxidase (GPX) activity, Amplex UltraRed, CM-H(2)DCFDA, Annexin V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and western blot assays. RESULTS:Enzymatic assays demonstrated limited GPX activity of the recombinant GPX7 protein. GPX7 exhibited a strong capacity to neutralise hydrogen peroxide (H(2)O(2)) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H(2)O(2)-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H(2)O(2), intracellular ROS, oxidative DNA damage and double-strand breaks, compared with controls (p<0.01). In addition, these cells demonstrated lower levels of ROS signalling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H(2)O(2)-induced oxidative stress. The knockdown of endogenous GPX7 in immortalised oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H(2)O(2), intracellular ROS, oxidative DNA damage, double-strand breaks, apoptosis, and ROS-dependent signalling (p<0.01). CONCLUSION:The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage, which are common risk factors for Barrett's oesophagus and OAC. 10.1136/gutjnl-2011-301078
    Anti-IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses (MATERIAL). A Placebo-controlled, Double-Blind Study. Sabogal Piñeros Yanaika S,Bal Suzanne M,van de Pol Marianne A,Dierdorp Barbara S,Dekker Tamara,Dijkhuis Annemiek,Brinkman Paul,van der Sluijs Koen F,Zwinderman Aeilko H,Majoor Christof J,Bonta Peter I,Ravanetti Lara,Sterk Peter J,Lutter René American journal of respiratory and critical care medicine RATIONALE:Eosinophils drive pathophysiology in stable and exacerbating eosinophilic asthma, and therefore treatment is focused on the reduction of eosinophil numbers. Mepolizumab, a humanized monoclonal antibody that neutralizes IL-5 and efficiently attenuates eosinophils, proved clinically effective in severe eosinophilic asthma but not in mild asthma. OBJECTIVES:To study the effect of mepolizumab on virus-induced immune responses in mild asthma. METHODS:Patients with mild asthma, steroid-naive and randomized for eosinophil numbers, received 750 mg mepolizumab intravenously in a placebo-controlled double-blind trial, 2 weeks after which patients were challenged with rhinovirus (RV) 16. FEV, FVC, fractional exhaled nitric oxide, symptom scores (asthma control score), viral load (PCR), eosinophil numbers, humoral (luminex, ELISA), and cellular (flow cytometry) immune parameters in blood, BAL fluid, and sputum, before and after mepolizumab and RV16, were assessed. MEASUREMENTS AND MAIN RESULTS:Mepolizumab attenuated baseline blood eosinophils and their activation, attenuated trendwise sputum eosinophils, and enhanced circulating natural killer cells. Mepolizumab did not affect FEV, FVC, and fractional exhaled nitric oxide, neither at baseline nor after RV16. On RV16 challenge mepolizumab did not prevent eosinophil activation but did enhance local B lymphocytes and macrophages and reduce neutrophils and their activation. Mepolizumab also enhanced secretory IgA and reduced tryptase in BAL fluid. Finally, mepolizumab affected particularly RV16-induced macrophage inflammatory protein-3a, vascular endothelial growth factor-A, and IL-1RA production in BAL fluid. CONCLUSIONS:Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-induced immune responses in mild asthma. Although these latter effects likely are caused by attenuated eosinophil numbers, we cannot exclude a role for basophils. Clinical trial registered with www.clinicaltrials.gov (NCT 01520051). 10.1164/rccm.201803-0461OC
    Impaired Cytomegalovirus Immunity in Idiopathic Pulmonary Fibrosis Lung Transplant Recipients with Short Telomeres. American journal of respiratory and critical care medicine RATIONALE:Cytomegalovirus (CMV)-related morbidities remain one of the most common complications after lung transplantation and have been linked to allograft dysfunction, but the factors that predict high risk for CMV complications and effective immunity are incompletely understood. OBJECTIVES:To determine if short telomeres in idiopathic pulmonary fibrosis (IPF) lung transplant recipients (LTRs) predict the risk for CMV-specific T-cell immunity and viral control. METHODS:We studied IPF-LTRs (n = 42) and age-matched non-IPF-LTRs (n = 42) and assessed CMV outcomes. We measured lymphocyte telomere length and DNA sequencing, and assessed CMV-specific T-cell immunity in LTRs at high risk for CMV events, using flow cytometry and fluorescence in situ hybridization. MEASUREMENTS AND MAIN RESULTS:We identified a high prevalence of relapsing CMV viremia in IPF-LTRs compared with non-IPF-LTRs (69% vs. 31%; odds ratio, 4.98; 95% confidence interval, 1.95-12.50; P < 0.001). Within this subset, IPF-LTRs who had short telomeres had the highest risk of CMV complications (P < 0.01) including relapsing-viremia episodes, end-organ disease, and CMV resistance to therapy, as well as shorter time to viremia versus age-matched non-IPF control subjects (P < 0.001). The short telomere defect in IPF-LTRs was associated with significantly impaired CMV-specific proliferative responses, T-cell effector functions, and induction of the major type-1 transcription factor T-bet (T-box 21;TBX21). CONCLUSIONS:Because the short telomere defect has been linked to the pathogenesis of IPF in some cases, our data indicate that impaired CMV immunity may be a systemic manifestation of telomere-mediated disease in these patients. Identifying this high-risk subset of LTRs has implications for risk assessment, management, and potential strategies for averting post-transplant CMV morbidities. 10.1164/rccm.201805-0825OC
    Monocytes control second-phase neutrophil emigration in established lipopolysaccharide-induced murine lung injury. Dhaliwal Kevin,Scholefield Emma,Ferenbach David,Gibbons Michael,Duffin Rodger,Dorward David A,Morris Andrew Conway,Humphries Duncan,MacKinnon Alison,Wilkinson Tom S,Wallace William A H,van Rooijen Nico,Mack Matthias,Rossi Adriano G,Davidson Donald J,Hirani Nik,Hughes Jeremy,Haslett Chris,Simpson A John American journal of respiratory and critical care medicine RATIONALE:Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES:To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS:In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS:PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS:These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI. 10.1164/rccm.201112-2132OC
    Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells. Chen Chia-Lin,Tsukamoto Hidekazu,Liu Jian-Chang,Kashiwabara Claudine,Feldman Douglas,Sher Linda,Dooley Steven,French Samuel W,Mishra Lopa,Petrovic Lydia,Jeong Joseph H,Machida Keigo The Journal of clinical investigation Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133(+) TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG-dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2(+/-) mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC. 10.1172/JCI65859
    Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. White Yvonne A R,Woods Dori C,Takai Yasushi,Ishihara Osamu,Seki Hiroyuki,Tilly Jonathan L Nature medicine Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. 10.1038/nm.2669
    Reprogramming of T cells from human peripheral blood. Loh Yuin-Han,Hartung Odelya,Li Hu,Guo Chunguang,Sahalie Julie M,Manos Philip D,Urbach Achia,Heffner Garrett C,Grskovic Marica,Vigneault Francois,Lensch M William,Park In-Hyun,Agarwal Suneet,Church George M,Collins James J,Irion Stefan,Daley George Q Cell stem cell 10.1016/j.stem.2010.06.004
    Signaling through BMPR-IA regulates quiescence and long-term activity of neural stem cells in the adult hippocampus. Mira Helena,Andreu Zoraida,Suh Hoonkyo,Lie D Chichung,Jessberger Sebastian,Consiglio Antonella,San Emeterio Juana,Hortigüela Rafael,Marqués-Torrejón María Angeles,Nakashima Kinichi,Colak Dilek,Götz Magdalena,Fariñas Isabel,Gage Fred H Cell stem cell Neural stem cells (NSCs) in the adult hippocampus divide infrequently, and the molecules that modulate their quiescence are largely unknown. Here, we show that bone morphogenetic protein (BMP) signaling is active in hippocampal NSCs, downstream of BMPR-IA. BMPs reversibly diminish proliferation of cultured NSCs while maintaining their undifferentiated state. In vivo, acute blockade of BMP signaling in the hippocampus by intracerebral infusion of Noggin first recruits quiescent NSCs into the cycle and increases neurogenesis; subsequently, it leads to decreased stem cell division and depletion of precursors and newborn neurons. Consistently, selective ablation of Bmpr1a in hippocampal NSCs, or inactivation of BMP canonical signaling in conditional Smad4 knockout mice, transiently enhances proliferation but later leads to a reduced number of precursors, thereby limiting neuronal birth. BMPs are therefore required to balance NSC quiescence/proliferation and to prevent loss of the stem cell activity that supports continuous neurogenesis in the mature hippocampus. 10.1016/j.stem.2010.04.016
    An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation of hematopoietic stem cell activity. Hope Kristin J,Cellot Sonia,Ting Stephen B,MacRae Tara,Mayotte Nadine,Iscove Norman N,Sauvageau Guy Cell stem cell In this study, we describe an in vivo RNA interference functional genetics approach to evaluate the role of 20 different conserved polarity factors and fate determinants in mouse hematopoietic stem cell (HSC) activity. In total, this screen revealed three enhancers and one suppressor of HSC-derived reconstitution. Pard6a, Prkcz, and Msi2 shRNA-mediated depletion significantly impaired HSC repopulation. An in vitro promotion of differentiation was observed after the silencing of these genes, consistent with their function in regulating HSC self-renewal. Conversely, Prox1 knockdown led to in vivo accumulation of primitive and differentiated cells. HSC activity was also enhanced in vitro when Prox1 levels were experimentally reduced, identifying it as a potential antagonist of self-renewal. HSC engineered to overexpress Msi2 or Prox1 showed the reverse phenotype to those transduced with corresponding shRNA vectors. Gene expression profiling studies identified a number of known HSC and cell cycle regulators as potential downstream targets to Msi2 and Prox1. 10.1016/j.stem.2010.06.007
    Primary tumor genotype is an important determinant in identification of lung cancer propagating cells. Curtis Stephen J,Sinkevicius Kerstin W,Li Danan,Lau Allison N,Roach Rebecca R,Zamponi Raffaella,Woolfenden Amber E,Kirsch David G,Wong Kwok-Kin,Kim Carla F Cell stem cell Successful cancer therapy requires the elimination or incapacitation of all tumor cells capable of regenerating a tumor. Therapeutic advances therefore necessitate the characterization of the cells that are able to propagate a tumor in vivo. We show an important link between tumor genotype and isolation of tumor-propagating cells (TPCs). Three mouse models of the most common form of human lung cancer each had TPCs with a unique cell-surface phenotype. The cell-surface marker Sca1 did not enrich for TPCs in tumors initiated with oncogenic Kras, and only Sca1-negative cells propagated EGFR mutant tumors. In contrast, Sca1-positive cells were enriched for tumor-propagating activity in Kras tumors with p53 deficiency. Primary tumors that differ in genotype at just one locus can therefore have tumor-propagating cell populations with distinct markers. Our studies show that the genotype of tumor samples must be considered in studies to identify, characterize, and target tumor-propagating cells. 10.1016/j.stem.2010.05.021
    Progression of Disease Within 24 Months in Follicular Lymphoma Is Associated With Reduced Intratumoral Immune Infiltration. Tobin Joshua W D,Keane Colm,Gunawardana Jay,Mollee Peter,Birch Simone,Hoang Thanh,Lee Justina,Li Li,Huang Li,Murigneux Valentine,Fink J Lynn,Matigian Nicholas,Vari Frank,Francis Santiyagu,Kridel Robert,Weigert Oliver,Haebe Sarah,Jurinovic Vindi,Klapper Wolfram,Steidl Christian,Sehn Laurie H,Law Soi-Cheng,Wykes Michelle N,Gandhi Maher K Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Understanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL. PATIENTS AND METHODS:Digital gene expression using a custom code set-five immune effector, six immune checkpoint, one macrophage molecules-was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more. RESULTS:Immune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltration (ie, high PD-L2) FL biopsies from immune infiltration (ie, low PD-L2) tumors. Immune infiltration tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltration subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile. CONCLUSION:Assessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24. 10.1200/JCO.18.02365
    Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Samavarchi-Tehrani Payman,Golipour Azadeh,David Laurent,Sung Hoon-Ki,Beyer Tobias A,Datti Alessandro,Woltjen Knut,Nagy Andras,Wrana Jeffrey L Cell stem cell Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP: 10.1016/j.stem.2010.04.015
    microRNA-181a-5p antisense oligonucleotides attenuate osteoarthritis in facet and knee joints. Nakamura Akihiro,Rampersaud Yoga Raja,Nakamura Sayaka,Sharma Anirudh,Zeng Fanxing,Rossomacha Evgeny,Ali Shabana Amanda,Krawetz Roman,Haroon Nigil,Perruccio Anthony V,Mahomed Nizar N,Gandhi Rajiv,Rockel Jason S,Kapoor Mohit Annals of the rheumatic diseases OBJECTIVES:We recently identified microRNA-181a-5p (miR-181a-5p) as a critical mediator involved in the destruction of lumbar facet joint (FJ) cartilage. In this study, we tested if locked nucleic acid (LNA) miR-181a-5p antisense oligonucleotides (ASO) could be used as a therapeutic to limit articular cartilage degeneration. METHODS:We used a variety of experimental models consisting of both human samples and animal models of FJ and knee osteoarthritis (OA) to test the effects of LNA-miR-181a-5p ASO on articular cartilage degeneration. Histopathological analysis including immunohistochemistry and in situ hybridisation were used to detect key OA catabolic markers and microRNA, respectively. Apoptotic/cell death markers were evaluated by flow cytometry. qPCR and immunoblotting were applied to quantify gene and protein expression. RESULTS:miR-181a-5p expression was increased in human FJ OA and knee OA cartilage as well as injury-induced FJ OA (rat) and trauma-induced knee OA (mouse) cartilage compared with control cartilage, correlating with classical OA catabolic markers in human, rat and mouse cartilage. We demonstrated that LNA-miR-181a-5p ASO in rat and mouse chondrocytes reduced the expression of cartilage catabolic and chondrocyte apoptotic/cell death markers in vitro. Treatment of OA-induced rat FJ or mouse knee joints with intra-articular injections of in vivo grade LNA-miR-181a-5p ASO attenuated cartilage destruction, and the expression of catabolic, hypertrophic, apoptotic/cell death and type II collagen breakdown markers. Finally, treatment of LNA-miR-181a-5p ASO in cultures of human knee OA chondrocytes (in vitro) and cartilage explants (ex vivo) further demonstrated its cartilage protective effects. CONCLUSIONS:Our data demonstrate, for the first time, that LNA-miR-181a-5p ASO exhibit cartilage-protective effects in FJ and knee OA. 10.1136/annrheumdis-2018-213629
    Effect of increased exercise in school children on physical fitness and endothelial progenitor cells: a prospective randomized trial. Walther Claudia,Gaede Luise,Adams Volker,Gelbrich Götz,Leichtle Alexander,Erbs Sandra,Sonnabend Melanie,Fikenzer Kati,Körner Antje,Kiess Wieland,Bruegel Mathias,Thiery Joachim,Schuler Gerhard Circulation BACKGROUND:The aim of this prospective, randomized study was to examine whether additional school exercise lessons would result in improved peak oxygen uptake (primary end point) and body mass index-standard deviation score, motor and coordinative abilities, circulating progenitor cells, and high-density lipoprotein cholesterol (major secondary end points). METHODS AND RESULTS:Seven sixth-grade classes (182 children, aged 11.1+/-0.7 years) were randomized to an intervention group (4 classes with 109 students) with daily school exercise lessons for 1 year and a control group (3 classes with 73 students) with regular school sports twice weekly. The significant effects of intervention estimated from ANCOVA adjusted for intraclass correlation were the following: increase of peak o(2) (3.7 mL/kg per minute; 95% confidence interval, 0.3 to 7.2) and increase of circulating progenitor cells evaluated by flow cytometry (97 cells per 1 x 10(6) leukocytes; 95% confidence interval, 13 to 181). No significant difference was seen for body mass index-standard deviation score (-0.08; 95% confidence interval, -0.28 to 0.13); however, there was a trend to reduction of the prevalence of overweight and obese children in the intervention group (from 12.8% to 7.3%). No treatment effect was seen for motor and coordinative abilities (4; 95% confidence interval, -1 to 8) and high-density lipoprotein cholesterol (0.03 mmol/L; 95% confidence interval, -0.08 to 0.14). CONCLUSIONS:Regular physical activity by means of daily school exercise lessons has a significant positive effect on physical fitness (o(2)max). Furthermore, the number of circulating progenitor cells can be increased, and there is a positive trend in body mass index-standard deviation score reduction and motor ability improvement. Therefore, we conclude that primary prevention by means of increasing physical activity should start in childhood. CLINICAL TRIAL REGISTRATION:URL: http://www.clinicaltrials.gov. Identifier: NCT00176371. 10.1161/CIRCULATIONAHA.109.865808
    Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown. Bäumer Nicole,Appel Neele,Terheyden Lisa,Buchholz Frank,Rossig Claudia,Müller-Tidow Carsten,Berdel Wolfgang E,Bäumer Sebastian Nature protocols Knockdown of genes by RNA interference (RNAi) in vitro requires methods of transfection or transduction, both of which have limited impact in vivo. As a virus-free approach, we chemically coupled cell surface receptors internalizing antibodies to the short interfering RNA (siRNA) carrier peptide protamine using the bispecific cross-linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone to carry siRNA. This complex can efficiently find, bind and internalize into receptor-positive cells in vitro and in vivo, which can be checked by flow cytometry, fluorescence microscopy and western blotting. This method obtains results similar to those of siRNA targeting molecules engineered by genetic fusions between receptor-binding and siRNA carrier units, with the advantage of using readily available purified proteins without the need for engineering, expression and purification of respective constructs. The procedure for coupling the complex takes ∼ 2 d, and the functional assays take ∼ 2 weeks. 10.1038/nprot.2015.137
    Foxp-mediated suppression of N-cadherin regulates neuroepithelial character and progenitor maintenance in the CNS. Rousso David L,Pearson Caroline Alayne,Gaber Zachary B,Miquelajauregui Amaya,Li Shanru,Portera-Cailliau Carlos,Morrisey Edward E,Novitch Bennett G Neuron Neuroepithelial attachments at adherens junctions are essential for the self-renewal of neural stem and progenitor cells and the polarized organization of the developing central nervous system. The balance between stem cell maintenance and differentiation depends on the precise assembly and disassembly of these adhesive contacts, but the gene regulatory mechanisms orchestrating this process are not known. Here, we demonstrate that two Forkhead transcription factors, Foxp2 and Foxp4, are progressively expressed upon neural differentiation in the spinal cord. Elevated expression of either Foxp represses the expression of a key component of adherens junctions, N-cadherin, and promotes the detachment of differentiating neurons from the neuroepithelium. Conversely, inactivation of Foxp2 and Foxp4 function in both chick and mouse results in a spectrum of neural tube defects associated with neuroepithelial disorganization and enhanced progenitor maintenance. Together, these data reveal a Foxp-based transcriptional mechanism that regulates the integrity and cytoarchitecture of neuroepithelial progenitors. 10.1016/j.neuron.2012.02.024
    Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus. Hollinshead Michael,Johns Helen L,Sayers Charlotte L,Gonzalez-Lopez Claudia,Smith Geoffrey L,Elliott Gillian The EMBO journal Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)-infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface. Consistent with this, capsids did not colocalise with either the trans-Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to <1%, resulting in aberrant localisation of capsids. These results suggest that endocytosis from the PM into endocytic tubules provides the main source of membrane for HSV1, and reveal a new mechanism for virus exploitation of the endocytic pathway. 10.1038/emboj.2012.262
    Nuclear envelope-associated dynein drives prophase centrosome separation and enables Eg5-independent bipolar spindle formation. Raaijmakers Jonne A,van Heesbeen Roy G H P,Meaders Johnathan L,Geers Erica F,Fernandez-Garcia Belen,Medema René H,Tanenbaum Marvin E The EMBO journal The microtubule motor protein kinesin-5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti-cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5-independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)-associated dynein that drives centrosome separation in prophase. This NE-dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5-dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin-5 inhibitors. 10.1038/emboj.2012.272
    RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids. Abdullah Zeinab,Schlee Martin,Roth Susanne,Mraheil Mobarak Abu,Barchet Winfried,Böttcher Jan,Hain Torsten,Geiger Sergej,Hayakawa Yoshihiro,Fritz Jörg H,Civril Filiz,Hopfner Karl-Peter,Kurts Christian,Ruland Jürgen,Hartmann Gunther,Chakraborty Trinad,Knolle Percy A The EMBO journal Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria. 10.1038/emboj.2012.274
    Nuclear export of histone deacetylase 7 during thymic selection is required for immune self-tolerance. Kasler Herbert G,Lim Hyung W,Mottet Denis,Collins Amy M,Lee Intelly S,Verdin Eric The EMBO journal Histone deacetylase 7 (HDAC7) is a T-cell receptor (TCR) signal-dependent regulator of differentiation that is highly expressed in CD4/CD8 double-positive (DP) thymocytes. Here, we examine the effect of blocking TCR-dependent nuclear export of HDAC7 during thymic selection, through expression of a signal-resistant mutant of HDAC7 (HDAC7-ΔP) in thymocytes. We find that HDAC7-ΔP transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection-associated gene expression programme in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self-tolerance. 10.1038/emboj.2012.295
    IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Krausgruber Thomas,Blazek Katrina,Smallie Tim,Alzabin Saba,Lockstone Helen,Sahgal Natasha,Hussell Tracy,Feldmann Marc,Udalova Irina A Nature immunology Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor. 10.1038/ni.1990
    Fate mapping of IL-17-producing T cells in inflammatory responses. Hirota Keiji,Duarte João H,Veldhoen Marc,Hornsby Eve,Li Ying,Cua Daniel J,Ahlfors Helena,Wilhelm Christoph,Tolaini Mauro,Menzel Ursula,Garefalaki Anna,Potocnik Alexandre J,Stockinger Brigitta Nature immunology Here we describe a reporter mouse strain designed to map the fate of cells that have activated interleukin 17A (IL-17A). We found that IL-17-producing helper T cells (T(H)17 cells) had distinct plasticity in different inflammatory settings. Chronic inflammatory conditions in experimental autoimmune encephalomyelitis (EAE) caused a switch to alternative cytokines in T(H)17 cells, whereas acute cutaneous infection with Candida albicans did not result in the deviation of T(H)17 cells to the production of alternative cytokines, although IL-17A production was shut off in the course of the infection. During the development of EAE, interferon-γ (IFN-γ) and other proinflammatory cytokines in the spinal cord were produced almost exclusively by cells that had produced IL-17 before their conversion by IL-23 ('ex-T(H)17 cells'). Thus, this model allows the actual functional fate of effector T cells to be related to T(H)17 developmental origin regardless of IL-17 expression. 10.1038/ni.1993
    p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis. Matin Rubeta N,Chikh Anissa,Chong Stephanie Law Pak,Mesher David,Graf Manuela,Sanza' Paolo,Senatore Valentina,Scatolini Maria,Moretti Francesca,Leigh Irene M,Proby Charlotte M,Costanzo Antonio,Chiorino Giovanna,Cerio Rino,Harwood Catherine A,Bergamaschi Daniele The Journal of experimental medicine The role of apoptosis in melanoma pathogenesis and chemoresistance is poorly characterized. Mutations in TP53 occur infrequently, yet the TP53 apoptotic pathway is often abrogated. This may result from alterations in TP53 family members, including the TP53 homologue TP63. Here we demonstrate that TP63 has an antiapoptotic role in melanoma and is responsible for mediating chemoresistance. Although p63 was not expressed in primary melanocytes, up-regulation of p63 mRNA and protein was observed in melanoma cell lines and clinical samples, providing the first evidence of significant p63 expression in this lineage. Upon genotoxic stress, endogenous p63 isoforms were stabilized in both nuclear and mitochondrial subcellular compartments. Our data provide evidence of a physiological interaction between p63 with p53 whereby translocation of p63 to the mitochondria occurred through a codependent process with p53, whereas accumulation of p53 in the nucleus was prevented by p63. Using RNA interference technology, both isoforms of p63 (TA and ΔNp63) were demonstrated to confer chemoresistance, revealing a novel oncogenic role for p63 in melanoma cells. Furthermore, expression of p63 in both primary and metastatic melanoma clinical samples significantly correlated with melanoma-specific deaths in these patients. Ultimately, these observations provide a possible explanation for abrogation of the p53-mediated apoptotic pathway in melanoma, implicating novel approaches aimed at sensitizing melanoma to therapeutic agents. 10.1084/jem.20121439
    Targeting of porous hybrid silica nanoparticles to cancer cells. Rosenholm Jessica M,Meinander Annika,Peuhu Emilia,Niemi Rasmus,Eriksson John E,Sahlgren Cecilia,Lindén Mika ACS nano Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of poly(ethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in many cancer cells as compared to normal cells, folic acid was used as the targeting ligand. The internalization of the particles in cell lines expressing different levels of folate receptors was studied. Flow cytometry was used to quantify the mean number of nanoparticles internalized per cell. Five times more particles were internalized by cancer cells expressing folate receptors as compared to the normal cells expressing low levels of the receptor. Not only the number of nanoparticles internalized per cell, but also the fraction of cells that had internalized nanoparticles was higher. The total number of particles internalized by the cancer cells was, therefore, about an order of magnitude higher than the total number of particles internalized by normal cells, a difference high enough to be of significant biological importance. In addition, the biospecifically tagged hybrid PEI-silica particles were shown to be noncytotoxic and able to specifically target folate receptor-expressing cancer cells also under coculture conditions. 10.1021/nn800781r
    Salmonella induces flagellin- and MyD88-dependent migration of bacteria-capturing dendritic cells into the gut lumen. Arques Juan L,Hautefort Isabelle,Ivory Kamal,Bertelli Eugenio,Regoli Marì,Clare Simon,Hinton Jay C D,Nicoletti Claudio Gastroenterology BACKGROUND & AIMS:Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella, by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella. METHODS:The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-DeltaSalmonella pathogenicity island (SPI) 1 or Escherichia coli DH5alpha by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (DeltafliC DeltafljB)- and SPI1-SPI2 (DeltaSPI1 DeltassrA)-deficient Salmonella, flagellated E coli K12, and MyD88 mice. RESULTS:Salmonella DeltaSPI1 induced migration of CD11c(+)CX(3)CR1(+)MHCII(+)CD11b(-)CD8alpha(-) DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella, soluble flagellin, and E coli DH5alpha or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella-induced damage of the epithelium. CONCLUSIONS:DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella-capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella-capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection. 10.1053/j.gastro.2009.04.010
    Optimization of gene expression through divergent mutational paths. Chou Hsin-Hung,Marx Christopher J Cell reports Adaptation under similar selective pressure often leads to comparable phenotypes. A longstanding question is whether such phenotypic repeatability entails similar (parallelism) or different genotypic changes (convergence). To better understand this, we characterized mutations that optimized expression of a plasmid-borne metabolic pathway during laboratory evolution of a bacterium. Expressing these pathway genes was essential for growth but came with substantial costs. Starting from overexpression, replicate populations founded by this bacterium all evolved to reduce expression. Despite this phenotypic repetitiveness, the underlying mutational spectrum was highly diverse. Analysis of these plasmid mutations identified three distinct means to modulate gene expression: (1) reducing the gene copy number, (2) lowering transcript stability, and (3) integration of the pathway-bearing plasmid into the host genome. Our study revealed diverse molecular changes beneath convergence to a simple phenotype. This complex genotype-phenotype mapping presents a challenge to inferring genetic evolution based solely on phenotypic changes. 10.1016/j.celrep.2011.12.003
    Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nakahira Kiichi,Haspel Jeffrey Adam,Rathinam Vijay A K,Lee Seon-Jin,Dolinay Tamas,Lam Hilaire C,Englert Joshua A,Rabinovitch Marlene,Cernadas Manuela,Kim Hong Pyo,Fitzgerald Katherine A,Ryter Stefan W,Choi Augustine M K Nature immunology Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity. 10.1038/ni.1980
    Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Efe Jem A,Hilcove Simon,Kim Janghwan,Zhou Hongyan,Ouyang Kunfu,Wang Gang,Chen Ju,Ding Sheng Nature cell biology Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm. 10.1038/ncb2164
    Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5. Yang Xiang-Ping,Ghoreschi Kamran,Steward-Tharp Scott M,Rodriguez-Canales Jaime,Zhu Jinfang,Grainger John R,Hirahara Kiyoshi,Sun Hong-Wei,Wei Lai,Vahedi Golnaz,Kanno Yuka,O'Shea John J,Laurence Arian Nature immunology Interleukin 2 (IL-2), a cytokine linked to human autoimmune disease, limits IL-17 production. Here we found that deletion of the gene encoding the transcription factor STAT3 in T cells abrogated IL-17 production and attenuated autoimmunity associated with IL-2 deficiency. Whereas STAT3 induced IL-17 and the transcription factor RORγt and inhibited the transcription factor Foxp3, IL-2 inhibited IL-17 independently of Foxp3 and RORγt. STAT3 and STAT5 bound to multiple common sites across the locus encoding IL-17. The induction of STAT5 binding by IL-2 was associated with less binding of STAT3 at these sites and the inhibition of associated active epigenetic marks. 'Titration' of the relative activation of STAT3 and STAT5 modulated the specification of cells to the IL-17-producing helper T cell (T(H)17 cell) subset. Thus, the balance rather than the absolute magnitude of these signals determined the propensity of cells to make a key inflammatory cytokine. 10.1038/ni.1995
    Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint. Yang Chunying,Tang Xi,Guo Xiaojing,Niikura Yohei,Kitagawa Katsumi,Cui Kemi,Wong Stephen T C,Fu Li,Xu Bo Molecular cell The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. 10.1016/j.molcel.2011.09.016
    PGC-1α, a key modulator of p53, promotes cell survival upon metabolic stress. Sen Nirmalya,Satija Yatendra Kumar,Das Sanjeev Molecular cell Metabolic stress results in p53 activation, which can trigger cell-cycle arrest, ROS clearance, or apoptosis. However, what determines the p53-mediated cell fate decision upon metabolic stress is not very well understood. We show here that PGC-1α binds to p53 and modulates its transactivation function, resulting in preferential transactivation of proarrest and metabolic target genes. Thus glucose starvation results in p53-dependent cell-cycle arrest and ROS clearance, but abrogation of PGC-1α expression results in extensive apoptosis. Additionally, prolonged starvation results in PGC-1α degradation concomitant with induction of apoptosis. We have also identified RNF2, a Polycomb group (PcG) protein, as the cognate E3 ubiquitin ligase. Starvation of mice where PGC-1α expression is abrogated results in loss of p53-mediated ROS clearance, enhanced p53-dependent apoptosis, and consequent severe liver atrophy. These findings provide key insights into the role of PGC-1α in regulating p53-mediated cell fate decisions in response to metabolic stress. 10.1016/j.molcel.2011.08.044
    Wheat Consumption Aggravates Colitis in Mice via Amylase Trypsin Inhibitor-mediated Dysbiosis. Pickert Geethanjali,Wirtz Stefan,Matzner Johannes,Ashfaq-Khan Muhammad,Heck Rosario,Rosigkeit Sebastian,Thies Dorothe,Surabattula Rambabu,Ehmann Dirk,Wehkamp Jan,Aslam Misbah,He Guiwei,Weigert Andreas,Foerster Friedrich,Klotz Luisa,Frick Julia-Stefanie,Becker Christoph,Bockamp Ernesto,Schuppan Detlef Gastroenterology BACKGROUND & AIMS:Wheat has become the world's major staple and its consumption correlates with prevalence of noncommunicable disorders such as inflammatory bowel diseases. Amylase trypsin inhibitors (ATIs), a component of wheat, activate the intestine's innate immune response via toll-like receptor 4 (TLR4). We investigated the effects of wheat and ATIs on severity of colitis and fecal microbiota in mice. METHODS:C57BL/6 wild-type and Tlr4 mice were fed wheat- or ATI-containing diets or a wheat-free (control) diet and then given dextran sodium sulfate to induce colitis; we also studied Il10 mice, which develop spontaneous colitis. Changes in fecal bacteria were assessed by taxa-specific quantitative polymerase chain reaction and 16S ribosomal RNA metagenomic sequencing. Feces were collected from mice on wheat-containing, ATI-containing, control diets and transplanted to intestines of mice with and without colitis on control or on ATI-containing diets. Intestinal tissues were collected and analyzed by histology, immunohistochemistry, and flow cytometry. Bacteria with reported immunomodulatory effects were incubated with ATIs and analyzed in radial diffusion assays. RESULTS:The wheat- or ATI-containing diets equally increased inflammation in intestinal tissues of C57BL/6 mice with colitis, compared with mice on control diets. The ATI-containing diet promoted expansion of taxa associated with development of colitis comparable to the wheat-containing diet. ATIs inhibited proliferation of specific human commensal bacteria in radial diffusion assays. Transplantation of microbiota from feces of mice fed the wheat- or ATI-containing diets to intestines of mice on control diets increased the severity of colitis in these mice. The ATI-containing diet did not increase the severity of colitis in Tlr4 mice. CONCLUSIONS:Consumption of wheat or wheat ATIs increases intestinal inflammation in mice with colitis, via TLR4, and alters their fecal microbiota. Wheat-based, ATI-containing diets therefore activate TLR4 signaling and promote intestinal dysbiosis. 10.1053/j.gastro.2020.03.064
    Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop. Aceto Nicola,Sausgruber Nina,Brinkhaus Heike,Gaidatzis Dimos,Martiny-Baron Georg,Mazzarol Giovanni,Confalonieri Stefano,Quarto Micaela,Hu Guang,Balwierz Piotr J,Pachkov Mikhail,Elledge Stephen J,van Nimwegen Erik,Stadler Michael B,Bentires-Alj Mohamed Nature medicine New cancer therapies are likely to arise from an in-depth understanding of the signaling networks influencing tumor initiation, progression and metastasis. We show a fundamental role for Src-homology 2 domain-containing phosphatase 2 (SHP2) in these processes in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancers. Knockdown of SHP2 eradicated breast tumor-initiating cells in xenograft models, and SHP2 depletion also prevented invasion in three-dimensional cultures and in a transductal invasion assay in vivo. Notably, SHP2 knockdown in established breast tumors blocked their growth and reduced metastasis. Mechanistically, SHP2 activated stemness-associated transcription factors, including v-myc myelocytomatosis viral oncogene homolog (c-Myc) and zinc finger E-box binding homeobox 1 (ZEB1), which resulted in the repression of let-7 microRNA and the expression of a set of 'SHP2 signature' genes. We found these genes to be simultaneously activated in a large subset of human primary breast tumors that are associated with invasive behavior and poor prognosis. These results provide new insights into the signaling cascades influencing tumor-initiating cells as well as a rationale for targeting SHP2 in breast cancer. 10.1038/nm.2645
    Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia. Schenk Tino,Chen Weihsu Claire,Göllner Stefanie,Howell Louise,Jin Liqing,Hebestreit Katja,Klein Hans-Ulrich,Popescu Andreea C,Burnett Alan,Mills Ken,Casero Robert A,Marton Laurence,Woster Patrick,Minden Mark D,Dugas Martin,Wang Jean C Y,Dick John E,Müller-Tidow Carsten,Petrie Kevin,Zelent Arthur Nature medicine Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4(me2)) across the genome, but it did increase H3K4(me2) and expression of myeloid-differentiation-associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML. 10.1038/nm.2661
    Immunomodulatory role of proteinase-activated receptor-2. Crilly Anne,Palmer Helen,Nickdel Mohammad B,Dunning Lynette,Lockhart John C,Plevin Robin,McInnes Iain B,Ferrell William R Annals of the rheumatic diseases OBJECTIVE:Proteinase-activated receptor-2 (PAR(2)) has been implicated in inflammatory articular pathology. Using the collagen-induced arthritis model (CIA) the authors have explored the capacity of PAR(2) to regulate adaptive immune pathways that could promote autoimmune mediated articular damage. METHODS:Using PAR(2) gene deletion and other approaches to inhibit or prevent PAR(2) activation, the development and progression of CIA were assessed via clinical and histological scores together with ex vivo immune analyses. RESULTS:The progression of CIA, assessed by arthritic score and histological assessment of joint damage, was significantly (p<0.0001) abrogated in PAR(2) deficient mice or in wild-type mice administered either a PAR(2) antagonist (ENMD-1068) or a PAR(2) neutralising antibody (SAM11). Lymph node derived cell suspensions from PAR(2) deficient mice were found to produce significantly less interleukin (IL)-17 and IFNγ in ex vivo recall collagen stimulation assays compared with wild-type littermates. In addition, substantial inhibition of TNFα, IL-6, IL-1β and IL-12 along with GM-CSF and MIP-1α was observed. However, spleen and lymph node histology did not differ between groups nor was any difference detected in draining lymph node cell subsets. Anticollagen antibody titres were significantly lower in PAR(2) deficient mice. CONCLUSION:These data support an important role for PAR(2) in the pathogenesis of CIA and suggest an immunomodulatory role for this receptor in an adaptive model of inflammatory arthritis. PAR(2) antagonism may offer future potential for the management of inflammatory arthritides in which a proteinase rich environment prevails. 10.1136/annrheumdis-2011-200869
    Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice. Yarandi Shadi S,Kulkarni Subhash,Saha Monalee,Sylvia Kristyn E,Sears Cynthia L,Pasricha Pankaj J Gastroenterology BACKGROUND & AIMS:The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice. METHODS:We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creER:tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creER:tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry. RESULTS:Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice. CONCLUSIONS:In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors. 10.1053/j.gastro.2020.03.050
    Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses. Chen Bo,Vousden Katherine A,Naiman Brian,Turman Sean,Sun Hong,Wang Shu,Vinall Lisa M K,Kemp Benjamin P,Kasturiangan Srinath,Rees D Gareth,Grant Ethan,Hinrichs Mary Jane,Eck Steven,DiGiandomenico Antonio,Jack Borrok M,Ly Neang,Xiong Ximing,Gonzalez Carlos,Morehouse Christopher,Wang Yue,Zhou Yebin,Cann Jennifer,Zhao Weiguang,Koelkebeck Holly,Okubo Koshu,Mayadas Tanya N,Howe David,Griffiths Janet,Kolbeck Roland,Herbst Ronald,Sims Gary P Annals of the rheumatic diseases OBJECTIVE:Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS:VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS:We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS:VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases. 10.1136/annrheumdis-2018-213523
    Stitched α-helical peptides via bis ring-closing metathesis. Hilinski Gerard J,Kim Young-Woo,Hong Jooyeon,Kutchukian Peter S,Crenshaw Charisse M,Berkovitch Shaunna S,Chang Andrew,Ham Sihyun,Verdine Gregory L Journal of the American Chemical Society Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides. 10.1021/ja505141j
    Triple-Labeling of Polymer-Coated Quantum Dots and Adsorbed Proteins for Tracing their Fate in Cell Cultures. Carrillo-Carrion Carolina,Bocanegra Ana I,Arnaiz Blanca,Feliu Neus,Zhu Dingcheng,Parak Wolfgang J ACS nano Colloidal CdSe/ZnS quantum dots were water solubilized by overcoating with an amphiphilic polymer. Human serum albumin (HSA) as a model protein was either adsorbed or chemically linked to the surface of the polymer-coated quantum dots. As the quantum dots are intrinsically fluorescent, and as the polymer coating and the HSA were fluorescent labeled, the final nanoparticle had three differently fluorescent components: the quantum dot core, the polymer shell, and the human serum albumin corona. Cells were incubated with these hybrid nanoparticles, and after removal of non-internalized nanoparticles, exocytosis of the three components of the nanoparticles was observed individually by flow cytometry and confocal microscopy. The data indicate that HSA is partly transported with the underlying polymer-coated quantum dots into cells. Upon desorption of proteins, those initially adsorbed to the quantum dots remain longer inside cells compared to free proteins. Part of the polymer shell is released from the quantum dots by enzymatic degradation, which is on a slower time scale than protein desorption. Data are quantitatively analyzed, and experimental pitfalls, such as the impact of cell proliferation and fluorescence quenching, are discussed. 10.1021/acsnano.9b00728
    IL-17A produced by αβ T cells drives airway hyper-responsiveness in mice and enhances mouse and human airway smooth muscle contraction. Kudo Makoto,Melton Andrew C,Chen Chun,Engler Mary B,Huang Katherine E,Ren Xin,Wang Yanli,Bernstein Xin,Li John T,Atabai Kamran,Huang Xiaozhu,Sheppard Dean Nature medicine Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle. 10.1038/nm.2684
    Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation. Hill David A,Siracusa Mark C,Abt Michael C,Kim Brian S,Kobuley Dmytro,Kubo Masato,Kambayashi Taku,Larosa David F,Renner Ellen D,Orange Jordan S,Bushman Frederic D,Artis David Nature medicine Commensal bacteria that colonize mammalian barrier surfaces are reported to influence T helper type 2 (T(H)2) cytokine-dependent inflammation and susceptibility to allergic disease, although the mechanisms that underlie these observations are poorly understood. In this report, we find that deliberate alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum IgE concentrations, increased steady-state circulating basophil populations and exaggerated basophil-mediated T(H)2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Furthermore, B cell-intrinsic expression of myeloid differentiation factor 88 (MyD88) was required to limit serum IgE concentrations and circulating basophil populations in mice. Commensal-derived signals were found to influence basophil development by limiting proliferation of bone marrow-resident precursor populations. Collectively, these results identify a previously unrecognized pathway through which commensal-derived signals influence basophil hematopoiesis and susceptibility to T(H)2 cytokine-dependent inflammation and allergic disease. 10.1038/nm.2657
    Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Ajami Bahareh,Bennett Jami L,Krieger Charles,McNagny Kelly M,Rossi Fabio M V Nature neuroscience In multiple sclerosis and the experimental autoimmune encephalitis (EAE) mouse model, two pools of morphologically indistinguishable phagocytic cells, microglia and inflammatory macrophages, accrue from proliferating resident precursors and recruitment of blood-borne progenitors, respectively. Whether these cell types are functionally equivalent is hotly debated, but is challenging to address experimentally. Using a combination of parabiosis and myeloablation to replace circulating progenitors without affecting CNS-resident microglia, we found a strong correlation between monocyte infiltration and progression to the paralytic stage of EAE. Inhibition of chemokine receptor-dependent recruitment of monocytes to the CNS blocked EAE progression, suggesting that these infiltrating cells are essential for pathogenesis. Finally, we found that, although microglia can enter the cell cycle and return to quiescence following remission, recruited monocytes vanish, and therefore do not ultimately contribute to the resident microglial pool. In conclusion, we identified two distinct subsets of myelomonocytic cells with distinct roles in neuroinflammation and disease progression. 10.1038/nn.2887
    Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers. Dieu-Nosjean Marie-Caroline,Giraldo Nicolas A,Kaplon Hélène,Germain Claire,Fridman Wolf Herman,Sautès-Fridman Catherine Immunological reviews The characterization of the microenvironment of human tumors led to the description of tertiary lymphoid structures (TLS) characterized by mature dendritic cells in a T-cell zone adjacent to B-cell follicle including a germinal center. TLS represent sites of lymphoid neogenesis that develop in most solid cancers. Analysis of the current literature shows that the TLS presence is associated with a favorable clinical outcome for cancer patients, regardless of the approach used to quantify TLS and the stage of the disease. Using several approaches that combine immunohistochemistry, gene expression assays, and flow cytometry on large series of lung tumors, our work demonstrated that TLS are important sites for the initiation and/or maintenance of the local and systemic T- and B-cell responses against tumors. Surrounded by high endothelial venules, they represent a privileged area for the recruitment of lymphocytes into tumors and generation of central-memory T and B cells that circulate and limit cancer progression. TLS can be considered as a novel biomarker to stratify the overall survival risk of untreated cancer patients and as a marker of efficient immunotherapies. The induction and manipulation of cancer-associated TLS using drug agonists and/or biotherapies should open new avenues to treat cancer patients. 10.1111/imr.12405
    Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet (London, England) BACKGROUND:A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. METHODS:We did a dose-escalation, single-centre, open-label, non-randomised, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults aged between 18 and 60 years were sequentially enrolled and allocated to one of three dose groups (5 × 10, 1 × 10, and 1·5 × 10 viral particles) to receive an intramuscular injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralising antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralisation and pseudovirus neutralisation tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127. FINDINGS:Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36·3 years) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the analysis. At least one adverse reaction within the first 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]. Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralising antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination. INTERPRETATION:The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation. FUNDING:National Key R&D Program of China, National Science and Technology Major Project, and CanSino Biologics. 10.1016/S0140-6736(20)31208-3
    Catalase-Integrated Hyaluronic Acid as Nanocarriers for Enhanced Photodynamic Therapy in Solid Tumor. Phua Soo Zeng Fiona,Yang Guangbao,Lim Wei Qi,Verma Apoorva,Chen Hongzhong,Thanabalu Thirumaran,Zhao Yanli ACS nano Photodynamic therapy (PDT) as a treatment method has many advantages such as minimal invasiveness, repeatable dosage, and low systemic toxicity. Issues with conventional PDT agents include the limited availability of endogenous oxygen and difficulty in accumulation at the tumor site, which has hindered the successful treatment of tumors. Herein, we developed catalase-encapsulated hyaluronic-acid-based nanoparticles loaded with adamantane-modified photosensitizer for enhanced PDT of solid tumors. Chlorin e6 (Ce6) as the photosensitizer was modified with adamantane to yield adamantane-modified Ce6 (aCe6). The obtained nanosystem (HA-CAT@aCe6) could target overly expressed CD44 receptors on cancer cells, supplying oxygen by converting endogenous hydrogen peroxide (HO) to oxygen, and improving PDT efficacy upon light irradiation. HA-CAT@aCe6 nanoparticles showed high colloidal stability and monodispersity in aqueous solution. The uptake and targeting property of HA-CAT@aCe6 were demonstrated by confocal microscopy and flow cytometry in the MDA-MB-231 cell line possessing overly expressed CD44 receptors. The encapsulated catalase was able to decompose the endogenous HO to generate O in situ for relieving hypoxia in cells incubated under hypoxic conditions. Cell viability assays indicated that HA-CAT@aCe6 possessed minimal cytotoxicity in the dark, while presenting high cellular toxicity under 660 nm light irradiation at normoxic conditions. As a result of the catalase capability in relieving hypoxia, HA-CAT@aCe6 also exhibited high cellular cytotoxicity under hypoxic condition. In vivo experiments revealed selective tumor accumulation of HA-CAT@aCe6 in MDA-MB-231 tumor bearing nude mice. Significant tumor regression was observed after intravenous injection of HA-CAT@aCe6 under light irradiation in comparison to the control system without loading catalase. Thus, HA-CAT@aCe6 demonstrated a great potential in overcoming hypoxia for targeted PDT. 10.1021/acsnano.9b01087
    Impact of heterogeneity of human peripheral blood monocyte subsets on myocardial salvage in patients with primary acute myocardial infarction. Tsujioka Hiroto,Imanishi Toshio,Ikejima Hideyuki,Kuroi Akio,Takarada Shigeho,Tanimoto Takashi,Kitabata Hironori,Okochi Keishi,Arita Yu,Ishibashi Kohei,Komukai Kenichi,Kataiwa Hideaki,Nakamura Nobuo,Hirata Kumiko,Tanaka Atsuhsi,Akasaka Takashi Journal of the American College of Cardiology OBJECTIVES:We examined whether distinct monocyte subsets contribute in specific ways to myocardial salvage in patients with acute myocardial infarction (AMI). BACKGROUND:Recent studies have shown that monocytes in human peripheral blood are heterogeneous. METHODS:We studied 36 patients with primary AMI. Peripheral blood sampling was performed 1, 2, 3, 4, 5, 8, and 12 days after AMI onset. Two monocyte subsets (CD14(+)CD16(-) and CD14(+)CD16(+)) were measured by flow cytometry. The extent of myocardial salvage 7 days after AMI was evaluated by cardiovascular magnetic resonance imaging as the difference between myocardium at risk (T2-weighted hyperintense lesion) and myocardial necrosis (delayed gadolinium enhancement). Cardiovascular magnetic resonance imaging was also performed 6 months after AMI. RESULTS:Circulating CD14(+)CD16(-) and CD14(+)CD16(+) monocytes increased in AMI patients, peaking on days 3 and 5 after onset, respectively. Importantly, the peak levels of CD14(+)CD16(-) monocytes, but not those of CD14(+)CD16(+) monocytes, were significantly negatively associated with the extent of myocardial salvage. We also found that the peak levels of CD14(+)CD16(-) monocytes, but not those of CD14(+)CD16(+) monocytes, were negatively correlated with recovery of left ventricular ejection fraction 6 months after infarction. CONCLUSIONS:The peak levels of CD14(+)CD16(-) monocytes affect both the extent of myocardial salvage and the recovery of left ventricular function after AMI, indicating that the manipulation of monocyte heterogeneity could be a novel therapeutic target for salvaging ischemic damage. 10.1016/j.jacc.2009.04.021
    A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity. Liang Joe C,Chang Andrew L,Kennedy Andrew B,Smolke Christina D Nucleic acids research Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 10(6) variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks. 10.1093/nar/gks636
    OCT4/SOX2-independent Nanog autorepression modulates heterogeneous Nanog gene expression in mouse ES cells. Navarro Pablo,Festuccia Nicola,Colby Douglas,Gagliardi Alessia,Mullin Nicholas P,Zhang Wensheng,Karwacki-Neisius Violetta,Osorno Rodrigo,Kelly David,Robertson Morag,Chambers Ian The EMBO journal NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self-renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self-renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog-centred network using inducible NANOG gain- and loss-of-function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2-independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog-centred autorepressive loop. Therefore, cellular variability in self-renewal efficiency is an emergent property of the pluripotency gene regulatory network. 10.1038/emboj.2012.321
    Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness. Lapouge Gaëlle,Beck Benjamin,Nassar Dany,Dubois Christine,Dekoninck Sophie,Blanpain Cédric The EMBO journal Cancer stem cells have been described in various cancers including squamous tumours of the skin by their ability to reform secondary tumours upon transplantation into immunodeficient mice. Here, we used transplantation of limiting dilution of different populations of FACS-isolated tumour cells from four distinct mouse models of squamous skin tumours to investigate the frequency of tumour propagating cells (TPCs) at different stages of tumour progression. We found that benign papillomas, despite growing rapidly in vivo and being clonogenic in vitro, reformed secondary tumours upon transplantation at very low frequency and only when tumour cells were co-transplanted together with tumour-associated fibroblasts or endothelial cells. In two models of skin squamous cell carcinoma (SCC), TPCs increased with tumour invasiveness. Interestingly, the frequency of TPCs increased in CD34(HI) but not in CD34(LO) SCC cells with serial transplantations, while the two populations initially gave rise to secondary tumours with the same frequency. Our results illustrate the progressive increase of squamous skin TPCs with tumour progression and invasiveness and reveal that serial transplantation may be required to define the long-term renewal potential of TPCs. 10.1038/emboj.2012.312
    Distinct signalling pathways control Toxoplasma egress and host-cell invasion. Lourido Sebastian,Tang Keliang,Sibley L David The EMBO journal Calcium signalling coordinates motility, cell invasion, and egress by apicomplexan parasites, yet the key mediators that transduce these signals remain largely unknown. One underlying assumption is that invasion into and egress from the host cell depend on highly similar systems to initiate motility. Using a chemical-genetic approach to specifically inhibit select calcium-dependent kinases (CDPKs), we instead demonstrate that these pathways are controlled by different kinases: both TgCDPK1 and TgCDPK3 were required during ionophore-induced egress, but only TgCDPK1 was required during invasion. Similarly, microneme secretion, which is necessary for motility during both invasion and egress, universally depended on TgCDPK1, but only exhibited TgCDPK3 dependence when triggered by certain stimuli. We also demonstrate that egress likely comes under a further level of control by cyclic GMP-dependent protein kinase and that its activation can induce egress and partially compensate for the inhibition of TgCDPK3. These results demonstrate that separate signalling pathways are integrated to regulate motility in response to the different signals that promote invasion or egress during infection by Toxoplasma gondii. 10.1038/emboj.2012.299
    Control of the development of CD8αα+ intestinal intraepithelial lymphocytes by TGF-β. Konkel Joanne E,Maruyama Takashi,Carpenter Andrea C,Xiong Yumei,Zamarron Brian F,Hall Bradford E,Kulkarni Ashok B,Zhang Pin,Bosselut Remy,Chen WanJun Nature immunology The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell-specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4⁻CD8⁻TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells. 10.1038/ni.1997
    The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Cretney Erika,Xin Annie,Shi Wei,Minnich Martina,Masson Frederick,Miasari Maria,Belz Gabrielle T,Smyth Gordon K,Busslinger Meinrad,Nutt Stephen L,Kallies Axel Nature immunology Regulatory T cells (T(reg) cells) are required for peripheral tolerance. Evidence indicates that T(reg) cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of T(reg) cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector T(reg) cells. Thus, our study defines a differentiation pathway that leads to the acquisition of T(reg) cell effector functions and requires both IRF4 and Blimp-1. 10.1038/ni.2006
    Maturation of effector regulatory T cells. Ohkura Naganari,Sakaguchi Shimon Nature immunology 10.1038/ni0411-283
    Notch Signaling Mediates Differentiation in Barrett's Esophagus and Promotes Progression to Adenocarcinoma. Gastroenterology BACKGROUND & AIMS:Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis. METHODS:We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5 (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB. RESULTS:Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5 cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5 cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5 cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation. CONCLUSIONS:Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE. 10.1053/j.gastro.2020.04.033
    Effect of nanoparticles on the cell life cycle. Mahmoudi Morteza,Azadmanesh Kayhan,Shokrgozar Mohammad A,Journeay W Shane,Laurent Sophie Chemical reviews 10.1021/cr1003166
    NFIL3 mutations alter immune homeostasis and sensitise for arthritis pathology. Schlenner Susan,Pasciuto Emanuela,Lagou Vasiliki,Burton Oliver,Prezzemolo Teresa,Junius Steffie,Roca Carlos P,Seillet Cyril,Louis Cynthia,Dooley James,Luong Kylie,Van Nieuwenhove Erika,Wicks Ian P,Belz Gabrielle,Humblet-Baron Stéphanie,Wouters Carine,Liston Adrian Annals of the rheumatic diseases OBJECTIVES: is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans. METHODS:Here, we characterised a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model. RESULTS:The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a preclinical model, where Nfil3-deficient mice upregulated IL-1β production, with more severe arthritis symptoms on disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1β as a pivotal cytokine. CONCLUSIONS:NFIL3 mutation can sensitise for arthritis development, in mice and humans, and rewires the innate immune system for IL-1β over-production. 10.1136/annrheumdis-2018-213764
    Molecular profile and proangiogenic activity of the adipose-derived stromal vascular fraction used as an autologous innovative medicinal product in patients with systemic sclerosis. Magalon Jérémy,Velier Mélanie,Simoncini Stéphanie,François Pauline,Bertrand Baptiste,Daumas Aurélie,Benyamine Audrey,Boissier Romain,Arnaud Laurent,Lyonnet Luc,Fernandez Samantha,Dignat-George Françoise,Casanova Dominique,Guillet Benjamin,Granel Brigitte,Paul Pascale,Sabatier Florence Annals of the rheumatic diseases OBJECTIVE:The autologous stromal vascular fraction (SVF) from adipose tissue is an alternative to cultured adipose-derived stem cells for use in regenerative medicine and represents a promising therapy for vasculopathy and hand disability in systemic sclerosis (SSc). However, the bioactivity of autologous SVF is not documented in this disease context. This study aimed to compare the molecular and functional profiles of the SVF-based medicinal product obtained from SSc and healthy subjects. METHODS:Good manufacturing practice (GMP)-grade SVF from 24 patients with SSc and 12 healthy donors (HD) was analysed by flow cytometry to compare the distribution of the CD45- and CD45+ haematopoietic cell subsets. The ability of SVF to form a vascular network was assessed using Matrigel in vivo assay. The transcriptomic and secretory profiles of the SSc-SVF were assessed by RNA sequencing and multiplex analysis, respectively, and were compared with the HD-SVF. RESULTS:The distribution of the leucocyte, endothelial, stromal, pericyte and transitional cell subsets was similar for SSc-SVF and HD-SVF. SSc-SVF retained its vasculogenic capacity, but the density of neovessels formed in SVF-loaded Matrigel implanted in nude mice was slightly decreased compared with HD-SVF. SSc-SVF displayed a differential molecular signature reflecting deregulation of angiogenesis, endothelial activation and fibrosis. CONCLUSIONS:Our study provides the first evidence that SSc does not compromise the vascular repair capacity of SVF, supporting its use as an innovative autologous biotherapy. The characterisation of the specific SSc-SVF molecular profile provides new perspectives for delineating markers of the potency of SVF and its targets for the treatment of SSc. 10.1136/annrheumdis-2018-214218
    Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease: PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development. Circulation BACKGROUND:Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. METHODS:Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. RESULTS:We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. CONCLUSIONS:This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need. 10.1161/CIRCULATIONAHA.119.043724
    Flow cytometry analysis of urothelial cell DNA content according to pathological and clinical data on 100 bladder tumors. Caratero C,Hijazi A,Caratero A,Mazerolles C,Rischmann P,Sarramon J P European urology DNA content of 100 bladder tumors (34 grade I, 42 grade II and 24 grade III, WHO classification) were studied by flow cytometry. Ten normal bladder samples were used as control. The 100 bladder tumors could then be separated into two groups. A first group of 60 tumors (60%) had a unimodal distribution with a diploid peak and a DNA index close to 1.0, 32 grade I, 22 grade II and 6 grade III tumors displayed this pattern as did the 10 normal bladders. The second group (40%) had a bimodal distribution with two peaks, the first one (diploid peak) with a DNA index of 1.0, the second (aneuploid peak) with a DNA index greater than 1.0. Two grade I, 20 grade II and 18 grade III tumors belonged to this group. Frequency of the aneuploid peak increased with tumor grade and infiltration progression. Hence 6% of grade I, 48% of grade II and 75% of grade III tumors showed an aneuploid peak as well as 8% of Pa, 46% of P1, 73% of P2 and 87.5% of P3 stage tumors. This study showed that a good correlation exists between flow-cytometric, pathological and clinical data.
    The Skin You Are In: Design-of-Experiments Optimization of Lipid Nanoparticle Self-Amplifying RNA Formulations in Human Skin Explants. Blakney Anna K,McKay Paul F,Ibarzo Yus Bárbara,Hunter Judith E,Dex Elizabeth A,Shattock Robin J ACS nano Messenger RNA (mRNA) is a promising tool for biotherapeutics, and self-amplifying mRNA (saRNA) is particularly advantageous, because it results in abundant protein expression and production is easily scalable. While mRNA therapeutics have been shown to be highly effective in small animals, the outcomes do not scale linearly when these formulations are translated to dose-escalation studies in humans. Here, we utilize a design of experiments (DoE) approach to optimize the formulation of saRNA lipid nanoparticles in human skin explants. We first observed that luciferase expression from saRNA peaked after 11 days in human skin. Using DoE inputs of complexing lipid identity, lipid nanoparticle dose, lipid concentration, particle concentration, and ratio of zwitterionic to cationic lipids, we optimized the saRNA-induced luciferase expression in skin explants. Lipid identity and lipid concentration were found to be significant parameters in the DoE model, and the optimized formulation resulted in ∼7-fold increase in luciferase expression, relative to initial 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) formulation. Using flow cytometry, we observed that optimized formulations delivered the saRNA to ∼2% of the resident cells in the human skin explants. Although immune cells comprise only 7% of the total population of cells in skin, immune cells were found to express ∼50% of the RNA. This study demonstrates the powerful combination of using a DoE approach paired with clinically relevant human skin explants to optimize nucleic acid formulations. We expect that this system will be useful for optimizing both formulation and molecular designs of clinically translational nucleic acid vaccines and therapeutics. 10.1021/acsnano.9b01774
    Minimal residual disease quantification is an independent predictor of progression-free and overall survival in chronic lymphocytic leukemia: a multivariate analysis from the randomized GCLLSG CLL8 trial. Böttcher Sebastian,Ritgen Matthias,Fischer Kirsten,Stilgenbauer Stephan,Busch Raymonde M,Fingerle-Rowson Günter,Fink Anna Maria,Bühler Andreas,Zenz Thorsten,Wenger Michael Karl,Mendila Myriam,Wendtner Clemens-Martin,Eichhorst Barbara F,Döhner Hartmut,Hallek Michael J,Kneba Michael Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:To determine the clinical significance of flow cytometric minimal residual disease (MRD) quantification in chronic lymphocytic leukemia (CLL) in addition to pretherapeutic risk factors and to compare the prognostic impact of MRD between the arms of the German CLL Study Group CLL8 trial. PATIENTS AND METHODS:MRD levels were prospectively quantified in 1,775 blood and bone marrow samples from 493 patients randomly assigned to receive fludarabine and cyclophosphamide (FC) or FC plus rituximab (FCR). Patients were categorized by MRD into low- (< 10(-4)), intermediate- (≥ 10(-4) to <10(-2)), and high-level (≥ 10(-2)) groups. RESULTS:Low MRD levels during and after therapy were associated with longer progression-free survival (PFS) and overall survival (OS; P < .0001). Median PFS is estimated at 68.7, 40.5, and 15.4 months for low, intermediate, and high MRD levels, respectively, when assessed 2 months after therapy. Compared with patients with low MRD, greater risks of disease progression were associated with intermediate and high MRD levels (hazard ratios, 2.49 and 14.7, respectively; both P < .0001). Median OS was 48.4 months in patients with high MRD and was not reached for lower MRD levels. MRD remained predictive for OS and PFS in multivariate analyses that included the most important pretherapeutic risk markers in CLL. PFS and OS did not differ between treatment arms within each MRD category. However, FCR induced low MRD levels more frequently than FC. CONCLUSION:MRD levels independently predict OS and PFS in CLL. Therefore, MRD quantification might serve as a surrogate marker to assess treatment efficacy in randomized trials before clinical end points can be evaluated. 10.1200/JCO.2011.36.9348
    Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Chao Mark P,Jaiswal Siddhartha,Weissman-Tsukamoto Rachel,Alizadeh Ash A,Gentles Andrew J,Volkmer Jens,Weiskopf Kipp,Willingham Stephen B,Raveh Tal,Park Christopher Y,Majeti Ravindra,Weissman Irving L Science translational medicine Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer. 10.1126/scitranslmed.3001375
    CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs. Sun Jie,Dodd Haley,Moser Emily K,Sharma Rahul,Braciale Thomas J Nature immunology Interleukin (IL)-10 is an important regulatory cytokine that can modulate excessive immune mediated injury. Several distinct cell types have been demonstrated to produce IL-10, including most recently CD8+ cytotoxic T lymphocytes (CTLs) responding to respiratory virus infection. Here we report that CD4+ T cell help in the form of IL-2 is required for IL-10 production by CTLs, but not for the induction of CTL effector cytokines. We show that IL-2 derived from CD4+ helper T cells cooperates with innate immune cell-derived IL-27 to amplify IL-10 production by CTLs through a Blimp-1-dependent mechanism. These findings reveal a previously unrecognized pathway that coordinates signals derived from innate and helper T cells to control the production of a regulatory cytokine by CTLs during acute viral infection. 10.1038/ni.1996
    RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota. Sawa Shinichiro,Lochner Matthias,Satoh-Takayama Naoko,Dulauroy Sophie,Bérard Marion,Kleinschek Melanie,Cua Daniel,Di Santo James P,Eberl Gérard Nature immunology Lymphoid cells that express the nuclear hormone receptor RORγt are involved in containment of the large intestinal microbiota and defense against pathogens through the production of interleukin 17 (IL-17) and IL-22. They include adaptive IL-17-producing helper T cells (T(H)17 cells), as well as innate lymphoid cells (ILCs) such as lymphoid tissue-inducer (LTi) cells and IL-22-producing NKp46+ cells. Here we show that in contrast to T(H)17 cells, both types of RORγt+ ILCs constitutively produced most of the intestinal IL-22 and that the symbiotic microbiota repressed this function through epithelial expression of IL-25. This function was greater in the absence of adaptive immunity and was fully restored and required after epithelial damage, which demonstrates a central role for RORγt+ ILCs in intestinal homeostasis. Our data identify a finely tuned equilibrium among intestinal symbionts, adaptive immunity and RORγt+ ILCs. 10.1038/ni.2002
    The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Delgoffe Greg M,Pollizzi Kristen N,Waickman Adam T,Heikamp Emily,Meyers David J,Horton Maureen R,Xiao Bo,Worley Paul F,Powell Jonathan D Nature immunology The kinase mTOR has emerged as an important regulator of the differentiation of helper T cells. Here we demonstrate that differentiation into the T(H)1 and T(H)17 subsets of helper T cells was selectively regulated by signaling from mTOR complex 1 (mTORC1) that was dependent on the small GTPase Rheb. Rheb-deficient T cells failed to generate T(H)1 and T(H)17 responses in vitro and in vivo and did not induce classical experimental autoimmune encephalomyelitis (EAE). However, they retained their ability to become T(H)2 cells. Alternatively, when mTORC2 signaling was deleted from T cells, they failed to generate T(H)2 cells in vitro and in vivo but preserved their ability to become T(H)1 and T(H)17 cells. Our data identify mechanisms by which two distinct signaling pathways downstream of mTOR regulate helper cell fate in different ways. These findings define a previously unknown paradigm that links T cell differentiation with selective metabolic signaling pathways. 10.1038/ni.2005
    Developing intestinal fortitude. Kappes Dietmar J Nature immunology 10.1038/ni0411-281
    Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway. Alzabin Saba,Abraham Sonya M,Taher Taher E,Palfreeman Andrew,Hull Dobrina,McNamee Kay,Jawad Ali,Pathan Ejaz,Kinderlerer Anne,Taylor Peter C,Williams Richard,Mageed Rizgar Annals of the rheumatic diseases OBJECTIVES:To establish if changes in Th1/Th17 cell populations previously reported in experimental arthritis occur in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor α (TNFα) agents, and whether the therapeutic response to anti-TNFα is compromised in patients and mice because of elevated Th17/IL-17 levels. Finally, to assess the efficacy of combined blockade of anti-TNFα and anti-IL-17 in experimental arthritis. METHODS:A longitudinal study of two independent cohorts (cohort 1, n=24; cohort 2, n=19) of patients with RA treated with anti-TNFα biological agents was carried out to assess their Th17/IL-17 levels before and after the start of anti-TNFα therapy. IL-12/23p40 production was assessed in plasma Peripheral blood lymphocytes (PBLs) and monocytes. Mice with collagen-induced arthritis (CIA) were treated with anti-TNFα alone, anti-IL17 alone or a combination of the two. Efficacy of treatment and response was assessed from changes in Disease Activity Score 28-erythrocyte sedimentation rate scores in patients, and in clinical scores and histological analysis in CIA. RESULTS:Significant increases in circulating Th17 cells were observed in patients after anti-TNFα therapy and this was accompanied by increased production of IL-12/23p40. There was an inverse relationship between baseline Th17 levels and the subsequent response of patients with RA to anti-TNFα therapy. In addition, PBLs from non-responder patients showed evidence of increased IL-17 production. Similarly, in anti-TNFα-treated mice, there was a strong correlation between IL-17 production and clinical score. Finally combined blockade of TNFα and IL-17 in CIA was more effective than monotherapy, particularly with respect to the duration of the therapeutic effect. CONCLUSIONS:These findings, which need to be confirmed in a larger cohort, suggest that a Th17-targeted therapeutic approach may be useful for anti-TNFα non-responder patients or as an adjunct to anti-TNFα therapy, provided that safety concerns can be addressed. 10.1136/annrheumdis-2011-201024
    Nuclear DNA distribution pattern of the parenchymal cells in adenocarcinomas of the pancreas and in chronic pancreatitis. A study of archival specimens using both image and flow cytometry. Weger A R,Falkmer U G,Schwab G,Glaser K,Kemmler G,Bodner E,Auer G U,Mikuz G Gastroenterology The nuclear DNA distribution pattern of the neoplastic parenchymal cells of 100 conventionally formalin-fixed and paraffin-embedded specimens from pancreatic adenocarcinomas and from 8 specimens of chronic pancreatitis was assessed by means of image cytometry. All material originated from pancreatic restrictions. Evaluable DNA histograms could be obtained for 77 carcinomas, and clinical data were available for 71 of these. In these 71 specimens, the nuclear DNA ploidy pattern was also investigated by means of flow cytometry. In 76 of the 77 cases, the image-cytometric DNA ploidy pattern obtained showed a "nondiploid" distribution with modal values as high as 8.5 c. In 21 cases, the neoplastic cells showed modal values in the "triploid" region. The analogous 71 flow-cytometric DNA histograms could only be evaluated in 50 cases because of excessively high amounts of background and/or excessively broad peaks. In 47 cases, the nuclear DNA histogram was nondiploid according to both techniques. The patients with carcinomas whose cell nuclei showed a triploid DNA distribution showed a significantly shorter survival time than those with tumor cell populations of nontriploid DNA distribution patterns. In the 8 specimens of chronic pancreatitis, the parenchymal cells were all equipped with nuclei showing diploid DNA distribution patterns. 10.1016/0016-5085(90)91253-3
    Let-7b and miR-495 stimulate differentiation and prevent metaplasia of pancreatic acinar cells by repressing HNF6. Prévot Pierre-Paul,Augereau Cécile,Simion Alexandru,Van den Steen Géraldine,Dauguet Nicolas,Lemaigre Frédéric P,Jacquemin Patrick Gastroenterology BACKGROUND & AIMS:Diseases of the exocrine pancreas are often associated with perturbed differentiation of acinar cells. MicroRNAs (miRNAs) regulate pancreas development, yet little is known about their contribution to acinar cell differentiation. We aimed to identify miRNAs that promote and control the maintenance of acinar differentiation. METHODS:We studied mice with pancreas- or acinar-specific inactivation of Dicer (Foxa3-Cre/Dicer(loxP/-) mice), combined (or not) with inactivation of hepatocyte nuclear factor (HNF) 6 (Foxa3-Cre/Dicer(loxP/-)/Hnf6-/- mice). The role of specific miRNAs in acinar differentiation was investigated by transfecting cultured cells with miRNA mimics or inhibitors. Pancreatitis-induced metaplasia was investigated in mice after administration of cerulein. RESULTS:Inhibition of miRNA synthesis in acini by inactivation of Dicer and pancreatitis-induced metaplasia were associated with repression of acinar differentiation and with induction of HNF6 and hepatic genes. The phenotype of Dicer-deficient acini depends on the induction of HNF6; overexpression of this factor in developing acinar cells is sufficient to repress acinar differentiation and to induce hepatic genes. Let-7b and miR-495 repress HNF6 and are expressed in developing acini. Their expression is inhibited in Dicer-deficient acini, as well as in pancreatitis-induced metaplasia. In addition, inhibiting let-7b and miR-495 in acinar cells results in similar effects to those found in Dicer-deficient acini and metaplastic cells, namely induction of HNF6 and hepatic genes and repression of acinar differentiation. CONCLUSIONS:Let-7b, miR-495, and their targets constitute a gene network that is required to establish and maintain pancreatic acinar cell differentiation. Additional studies of this network will increase our understanding of pancreatic diseases. 10.1053/j.gastro.2013.05.016
    Isolation and phenotypic characterization of colorectal cancer stem cells with organ-specific metastatic potential. Gao Wenchao,Chen Lu,Ma Zhenyu,Du Zunguo,Zhao Zhonghua,Hu Zhiqian,Li Qingquan Gastroenterology BACKGROUND & AIMS:Migrating cancer stem cells (MCSCs) are believed to form metastases. We sought to identify markers of MCSCs from human colorectal cancers (CRCs) and determine their roles in organ-specific metastasis. METHODS:To identify colorectal MCSCs that contribute to organ-specific metastasis, we developed a model of liver or lung metastasis using primary tumor cells from patients with CRC who had liver and lung metastases. Distinct organ-specific metastatic cells were isolated by 6 cycles of selecting for cells that formed liver and lung tumors after subcutaneous injection into mice. Microarray analysis was used to identify markers of the organ-specific MCSCs. We then measured levels of these markers in CRC cell lines and 128 CRC samples. We characterized the functional roles of these markers in organ-specific metastasis. RESULTS:We identified CD110 and CDCP1 as cell surface markers of MCSCs from human colorectal tumors that metastasized to liver and lung. We observed a distinct pattern of CD110 and CDCP1 in a panel of primary colorectal tumor samples and their matched liver or pulmonary metastases, indicating that these proteins might serve as biomarkers of organ-specific metastasis. Functional studies showed that thrombopoietin attracts CD110(+) CSCs and increases their self-renewal to promote formation of liver metastases. CDCP1 promoted adhesion of CRC cells to the lung endothelium. CONCLUSIONS:We isolated MCSCs from primary human CRCs and found that the CD110(+) and CDCP1(+) subpopulations mediate organ-specific metastasis. These findings might be used to aid in selection of patients for postoperative adjuvant therapy. 10.1053/j.gastro.2013.05.049
    Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Lawson Devon A,Bhakta Nirav R,Kessenbrock Kai,Prummel Karin D,Yu Ying,Takai Ken,Zhou Alicia,Eyob Henok,Balakrishnan Sanjeev,Wang Chih-Yang,Yaswen Paul,Goga Andrei,Werb Zena Nature Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated by stem-like cells that proliferate and differentiate to produce advanced metastatic disease. 10.1038/nature15260
    Secretory leukocyte protease inhibitor: a secreted pattern recognition receptor for mycobacteria. Gomez Sonia A,Argüelles Claudia L,Guerrieri Diego,Tateosian Nancy L,Amiano Nicolás O,Slimovich Rut,Maffia Paulo C,Abbate Eduardo,Musella Rosa M,Garcia Verónica E,Chuluyan H Eduardo American journal of respiratory and critical care medicine RATIONALE:Human secretory leukocyte protease inhibitor (SLPI) displays bactericidal activity against pathogens such as Escherichia coli and Streptococcus. Furthermore, it has been reported that murine SLPI shows potent antimycobacterial activity. OBJECTIVES:The aim of the present study was to investigate whether human recombinant SLPI not only kills mycobacteria but also acts as a pattern recognition receptor for the host immune system. METHODS:For the in vivo experiment, BALB/c mice were infected by intranasal instillation with Mycobacterium bovis BCG and viable BCG load in lung homogenates was later determined. For the in vitro experiments, SLPI was incubated overnight with a suspension of M. bovis BCG or the virulent strain Mycobacterium tuberculosis H37Rv, and the percentage survival as well as the binding of SLPI to mycobacteria was determined. Furthermore, bacteria phagocytosis was also determined by flow cytometry. MEASUREMENTS AND MAIN RESULTS:Intranasal SLPI treatment decreased the number of colony-forming units recovered from lung homogenates, indicating that SLPI interfered with M. bovis BCG infection. Moreover, SLPI decreased the viability of both M. bovis BCG and H37Rv. We demonstrated that SLPI attached to the surface of the mycobacteria by binding to pathogen-associated molecular pattern mannan-capped lipoarabinomannans and phosphatidylinositol mannoside. Furthermore, we found that in the sputum of patients with tuberculosis, mycobacteria were coated with endogenous SLPI. Finally, we showed that phagocytosis of SLPI-coated mycobacteria was faster than that of uncoated bacteria. CONCLUSIONS:The present results demonstrate for the first time that human SLPI kills mycobacteria and is a new pattern recognition receptor for them. 10.1164/rccm.200804-615OC
    Crucial role of Salmonella genomic island 1 master activator in the parasitism of IncC plasmids. Durand Romain,Huguet Kévin T,Rivard Nicolas,Carraro Nicolas,Rodrigue Sébastien,Burrus Vincent Nucleic acids research IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in the Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1's lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters of SGI1 and the helper IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential to elicit the excision and replication of SGI1 and destabilise the helper IncC plasmid. 10.1093/nar/gkab204
    RNAs competing for microRNAs mutually influence their fluctuations in a highly non-linear microRNA-dependent manner in single cells. Bosia Carla,Sgrò Francesco,Conti Laura,Baldassi Carlo,Brusa Davide,Cavallo Federica,Cunto Ferdinando Di,Turco Emilia,Pagnani Andrea,Zecchina Riccardo Genome biology BACKGROUND:Distinct RNA species may compete for binding to microRNAs (miRNAs). This competition creates an indirect interaction between miRNA targets, which behave as miRNA sponges and eventually influence each other's expression levels. Theoretical predictions suggest that not only the mean expression levels of targets but also the fluctuations around the means are coupled through miRNAs. This may result in striking effects on a broad range of cellular processes, such as cell differentiation and proliferation. Although several studies have reported the functional relevance of this mechanism of interaction, detailed experiments are lacking that study this phenomenon in controlled conditions by mimicking a physiological range. RESULTS:We used an experimental design based on two bidirectional plasmids and flow cytometry measurements of cotransfected mammalian cells. We validated a stochastic gene interaction model that describes how mRNAs can influence each other's fluctuations in a miRNA-dependent manner in single cells. We show that miRNA-target correlations eventually lead to either bimodal cell population distributions with high and low target expression states, or correlated fluctuations across targets when the pool of unbound targets and miRNAs are in near-equimolar concentration. We found that there is an optimal range of conditions for the onset of cross-regulation, which is compatible with 10-1000 copies of targets per cell. CONCLUSIONS:Our results are summarized in a phase diagram for miRNA-mediated cross-regulation that links experimentally measured quantities and effective model parameters. This phase diagram can be applied to in vivo studies of RNAs that are in competition for miRNA binding. 10.1186/s13059-017-1162-x
    Anti-B-Cell Maturation Antigen BiTE Molecule AMG 420 Induces Responses in Multiple Myeloma. Topp Max S,Duell Johannes,Zugmaier Gerhard,Attal Michel,Moreau Philippe,Langer Christian,Krönke Jan,Facon Thierry,Salnikov Alexey V,Lesley Robin,Beutner Karl,Kalabus James,Rasmussen Erik,Riemann Kathrin,Minella Alex C,Munzert Gerd,Einsele Hermann Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:The anti-B-cell maturation antigen BiTE molecule AMG 420 was assessed in patients with relapsed/refractory multiple myeloma. PATIENTS AND METHODS:In this first-in-human study, up to 10 cycles of AMG 420 were given (4-week infusions/6-week cycles). Patients had progression after ≥ 2 lines of prior therapy and no extramedullary disease. Minimal residual disease (MRD) response was defined as < 1 tumor cell/10 bone marrow cells by flow cytometry. RESULTS:Forty-two patients received AMG 420 at 0.2-800 μg/d. Median age was 65 years, and median disease duration was 5.2 years. Median exposure was 1 cycle (range, 1-10 cycles) and 7 cycles (range, 1-10 cycles) for responders. Patients discontinued for disease progression (n = 25), adverse events (AEs; n = 7), death (n = 4), completion of 10 cycles (n = 3), and consent withdrawal (n = 1). Two patients remain on treatment. There were 2 nontreatment-related deaths from AEs, influenza/aspergillosis and adenovirus-related hepatitis. Serious AEs (n = 20; 48%) included infections (n = 14) and polyneuropathy (n = 2); treatment-related serious AEs included 2 grade 3 polyneuropathies and 1 grade 3 edema. There were no grade ≥ 3 CNS toxicities or anti-AMG 420 antibodies. In this study, 800 μg/d was considered to not be tolerable because of 1 instance each of grade 3 cytokine release syndrome and grade 3 polyneuropathy, both of which resolved. The overall response rate was 31% (n = 13 of 42). At the maximum tolerated dose (MTD) of 400 μg/d, the response rate was 70% (n = 7 of 10). Of these, five patients experienced MRD-negative complete responses, and 1 had a partial response, and 1 had a very good partial response; all 7 patients responded during the first cycle, and some responses lasted > 1 year. CONCLUSION:In this study of AMG 420 in patients with relapsed/refractory multiple myeloma, the response rate was 70%, including 50% MRD-negative complete responses, at 400 μg/d, the MTD for this study. 10.1200/JCO.19.02657
    Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice. Armacki Milena,Polaschek Sandra,Waldenmaier Mareike,Morawe Mareen,Ruhland Claudia,Schmid Rebecca,Lechel André,Tharehalli Umesh,Steup Christoph,Bektas Yasin,Li Hongxia,Kraus Johann M,Kestler Hans A,Kruger Stephan,Ormanns Steffen,Walther Paul,Eiseler Tim,Seufferlein Thomas Gastroenterology BACKGROUND & AIMS:Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. METHODS:We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1 mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. RESULTS:Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6β4 into sEVs-a process that required CD82. CONCLUSIONS:Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice. 10.1053/j.gastro.2020.05.052
    Bile Acids Signal via TGR5 to Activate Intestinal Stem Cells and Epithelial Regeneration. Sorrentino Giovanni,Perino Alessia,Yildiz Ece,El Alam Gaby,Bou Sleiman Maroun,Gioiello Antimo,Pellicciari Roberto,Schoonjans Kristina Gastroenterology BACKGROUND & AIMS:Renewal and patterning of the intestinal epithelium is coordinated by intestinal stem cells (ISCs); dietary and metabolic factors provide signals to the niche that control ISC activity. Bile acids (BAs), metabolites in the gut, signal nutrient availability by activating the G protein-coupled bile acid receptor 1 (GPBAR1, also called TGR5). TGR5 is expressed in the intestinal epithelium, but it is not clear how its activation affects ISCs and regeneration of the intestinal epithelium. We studied the role of BAs and TGR5 in intestinal renewal, and regulation of ISC function in mice and intestinal organoids. METHODS:We derived intestinal organoids from wild-type mice and Tgr5 mice, incubated them with BAs or the TGR5 agonist INT-777, and monitored ISC function by morphologic analyses and colony-forming assays. We disrupted Tgr5 specifically in Lgr5-positive ISCs in mice (Tgr5 mice) and analyzed ISC number, proliferation, and differentiation by flow cytometry, immunofluorescence, and organoid assays. Tgr5 mice were given cholecystokinin; we measured the effects of BA release into the intestinal lumen and on cell renewal. We induced colitis in Tgr5 mice by administration of dextran sulfate sodium; disease severity was determined based on body weight, colon length, and histopathology analysis of colon biopsies. RESULTS:BAs and TGR5 agonists promoted growth of intestinal organoids. Administration of cholecystokinin to mice resulted in acute release of BAs into the intestinal lumen and increased proliferation of the intestinal epithelium. BAs and Tgr5 expression in ISCs were required for homeostatic intestinal epithelial renewal and fate specification, and for regeneration after colitis induction. Tgr5 mice developed more severe colitis than mice without Tgr5 disruption in ISCs. ISCs incubated with INT-777 increased activation of yes-associated protein 1 (YAP1) and of its upstream regulator SRC. Inhibitors of YAP1 and SRC prevented organoid growth induced by TGR5 activation. CONCLUSIONS:BAs promote regeneration of the intestinal epithelium via activation of TGR5 in ISCs, resulting in activation of SRC and YAP and activation of their target genes. Release of endogenous BAs in the intestinal lumen is sufficient to promote ISC renewal and drives regeneration in response to injury. 10.1053/j.gastro.2020.05.067
    Gut-derived lipopolysaccharide augments adipose macrophage accumulation but is not essential for impaired glucose or insulin tolerance in mice. Caesar Robert,Reigstad Christopher S,Bäckhed Helene Kling,Reinhardt Christoph,Ketonen Maria,Lundén Gunnel Östergren,Cani Patrice D,Bäckhed Fredrik Gut BACKGROUND:Obesity is associated with accumulation of macrophages in white adipose tissue (WAT), which contribute to the development of insulin resistance. Germ-free (GF) mice have reduced adiposity and are protected against diet-induced obesity, OBJECTIVE:To investigate whether the gut microbiota and, specifically, gut-derived lipopolysaccharide (LPS) promote WAT inflammation and contribute to impaired glucose metabolism. METHOD:Macrophage composition and expression of proinflammatory and anti-inflammatory markers were compared in WAT of GF, conventionally raised and Escherichia coli-monocolonised mice. Additionally, glucose and insulin tolerance in these mice was determined. RESULTS:The presence of a gut microbiota resulted in impaired glucose metabolism and increased macrophage accumulation and polarisation towards the proinflammatory M1 phenotype in WAT. Monocolonisation of GF mice for 4 weeks with E. coli W3110 or the isogenic strain MLK1067 (which expresses LPS with reduced immunogenicity) resulted in impaired glucose and insulin tolerance and promoted M1 polarisation of CD11b cells in WAT. However, colonisation with E. coli W3110 but not MLK1067 promoted macrophage accumulation and upregulation of proinflammatory and anti-inflammatory gene expression as well as JNK phosphorylation. CONCLUSION:Gut microbiota induced LPS-dependent macrophage accumulation in WAT, whereas impairment of systemic glucose metabolism was not dependent on LPS. These results indicate that macrophage accumulation in WAT does not always correlate with impaired glucose metabolism. 10.1136/gutjnl-2011-301689
    Hypomethylation of and is associated with type-I interferon-dependent expression in lupus CD8+ T cells. Miller Shaylynn,Tsou Pei-Suen,Coit Patrick,Gensterblum-Miller Elizabeth,Renauer Paul,Rohraff Dallas M,Kilian Nathan C,Schonfeld Mark,Sawalha Amr H Annals of the rheumatic diseases OBJECTIVE:We examined genome-wide DNA methylation changes in CD8+ T cells from patients with lupus and controls and investigated the functional relevance of some of these changes in lupus. METHODS:Genome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR. Inhibiting STAT1 and CIITA was performed using fludarabine and CIITA siRNA, respectively. RESULTS:Lupus CD8+ T cells had 188 hypomethylated CpG sites compared with healthy matched controls. Among the most hypomethylated were sites associated with . Genes involved in the type-I interferon response, including , were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. CIITA knockdown and STAT1 inhibition experiments revealed that HLA-DRB1 expression in lupus CD8+ T cells is dependent on CIITA and STAT1 signalling. Coincubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69 and cytokine production, in patients with lupus but not in healthy controls. This can be blocked by neutralising antibodies targeting HLA-DR. CONCLUSIONS:Lupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in patients with lupus. A possible pathogenic role for CD8+ T cells in lupus that is dependent on a high type-I interferon environment and epigenetic priming warrants further characterisation. 10.1136/annrheumdis-2018-214323
    Circulating follicular helper T cells are increased in systemic sclerosis and promote plasmablast differentiation through the IL-21 pathway which can be inhibited by ruxolitinib. Ricard Laure,Jachiet Vincent,Malard Florent,Ye Yishan,Stocker Nicolas,Rivière Sébastien,Senet Patricia,Monfort Jean-Benoit,Fain Olivier,Mohty Mohamad,Gaugler Béatrice,Mekinian Arsène Annals of the rheumatic diseases OBJECTIVES:Systemic sclerosis (SSc) is an autoimmune disease characterised by widespread fibrosis, microangiopathy and autoantibodies. Follicular helper T (Tfh) cells CD4CXCR5PD-1 cooperate with B lymphocytes to induce the differentiation of plasmocytes secreting immunoglobulins (Ig). Circulating Tfh (cTfh) cells are increased in several autoimmune diseases. However, there are no data about cTfh cells and their interaction with B cells in SSc. The aim of this study was to perform a quantitative and functional analysis of cTfh cells in SSc. METHODS:Using flow cytometry, we analysed cTfh cells from 50 patients with SSc and 32 healthy controls (HC). In vitro coculture experiments of sorted cTfh and B cells were performed for functional analysis. IgG and IgM production were measured by ELISA. RESULTS:We observed that cTfh cell numbers are increased in patients with SSc compared with HC. Furthermore, the increase in cTfh cells was more potent in patients with severe forms of SSc such as diffuse SSc and in the presence of arterial pulmonary hypertension. cTfh cells from patients with SSc present an activated Tfh phenotype, with high expression of BCL-6, increased capacity to produce IL-21 in comparison with healthy controls. In vitro, cTfh cells from patients with SSc had higher capacity to stimulate the differentiation of CD19CD27CD38 B cells and their secretion of IgG and IgM through the IL-21 pathway than Tfh cells from healthy controls. Blocking IL-21R or using the JAK1/2 inhibitor ruxolitinib reduced the Tfh cells' capacity to stimulate the plasmablasts and decreased the Ig production. CONCLUSIONS:Circulating Tfh cells are increased in SSc and correlate with SSc severity. The IL-21 pathway or JAK1/2 blockade by ruxolitinib could be a promising strategy in the treatment of SSc. 10.1136/annrheumdis-2018-214382
    Prediction of relapse or survival after resection in human hepatomas by DNA flow cytometry. Chiu J H,Kao H L,Wu L H,Chang H M,Lui W Y The Journal of clinical investigation To investigate the change of DNA content and the effect of synthetic phase (S-phase) fraction on hepatocytes and hepatomas, DNA content and S-phase fraction were measured by flow cytometry in human livers and hepatoma tissues. The ploidy status of nontumor parts of resected hepatoma, fetal liver, and focal nodular hyperplasia were diploid, similar to that of the normal liver. Three patterns of DNA ploidy in human hepatoma cells were newly classified, namely, pattern I, diploid tumors; pattern II, aneuploid tumors with single G0/G1 peak; and pattern III, aneuploid tumors with more than one G0/G1 peaks. Among the 130 resectable hepatomas measured for DNA ploidy status, 84 (64.6%) were pattern I, 20 (15.4%) pattern II, and 26 (20%) pattern III. Multivariate analyses for those 130 patients who underwent hepatic resection showed that, in addition to tumor size, DNA ploidy was another prognostic factor in predicting overall survival and disease-free survival. Patients with small tumors (less than 5 cm) had a significantly higher overall survival rate than those with large tumor (greater than 5 cm). Patients with pattern III hepatomas had a significantly lower overall survival rate and a higher recurrent rate than did those with pattern I or pattern II tumors. The S-phase fraction was a significant predictor of overall survival rate in patients with pattern II, but not with pattern I, tumors. We conclude that DNA flow-cytometric measurements of ploidy and S-phase fraction are potential important prognostic predictors in patients with resectable hepatomas. 10.1172/JCI115618
    ATP11C is critical for the internalization of phosphatidylserine and differentiation of B lymphocytes. Yabas Mehmet,Teh Charis E,Frankenreiter Sandra,Lal Dennis,Roots Carla M,Whittle Belinda,Andrews Daniel T,Zhang Yafei,Teoh Narci C,Sprent Jonathan,Tze Lina E,Kucharska Edyta M,Kofler Jennifer,Farell Geoffrey C,Bröer Stefan,Goodnow Christopher C,Enders Anselm Nature immunology Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell-deficiency syndrome in mice caused by mutations in Atp11c, which encodes a member of the P4 ATPase family thought to serve as 'flippases' that concentrate aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11C resulted in a lower rate of phosphatidylserine translocation in pro-B cells and much lower pre-B cell and B cell numbers despite expression of pre-rearranged immunoglobulin transgenes or enforced expression of the prosurvival protein Bcl-2 to prevent apoptosis and abolished pre-B cell population expansion in response to a transgene encoding interleukin 7. The only other abnormalities we noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. Our results identify an intimate connection between phospholipid transport and B lymphocyte function. 10.1038/ni.2011
    STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development. Limnander Andre,Depeille Philippe,Freedman Tanya S,Liou Jen,Leitges Michael,Kurosaki Tomohiro,Roose Jeroen P,Weiss Arthur Nature immunology Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca(2+)-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide-exchange factor RasGRP and depended on the concentration of the Ca(2+) sensor STIM1, which controls the magnitude of Ca(2+) entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca(2+)-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells. 10.1038/ni.2016
    The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells. Motomura Yasutaka,Kitamura Hiroshi,Hijikata Atsushi,Matsunaga Yuko,Matsumoto Koichiro,Inoue Hiromasa,Atarashi Koji,Hori Shohei,Watarai Hiroshi,Zhu Jinfang,Taniguchi Masaru,Kubo Masato Nature immunology The immunoregulatory cytokine interleukin 10 (IL-10) is expressed mainly by T helper type 2 (T(H)2) cells but also by T(H)1 cells during chronic infection. Here we observed plasticity in the expression of IL-10 and IL-13 after chronic T(H)1 stimulation; furthermore, the expression of Il10 and Il13 was regulated by the transcription factor E4BP4. Chronically stimulated E4BP4-deficient (Nfil3(-/-); called 'E4bp4(-/-)' here) T(H)1 cells, regulatory T cells (T(reg) cells) and natural killer T cells (NKT cells) had attenuated expression of IL-10 and IL-13. Enforced expression of E4bp4 initiated the production of IL-10 and IL-13 by conventional T(H)1 cells. E4bp4(-/-) T(H)2 cells showed impairment of IL-10 production with no effect on IL-13. Our results indicate that E4BP4 has multiple functions in controlling the plasticity of IL-13 in T(H)1 cells and IL-10 in T(H)1 cells, T(H)2 cells, T(reg) cells and NKT cells. 10.1038/ni.2020
    The E3 ligase Itch is a negative regulator of the homeostasis and function of hematopoietic stem cells. Rathinam Chozhavendan,Matesic Lydia E,Flavell Richard A Nature immunology Although hematopoietic stem cells (HSCs) are the most thoroughly characterized type of adult stem cell, the intricate molecular machinery that regulates their self-renewal properties remains elusive. Here we showed that the E3 ubiquitin ligase Itch negatively regulated the development and function of HSCs. Itch(-/-) mice had HSCs with enhanced frequency, competence and long-term repopulating activity. Itch-deficient HSCs showed accelerated proliferation rates and sustained progenitor properties, as well as more signaling by the transcription factor Notch1, due to more accumulation of activated Notch1. Knockdown of Notch1 in Itch-mutant HSCs resulted in reversion of the phenotype. Thus, we identify Itch as a previously unknown negative regulator of HSC homeostasis and function. 10.1038/ni.2021
    Personalized Transcriptomics Reveals Heterogeneous Immunophenotypes in Children with Viral Bronchiolitis. Jones Anya C,Anderson Denise,Galbraith Sally,Fantino Emmanuelle,Gutierrez Cardenas Diana,Read James F,Serralha Michael,Holt Barbara J,Strickland Deborah H,Sly Peter D,Bosco Anthony,Holt Patrick G American journal of respiratory and critical care medicine A subset of infants are hypersusceptible to severe/acute viral bronchiolitis (AVB), for reasons incompletely understood. To characterize the cellular/molecular mechanisms underlying infant AVB in circulating cells/local airway tissues. Peripheral blood mononuclear cells and nasal scrapings were obtained from infants (<18 mo) and children (≥18 mo to 5 yr) during AVB and after convalescence. Immune response patterns were profiled by multiplex analysis of plasma cytokines, flow cytometry, and transcriptomics (RNA-Seq). Molecular profiling of group-level data used a combination of upstream regulator and coexpression network analysis, followed by individual subject-level data analysis using personalized -of-1-pathways methodology. Group-level analyses demonstrated that infant peripheral blood mononuclear cell responses were dominated by monocyte-associated hyperupregulated type 1 IFN signaling/proinflammatory pathways (drivers: TNF [tumor necrosis factor], IL-6, TREM1 [triggering receptor expressed on myeloid cells 1], and IL-1B), versus a combination of inflammation (PTGER2 [prostaglandin E receptor 2] and IL-6) plus growth/repair/remodeling pathways (ERBB2 [erbb-b2 receptor tyrosine kinase 2], TGFB1 [transforming growth factor-β1], AREG [amphiregulin], and HGF [hepatocyte growth factor]) coupled with T-helper cell type 2 and natural killer cell signaling in children. Age-related differences were not attributable to differential steroid usage or variations in underlying viral pathogens. Nasal mucosal responses were comparable qualitatively in infants/children, dominated by IFN types 1-3, but the magnitude of upregulation was higher in infants (range, 6- to 48-fold) than children (5- to 17-fold). -of-1-pathways analysis confirmed differential upregulation of innate immunity in infants and natural killer cell networks in children, and additionally demonstrated covert AVB response subphenotypes that were independent of chronologic age. Dysregulated expression of IFN-dependent pathways after respiratory viral infections is a defining immunophenotypic feature of AVB-susceptible infants and a subset of children. Susceptible subjects seem to represent a discrete subgroup who cluster based on (slow) kinetics of postnatal maturation of innate immune competence. 10.1164/rccm.201804-0715OC
    Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Witte Martin D,Kallemeijn Wouter W,Aten Jan,Li Kah-Yee,Strijland Anneke,Donker-Koopman Wilma E,van den Nieuwendijk Adrianus M C H,Bleijlevens Boris,Kramer Gertjan,Florea Bogdan I,Hooibrink Berend,Hollak Carla E M,Ottenhoff Roelof,Boot Rolf G,van der Marel Gijsbert A,Overkleeft Herman S,Aerts Johannes M F G Nature chemical biology Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells. 10.1038/nchembio.466
    SPaRTAN, a computational framework for linking cell-surface receptors to transcriptional regulators. Ma Xiaojun,Somasundaram Ashwin,Qi Zengbiao,Hartman Douglas J,Singh Harinder,Osmanbeyoglu Hatice Ulku Nucleic acids research The identity and functions of specialized cell types are dependent on the complex interplay between signaling and transcriptional networks. Recently single-cell technologies have been developed that enable simultaneous quantitative analysis of cell-surface receptor expression with transcriptional states. To date, these datasets have not been used to systematically develop cell-context-specific maps of the interface between signaling and transcriptional regulators orchestrating cellular identity and function. We present SPaRTAN (Single-cell Proteomic and RNA based Transcription factor Activity Network), a computational method to link cell-surface receptors to transcription factors (TFs) by exploiting cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) datasets with cis-regulatory information. SPaRTAN is applied to immune cell types in the blood to predict the coupling of signaling receptors with cell context-specific TFs. Selected predictions are validated by prior knowledge and flow cytometry analyses. SPaRTAN is then used to predict the signaling coupled TF states of tumor infiltrating CD8+ T cells in malignant peritoneal and pleural mesotheliomas. SPaRTAN enhances the utility of CITE-seq datasets to uncover TF and cell-surface receptor relationships in diverse cellular states. 10.1093/nar/gkab745
    Association of Microvesicles With Graft Patency in Patients Undergoing CABG Surgery. Camera Marina,Brambilla Marta,Canzano Paola,Cavallotti Laura,Parolari Alessandro,Tedesco Calogero C,Zara Chiara,Rossetti Laura,Tremoli Elena Journal of the American College of Cardiology BACKGROUND:Graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (CABG). Biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. The prognostic value of microvesicles (MVs) for midterm graft patency has never been tested. OBJECTIVES:The aim of this study was to evaluate whether MV pre-operative signature (number, cellular origin, procoagulant phenotype) could predict midterm graft failure and to investigate potential functional role of MVs in graft occlusion. METHODS:This was a nested case-control substudy of the CAGE (CoronAry bypass grafting: factors related to late events and Graft patency) study that enrolled 330 patients undergoing elective CABG. Of these, 179 underwent coronary computed tomography angiography 18 months post-surgery showing 24% graft occlusion. Flow cytometry MV analysis was performed in 60 patients (30 per group with occluded [cases] and patent [control subjects] grafts) on plasma samples collected the day before surgery and at follow-up. RESULTS:Before surgery, cases had 2- and 4-fold more activated platelet-derived and tissue-factor positive MVs respectively than control subjects. The MV procoagulant capacity was also significantly greater. Altogether this MV signature properly classified graft occlusion (area under the curve 0.897 [95% confidence interval: 0.81 to 0.98]; p < 0.0001). By using an MV score (0 to 6), the odds ratio for occlusion for a score above 3 was 16.3 (95% confidence interval: 4.1 to 65.3; p < 0.0001). CONCLUSIONS:The pre-operative signature of MVs is independently associated with midterm graft occlusion in CABG patients and a cumulative MV score stratifies patients' risk. Because the MV signature mirrors platelet activation, patients with a high MV score could benefit from a personalized antiplatelet therapy. 10.1016/j.jacc.2020.03.073
    Human liver infiltrating γδ T cells are composed of clonally expanded circulating and tissue-resident populations. Hunter Stuart,Willcox Carrie R,Davey Martin S,Kasatskaya Sofya A,Jeffery Hannah C,Chudakov Dmitriy M,Oo Ye H,Willcox Benjamin E Journal of hepatology BACKGROUND & AIMS:γδ T cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ T cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear. Therefore, we aimed to elucidate the T cell receptor (TCR) diversity, immunophenotype and function of γδ T cells in the human liver. METHODS:We characterised the TCR repertoire, immunophenotype and function of human liver infiltrating γδ T cells, by TCR sequencing analysis, flow cytometry, in situ hybridisation and immunohistochemistry. We focussed on the predominant tissue-associated Vδ2 γδ subset, which is implicated in liver immunopathology. RESULTS:Intrahepatic Vδ2 γδ T cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T cells were predominantly CD27 effector lymphocytes, whereas naïve CD27, TCR-diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RA Vδ2 γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2 γδ T cell pool also included a phenotypically distinct CD45RA effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vδ2 γδ cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli. CONCLUSION:These findings suggest that the ability of Vδ2 γδ T cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues, such as the liver, which results in functionally distinct peripheral and liver-resident memory γδ T cell subsets. They also highlight the inherent functional plasticity within the Vδ2 γδ T cell compartment and provide information that could be used for the design of cellular therapies that suppress liver inflammation or combat liver cancer. LAY SUMMARY:γδ T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ T cells are largely excluded. Moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance. 10.1016/j.jhep.2018.05.007
    Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Wen Haitao,Gris Denis,Lei Yu,Jha Sushmita,Zhang Lu,Huang Max Tze-Han,Brickey Willie June,Ting Jenny P-Y Nature immunology High-fat diet (HFD) and inflammation are key contributors to insulin resistance and type 2 diabetes (T2D). Interleukin (IL)-1β plays a role in insulin resistance, yet how IL-1β is induced by the fatty acids in an HFD, and how this alters insulin signaling, is unclear. We show that the saturated fatty acid palmitate, but not unsaturated oleate, induces the activation of the NLRP3-ASC inflammasome, causing caspase-1, IL-1β and IL-18 production. This pathway involves mitochondrial reactive oxygen species and the AMP-activated protein kinase and unc-51-like kinase-1 (ULK1) autophagy signaling cascade. Inflammasome activation in hematopoietic cells impairs insulin signaling in several target tissues to reduce glucose tolerance and insulin sensitivity. Furthermore, IL-1β affects insulin sensitivity through tumor necrosis factor-independent and dependent pathways. These findings provide insights into the association of inflammation, diet and T2D. 10.1038/ni.2022
    Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus. Anderson Beverley H,Kasher Paul R,Mayer Josephine,Szynkiewicz Marcin,Jenkinson Emma M,Bhaskar Sanjeev S,Urquhart Jill E,Daly Sarah B,Dickerson Jonathan E,O'Sullivan James,Leibundgut Elisabeth Oppliger,Muter Joanne,Abdel-Salem Ghada M H,Babul-Hirji Riyana,Baxter Peter,Berger Andrea,Bonafé Luisa,Brunstom-Hernandez Janice E,Buckard Johannes A,Chitayat David,Chong Wui K,Cordelli Duccio M,Ferreira Patrick,Fluss Joel,Forrest Ewan H,Franzoni Emilio,Garone Caterina,Hammans Simon R,Houge Gunnar,Hughes Imelda,Jacquemont Sebastien,Jeannet Pierre-Yves,Jefferson Rosalind J,Kumar Ram,Kutschke Georg,Lundberg Staffan,Lourenço Charles M,Mehta Ramesh,Naidu Sakkubai,Nischal Ken K,Nunes Luís,Ounap Katrin,Philippart Michel,Prabhakar Prab,Risen Sarah R,Schiffmann Raphael,Soh Calvin,Stephenson John B P,Stewart Helen,Stone Jon,Tolmie John L,van der Knaap Marjo S,Vieira Jose P,Vilain Catheline N,Wakeling Emma L,Wermenbol Vanessa,Whitney Andrea,Lovell Simon C,Meyer Stefan,Livingston John H,Baerlocher Gabriela M,Black Graeme C M,Rice Gillian I,Crow Yanick J Nature genetics Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous γH2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the α-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-α primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity. 10.1038/ng.1084
    Neoadjuvant Chemotherapy Reinforces Antitumour T cell Response in Urothelial Urinary Bladder Cancer. Krantz David,Hartana Ciputra Adijaya,Winerdal Malin E,Johansson Markus,Alamdari Farhood,Jakubczyk Tomasz,Huge Ylva,Aljabery Firas,Palmqvist Karin,Zirakzadeh A Ali,Holmström Benny,Riklund Katrine,Sherif Amir,Winqvist Ola European urology Evidence indicates that neoadjuvant chemotherapy (NAC) may promote antitumour immune responses by activating T cells. The tumour-draining sentinel node (SN) is a key site to study tumour-specific T cell activation, being the primary immunological barrier against the tumour. In this prospective study, we set out to elucidate the effects of NAC on T cell subsets in the SNs of patients with muscle-invasive urothelial bladder cancer. We found that CD8 effector T (Teff) cell exhaustion was reduced after NAC treatment, while cytotoxicity was increased. Additionally, in complete responders (CR patients), these cells were functionally committed effectors, as displayed by epigenetic analysis. In CD4 Teffs, NAC treatment was associated with increased clonal expansion of tumour-specific SN-derived cells, as demonstrated by a specific cell reactivity assay. In contrast, we observed an attenuating effect of NAC on regulatory T cells (Tregs) with a dose-dependent decrease in Treg frequency and reduced effector molecule expression in the remaining Tregs. In addition, multicolour flow cytometry analysis revealed that CR patients had higher Teff to activated Treg ratio, promoting antitumoural T cell activation. These results suggest that NAC reinforces the antitumour immune response by activating the effector arm of the T cell compartment and diminishing the influence of suppressive Tregs. PATIENT SUMMARY: In this report, we analysed the effect of chemotherapy on immune cell subsets of 40 patients with advanced bladder cancer. We found that chemotherapy has a positive effect on immune effector T cells, whereas an opposite, diminishing effect was observed for immune-suppressive regulatory T cells. We conclude that chemotherapy reinforces the antitumour immune response in bladder cancer patients. 10.1016/j.eururo.2018.06.048
    IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis. Dasgupta Debanjali,Nakao Yasuhiko,Mauer Amy S,Thompson Jill M,Sehrawat Tejasav S,Liao Chieh-Yu,Krishnan Anuradha,Lucien Fabrice,Guo Qianqian,Liu Mengfei,Xue Fei,Fukushima Masanori,Katsumi Tomohiro,Bansal Aditya,Pandey Mukesh K,Maiers Jessica L,DeGrado Timothy,Ibrahim Samar H,Revzin Alexander,Pavelko Kevin D,Barry Michael A,Kaufman Randal J,Malhi Harmeet Gastroenterology BACKGROUND & AIMS:Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS:C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1α) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4μ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS:Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS:In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH. 10.1053/j.gastro.2020.06.031
    IL22BP Mediates the Antitumor Effects of Lymphotoxin Against Colorectal Tumors in Mice and Humans. Kempski Jan,Giannou Anastasios D,Riecken Kristoffer,Zhao Lilan,Steglich Babett,Lücke Jöran,Garcia-Perez Laura,Karstens Karl-Frederick,Wöstemeier Anna,Nawrocki Mikolaj,Pelczar Penelope,Witkowski Mario,Nilsson Sven,Konczalla Leonie,Shiri Ahmad Mustafa,Kempska Joanna,Wahib Ramez,Brockmann Leonie,Huber Philipp,Gnirck Ann-Christin,Turner Jan-Eric,Zazara Dimitra E,Arck Petra C,Stein Alexander,Simon Ronald,Daubmann Anne,Meiners Jan,Perez Daniel,Strowig Till,Koni Pandelakis,Kruglov Andrey A,Sauter Guido,Izbicki Jakob R,Guse Andreas H,Rösch Thomas,Lohse Ansgar W,Flavell Richard A,Gagliani Nicola,Huber Samuel Gastroenterology BACKGROUND & AIMS:Unregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice. METHODS:We obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf, Lta, and Ltb mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times. RESULTS:Levels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte-derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC. CONCLUSIONS:Lymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist. 10.1053/j.gastro.2020.06.033
    Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice. Gastroenterology BACKGROUND & AIMS:Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS:We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-Ab) or Rag1 mice (Rag1I-Ab); we used I-Ab mice as controls. Colitis was induced by administration of DSS, transfer of CD4CD45RB T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgACD138 plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS:Mice with IEC-specific loss of MHCII (I-Ab mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-Ab mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-Ab vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-Ab mice was 50% lower than that of SIgA from mAb mice. Fifty percent of I-Ab mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-Ab mice compared with I-Ab mice. CONCLUSIONS:In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria. 10.1053/j.gastro.2020.06.049
    Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease. Ramli Muhammad Nadzim Bin,Lim Yee Siang,Koe Chwee Tat,Demircioglu Deniz,Tng Weiquan,Gonzales Kevin Andrew Uy,Tan Cheng Peow,Szczerbinska Iwona,Liang Hongqing,Soe Einsi Lynn,Lu Zhiping,Ariyachet Chaiyaboot,Yu Ka Man,Koh Shu Hui,Yaw Lai Ping,Jumat Nur Halisah Binte,Lim John Soon Yew,Wright Graham,Shabbir Asim,Dan Yock Young,Ng Huck-Hui,Chan Yun-Shen Gastroenterology BACKGROUND & AIMS:There are few in vitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. METHODS:We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. RESULTS:Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. CONCLUSIONS:We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH. 10.1053/j.gastro.2020.06.010
    Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Feldhaus Michael J,Siegel Robert W,Opresko Lee K,Coleman James R,Feldhaus Jane M Weaver,Yeung Yik A,Cochran Jennifer R,Heinzelman Peter,Colby David,Swers Jeffrey,Graff Christilyn,Wiley H Steven,Wittrup K Dane Nature biotechnology A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications. 10.1038/nbt785
    Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation. Qiu Xiaolei,Brown Katharine,Hirschey Matthew D,Verdin Eric,Chen Danica Cell metabolism A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases. 10.1016/j.cmet.2010.11.015
    Immune inhibitory ligand CD200 induction by TLRs and NLRs limits macrophage activation to protect the host from meningococcal septicemia. Mukhopadhyay Subhankar,Plüddemann Annette,Hoe J Claire,Williams Kevin J,Varin Audrey,Makepeace Katherine,Aknin Marie-Laure,Bowdish Dawn M E,Smale Stephen T,Barclay A Neil,Gordon Siamon Cell host & microbe Macrophage activation is essential for protection against bacterial pathogens but needs to be regulated to prevent damage to the host. We show a key role for the immune inhibitory receptor CD200R and its ligand CD200 in the context of infection with the Gram-negative human pathogen Neisseria meningitidis. N. meningitidis induced CD200 but downregulated CD200R on macrophages in a manner dependent on Neisserial lipopolysaccharide, Toll-like receptor-4 (TLR-4), and the MyD88 pathway but independent of a known Neisserial receptor, scavenger receptor A (SR-A). Agonists of the pattern-recognition receptors nucleotide oligomerization domain 2 (NOD2) and NACHT-LRR protein 3 (NALP3) also induced CD200. The NF-κB member c-Rel was essential for TLR-, NOD2-, and NALP3-mediated induction of CD200. CD200(-/-) animals showed higher lethality in response to experimental meningococcal septicemia, induced higher levels of proinflammatory cytokines, and recruited increased numbers of activated leukocytes, despite comparable bacterial clearance. Thus CD200 is induced by TLR-, NOD2-, and NALP3-mediated pathways, limiting their function and protecting the host from excessive inflammation. 10.1016/j.chom.2010.08.005
    Granulocyte-colony stimulating factor reactivates human cytomegalovirus in a latently infected humanized mouse model. Smith M Shane,Goldman Devorah C,Bailey Alexis S,Pfaffle Dana L,Kreklywich Craig N,Spencer Doran B,Othieno Florence A,Streblow Daniel N,Garcia J Victor,Fleming William H,Nelson Jay A Cell host & microbe Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in organ transplant recipients. The use of granulocyte-colony stimulating factor (G-CSF)-mobilized stem cells from HCMV seropositive donors is suggested to double the risk of late-onset HCMV disease and chronic graft-versus-host disease in recipients when compared to conventional bone marrow transplantation with HCMV seropositive donors, although the etiology of the increased risk is unknown. To understand mechanisms of HCMV transmission in patients receiving G-CSF-mobilized blood products, we generated a NOD-scid IL2Rγ(c)(null)-humanized mouse model in which HCMV establishes latent infection in human hematopoietic cells. In this model, G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. In addition to establishing a humanized mouse model for systemic and latent HCMV infection, these results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients. 10.1016/j.chom.2010.08.001
    Stable and dynamic nucleosome states during a meiotic developmental process. Zhang Liye,Ma Hong,Pugh B Franklin Genome research The plasticity of chromatin organization as chromosomes undergo a full compendium of transactions including DNA replication, recombination, chromatin compaction, and changes in transcription during a developmental program is unknown. We generated genome-wide maps of individual nucleosome organizational states, including positions and occupancy of all nucleosomes, and H3K9 acetylation and H3K4, K36, K79 tri-methylation, during meiotic spore development (gametogenesis) in Saccharomyces. Nucleosome organization was remarkably constant as the genome underwent compaction. However, during an acute meiotic starvation response, nucleosomes were repositioned to alter the accessibility of select transcriptional start sites. Surprisingly, the majority of the meiotic programs did not use this nucleosome repositioning, but was dominated by antisense control. Histone modification states were also remarkably stable, being abundant at specific nucleosome positions at three-quarters of all genes, despite most genes being rarely transcribed. Our findings suggest that, during meiosis, the basic features of genomic chromatin organization are essentially a fixed property of chromosomes, but tweaked in a restricted and program-specific manner. 10.1101/gr.117465.110
    Myb-domain protein Teb1 controls histone levels and centromere assembly in fission yeast. Valente Luis P,Dehé Pierre-Marie,Klutstein Michael,Aligianni Sofia,Watt Stephen,Bähler Jürg,Cooper Julia Promisel The EMBO journal The TTAGGG motif is common to two seemingly unrelated dimensions of chromatin function-the vertebrate telomere repeat and the promoter regions of many Schizosaccharomyces pombe genes, including all of those encoding canonical histones. The essential S. pombe protein Teb1 contains two Myb-like DNA binding domains related to those found in telomere proteins and binds the human telomere repeat sequence TTAGGG. Here, we analyse Teb1 binding throughout the genome and the consequences of reduced Teb1 function. Chromatin immunoprecipitation (ChIP)-on-chip analysis reveals robust Teb1 binding at many promoters, notably including all of those controlling canonical histone gene expression. A hypomorphic allele, teb1-1, confers reduced binding and reduced levels of histone transcripts. Prompted by previously suggested connections between histone expression and centromere identity, we examined localization of the centromeric histone H3 variant Cnp1 and found reduced centromeric binding along with reduced centromeric silencing. These data identify Teb1 as a master regulator of histone levels and centromere identity. 10.1038/emboj.2012.339
    Ectopic lymphoid tissue alters the chemokine gradient, increases lymphocyte retention and exacerbates murine ileitis. McNamee Eóin N,Masterson Joanne C,Jedlicka Paul,Collins Colm B,Williams Ifor R,Rivera-Nieves Jesús Gut BACKGROUND:The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood. DESIGN:Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed. RESULTS:Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis. CONCLUSIONS:Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases. 10.1136/gutjnl-2011-301272
    Absolute quantification of somatic DNA alterations in human cancer. Carter Scott L,Cibulskis Kristian,Helman Elena,McKenna Aaron,Shen Hui,Zack Travis,Laird Peter W,Onofrio Robert C,Winckler Wendy,Weir Barbara A,Beroukhim Rameen,Pellman David,Levine Douglas A,Lander Eric S,Meyerson Matthew,Getz Gad Nature biotechnology We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity. 10.1038/nbt.2203
    Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice. Kagiwada Saya,Kurimoto Kazuki,Hirota Takayuki,Yamaji Masashi,Saitou Mitinori The EMBO journal Genome-wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication-coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re-evaluation of and provide novel insights into the mechanism of genome-wide DNA demethylation in PGCs. 10.1038/emboj.2012.331
    Single-cell analysis of RORα tracer mouse lung reveals ILC progenitors and effector ILC2 subsets. Ghaedi Maryam,Shen Zi Yi,Orangi Mona,Martinez-Gonzalez Itziar,Wei Lisa,Lu Xiaoxiao,Das Arundhoti,Heravi-Moussavi Alireza,Marra Marco A,Bhandoola Avinash,Takei Fumio The Journal of experimental medicine Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor-related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2- population different from conventional IL-18Rα-ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets. 10.1084/jem.20182293
    Salivary amylase-producing multiple myeloma: case report and review of the current literature. Sosnoff David R,Friend Rahsaan B,Berkovic Michael,Rasansky Ronald J,Hoffman Stephen M J Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10.1200/JCO.2012.46.4677
    High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Duda Katarzyna,Lonowski Lindsey A,Kofoed-Nielsen Michael,Ibarra Adriana,Delay Catherine M,Kang Qiaohua,Yang Zhang,Pruett-Miller Shondra M,Bennett Eric P,Wandall Hans H,Davis Gregory D,Hansen Steen H,Frödin Morten Nucleic acids research Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and ∼ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9. 10.1093/nar/gku251
    Continued response off treatment after BRAF inhibition in refractory hairy cell leukemia. Dietrich Sascha,Hüllein Jennifer,Hundemer Michael,Lehners Nicola,Jethwa Alexander,Capper David,Acker Till,Garvalov Boyan K,Andrulis Mindaugas,Blume Carolin,Schulte Christoph,Mandel Thomas,Meissner Julia,Fröhling Stefan,von Kalle Christof,Glimm Hanno,Ho Anthony D,Zenz Thorsten Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10.1200/JCO.2012.45.9495
    Complete remission after treatment with single-agent ofatumumab in a patient with high-risk leukemic mantle-cell lymphoma. Hunstig Friederike,Hammersen Jakob,Kunert Christa,Petersen Iver,Merz Hartmut,Glaser Anita,Teichgräber Ulf,Hochhaus Andreas,La Rosée Paul Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10.1200/JCO.2012.45.9438
    Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells. Drukker Micha,Tang Chad,Ardehali Reza,Rinkevich Yuval,Seita Jun,Lee Andrew S,Mosley Adriane R,Weissman Irving L,Soen Yoav Nature biotechnology To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4(+) cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny, APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors. 10.1038/nbt.2239
    Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages. Liao Wei,Lin Jian-Xin,Wang Lu,Li Peng,Leonard Warren J Nature immunology Helper T cells control host defense against pathogens. The receptors for interleukin 12 (IL-12), IL-4 and IL-6 are required for differentiation into the T(H)1, T(H)2 and T(H)17 subsets of helper T cells, respectively. IL-2 signaling via the transcription factor STAT5 controls T(H)2 differentiation by regulating both the T(H)2 cytokine gene cluster and expression of Il4ra, the gene encoding the IL-4 receptor α-chain. Here we show that IL-2 regulated T(H)1 differentiation, inducing STAT5-dependent expression of the IL-12 receptor β2-chain (IL-12Rβ2) and the transcription factor T-bet, with impaired human T(H)1 differentiation when IL-2 was blocked. T(H)1 differentiation was also impaired in mouse Il2(-/-) T cells but was restored by IL-12Rβ2 expression. Consistent with the inhibition of T(H)17 differentiation by IL-2, treatment with IL-2 resulted in lower expression of the genes encoding the IL-6 receptor α-chain (Il6ra) and the IL-6 signal transducer gp130 (encoded by Il6st), and retroviral transduction of Il6st augmented T(H)17 differentiation even when IL-2 was present. Thus, IL-2 influences helper T cell differentiation by modulating the expression of cytokine receptors to help specify and maintain differentiated states. 10.1038/ni.2030
    RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation. Codarri Laura,Gyülvészi Gabor,Tosevski Vinko,Hesske Lysann,Fontana Adriano,Magnenat Laurent,Suter Tobias,Becher Burkhard Nature immunology Although the role of the T(H)1 and T(H)17 subsets of helper T cells as disease mediators in autoimmune neuroinflammation remains a subject of some debate, none of their signature cytokines are essential for disease development. Here we report that interleukin 23 (IL-23) and the transcription factor RORγt drove expression of the cytokine GM-CSF in helper T cells, whereas IL-12, interferon-γ (IFN-γ) and IL-27 acted as negative regulators. Autoreactive helper T cells specifically lacking GM-CSF failed to initiate neuroinflammation despite expression of IL-17A or IFN-γ, whereas GM-CSF secretion by Ifng(-/-)Il17a(-/-) helper T cells was sufficient to induce experimental autoimmune encephalomyelitis (EAE). During the disease effector phase, GM-CSF sustained neuroinflammation via myeloid cells that infiltrated the central nervous system. Thus, in contrast to all other known helper T cell-derived cytokines, GM-CSF serves a nonredundant function in the initiation of autoimmune inflammation regardless of helper T cell polarization. 10.1038/ni.2027
    A BATF-ling connection between B cells and follicular helper T cells. Ellyard Julia I,Vinuesa Carola G Nature immunology The transcription factor BATF directly regulates key components of the formation and function of follicular helper T cells and antibody class switching in B cells. 10.1038/ni.2042
    Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Thiery Jerome,Keefe Dennis,Boulant Steeve,Boucrot Emmanuel,Walch Michael,Martinvalet Denis,Goping Ing Swie,Bleackley R Chris,Kirchhausen Tomas,Lieberman Judy Nature immunology How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release. 10.1038/ni.2050
    Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b. Rossi Riccardo L,Rossetti Grazisa,Wenandy Lynn,Curti Serena,Ripamonti Anna,Bonnal Raoul J P,Birolo Roberto Sciarretta,Moro Monica,Crosti Maria C,Gruarin Paola,Maglie Stefano,Marabita Francesco,Mascheroni Debora,Parente Valeria,Comelli Mario,Trabucchi Emilio,De Francesco Raffaele,Geginat Jens,Abrignani Sergio,Pagani Massimiliano Nature immunology MicroRNAs are small noncoding RNAs that regulate gene expression post-transcriptionally. Here we applied microRNA profiling to 17 human lymphocyte subsets to identify microRNA signatures that were distinct among various subsets and different from those of mouse lymphocytes. One of the signature microRNAs of naive CD4+ T cells, miR-125b, regulated the expression of genes encoding molecules involved in T cell differentiation, including IFNG, IL2RB, IL10RA and PRDM1. The expression of synthetic miR-125b and lentiviral vectors encoding the precursor to miR-125b in naive lymphocytes inhibited differentiation to effector cells. Our data provide an 'atlas' of microRNA expression in human lymphocytes, define subset-specific signatures and their target genes and indicate that the naive state of T cells is enforced by microRNA. 10.1038/ni.2057
    Randomized Phase II Study of First-Line Cladribine With Concurrent or Delayed Rituximab in Patients With Hairy Cell Leukemia. Chihara Dai,Arons Evgeny,Stetler-Stevenson Maryalice,Yuan Constance M,Wang Hao-Wei,Zhou Hong,Raffeld Mark,Xi Liqiang,Steinberg Seth M,Feurtado Julie,James Lacey,Wilson Wyndham,Braylan Raul C,Calvo Katherine R,Maric Irina,Dulau-Florea Alina,Kreitman Robert J Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Single-agent purine analog, usually cladribine, has been the standard first-line therapy of hairy cell leukemia (HCL) for 30 years. High complete remission (CR) rates often include minimal residual disease (MRD), leading to relapse and repeated treatments. Rituximab can clear MRD, but long-term results are unknown and optimal timing of rituximab undefined. PATIENTS AND METHODS:Patients were randomly assigned to first-line cladribine 0.15 mg/kg intravenously days 1-5 with 8 weekly doses of rituximab 375 mg/m begun either day 1 (concurrent, CDAR) or ≥ 6 months later (delayed) after detection of MRD in blood. MRD tests included blood and bone marrow (BM) flow cytometry, and BM immunohistochemistry. RESULTS:Sixty-eight patients with purine analog-naïve classic HCL were randomly assigned 1:1 to concurrent versus delayed arms. At 6 months after CDAR versus cladribine monotherapy, CR rates were 100% versus 88% ( = .11), MRD-free CR rates 97% versus 24% ( < .0001, primary end point), and blood MRD-free rates 100% versus 50% ( < .0001), respectively. At 96 months median follow-up, 94% versus 12% remained MRD free. Compared with CDAR, delayed rituximab after cladribine achieved lower rate (67% of 21 evaluable patients; = .0034) and durability ( = .0081, hazard radio favoring CDAR, 0.094) of MRD-free CR. Nevertheless, 12 patients in the delayed arm remained MRD free when restaged 6-104 (median, 78) months after last delayed rituximab treatment. Compared with cladribine monotherapy, CDAR led to brief grade 3/4 thrombocytopenia (59% 9%; < .0001) and platelet transfusions without bleeding (35% 0%; = .0002), but higher neutrophil ( = .017) and platelet ( = .0015) counts at 4 weeks. CONCLUSION:Achieving MRD-free CR of HCL after first-line cladribine is greatly enhanced by concurrent rituximab and less so by delayed rituximab. Longer follow-up will determine if MRD-free survival leads to less need for additional therapy or cure of HCL. 10.1200/JCO.19.02250
    Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay. Eirew Peter,Stingl John,Eaves Connie J Nature protocols Methods to identify and enumerate primitive, and typically rare, undifferentiated cells in normal tissue using functional endpoints are powerful tools for acquiring insights into the mechanisms that regulate normal tissue stem cell turnover and differentiation. In this paper, we describe a xenotransplantation-based protocol that allows mammary stem cells with in vivo tissue regenerative properties to be specifically detected and quantified among the heterogeneous cell populations obtained from dissociated normal human mammary tissue. This methodology involves implanting a collagen gel containing the test cells in combination with supportive fibroblasts under the kidney capsule of highly immune-deficient, hormone-supplemented mice and then, 4 weeks later, searching for regenerated human cells with in vitro clonogenic activity. Quantification of the input human mammary stem cells is achieved using standard limiting dilution transplant approaches. This approach circumvents the need to modify the mouse mammary fat pad, and is objective, rapid (∼5 weeks) and economical to perform. 10.1038/nprot.2010.148
    Membrane redistributions through multi-intercellular exchanges and serial trogocytosis. Alegre Estibaliz,Howangyin Kiave-Yune,Favier Benoit,Baudhuin Jeremy,Lesport Emilie,Daouya Marina,Gonzalez Alvaro,Carosella Edgardo D,Lemaoult Joel Cell research Trogocytosis is a rapid transfer between cells of membranes and associated proteins. Trogocytic exchanges have been investigated between different cell types, mainly in two-cell systems, involving one donor and one acceptor cell type. Here, we studied trogocytosis in a more complex system, involving not only several immune cell subsets but also multiple tumor cells. We show that CD4(+) T cells, CD8(+) T cells and monocytes can acquire membrane patches and the intact proteins they contain from different tumor cells by multiple simultaneous trogocytoses. The trogocytic capabilities of CD4(+) and CD8(+) T cells were found to be similar, but inferior to that of autologous monocytes. Activated peripheral-blood mononuclear cells (PBMCs) may also exchange membranes between themselves in an all-autologous system. For this reason, monocytes are capable of acquiring membranes from multiple tumor cell sources, and transfer them again to autologous T cells, along with some of their own membranes (serial trogocytosis). Our data illustrate the extent of membrane exchanges between autologous activated immune effector cells and their environment, and how the cellular content of the local environment, including "bystander" cells, may impact the functions of immune effector cells. 10.1038/cr.2010.136
    Increased Production of LIGHT by T Cells in Eosinophilic Esophagitis Promotes Differentiation of Esophageal Fibroblasts Toward an Inflammatory Phenotype. Manresa Mario C,Chiang Austin W T,Kurten Richard C,Dohil Ranjan,Brickner Howard,Dohil Lucas,Herro Rana,Akuthota Praveen,Lewis Nathan E,Croft Michael,Aceves Seema S Gastroenterology BACKGROUND & AIMS:Eosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFβ1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE. METHODS:We analyzed publicly available esophageal CD3 T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFβ1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry. RESULTS:LIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFβ1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFβ1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1. CONCLUSIONS:T cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFβ1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE. 10.1053/j.gastro.2020.07.035
    RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3. Karmakar Saswati,Rauth Sanchita,Nallasamy Palanisamy,Perumal Naveenkumar,Nimmakayala Rama Krishna,Leon Frank,Gupta Rohitesh,Barkeer Srikanth,Venkata Ramakanth Chirravuri,Raman Venu,Rachagani Satyanarayana,Ponnusamy Moorthy P,Batra Surinder K Gastroenterology BACKGROUND & AIMS:It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. METHODS:Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. RESULTS:Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and β-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). CONCLUSIONS:In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer. 10.1053/j.gastro.2020.07.053
    Tracking of Inhaled Near-Infrared Fluorescent Nanoparticles in Lungs of SKH-1 Mice with Allergic Airway Inflammation. Markus M Andrea,Napp Joanna,Behnke Thomas,Mitkovski Miso,Monecke Sebastian,Dullin Christian,Kilfeather Stephen,Dressel Ralf,Resch-Genger Ute,Alves Frauke ACS nano Molecular imaging of inflammatory lung diseases, such as asthma, has been limited to date. The recruitment of innate immune cells to the airways is central to the inflammation process. This study exploits these cells for imaging purposes within the lung, using inhaled polystyrene nanoparticles loaded with the near-infrared fluorescence dye Itrybe (Itrybe-NPs). By means of in vivo and ex vivo fluorescence reflectance imaging of an ovalbumin-based allergic airway inflammation (AAI) model in hairless SKH-1 mice, we show that subsequent to intranasal application of Itrybe-NPs, AAI lungs display fluorescence intensities significantly higher than those in lungs of control mice for at least 24 h. Ex vivo immunofluorescence analysis of lung tissue demonstrates the uptake of Itrybe-NPs predominantly by CD68(+)CD11c(+)ECF-L(+)MHCII(low) cells, identifying them as alveolar M2 macrophages in the peribronchial and alveolar areas. The in vivo results were validated by confocal microscopy, overlapping tile analysis, and flow cytometry, showing an amount of Itrybe-NP-containing macrophages in lungs of AAI mice significantly larger than that in controls. A small percentage of NP-containing cells were identified as dendritic cells. Flow cytometry of tracheobronchial lymph nodes showed that Itrybe-NPs were negligible in lung draining lymph nodes 24 h after inhalation. This imaging approach may advance preclinical monitoring of AAI in vivo over time and aid the investigation of the role that macrophages play during lung inflammation. Furthermore, it allows for tracking of inhaled nanoparticles and can hence be utilized for studies of the fate of potential new nanotherapeutics. 10.1021/acsnano.5b04026
    Aryl Hydrocarbon Receptor Ligands in Cigarette Smoke Induce Production of Interleukin-22 to Promote Pancreatic Fibrosis in Models of Chronic Pancreatitis. Xue Jing,Zhao Qinglan,Sharma Vishal,Nguyen Linh P,Lee Yvonne N,Pham Kim L,Edderkaoui Mouad,Pandol Stephen J,Park Walter,Habtezion Aida Gastroenterology BACKGROUND & AIMS:Cigarette smoke has been identified as an independent risk factor for chronic pancreatitis (CP). Little is known about the mechanisms by which smoking promotes development of CP. We assessed the effects of aryl hydrocarbon receptor (AhR) ligands found in cigarette smoke on immune cell activation in humans and pancreatic fibrosis in animal models of CP. METHODS:We obtained serum samples from patients with CP treated at Stanford University hospital and healthy individuals (controls) and isolated CD4 T cells. Levels of interleukin-22 (IL22) were measured by enzyme-linked immunosorbent assay and smoking histories were collected. T cells from healthy nonsmokers and smokers were stimulated and incubated with AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene) or antagonists and analyzed by flow cytometry. Mice were given intraperitoneal injections of caerulein or saline, with or without lipopolysaccharide, to induce CP. Some mice were given intraperitoneal injections of AhR agonists at the start of caerulein injection, with or without an antibody against IL22 (anti-IL22) starting 2 weeks after the first caerulein injection, or recombinant mouse IL22 or vehicle (control) intraperitoneally 4 weeks after the first caerulein injection. Mice were exposed to normal air or cigarette smoke for 6 h/d for 7 weeks and expression of AhR gene targets was measured. Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. Pancreatic stellate cells and T cells were isolated and studied using immunoblot, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent analyses. RESULTS:Mice given AhR agonists developed more severe pancreatic fibrosis (based on decreased pancreas size, histology, and increased expression of fibrosis-associated genes) than mice not given agonists after caerulein injection. In mice given saline instead of caerulein, AhR ligands did not induce fibrosis. Pancreatic T cells from mice given AhR agonists and caerulein were activated and expressed IL22, but not IL17 or interferon gamma. Human T cells exposed to AhR agonists up-regulated expression of IL22. In mice given anti-IL22, pancreatic fibrosis did not progress, whereas mice given recombinant IL22 had a smaller pancreas and increased fibrosis. Pancreatic stellate cells isolated from mouse and human pancreata expressed the IL22 receptor IL22RA1. Incubation of the pancreatic stellate cells with IL22 induced their expression of the extracellular matrix genes fibronectin 1 and collagen type I α1 chain, but not α2 smooth muscle actin or transforming growth factor-β. Serum samples from smokers had significantly higher levels of IL22 than those from nonsmokers. CONCLUSIONS:AhR ligands found in cigarette smoke increase the severity of pancreatic fibrosis in mouse models of pancreatitis via up-regulation of IL22. This pathway might be targeted for treatment of CP and serve as a biomarker of disease. 10.1053/j.gastro.2016.09.064
    NKp46 is a diagnostic biomarker and may be a therapeutic target in gastrointestinal T-cell lymphoproliferative diseases: a CELAC study. Cheminant Morgane,Bruneau Julie,Malamut Georgia,Sibon David,Guegan Nicolas,van Gils Tom,Cording Sascha,Trinquand Amélie,Verkarre Virginie,Lhermitte Ludovic,Brousse Nicole,Jannot Anne-Sophie,Khater Sherine,Frenzel Laurent,Delarue Richard,Suarez Felipe,Marçais Ambroise,Mulder Chris Jj,Macintyre Elizabeth,Asnafi Vahid,Pouyet Laurent,Bonnafous Cécile,Lhospice Florence,Molina Thierry Jo,Meresse Bertrand,Cellier Christophe,Cerf-Bensussan Nadine,Hermine Olivier, Gut OBJECTIVES:Primary GI T-cell lymphoproliferative diseases (T-LPD) are heterogeneous entities, which raise difficult diagnosis and therapeutic challenges. We have recently provided evidences that lymphomas complicating coeliac disease (CD) arise from innate-like lymphocytes, which may carry NK receptors (NKRs). DESIGN:NKRs expression was compared by flow cytometry in intraepithelial lymphocytes (IEL) from CD, type I or type II refractory CD (RCD). NKp46 was next assessed by immunohistochemistry in paraffin-embedded biopsies from 204 patients with CD, RCDI, RCDII or GI T-cell lymphomas and from a validation cohort of 61 patients. The cytotoxic properties of an anti-NKp46 monoclonal antibody conjugated to pyrrolobenzodiazepine (PBD) was tested in human primary tumour cells isolated from fresh duodenal biopsies. RESULTS:NKp46 (but not CD94, NKG2A, NKG2C, NKG2D) was significantly more expressed by malignant RCDII IEL than by normal IEL in CD and RCDI. In paraffin biopsies, detection of >25 NKp46+ IEL per 100 epithelial cells discriminated RCDII from CD and RCDI. NKp46 was also detected in enteropathy-associated T-cell lymphomas (EATL, 24/29) and in monomorphic epitheliotropic intestinal T-cell lymphomas (MEITL, 4/4) but not in indolent T-LPD (0/15). Treatment with anti-NKp46-PBD could efficiently and selectively kill human NKp46+ primary IEL . CONCLUSION:NKp46 is a novel biomarker useful for diagnosis and therapeutic stratification of GI T-LPD. Strong preclinical rationale identifies anti-NKp46-PBD as a promising therapy for RCDII, EATL and MEITL. 10.1136/gutjnl-2018-317371
    The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF. El-Behi Mohamed,Ciric Bogoljub,Dai Hong,Yan Yaping,Cullimore Melissa,Safavi Farinaz,Zhang Guang-Xian,Dittel Bonnie N,Rostami Abdolmohamad Nature immunology Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) require exposure to IL-23 to become encephalitogenic, but the mechanism by which IL-23 promotes their pathogenicity is not known. Here we found that IL-23 induced production of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in T(H)17 cells and that GM-CSF had an essential role in their encephalitogenicity. Our findings identify a chief mechanism that underlies the important role of IL-23 in autoimmune diseases. IL-23 induced a positive feedback loop whereby GM-CSF secreted by T(H)17 cells stimulated the production of IL-23 by antigen-presenting cells. Such cross-regulation of IL-23 and GM-CSF explains the similar pattern of resistance to autoimmunity when either of the two cytokines is absent and identifies T(H)17 cells as a crucial source of GM-CSF in autoimmune inflammation. 10.1038/ni.2031
    Transcription factor Foxp1 exerts essential cell-intrinsic regulation of the quiescence of naive T cells. Feng Xiaoming,Wang Haikun,Takata Hiroshi,Day Timothy J,Willen Jessica,Hu Hui Nature immunology The molecular mechanisms that underlie T cell quiescence are poorly understood. Here we report that mature naive CD8(+) T cells lacking the transcription factor Foxp1 gained effector phenotype and function and proliferated directly in response to interleukin 7 (IL-7) in vitro. Foxp1 repressed expression of the IL-7 receptor α-chain (IL-7Rα) by antagonizing Foxo1 and negatively regulated signaling by the kinases MEK and Erk. Acute deletion of Foxp1 induced naive T cells to gain an effector phenotype and proliferate in lympho-replete mice. Foxp1-deficient naive CD8(+) T cells proliferated even in lymphopenic mice deficient in major histocompatibility complex class I. Our results demonstrate that Foxp1 exerts essential cell-intrinsic regulation of naive T cell quiescence, providing direct evidence that lymphocyte quiescence is achieved through actively maintained mechanisms that include transcriptional regulation. 10.1038/ni.2034
    Genetic analysis of basophil function in vivo. Sullivan Brandon M,Liang Hong-Erh,Bando Jennifer K,Wu Davina,Cheng Laurence E,McKerrow James K,Allen Christopher D C,Locksley Richard M Nature immunology Contributions by basophils to allergic and helminth immunity remain incompletely defined. Using sensitive interleukin 4 (Il4) reporter alleles, we demonstrate here that basophil IL-4 production occurs by a CD4(+) T cell-dependent process restricted to the peripheral tissues affected. We genetically marked and achieved specific deletion of basophils and found that basophils did not mediate T helper type 2 (T(H)2) priming in vivo. Two-photon imaging confirmed that basophils did not interact with antigen-specific T cells in lymph nodes but engaged in prolonged serial interactions with T cells in lung tissues. Although targeted deletion of IL-4 and IL-13 in either CD4(+) T cells or basophils had a minimal effect on worm clearance, deletion from both lineages demonstrated a nonredundant role for basophil cytokines in primary helminth immunity. 10.1038/ni.2036
    The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Ise Wataru,Kohyama Masako,Schraml Barbara U,Zhang Tingting,Schwer Bjoern,Basu Uttiya,Alt Frederick W,Tang Jun,Oltz Eugene M,Murphy Theresa L,Murphy Kenneth M Nature immunology The transcription factor BATF controls the differentiation of interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) by regulating expression of the transcription factor RORγt itself and RORγt target genes such as Il17. Here we report the mechanism by which BATF controls in vivo class-switch recombination (CSR). In T cells, BATF directly controlled expression of the transcription factors Bcl-6 and c-Maf, both of which are needed for development of follicular helper T cells (T(FH) cells). Restoring T(FH) cell activity to Batf(-/-) T cells in vivo required coexpression of Bcl-6 and c-Maf. In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (I(H)-C(H)). Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo. 10.1038/ni.2037
    Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway. Marcet Brice,Chevalier Benoît,Luxardi Guillaume,Coraux Christelle,Zaragosi Laure-Emmanuelle,Cibois Marie,Robbe-Sermesant Karine,Jolly Thomas,Cardinaud Bruno,Moreilhon Chimène,Giovannini-Chami Lisa,Nawrocki-Raby Béatrice,Birembaut Philippe,Waldmann Rainer,Kodjabachian Laurent,Barbry Pascal Nature cell biology Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors. 10.1038/ncb2241
    Group 2 Innate Lymphoid Cells Attenuate Inflammatory Arthritis and Protect from Bone Destruction in Mice. Omata Yasunori,Frech Michael,Primbs Tatjana,Lucas Sébastien,Andreev Darja,Scholtysek Carina,Sarter Kerstin,Kindermann Markus,Yeremenko Nataliya,Baeten Dominique L,Andreas Nico,Kamradt Thomas,Bozec Aline,Ramming Andreas,Krönke Gerhard,Wirtz Stefan,Schett Georg,Zaiss Mario M Cell reports Group 2 innate lymphoid cells (ILC2s) were detected in the peripheral blood and the joints of rheumatoid arthritis (RA) patients, serum-induced arthritis (SIA), and collagen-induced arthritis (CIA) using flow cytometry. Circulating ILC2s were significantly increased in RA patients compared with healthy controls and inversely correlated with disease activity. Induction of arthritis in mice led to a fast increase in ILC2 number. To elucidate the role of ILC2 in arthritis, loss- and gain-of-function mouse models for ILC2 were subjected to arthritis. Reduction of ILC2 numbers in RORα/GATA3 and Tie2/RORα mice significantly exacerbated arthritis. Increasing ILC2 numbers in mice by IL-25/IL-33 mini-circles or IL-2/IL-2 antibody complex and the adoptive transfer of wild-type (WT) ILC2s significantly attenuated arthritis by affecting the initiation phase. In addition, adoptive transfer of IL-4/13-competent WT but not IL-4/13 ILC2s and decreased cytokine secretion by macrophages. These data show that ILC2s have immune-regulatory functions in arthritis. 10.1016/j.celrep.2018.06.005
    Characterization of seven kidney tumors by flow cytometry: analysis of cell cycle, DNA content and P-glycoprotein expression. Lizard G,Roignot P,Dusserre-Guion L,Morlevat F,Michiels-Marzais D,Ferry N,Trémeaux J C European urology Seven kidney tumors obtained from patients aged from 5 to 76 years were analyzed by flow cytometry for cell cycle, DNA content and P-glycoprotein expression involved in multidrug resistance. The DNA index seems to be an important criterion since all the tumors were aneuploid. In a case of clear cell carcinoma, two aneuploid clones were identified. In 5 cases of kidney tumors a high proportion of cells in proliferation (S + (G2 + M)) was observed; it was comprised between 13 and 33%. As for P-glycoprotein it was detected only in few tumor cells (5-15%) respectively in a case of clear cell carcinoma and in a case of Wilms' tumor.
    Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity. Abbas Hussein A,Maccio Daniela R,Coskun Suleyman,Jackson James G,Hazen Amy L,Sills Tiffany M,You M James,Hirschi Karen K,Lozano Guillermina Cell stem cell Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis. 10.1016/j.stem.2010.09.013
    Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Moeller Antje,Gilpin Sarah E,Ask Kjetil,Cox Gerard,Cook Deborah,Gauldie Jack,Margetts Peter J,Farkas Laszlo,Dobranowski Julian,Boylan Colm,O'Byrne Paul M,Strieter Robert M,Kolb Martin American journal of respiratory and critical care medicine RATIONALE:The clinical management of idiopathic pulmonary fibrosis (IPF) remains a major challenge due to lack of effective drug therapy or accurate indicators for disease progression. Fibrocytes are circulating mesenchymal cell progenitors that are involved in tissue repair and fibrosis. OBJECTIVES:To test the hypothesis that assay of these cells may provide a biomarker for activity and progression of IPF. METHODS:Fibrocytes were defined as cells positive for CD45 and collagen-1 by flow cytometry and quantified in patients with stable IPF and during acute exacerbation of the disease. We investigated the clinical and prognostic value of fibrocyte counts by comparison with standard clinical parameters and survival. We used healthy age-matched volunteers and patients with acute respiratory distress syndrome as control subjects. MEASUREMENTS AND MAIN RESULTS:Fibrocytes were significantly elevated in patients with stable IPF (n = 51), with a further increase during acute disease exacerbation (n = 7; P < 0.001 vs. control subjects). Patients with acute respiratory distress syndrome (n = 10) were not different from healthy control subjects or stable patients with IPF. Fibrocyte numbers were not correlated with lung function or radiologic severity scores, but they were an independent predictor of early mortality. The mean survival of patients with fibrocytes higher than 5% of total blood leukocytes was 7.5 months compared with 27 months for patients with less than 5% (P < 0.0001). CONCLUSIONS:Fibrocytes are an indicator for disease activity of IPF and might be useful as a clinical marker for disease progression. This study suggests that quantification of circulating fibrocytes may allow prediction of early mortality in patients with IPF. 10.1164/rccm.200810-1534OC
    Peripheral CD4+ T cell subsets and antibody response in COVID-19 convalescent individuals. Gong Fang,Dai Yaping,Zheng Ting,Cheng Liang,Zhao Dan,Wang Hao,Liu Min,Pei Hao,Jin Tengchuan,Yu Di,Zhou Pengcheng The Journal of clinical investigation BACKGROUNDMarked progress is achieved in understanding the physiopathology of coronavirus disease 2019 (COVID-19), which caused a global pandemic. However, the CD4+ T cell population critical for antibody response in COVID-19 is poorly understood.METHODSIn this study, we provided a comprehensive analysis of peripheral CD4+ T cells from 13 COVID-19 convalescent patients, defined as confirmed free of SARS-CoV-2 for 2 to 4 weeks, using flow cytometry and magnetic chemiluminescence enzyme antibody immunoassay. The data were correlated with clinical characteristics.RESULTSWe observed that, relative to healthy individuals, convalescent patients displayed an altered peripheral CD4+ T cell spectrum. Specifically, consistent with other viral infections, cTfh1 cells associated with SARS-CoV-2-targeting antibodies were found in COVID-19 covalescent patients. Individuals with severe disease showed higher frequencies of Tem and Tfh-em cells but lower frequencies of Tcm, Tfh-cm, Tfr, and Tnaive cells, compared with healthy individuals and patients with mild and moderate disease. Interestingly, a higher frequency of cTfh-em cells correlated with a lower blood oxygen level, recorded at the time of admission, in convalescent patients. These observations might constitute residual effects by which COVID-19 can impact the homeostasis of CD4+ T cells in the long-term and explain the highest ratio of class-switched virus-specific antibody producing individuals found in our severe COVID-19 cohort.CONCLUSIONOur study demonstrated a close connection between CD4+ T cells and antibody production in COVID-19 convalescent patients.FUNDINGSix Talent Peaks Project in Jiangsu Province and the National Natural Science Foundation of China (NSFC). 10.1172/JCI141054
    mTOR activation induces tumor suppressors that inhibit leukemogenesis and deplete hematopoietic stem cells after Pten deletion. Lee Jae Y,Nakada Daisuke,Yilmaz Omer H,Tothova Zuzana,Joseph Nancy M,Lim Megan S,Gilliland D Gary,Morrison Sean J Cell stem cell Pten deficiency depletes hematopoietic stem cells (HSCs) but expands leukemia-initiating cells, and the mTOR inhibitor, rapamycin, blocks these effects. Understanding the opposite effects of mTOR activation on HSCs versus leukemia-initiating cells could improve antileukemia therapies. We found that the depletion of Pten-deficient HSCs was not caused by oxidative stress and could not be blocked by N-acetyl-cysteine. Instead, Pten deletion induced, and rapamycin attenuated, the expression of p16(Ink4a) and p53 in HSCs, and p19(Arf) and p53 in other hematopoietic cells. p53 suppressed leukemogenesis and promoted HSC depletion after Pten deletion. p16(Ink4a) also promoted HSC depletion but had a limited role suppressing leukemogenesis. p19(Arf) strongly suppressed leukemogenesis but did not deplete HSCs. Secondary mutations attenuated this tumor suppressor response in some leukemias that arose after Pten deletion. mTOR activation therefore depletes HSCs by a tumor suppressor response that is attenuated by secondary mutations in leukemogenic clones. 10.1016/j.stem.2010.09.015
    Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B. Kennedy Patrick T F,Sandalova Elena,Jo Juandy,Gill Upkar,Ushiro-Lumb Ines,Tan Anthony T,Naik Sandhia,Foster Graham R,Bertoletti Antonio Gastroenterology BACKGROUND & AIMS:Chronic hepatitis B (CHB) infection acquired perinatally or in early childhood has been associated with a prolonged phase of immune tolerance from viral exposure into early adulthood. The immune-tolerant phase of the disease is characterized by high levels of hepatitis B virus (HBV) DNA and normal liver biochemistry, with minimal or no fibrosis. We investigated whether the age of patients with CHB affects their antiviral immunity and whether children and young adults have a veritable state of immunologic tolerance. METHODS:We isolated T cells from different age groups of patients with CHB and used flow cytometric methods to measure production of effector and inflammatory cytokines (interferon, tumor necrosis factor, interleukin [IL]-17A, IL-22, and IL-8), T-helper (Th)2 cytokines (IL-10, IL-4), Th1 cytokines (IL-2 and IL-21), and the CC chemokine CCL3 (MIP-1). We also measured markers of T-cell exhaustion or inhibition (PD-1, LAG-3, TIM3, LAIR-1, and CTLA-4) and HBV-specific T cells. RESULTS:Young patients with CHB have a Th1-cell cytokine profile and a partial profile of T-cell exhaustion. Direct quantification of the HBV-specific T-cell response showed that young patients with CHB have more HBV-specific T cells with the ability to proliferate and produce cytokines than adult patients with CHB. CONCLUSIONS:HBV infection in younger patients is not associated with an immune profile of T-cell tolerance. On the contrary, children and young adults with chronic HBV infection have an HBV-specific immune profile that is less compromised than that observed in older patients. 10.1053/j.gastro.2012.06.009
    S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants. Willers Maike,Ulas Thomas,Völlger Lena,Vogl Thomas,Heinemann Anna S,Pirr Sabine,Pagel Julia,Fehlhaber Beate,Halle Olga,Schöning Jennifer,Schreek Sabine,Löber Ulrike,Essex Morgan,Hombach Peter,Graspeuntner Simon,Basic Marijana,Bleich Andre,Cloppenborg-Schmidt Katja,Künzel Sven,Jonigk Danny,Rupp Jan,Hansen Gesine,Förster Reinhold,Baines John F,Härtel Christoph,Schultze Joachim L,Forslund Sofia K,Roth Johannes,Viemann Dorothee Gastroenterology BACKGROUND & AIMS:After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. METHODS:We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9 mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS:Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. CONCLUSION:S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years. 10.1053/j.gastro.2020.08.019
    Group 2 Innate Lymphoid Cells Coordinate Damage Response in the Stomach. Meyer Anne R,Engevik Amy C,Madorsky Toni,Belmont Erika,Stier Matthew T,Norlander Allison E,Pilkinton Mark A,McDonnell Wyatt J,Weis Jared A,Jang Bogun,Mallal Simon A,Peebles R Stokes,Goldenring James R Gastroenterology BACKGROUND & AIMS:Severe injury to the lining of the stomach leads to changes in the epithelium (reprogramming) that protect and promote repair of the tissue, including development of spasmolytic polypeptide-expressing metaplasia (SPEM) and tuft and foveolar cell hyperplasia. Acute gastric damage elicits a type-2 inflammatory response that includes production of type-2 cytokines and infiltration by eosinophils and alternatively activated macrophages. Stomachs of mice that lack interleukin 33 (IL33) or interleukin 13 (IL13) did not undergo epithelial reprogramming after drug-induced injury. We investigated the role of group 2 innate lymphoid cells (ILC2s) in gastric epithelial repair. METHODS:Acute gastric injury was induced in C57BL/6J mice (wild-type and RAG1 knockout) by administration of L635. We isolated ILC2s by flow cytometry from stomachs of mice that were and were not given L635 and performed single-cell RNA sequencing. ILC2s were depleted from wild-type and RAG1-knockout mice by administration of anti-CD90.2. We assessed gastric cell lineages, markers of metaplasia, inflammation, and proliferation. Gastric tissue microarrays from patients with gastric adenocarcinoma were analyzed by immunostaining. RESULTS:There was a significant increase in the number of GATA3-positive ILC2s in stomach tissues from wild-type mice after L635-induced damage, but not in stomach tissues from IL33-knockout mice. We characterized a marker signature of gastric mucosal ILC2s and identified a transcription profile of metaplasia-associated ILC2s, which included changes in expression of Il5, Il13, Csf2, Pd1, and Ramp3; these changes were validated by quantitative polymerase chain reaction and immunocytochemistry. Depletion of ILC2s from mice blocked development of metaplasia after L635-induced injury in wild-type and RAG1-knockout mice and prevented foveolar and tuft cell hyperplasia and infiltration or activation of macrophages after injury. Numbers of ILC2s were increased in stomach tissues from patients with SPEM compared with patients with normal corpus mucosa. CONCLUSIONS:In analyses of stomach tissues from mice with gastric tissue damage and patients with SPEM, we found evidence of type 2 inflammation and increased numbers of ILC2s. Our results suggest that ILC2s coordinate the metaplastic response to severe gastric injury. 10.1053/j.gastro.2020.08.051
    Recognition of β-linked self glycolipids mediated by natural killer T cell antigen receptors. Pellicci Daniel G,Clarke Andrew J,Patel Onisha,Mallevaey Thierry,Beddoe Travis,Le Nours Jérôme,Uldrich Adam P,McCluskey James,Besra Gurdyal S,Porcelli Steven A,Gapin Laurent,Godfrey Dale I,Rossjohn Jamie Nature immunology The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to β-linked antigens. 10.1038/ni.2076
    Type V collagen-induced tolerance prevents airway hyperresponsiveness. Lott Jeremy M,Sehra Sarita,Mehrotra Purvi,Mickler Elizabeth A,Fisher Amanda J,Zhang Wenwu,Presson Robert G,Busk Michael F,Goenka Shreevrat,Gunst Susan J,Kaplan Mark H,Wilkes David S,Wenzel Sally E American journal of respiratory and critical care medicine 10.1164/ajrccm.187.4.454
    A motor-driven mechanism for cell-length sensing. Rishal Ida,Kam Naaman,Perry Rotem Ben-Tov,Shinder Vera,Fisher Elizabeth M C,Schiavo Giampietro,Fainzilber Mike Cell reports Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing.Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development.Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1.Thus, molecular motors critically influence cell length sensing and growth control. 10.1016/j.celrep.2012.05.013
    Isolating and engineering human antibodies using yeast surface display. Chao Ginger,Lau Wai L,Hackel Benjamin J,Sazinsky Stephen L,Lippow Shaun M,Wittrup K Dane Nature protocols This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest. 10.1038/nprot.2006.94
    Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity. Hutton Colin,Heider Felix,Blanco-Gomez Adrian,Banyard Antonia,Kononov Alexander,Zhang Xiaohong,Karim Saadia,Paulus-Hock Viola,Watt Dale,Steele Nina,Kemp Samantha,Hogg Elizabeth K J,Kelly Joanna,Jackstadt Rene-Filip,Lopes Filipa,Menotti Matteo,Chisholm Luke,Lamarca Angela,Valle Juan,Sansom Owen J,Springer Caroline,Malliri Angeliki,Marais Richard,Pasca di Magliano Marina,Zelenay Santiago,Morton Jennifer P,Jørgensen Claus Cancer cell Fibroblasts display extensive transcriptional heterogeneity, yet functional annotation and characterization of their heterocellular relationships remains incomplete. Using mass cytometry, we chart the stromal composition of 18 murine tissues and 5 spontaneous tumor models, with an emphasis on mesenchymal phenotypes. This analysis reveals extensive stromal heterogeneity across tissues and tumors, and identifies coordinated relationships between mesenchymal and immune cell subsets in pancreatic ductal adenocarcinoma. Expression of CD105 demarks two stable and functionally distinct pancreatic fibroblast lineages, which are also identified in murine and human healthy tissues and tumors. Whereas CD105-positive pancreatic fibroblasts are permissive for tumor growth in vivo, CD105-negative fibroblasts are highly tumor suppressive. This restrictive effect is entirely dependent on functional adaptive immunity. Collectively, these results reveal two functionally distinct pancreatic fibroblast lineages and highlight the importance of mesenchymal and immune cell interactions in restricting tumor growth. 10.1016/j.ccell.2021.06.017
    Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Narsinh Kazim H,Jia Fangjun,Robbins Robert C,Kay Mark A,Longaker Michael T,Wu Joseph C Nature protocols Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks. 10.1038/nprot.2010.173
    In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Lo Celso Cristina,Lin Charles P,Scadden David T Nature protocols In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ∼1 week. 10.1038/nprot.2010.168
    IL-23 induces spondyloarthropathy by acting on ROR-γt+ CD3+CD4-CD8- entheseal resident T cells. Sherlock Jonathan P,Joyce-Shaikh Barbara,Turner Scott P,Chao Cheng-Chi,Sathe Manjiri,Grein Jeff,Gorman Daniel M,Bowman Edward P,McClanahan Terrill K,Yearley Jennifer H,Eberl Gérard,Buckley Christopher D,Kastelein Robert A,Pierce Robert H,Laface Drake M,Cua Daniel J Nature medicine The spondyloarthropathies are a group of rheumatic diseases that are associated with inflammation at anatomically distal sites, particularly the tendon-bone attachments (entheses) and the aortic root. Serum concentrations of interleukin-23 (IL-23) are elevated and polymorphisms in the IL-23 receptor are associated with ankyosing spondylitis, however, it remains unclear whether IL-23 acts locally at the enthesis or distally on circulating cell populations. We show here that IL-23 is essential in enthesitis and acts on previously unidentified IL-23 receptor (IL-23R)(+), RAR-related orphan receptor γt (ROR-γt)(+)CD3(+)CD4(-)CD8(-), stem cell antigen 1 (Sca1)(+) entheseal resident T cells. These cells allow entheses to respond to IL-23 in vitro-in the absence of further cellular recruitment--and to elaborate inflammatory mediators including IL-6, IL-17, IL-22 and chemokine (C-X-C motif) ligand 1 (CXCL1). Notably, the in vivo expression of IL-23 is sufficient to phenocopy the human disease, with the specific and characteristic development of enthesitis and entheseal new bone formation in the initial complete absence of synovitis. As in the human condition, inflammation also develops in vivo at the aortic root and valve, which are structurally similar to entheses. The presence of these entheseal resident cells and their production of IL-22, which activates signal transducer and activator of transcription 3 (STAT3)-dependent osteoblast-mediated bone remodeling, explains why dysregulation of IL-23 results in inflammation at this precise anatomical site. 10.1038/nm.2817
    The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Hanna Richard N,Carlin Leo M,Hubbeling Harper G,Nackiewicz Dominika,Green Angela M,Punt Jennifer A,Geissmann Frederic,Hedrick Catherine C Nature immunology The transcription factors that regulate differentiation into the monocyte subset in bone marrow have not yet been identified. Here we found that the orphan nuclear receptor NR4A1 controlled the differentiation of Ly6C- monocytes. Ly6C- monocytes, which function in a surveillance role in circulation, were absent from Nr4a1-/- mice. Normal numbers of myeloid progenitor cells were present in Nr4a1-/- mice, which indicated that the defect occurred during later stages of monocyte development. The defect was cell intrinsic, as wild-type mice that received bone marrow from Nr4a1-/- mice developed fewer patrolling monocytes than did recipients of wild-type bone marrow. The Ly6C- monocytes remaining in the bone marrow of Nr4a1-/- mice were arrested in S phase of the cell cycle and underwent apoptosis. Thus, NR4A1 functions as a master regulator of the differentiation and survival of 'patrolling' Ly6C- monocytes. 10.1038/ni.2063
    Autocrine IL-2 is required for secondary population expansion of CD8(+) memory T cells. Feau Sonia,Arens Ramon,Togher Susan,Schoenberger Stephen P Nature immunology Two competing theories have been put forward to explain the role of CD4(+) T cells in priming CD8(+) memory T cells: one proposes paracrine secretion of interleukin 2 (IL-2); the other proposes the activation of antigen-presenting cells (APCs) via the costimulatory molecule CD40 and its ligand CD40L. We investigated the requirement for IL-2 by the relevant three cell types in vivo and found that CD8(+) T cells, rather than CD4(+) T cells or dendritic cells (DCs), produced the IL-2 necessary for CD8(+) T cell memory. Il2(-/-) CD4(+) T cells were able to provide help only if their ability to transmit signals via CD40L was intact. Our findings reconcile contradictory elements implicit in each model noted above by showing that CD4(+) T cells activate APCs through a CD40L-dependent mechanism to enable autocrine production of IL-2 in CD8(+) memory T cells. 10.1038/ni.2079
    Sequential therapy with fludarabine, high-dose cyclophosphamide, and rituximab in previously untreated patients with chronic lymphocytic leukemia produces high-quality responses: molecular remissions predict for durable complete responses. Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Modern combination strategies are active in chronic lymphocytic leukemia (CLL) but can have significant myelosuppression and immunosuppression that may require dose attenuation for safety. We explored a sequential treatment strategy to allow safe delivery of active agents at full doses. Previously, we studied sequential therapy with fludarabine followed by cyclophosphamide (F-->C). In that study, cyclophosphamide consolidation improved the frequency of complete response (CR) four-fold. Subsequently, rituximab was added to this regimen (F-->C-->R). PATIENTS AND METHODS:Thirty-six previously untreated CLL patients received therapy with fludarabine 25 mg/m(2) on days 1 through 5 every 4 weeks for six cycles, followed by consolidation with cyclophosphamide 3,000 mg/m(2) administered every 3 weeks for three cycles, followed by consolidation with weekly rituximab 375 mg/m(2) for four cycles. Evaluation for minimal residual disease included flow cytometry and a highly sensitive clonotypic polymerase chain reaction (PCR). The median age was 59 years (range, 37 to 71 years), 61% of patients had high-risk disease, and 58% had unmutated IgV(H) genes. RESULTS:There were 32 responses (89%), including 22 CRs (61%). Consolidation with cyclophosphamide improved responses in 13 patients (36%); nine patients (25%) further improved their response with rituximab. Twenty patients (56%) achieved flow cytometric CRs, and 12 patients (33%) achieved a molecular CR (PCR negative). Patients achieving molecular CRs had an excellent prognosis with a plateau in the response duration curve, and 90% remain in clinical CR at 5 years. For the entire group, 5-year survival rate is 71% compared with a rate of 48% with our prior F-->C regimen (P = .10). CONCLUSION:Sequential therapy with F-->C-->R yields improvement in quality of response, with many patients achieving a PCR-negative state. 10.1200/JCO.2008.16.4459
    Prolonged adaptive immune activation in COVID-19: implications for maintenance of long-term immunity? Mudd Philip A,Remy Kenneth E The Journal of clinical investigation Ongoing observational clinical research has prioritized understanding the human immune response to SARS-CoV-2 during the coronavirus disease 2019 (COVID-19) pandemic. Several recent studies suggest that immune dysregulation with early and prolonged adaptive immune system activation can result in cellular exhaustion. In this issue of the JCI, Files et al. compared cellular immune phenotypes during the first two months of COVID-19 in hospitalized and less severe, non-hospitalized patients. The authors utilized flow cytometry to analyze circulating peripheral blood mononuclear cells. Both patient cohorts maintained B and T cell phenotypes consistent with activation and cellular exhaustion throughout the first two months of infection. Additionally, follow-up samples from the non-hospitalized patient cohort showed that activation markers and cellular exhaustion increased over time. These findings illustrate the persistent nature of the adaptive immune system changes that have been noted in COVID-19 and suggest longer term effects that may shape the maintenance of immunity to SARS-CoV-2. 10.1172/JCI143928
    Lung megakaryocytes are immune modulatory cells. Pariser Daphne N,Hilt Zachary T,Ture Sara K,Blick-Nitko Sara K,Looney Mark R,Cleary Simon J,Roman-Pagan Estheany,Saunders Jerry,Georas Steve N,Veazey Janelle,Madere Ferralita,Santos Laura Tesoro,Arne Allison,Huynh Nguyen Pt,Livada Alison C,Guerrero-Martin Selena M,Lyons Claire,Metcalf-Pate Kelly A,McGrath Kathleen E,Palis James,Morrell Craig N The Journal of clinical investigation Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment. 10.1172/JCI137377
    Phase I/II Study of CPX-351 Followed by Fludarabine, Cytarabine, and Granulocyte-Colony Stimulating Factor for Children With Relapsed Acute Myeloid Leukemia: A Report From the Children's Oncology Group. Cooper Todd M,Absalon Michael J,Alonzo Todd A,Gerbing Robert B,Leger Kasey J,Hirsch Betsy A,Pollard Jessica,Razzouk Bassem I,Aplenc Richard,Kolb E Anders Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Effective regimens are needed for children with relapsed acute myeloid leukemia (AML). AAML1421 is a phase I/II study of CPX-351, a liposomal preparation of daunorubicin and cytarabine. AAML1421 sought to determine the recommended phase II dose (RP2D) of CPX-351 and the response rate after up to 2 cycles of therapy. PATIENTS AND METHODS:Children > 1 and ≤ 21 years of age with relapsed/refractory AML were eligible for dose finding; those in first relapse were eligible for the efficacy phase. Dose-limiting toxicity (DLT) assessment occurred during cycle 1. Two cycles of therapy were offered (cycle 1: CPX-351; cycle 2: FLAG [fludarabine 30 mg/m/dose on days 1-5; cytarabine 2,000 mg/m/dose on days 1-5; and granulocyte-colony stimulating factor 5 µg/kg/dose, days 1-5 and day 15 through absolute neutrophil count > 500/µL]). Response was assessed after each cycle. RESULTS:Thirty-eight patients enrolled: 6 in the dose-finding phase and 32 in the efficacy phase. During dose finding, 1/6 patients experienced a DLT (grade 3 decrease in ejection fraction). The RP2D was 135 units/m on days 1, 3, and 5. Toxicities of grade ≥ 3 during cycle 1 included fever/neutropenia (45%), infection (47%), and rash (40%). There was no toxic mortality. Best responses included 20 complete response (CR; 54%), 5 CR with partial recovery of platelet count (CRp; 14%), and 5 CR with incomplete blood count recovery (14%). Twenty-one of 25 with CR/CRp had no detectable residual disease (RD; 84%) by flow cytometry. Hematopoietic stem cell transplantation (HSCT) was used as consolidation in 29/30 responders (96.7%); 20/25 (80%) had no RD before HSCT. CONCLUSION:The RP2D of CPX-351 is 135 units/m/dose on days 1, 3, and 5. Toxicity was manageable, and protocol therapy was effective. Response rates are superior to prior published North American cooperative group clinical trials for children with AML in first relapse. 10.1200/JCO.19.03306
    An essential role of the transcription factor GATA-3 for the function of regulatory T cells. Wang Yunqi,Su Maureen A,Wan Yisong Y Immunity Forkhead Box P3 (Foxp3)-expressing regulatory T (Treg) cells are central to maintaining self-tolerance and immune homeostasis. How Treg cell function and Foxp3 expression are regulated is an important question under intensive investigation. Here, we have demonstrated an essential role for the transcription factor GATA-3, a previously recognized Th2 cell master regulator, in controlling Treg cell function. Treg cell-specific GATA-3 deletion led to a spontaneous inflammatory disorder in mice. GATA-3-null Treg cells were defective in peripheral homeostasis and suppressive function, gained Th17 cell phenotypes, and expressed reduced amounts of Foxp3. In addition, GATA-3 controlled Foxp3 expression by binding to and promoting the activity of cis-acting elements of Foxp3. Furthermore, the combined function of GATA-3 and Foxp3 was essential for Foxp3 expression. These findings provide insights into immune regulatory mechanisms and uncover a critical function of GATA-3 in Treg cells and immune tolerance. 10.1016/j.immuni.2011.08.012
    APOBEC3B interaction with PRC2 modulates microenvironment to promote HCC progression. Wang Duowei,Li Xianjing,Li Jiani,Lu Yuan,Zhao Sen,Tang Xinying,Chen Xin,Li Jiaying,Zheng Yan,Li Shuran,Sun Rui,Yan Ming,Yu Decai,Cao Guangwen,Yang Yong Gut OBJECTIVE:APOBEC3B (A3B), a cytidine deaminase acting as a contributor to the APOBEC mutation pattern in many kinds of tumours, is upregulated in patients with hepatocellular carcinoma (HCC). However, APOBEC mutation patterns are absent in HCC. The mechanism of how A3B affects HCC progression remains elusive. DESIGN:A3B -promoter luciferase reporter and other techniques were applied to elucidate mechanisms of A3B upregulation in HCC. A3B overexpression and knockdown cell models, immunocompetent and immune-deficient mouse HCC model were conducted to investigate the influence of A3B on HCC progression. RNA-seq, flow cytometry and other techniques were conducted to analyse how A3B modulated the cytokine to enhance the recruitment of myeloid--derived suppressor cells (MDSCs) and tumour--associated macrophages (TAMs). RESULTS:A3B upregulation through non-classical nuclear factor-κB (NF-κB)signalling promotes HCC growth in immunocompetent mice, associated with an increase of MDSCs, TAMs and programmed cell death1 (PD1) exprssed CD8 T cells. A CCR2 antagonist suppressed TAMs and MDSCs infiltration and delayed tumour growth in A3B and A3B- expressing mouse tumours. Mechanistically, A3B upregulation in HCC depresses global H3K27me3 abundance via interaction with polycomb repressor complex 2 (PRC2) and reduces an occupancy of H3K27me3 on promoters of the chemokine CCL2 to recruit massive TAMs and MDSCs. CONCLUSION:Our observations uncover a deaminase-independent role of the A3B in modulating the HCC microenvironment and demonstrate a proof for the concept of targeting A3B in HCC immunotherapy. 10.1136/gutjnl-2018-317601
    The signaling adaptor Eps8 is an essential actin capping protein for dendritic cell migration. Frittoli Emanuela,Matteoli Gianluca,Palamidessi Andrea,Mazzini Elisa,Maddaluno Luigi,Disanza Andrea,Yang Changsong,Svitkina Tatyana,Rescigno Maria,Scita Giorgio Immunity Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D in vitro and are delayed in reaching the draining lymph node (DLN) in vivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration. 10.1016/j.immuni.2011.07.007
    Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes. Kumar Rashmi,Ferez María,Swamy Mahima,Arechaga Ignacio,Rejas María Teresa,Valpuesta Jose M,Schamel Wolfgang W A,Alarcon Balbino,van Santen Hisse M Immunity Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory. 10.1016/j.immuni.2011.08.010
    DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer. Bailey Jennifer M,Alsina Janivette,Rasheed Zeshaan A,McAllister Florencia M,Fu Ya-Yuan,Plentz Ruben,Zhang Hao,Pasricha Pankaj J,Bardeesy Nabeel,Matsui William,Maitra Anirban,Leach Steven D Gastroenterology BACKGROUND & AIMS:As in other tumor types, progression of pancreatic cancer may require a functionally unique population of cancer stem cells. Although such cells have been identified in many invasive cancers, it is not clear whether they emerge during early or late stages of tumorigenesis. Using mouse models and human pancreatic cancer cell lines, we investigated whether preinvasive pancreatic neoplasia contains a subpopulation of cells with distinct morphologies and cancer stem cell-like properties. METHODS:Pancreatic tissue samples were collected from the KC(Pdx1), KPC(Pdx1), and KC(iMist1) mouse models of pancreatic intraepithelial neoplasia (PanIN) and analyzed by confocal and electron microscopy, lineage tracing, and fluorescence-activated cell sorting. Subpopulations of human pancreatic ductal adenocarcinoma (PDAC) cells were similarly analyzed and also used in complementary DNA microarray analyses. RESULTS:The microtubule regulator DCLK1 marked a morphologically distinct and functionally unique population of pancreatic cancer-initiating cells. These cells displayed morphological and molecular features of gastrointestinal tuft cells. Cells that expressed DCLK1 also expressed high levels of ATAT1, HES1, HEY1, IGF1R, and ABL1, and manipulation of these pathways in PDAC cell lines inhibited their clonogenic potential. Pharmacological inhibition of γ-secretase activity reduced the abundance of these cells in murine PanIN in a manner that correlated with inhibition of PanIN progression. CONCLUSIONS:Human PDAC cells and pancreatic neoplasms in mice contain morphologically and functionally distinct subpopulations that have cancer stem cell-like properties. These populations can be identified at the earliest stages of pancreatic tumorigenesis and provide new cellular and molecular targets for pancreatic cancer treatment and/or chemoprevention. 10.1053/j.gastro.2013.09.050
    Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Beyer Marc,Thabet Yasser,Müller Roman-Ulrich,Sadlon Timothy,Classen Sabine,Lahl Katharina,Basu Samik,Zhou Xuyu,Bailey-Bucktrout Samantha L,Krebs Wolfgang,Schönfeld Eva A,Böttcher Jan,Golovina Tatiana,Mayer Christian T,Hofmann Andrea,Sommer Daniel,Debey-Pascher Svenja,Endl Elmar,Limmer Andreas,Hippen Keli L,Blazar Bruce R,Balderas Robert,Quast Thomas,Waha Andreas,Mayer Günter,Famulok Michael,Knolle Percy A,Wickenhauser Claudia,Kolanus Waldemar,Schermer Bernhard,Bluestone Jeffrey A,Barry Simon C,Sparwasser Tim,Riley James L,Schultze Joachim L Nature immunology Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality. 10.1038/ni.2084
    Immune complex-induced inhibition of osteoclastogenesis is mediated via activating but not inhibitory Fcγ receptors on myeloid precursor cells. Grevers Lilyanne C,de Vries Teun J,Everts Vincent,Verbeek J Sjef,van den Berg Wim B,van Lent Peter L E M Annals of the rheumatic diseases OBJECTIVE:To investigate the role of Fcγ receptors (FcγRs) in osteoclastogenesis and osteoclast function. METHODS:Bone destruction was analysed in arthritic knee joints of several FcγR-knockout mouse strains. Unfractionated bone marrow cells were differentiated in vitro towards osteoclasts in the absence or presence of immune complexes (ICs) and stimulated thereafter for 24 h with tumour necrosis factor α (TNFα) or lipopolysaccharide (LPS). In addition, mature osteoclasts were stimulated with ICs. Experiments were analysed for osteoclast formation, bone resorption and the expression of FcγRs and osteoclast markers. RESULTS:Bone destruction was significantly increased in arthritic knee joints of FcγRIIB-deficient mice. All FcγR classes were highly expressed on osteoclast precursors. Expression of the inhibitory FcγRIIB was similar on mature osteoclasts compared to macrophages, whereas activating FcγR levels were significantly lower. IC stimulation of mature osteoclasts did not affect their number or their bone resorptive capacity. ICs significantly inhibited differentiation of unfractionated bone marrow cells towards osteoclasts, bone resorption and expression of osteoclast markers. In the presence of ICs, osteoclastogenesis of FcγRIIB(-/-) precursors and bone resorption remained inhibited. In contrast, ICs could not inhibit osteoclast formation or bone resorption of FcRγ-chain(-/-) precursors. When IC-inhibited osteoclastogenesis was followed by stimulation with TNFα or LPS, the inhibitory effects of ICs were overruled. CONCLUSION:Activating FcγRs mediate IC-induced inhibition of osteoclastogenesis, which might be overruled in the presence of proinflammatory mediators. This suggests that the balance of FcγR-mediated inflammation, through proinflammatory cytokine production, as well as the direct inhibitory effect of ICs on osteoclastogenesis determines the net effect on bone loss. 10.1136/annrheumdis-2012-201568
    Overexpression of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion through a cdc25A-independent pathway in medulloblastoma. Lau Kin-Mang,Chan Queeny Kwan Yi,Pang Jesse C S,Ma Fanny Man-Ting,Li Kay K W,Yeung Walter Wai,Cheng Alfred S L,Feng Hai,Chung Nellie Y F,Li Hiu-Ming,Zhou Liangfu,Wang Yin,Mao Ying,Ng Ho-Keung Acta neuropathologica Overexpression of high mobility group AT-hook 1 (HMGA1) is common in human cancers. Little is known about the mechanisms underlying its deregulation and downstream targets, and information about its clinical and biological significance in medulloblastoma (MB) is lacking. Here, we demonstrated frequent genomic gain at 6p21.33-6p21.31 with copy number increase leading to overexpression of HMGA1 in MB. The overexpression correlated with a high proliferation index and poor prognosis. Moreover, we found that hsa-miR-124a targeted 3'UTR of HMGA1 and negatively modulated the expression in MB cells, indicating that loss/downregulation of hsa-miR-124a reported in our previous study could contribute to the overexpression. Regarding the biological significance of HMGA1, siRNA knockdown and ectopic expression studies revealed the crucial roles of HMGA1 in controlling MB cell growth and migration/invasion through modulation of apoptosis and formation of filopodia and stress fibers, respectively. Furthermore, we identified cdc25A as a target of HMGA1 and showed that physical interaction between HMGA1 and the cdc25A promoter is required for transcriptional upregulation. In clinical samples, HMGA1 and cdc25A were concordantly overexpressed. Functionally, cdc25A is involved in the HMGA1-mediated control of MB cell growth. Finally, netropsin, which competes with HMGA1 in DNA binding, reduced the expression of cdc25A by suppression of its promoter activity and inhibited in vitro and in vivo intracranial MB cell growth. In conclusion, our results delineate the mechanisms underlying the deregulation and reveal the functional significance of HMGA1 in controlling MB cell growth and migration/invasion. Importantly, the results highlight the therapeutic potential of targeting HMGA1 in MB patients. 10.1007/s00401-011-0934-8
    PD-1 blockade improves Kupffer cell bacterial clearance in acute liver injury. Triantafyllou Evangelos,Gudd Cathrin Lc,Mawhin Marie-Anne,Husbyn Hannah C,Trovato Francesca M,Siggins Matthew K,O'Connor Thomas,Kudo Hiromi,Mukherjee Sujit K,Wendon Julia A,Bernsmeier Christine,Goldin Robert D,Botto Marina,Khamri Wafa,McPhail Mark Jw,Possamai Lucia A,Woollard Kevin J,Antoniades Charalambos G,Thursz Mark R The Journal of clinical investigation Patients with acute liver failure (ALF) have systemic innate immune suppression and increased susceptibility to infections. Programmed cell death 1 (PD-1) expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of the programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) pathway in regulating Kupffer cell (KC) inflammatory and antimicrobial responses in acetaminophen-induced (APAP-induced) acute liver injury. Using intravital imaging and flow cytometry, we found impaired KC bacterial clearance and systemic bacterial dissemination in mice with liver injury. We detected increased PD-1 and PD-L1 expression in KCs and lymphocyte subsets, respectively, during injury resolution. Gene expression profiling of PD-1+ KCs revealed an immune-suppressive profile and reduced pathogen responses. Compared with WT mice, PD-1-deficient mice and anti-PD-1-treated mice with liver injury showed improved KC bacterial clearance, a reduced tissue bacterial load, and protection from sepsis. Blood samples from patients with ALF revealed enhanced PD-1 and PD-L1 expression by monocytes and lymphocytes, respectively, and that soluble PD-L1 plasma levels could predict outcomes and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for the PD-1/PD-L1 axis in suppressing KC and monocyte antimicrobial responses after liver injury and identifies anti-PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF. 10.1172/JCI140196
    Rapid isolation of single malaria parasite-infected red blood cells by cell sorting. Miao Jun,Cui Liwang Nature protocols Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples. 10.1038/nprot.2010.185
    Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Quah Ben J C,Warren Hilary S,Parish Christopher R Nature protocols This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry. 10.1038/nprot.2007.296
    Automated sorting of live C. elegans using laFACS. Fernandez Anita G,Mis Emily K,Bargmann Bastiaan O R,Birnbaum Kenneth D,Piano Fabio Nature methods 10.1038/nmeth.f.304
    Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration. Braun Asolina,Worbs Tim,Moschovakis G Leandros,Halle Stephan,Hoffmann Katharina,Bölter Jasmin,Münk Anika,Förster Reinhold Nature immunology Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system. 10.1038/ni.2085
    Diabetes impairs stem cell and proangiogenic cell mobilization in humans. Fadini Gian Paolo,Albiero Mattia,Vigili de Kreutzenberg Saula,Boscaro Elisa,Cappellari Roberta,Marescotti Mariacristina,Poncina Nicol,Agostini Carlo,Avogaro Angelo Diabetes care OBJECTIVE:Diabetes mellitus (DM) increases cardiovascular risk, at least in part, through shortage of vascular regenerative cells derived from the bone marrow (BM). In experimental models, DM causes morphological and functional BM alterations, but information on BM function in human DM is missing. Herein, we sought to assay mobilization of stem and proangiogenic cells in subjects with and without DM. RESEARCH DESIGN AND METHODS:In a prospective trial (NCT01102699), we tested BM responsiveness to 5 μg/kg human recombinant granulocyte colony-stimulating factor (hrG-CSF) in 24 individuals with DM (10 type 1 and 14 type 2) and 14 individuals without DM. Before and 24 h after hrG-CSF, we quantified circulating stem/progenitor cells and total and differential white blood cell counts. We also evaluated in vivo the proangiogenic capacity of peripheral blood mononuclear cells using the Matrigel plug assay. RESULTS:In response to hrG-CSF, levels of CD34(+) cells and other progenitor cell phenotypes increased in subjects without DM. Patients with DM had significantly impaired mobilization of CD34(+), CD133(+), and CD34(+)CD133(+) hematopoietic stem cells and CD133(+)KDR(+) endothelial progenitors, independently of potential confounders. The in vivo angiogenic capacity of peripheral blood mononuclear cells significantly increased after hrG-CSF in control subjects without DM, but not in patients with DM. DM was also associated with the inability to upregulate CD26/DPP-4 on CD34(+) cells, which is required for the mobilizing effect of granulocyte colony-stimulating factor. CONCLUSIONS:Stem and proangiogenic cell mobilization in response to hrG-CSF is impaired in DM, possibly because of maladaptive CD26/DPP-4 regulation. These alterations may hamper tissue repair and favor the development of cardiovascular complications. 10.2337/dc12-1084
    Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. Parmar Kush M,Larman H Benjamin,Dai Guohao,Zhang Yuzhi,Wang Eric T,Moorthy Sripriya N,Kratz Johannes R,Lin Zhiyong,Jain Mukesh K,Gimbrone Michael A,García-Cardeña Guillermo The Journal of clinical investigation In the face of systemic risk factors, certain regions of the arterial vasculature remain relatively resistant to the development of atherosclerotic lesions. The biomechanically distinct environments in these arterial geometries exert a protective influence via certain key functions of the endothelial lining; however, the mechanisms underlying the coordinated regulation of specific mechano-activated transcriptional programs leading to distinct endothelial functional phenotypes have remained elusive. Here, we show that the transcription factor Kruppel-like factor 2 (KLF2) is selectively induced in endothelial cells exposed to a biomechanical stimulus characteristic of atheroprotected regions of the human carotid and that this flow-mediated increase in expression occurs via a MEK5/ERK5/MEF2 signaling pathway. Overexpression and silencing of KLF2 in the context of flow, combined with findings from genome-wide analyses of gene expression, demonstrate that the induction of KLF2 results in the orchestrated regulation of endothelial transcriptional programs controlling inflammation, thrombosis/hemostasis, vascular tone, and blood vessel development. Our data also indicate that KLF2 expression globally modulates IL-1beta-mediated endothelial activation. KLF2 therefore serves as a mechano-activated transcription factor important in the integration of multiple endothelial functions associated with regions of the arterial vasculature that are relatively resistant to atherogenesis. 10.1172/JCI24787
    Regeneration Defects in Yap and Taz Mutant Mouse Livers Are Caused by Bile Duct Disruption and Cholestasis. Verboven Elisabeth,Moya Iván M,Sansores-Garcia Leticia,Xie Jun,Hillen Hanne,Kowalczyk Weronika,Vella Gerlanda,Verhulst Stefaan,Castaldo Stéphanie A,Algueró-Nadal Ana,Romanelli Lucia,Mercader-Celma Cristina,Souza Natália A,Soheily Soheil,Van Huffel Leen,Van Brussel Thomas,Lambrechts Diether,Roskams Tania,Lemaigre Frédéric P,Bergers Gabrielle,van Grunsven Leo A,Halder Georg Gastroenterology BACKGROUND AND AIMS:The Hippo pathway and its downstream effectors YAP and TAZ (YAP/TAZ) are heralded as important regulators of organ growth and regeneration. However, different studies provided contradictory conclusions about their role during regeneration of different organs, ranging from promoting proliferation to inhibiting it. Here we resolve the function of YAP/TAZ during regeneration of the liver, where Hippo's role in growth control has been studied most intensely. METHODS:We evaluated liver regeneration after carbon tetrachloride toxic liver injury in mice with conditional deletion of Yap/Taz in hepatocytes and/or biliary epithelial cells, and measured the behavior of different cell types during regeneration by histology, RNA sequencing, and flow cytometry. RESULTS:We found that YAP/TAZ were activated in hepatocytes in response to carbon tetrachloride toxic injury. However, their targeted deletion in adult hepatocytes did not noticeably impair liver regeneration. In contrast, Yap/Taz deletion in adult bile ducts caused severe defects and delay in liver regeneration. Mechanistically, we showed that Yap/Taz mutant bile ducts degenerated, causing cholestasis, which stalled the recruitment of phagocytic macrophages and the removal of cellular corpses from injury sites. Elevated bile acids activated pregnane X receptor, which was sufficient to recapitulate the phenotype observed in mutant mice. CONCLUSIONS:Our data show that YAP/TAZ are practically dispensable in hepatocytes for liver development and regeneration. Rather, YAP/TAZ play an indirect role in liver regeneration by preserving bile duct integrity and securing immune cell recruitment and function. 10.1053/j.gastro.2020.10.035
    Inhibition of colon carcinogenesis by 2-methoxy-5-amino-N-hydroxybenzamide, a novel derivative of mesalamine. Stolfi Carmine,Sarra Massimiliano,Caruso Roberta,Fantini Massimo Claudio,Fina Daniele,Pellegrini Roberto,Palmieri Giampiero,Macdonald Thomas T,Pallone Francesco,Monteleone Giovanni Gastroenterology BACKGROUND & AIMS:Mesalamine has been reported to protect against inflammatory bowel disease-related colorectal cancer (CRC), but several drug-related issues have limited its use in chemopreventive programs. We evaluated the antineoplastic properties of mesalamine derivatives using in vitro and in vivo models of CRC. METHODS:CRC cell proliferation and cell-cycle progression were evaluated by flow cytometry after exposure to mesalamine or mesalamine derivatives. Cyclins, cyclin-dependent kinases, and endoplasmic reticulum stress-related molecules were examined by immunoblotting. The in vivo antineoplastic effect of 2-methoxy-5-amino-N-hydroxybenzamide (2-14) was evaluated in a syngenic, CT26-derived xenograft mouse model of CRC and in the azoxymethane/dextran sulfate sodium-induced mouse model of colitis-associated CRC. RESULTS:The mesalamine derivative 2-14 was 10-fold more potent than mesalamine in inhibiting CRC cell proliferation. After exposure to 2-14, cyclin D1 expression was reduced and G0/G1 phase cells accumulated. These events were preceded by activation of eukaryotic translation initiation factor 2-alpha kinase 3 (pancreatic endoplasmic reticulum eIF2alpha kinase), phosphorylation of eukaryotic translation initiation factor 2alpha, induction of activating transcription factor 4, and splicing of X-box binding protein 1 messenger RNA, events that define endoplasmic reticulum stress. Silencing of PERK restored cyclin D1 levels, allowing cells to overcome the cell-cycle block induced by 2-14. Mice injected with 2-14 developed fewer CRC xenograft-derived tumors. Moreover, 2-14 injection reduced the development of neoplastic lesions induced by azoxymethane and dextran sulfate sodium in mice. CONCLUSIONS:The mesalamine derivative 2-14 inhibited CRC cell proliferation in vitro and prevented CRC progression in mouse models. 10.1053/j.gastro.2009.08.062
    Pathogenic bacteria and dead cells are internalized by a unique subset of Peyer's patch dendritic cells that express lysozyme. Lelouard Hugues,Henri Sandrine,De Bovis Béatrice,Mugnier Bénédicte,Chollat-Namy Alexandre,Malissen Bernard,Méresse Stéphane,Gorvel Jean-Pierre Gastroenterology BACKGROUND & AIMS:Lysozyme has an important role in preventing bacterial infection. In the gastrointestinal tract, lysozyme is thought to be mainly expressed by Paneth cells of the crypt epithelium. We investigated its expression in the Peyer's patch, a major intestinal site of antigen sampling and pathogen entry. METHODS:We performed immunostaining on normal and Salmonella Typhimurium-infected intestinal samples and analyzed them by confocal microscopy and flow cytometry. RESULTS:In Peyer's patch of mouse, rat, and human, lysozyme was strongly expressed in the germinal center of follicles by tingible body macrophages and in the subepithelial dome by a subset of myeloid dendritic cells (DC). Among DC subsets from mouse Peyer's patches, these lysozyme-expressing DC displayed the highest surface expression of class II major histocompatibility complex and costimulatory molecules; they were the most efficient at capturing microspheres in vitro. Moreover, they were the main DC subset involved in bacterial pathogen uptake and in dead cell clearance, including M cells. CONCLUSIONS:The subepithelial dome of Peyer's patches contains a unique population of intestinal DC that secretes high levels of lysozyme and internalizes bacteria and dead cells. 10.1053/j.gastro.2009.09.051
    Smed-myb-1 Specifies Early Temporal Identity during Planarian Epidermal Differentiation. Zhu Shu Jun,Pearson Bret J Cell reports The planarian epidermis provides an excellent model to explore adult stem cell (ASC) lineage development due to well-characterized and distinct spatiotemporal phases during lineage progression. Using flow cytometry-isolated cells enriched in epidermal progenitors, we performed transcriptional profiling and RNAi screening to uncover regulators of epidermal differentiation. We identified a MYB-type transcription factor (Smed-myb-1) required for the specification of the first temporal phase of post-mitotic maturation. Knockdown of myb-1 abolished the early progenitor phase of differentiation without ceasing production of subsequent epidermal progenitor states or homeostatic turnover and regeneration of the epidermis. Further examination revealed accelerated maturation of ASC descendants, with premature entry into subsequent progeny phases and, ultimately, the epidermis. These results demonstrate that a spatiotemporal shift in lineage progression occurs in the absence of the early progenitor state after myb-1 RNAi, and identify myb-1 as a critical regulator of the early temporal window in stepwise specification during planarian epidermal differentiation. 10.1016/j.celrep.2018.09.011
    Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis. Affandi Alsya J,Carvalheiro Tiago,Ottria Andrea,Broen Jasper Ca,Bossini-Castillo Lara,Tieland Ralph G,Bon Lenny van,Chouri Eleni,Rossato Marzia,Mertens Jorre S,Garcia Samuel,Pandit Aridaman,de Kroon Laurie Mg,Christmann Romy B,Martin Javier,van Roon Joel Ag,Radstake Timothy Rdj,Marut Wioleta Annals of the rheumatic diseases OBJECTIVES:Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology. METHODS:In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, -Cre: mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model. RESULTS:Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the gene, which is associated with polymorphism rs6672420, correlates with lower expression and SSc susceptibility. Hypoxia is another factor that decreases level in pDC. Mouse pDCs deficient of show enhanced maturation markers on CpG stimulation. In vivo, deletion of in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis. CONCLUSIONS:We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of in SSc model further underscoring the role of pDCs in this disease. 10.1136/annrheumdis-2018-214991
    Klf4 methylated by Prmt1 restrains the commitment of primitive endoderm. Nucleic acids research The second cell fate decision in the early stage of mammalian embryonic development is pivotal; however, the underlying molecular mechanism is largely unexplored. Here, we report that Prmt1 acts as an important regulator in primitive endoderm (PrE) formation. First, Prmt1 depletion promotes PrE gene expression in mouse embryonic stem cells (ESCs). Single-cell RNA sequencing and flow cytometry assays demonstrated that Prmt1 depletion in mESCs contributes to an emerging cluster, where PrE genes are upregulated significantly. Furthermore, the efficiency of extraembryonic endoderm stem cell induction increased in Prmt1-depleted ESCs. Second, the pluripotency factor Klf4 methylated at Arg396 by Prmt1 is required for recruitment of the repressive mSin3a/HDAC complex to silence PrE genes. Most importantly, an embryonic chimeric assay showed that Prmt1 inhibition and mutated Klf4 at Arg 396 induce the integration of mouse ESCs into the PrE lineage. Therefore, we reveal a regulatory mechanism for cell fate decisions centered on Prmt1-mediated Klf4 methylation. 10.1093/nar/gkac054
    Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. Deng Liufu,Liang Hua,Burnette Byron,Beckett Michael,Darga Thomas,Weichselbaum Ralph R,Fu Yang-Xin The Journal of clinical investigation High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death-ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti-PD-L1 enhanced the efficacy of IR through a cytotoxic T cell-dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti-PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy. 10.1172/JCI67313
    Targeting the cell cycle inhibitor p57Kip2 promotes adult human β cell replication. Avrahami Dana,Li Changhong,Yu Ming,Jiao Yang,Zhang Jia,Naji Ali,Ziaie Seyed,Glaser Benjamin,Kaestner Klaus H The Journal of clinical investigation Children with focal hyperinsulinism of infancy display a dramatic, non-neoplastic clonal expansion of β cells that have undergone mitotic recombination, resulting in paternal disomy of part of chromosome 11. This disomic region contains imprinted genes, including the gene encoding the cell cycle inhibitor p57Kip2 (CDKN1C), which is silenced as a consequence of the recombination event. We hypothesized that targeting p57Kip2 could stimulate adult human β cell replication. Indeed, when we suppressed CDKN1C expression in human islets obtained from deceased adult organ donors and transplanted them into hyperglycemic, immunodeficient mice, β cell replication increased more than 3-fold. The newly replicated cells retained properties of mature β cells, including the expression of β cell markers such as insulin, PDX1, and NKX6.1. Importantly, these newly replicated cells demonstrated normal glucose-induced calcium influx, further indicating β cell functionality. These findings provide a molecular explanation for the massive β cell replication that occurs in children with focal hyperinsulinism. These data also provided evidence that β cells from older humans, in which baseline replication is negligible, can be coaxed to re-enter and complete the cell cycle while maintaining mature β cell properties. Thus, controlled manipulation of this pathway holds promise for the expansion of β cells in patients with type 2 diabetes. 10.1172/JCI69519