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    IL-17A secreted from lymphatic endothelial cells promotes tumorigenesis by upregulation of PD-L1 in hepatoma stem cells. Wei Yuanyan,Shi Danfang,Liang Ziwei,Liu Yuming,Li Yinan,Xing Yang,Liu Weitao,Ai Zhilong,Zhuang Jianhui,Chen Xiaoning,Gao Qiang,Jiang Jianhai Journal of hepatology BACKGROUND & AIMS:The microenvironment regulates hepatoma stem cell behavior. However, the contributions of lymphatic endothelial cells to the hepatoma stem cell niche remain largely unknown; we aimed to analyze this contribution and elucidate the mechanisms behind it. METHODS:Associations between lymphatic endothelial cells and CD133 hepatoma stem cells were analyzed by immunofluorescence and adhesion assays; with the effects of their association on IL-17A expression examined using western blot, quantitative reverse transcription PCR and luciferase reporter assay. The effects of IL-17A on the self-renewal and tumorigenesis of hepatoma stem cells were examined using sphere and tumor formation assays. The role of IL-17A in immune escape by hepatoma stem cells was examined using flow cytometry. The expression of IL-17A in hepatoma tissues was examined using immunohistochemistry. RESULTS:CD133 hepatoma stem cells preferentially interact with lymphatic endothelial cells. The interaction between the mannose receptor and high-mannose type N-glycans mediates the interaction between CD133 hepatoma stem cells and lymphatic endothelial cells. This interaction activates cytokine IL-17A expression in lymphatic endothelial cells. IL-17A promotes the self-renewal of hepatoma stem cells. It also promotes their immune escape, partly through upregulation of PD-L1. CONCLUSION:Interactions between lymphatic endothelial cells and hepatoma stem cells promote the self-renewal and immune escape of hepatoma stem cells, by activating IL-17A signaling. Thus, inhibiting IL-17A signaling may be a promising approach for hepatoma treatment. LAY SUMMARY:The microenvironment is crucial for the self-renewal and development of hepatoma stem cells, which lead to the development of liver cancer. Lymphatic endothelial cells are an important component of this niche microenvironment, helping hepatoma stem cells to self-renew and escape immune attack, by upregulating IL-17A signaling. Thus, targeting IL-17A signaling is a potential strategy for the treatment of hepatoma. 10.1016/j.jhep.2019.08.034
    Derivation of iPSCs in stirred suspension bioreactors. Shafa Mehdi,Day Brad,Yamashita Akihiro,Meng Guoliang,Liu Shiying,Krawetz Roman,Rancourt Derrick E Nature methods Induced pluripotent stem cells (iPSCs) are typically derived in adherent culture. Here we report fast and efficient derivation of mouse iPSCs in stirred suspension bioreactors, with and without the use of c-Myc. Suspension-reprogrammed cells expressed pluripotency markers, showed multilineage differentiation in vitro and in vivo, and contributed to the germline in chimeric mice. Suspension reprogramming has the potential to accelerate and standardize iPSC research. 10.1038/nmeth.1973
    Ryanodine Receptor Calcium Leak in Circulating B-Lymphocytes as a Biomarker in Heart Failure. Kushnir Alexander,Santulli Gaetano,Reiken Steven R,Coromilas Ellie,Godfrey Sarah J,Brunjes Danielle L,Colombo Paolo C,Yuzefpolskaya Melana,Sokol Seth I,Kitsis Richard N,Marks Andrew R Circulation BACKGROUND:Advances in congestive heart failure (CHF) management depend on biomarkers for monitoring disease progression and therapeutic response. During systole, intracellular Ca is released from the sarcoplasmic reticulum into the cytoplasm through type-2 ryanodine receptor/Ca release channels. In CHF, chronically elevated circulating catecholamine levels cause pathological remodeling of type-2 ryanodine receptor/Ca release channels resulting in diastolic sarcoplasmic reticulum Ca leak and decreased myocardial contractility. Similarly, skeletal muscle contraction requires sarcoplasmic reticulum Ca release through type-1 ryanodine receptors (RyR1), and chronically elevated catecholamine levels in CHF cause RyR1-mediated sarcoplasmic reticulum Ca leak, contributing to myopathy and weakness. Circulating B-lymphocytes express RyR1 and catecholamine-responsive signaling cascades, making them a potential surrogate for defects in intracellular Ca handling because of leaky RyR channels in CHF. METHODS:Whole blood was collected from patients with CHF, CHF following left-ventricular assist device implant, and controls. Blood was also collected from mice with ischemic CHF, ischemic CHF+S107 (a drug that specifically reduces RyR channel Ca leak), and wild-type controls. Channel macromolecular complex was assessed by immunostaining RyR1 immunoprecipitated from lymphocyte-enriched preparations. RyR1 Ca leak was assessed using flow cytometry to measure Ca fluorescence in B-lymphocytes in the absence and presence of RyR1 agonists that empty RyR1 Ca stores within the endoplasmic reticulum. RESULTS:Circulating B-lymphocytes from humans and mice with CHF exhibited remodeled RyR1 and decreased endoplasmic reticulum Ca stores, consistent with chronic intracellular Ca leak. This Ca leak correlated with circulating catecholamine levels. The intracellular Ca leak was significantly reduced in mice treated with the Rycal S107. Patients with CHF treated with left-ventricular assist devices exhibited a heterogeneous response. CONCLUSIONS:In CHF, B-lymphocytes exhibit remodeled leaky RyR1 channels and decreased endoplasmic reticulum Ca stores consistent with chronic intracellular Ca leak. RyR1-mediated Ca leak in B-lymphocytes assessed using flow cytometry provides a surrogate measure of intracellular Ca handling and systemic sympathetic burden, presenting a novel biomarker for monitoring response to pharmacological and mechanical CHF therapy. 10.1161/CIRCULATIONAHA.117.032703
    High expression of the very late antigen-4 integrin independently predicts reduced risk of relapse and improved outcome in pediatric acute myeloid leukemia: a report from the children's oncology group. Walter Roland B,Alonzo Todd A,Gerbing Robert B,Ho Phoenix A,Smith Franklin O,Raimondi Susana C,Hirsch Betsy A,Gamis Alan S,Franklin Janet L,Hurwitz Craig A,Loken Michael R,Meshinchi Soheil Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:To evaluate the prognostic significance of the integrin cell adhesion molecule very late antigen-4 (VLA-4) in acute myeloid leukemia (AML). PATIENTS AND METHODS:We prospectively quantified VLA-4 expression in 216 patients enrolled onto COG-AAML03P1 by flow cytometry and correlated expression levels with disease characteristics and clinical outcome. RESULTS:VLA-4 mean fluorescence intensity (MFI) varied 35-fold (range, 30 to 1,110; median, 219.5). High VLA-4 expression (> median MFI), compared with low expression, was associated with younger age (7.1 v 12.1 years, respectively; P < .001), lower FLT3 internal tandem duplication prevalence (4% v 21%, respectively; P < .001), and higher likelihood of extramedullary disease (16% v 5%, respectively; P = .013). In low- and high-expression groups, rates of remission (89% v 80%, respectively; P = .137) and minimal residual disease (29% v 25%, respectively; P = .700) were similar. Patients with low VLA-4 expression, compared with high expression, had a higher relapse rate (RR; 44% +/- 10% v 24% +/- 9%, respectively; P = .011) and lower disease-free survival (DFS; 48% +/- 11% v 67% +/- 10%, respectively; P = .023) after 3 years. Multivariate analyses showed that low VLA-4 expression was an independent adverse prognostic factor for DFS (hazard ratio [HR] = 1.98; P = .038) and RR (HR = 2.77; P = .009). Subgroup analyses indicated that the prognostic role of VLA-4 expression was most prominent in patients with standard-risk AML, in whom low VLA-4 expression was associated with inferior DFS (34% +/- 16% v 69% +/- 14% for high expression; P = .011) and higher RR (61% +/- 16% v 26% +/- 14% for high expression; P = .009). A similar trend was seen in low-risk but not high-risk patients. CONCLUSION:High VLA-4 expression is associated with better clinical outcome in pediatric AML and is an independent predictor of relapse that may refine our abilities to stratify patients without identifiable cytogenetic or molecular risk factors. 10.1200/JCO.2009.27.5693
    An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP. Li Xiaobo,Zhu Yuanyuan,Zhang Xiao,An Xiang,Weng Mingjiao,Shi Jiaqi,Wang Song,Liu Caiqi,Luo Shengnan,Zheng Tongsen The Journal of clinical investigation It has been revealed that 2'3'-cyclic-GMP-AMP (cGAMP), a second messenger that activates the antiviral stimulator of IFN genes (STING), elicits an antitumoral immune response. Since cGAMP cannot cross the cell membrane, it is not clear how intracellular STING has been activated by extracellular cGAMP until SLC19A1 was identified as an importer to transport extracellular cGAMP into the cytosol. However, SLC19A1-deficient cells also sense extracellular cGAMP, suggesting the presence of mechanisms other than the facilitating transporters for STING sensing extracellular cGAMP. Here, using immunoprecipitation, immunofluorescence, and flow cytometry, we identified an alternatively spliced STING isoform, plasmatic membrane STING (pmSTING), that localized in the plasma membrane with its C-terminus outside the cell, due to a lack of 1 transmembrane domain in its N-terminus compared with canonical STING. Further studies showed that extracellular cGAMP not only promoted the dimerization of pmSTING and interaction of pmSTING with TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3), but also enhanced the phosphorylation of TBK1 and IRF3 and the production of IFN in pmSTING-transfected cells. Additionally, we also identified similar pmSTING isoforms in other species including human. This study suggests a conserved role for pmSTING in sensing extracellular cGAMP and provides insight into the role of cGAMP as an immunotransmitter. 10.1172/JCI144339
    Leukemia stemness signatures step toward the clinic. Becker Michael W,Jordan Craig T Cell stem cell A recent Nature Medicine study by Eppert et al. (2011) describes analyses of functionally defined leukemia stem cell populations that provide new insights on the biology of human tumor populations and the potential use of stem cell-associated gene signatures for prognosis. 10.1016/j.stem.2011.08.006
    DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation. Jabara Haifa H,McDonald Douglas R,Janssen Erin,Massaad Michel J,Ramesh Narayanaswamy,Borzutzky Arturo,Rauter Ingrid,Benson Halli,Schneider Lynda,Baxi Sachin,Recher Mike,Notarangelo Luigi D,Wakim Rima,Dbaibo Ghassan,Dasouki Majed,Al-Herz Waleed,Barlan Isil,Baris Safa,Kutukculer Necil,Ochs Hans D,Plebani Alessandro,Kanariou Maria,Lefranc Gerard,Reisli Ismail,Fitzgerald Katherine A,Golenbock Douglas,Manis John,Keles Sevgi,Ceja Reuben,Chatila Talal A,Geha Raif S Nature immunology The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells. 10.1038/ni.2305
    CX₃CL1 (fractalkine) and its receptor CX₃CR1 regulate atopic dermatitis by controlling effector T cell retention in inflamed skin. Staumont-Sallé Delphine,Fleury Sébastien,Lazzari Anne,Molendi-Coste Olivier,Hornez Nicolas,Lavogiez Céline,Kanda Akira,Wartelle Julien,Fries Anissa,Pennino Davide,Mionnet Cyrille,Prawitt Janne,Bouchaert Emmanuel,Delaporte Emmanuel,Glaichenhaus Nicolas,Staels Bart,Julia Valérie,Dombrowicz David The Journal of experimental medicine Atopic dermatitis (AD) is a chronic allergic dermatosis characterized by epidermal thickening and dermal inflammatory infiltrates with a dominant Th2 profile during the acute phase, whereas a Th1 profile is characteristic of the chronic stage. Among chemokines and chemokine receptors associated with inflammation, increased levels of CX3CL1 (fractalkine) and its unique receptor, CX3CR1, have been observed in human AD. We have thus investigated their role and mechanism of action in experimental models of AD and psoriasis. AD pathology and immune responses, but not psoriasis, were profoundly decreased in CX3CR1-deficient mice and upon blocking CX3CL1-CX3CR1 interactions in wild-type mice. CX3CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX3CR1 expression by both Th2 and Th1 cells was required to induce AD. Surprisingly, unlike in allergic asthma, where CX3CL1 and CX3CR1 regulate the pathology by controlling effector CD4(+) T cell survival within inflamed tissues, adoptive transfer experiments established CX3CR1 as a key regulator of CD4(+) T cell retention in inflamed skin, indicating a new function for this chemokine receptor. Therefore, although CX3CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases. 10.1084/jem.20121350
    12-Hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor. Liu Min,Saeki Kazuko,Matsunobu Takehiko,Okuno Toshiaki,Koga Tomoaki,Sugimoto Yukihiko,Yokoyama Chieko,Nakamizo Satoshi,Kabashima Kenji,Narumiya Shuh,Shimizu Takao,Yokomizo Takehiko The Journal of experimental medicine Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. As mouse BLT2 is highly expressed in epidermal keratinocytes, we investigated the role of the 12-HHT/BLT2 axis in skin wound healing processes. 12-HHT accumulated in the wound fluid in mice, and BLT2-deficient mice exhibited impaired re-epithelialization and delayed wound closure after skin punching. Aspirin administration reduced 12-HHT production and resulted in delayed wound closure in wild-type mice, which was abrogated in BLT2-deficient mice. In vitro scratch assay using primary keratinocytes and a keratinocyte cell line also showed that the 12-HHT/BLT2 axis accelerated wound closure through the production of tumor necrosis factor α (TNF) and matrix metalloproteinases (MMPs). A synthetic BLT2 agonist accelerated wound closure in cultured cells as well as in C57BL/6J and diabetic mice. These results identify a novel mechanism underlying the action of the 12-HHT/BLT2 axis in epidermal keratinocytes and accordingly suggest the use of BLT2 agonists as therapeutic agents to accelerate wound healing, particularly for intractable wounds, such as diabetic ulcers. 10.1084/jem.20132063
    B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection. Nothelfer Katharina,Arena Ellen T,Pinaud Laurie,Neunlist Michel,Mozeleski Brian,Belotserkovsky Ilia,Parsot Claude,Dinadayala Premkumar,Burger-Kentischer Anke,Raqib Rubhana,Sansonetti Philippe J,Phalipon Armelle The Journal of experimental medicine Antibody-mediated immunity to Shigella, the causative agent of bacillary dysentery, requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. We show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and occasionally invades B lymphocytes. The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo. In addition to cell death occurring in Shigella-invaded CL-01 B lymphocytes, we provide evidence that the T3SA needle tip protein IpaD can induce cell death in noninvaded cells. IpaD binds to and induces B cell apoptosis via TLR2, a signaling receptor thus far considered to result in activation of B lymphocytes. The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding. Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals. This study therefore adds direct B lymphocyte targeting to the diversity of mechanisms used by Shigella to dampen the host immune response. 10.1084/jem.20130914
    Fgd5 identifies hematopoietic stem cells in the murine bone marrow. Gazit Roi,Mandal Pankaj K,Ebina Wataru,Ben-Zvi Ayal,Nombela-Arrieta César,Silberstein Leslie E,Rossi Derrick J The Journal of experimental medicine Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence. 10.1084/jem.20130428
    CD10+ pancreatic stellate cells enhance the progression of pancreatic cancer. Ikenaga Naoki,Ohuchida Kenoki,Mizumoto Kazuhiro,Cui Lin,Kayashima Tadashi,Morimatsu Katsuya,Moriyama Taiki,Nakata Kohei,Fujita Hayato,Tanaka Masao Gastroenterology BACKGROUND & AIMS:Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer by producing extracellular matrix and soluble factors. However, the functional heterogeneity of PSCs has not been identified until now. Detailed characterization of the PSCs in human pancreatic cancer would provide a set of potential targets for stroma-directed therapy. METHODS:We isolated PSCs from fresh pancreatic ductal adenocarcinoma tissue and sorted them by flow cytometry according to cell surface expression of CD10, which is a stromal prognostic marker for various tumors. We analyzed the functional differences between CD10(+) PSCs and CD10(-) PSCs. RESULTS:Immunohistochemical analysis showed that the frequency of CD10 expression by PSCs was markedly higher in tumor tissue than in normal tissue (33.7% vs 0%, respectively, P = .028). In pancreatic ductal adenocarcinoma, CD10 expression by PSCs was associated with positive nodal metastases (P = .011) and a shorter survival time (P < .001). In vitro coculture experiments showed that CD10(+) PSCs promoted the invasiveness of pancreatic cancer cell lines, SUIT-2 and Panc-1 cells more intensively than CD10(-) PSCs. CD10(+) PSCs significantly increased the tumor growth and invasiveness of SUIT-2 cells in a murine cotransplantation model. CD10(+) PSCs secreted higher levels of matrix metalloproteinase 3 than CD10(-) PSCs, and knockdown of matrix metalloproteinase 3 in cocultured PSCs reduced the invasion of SUIT-2 and Panc-1 cells. CONCLUSIONS:CD10(+) PSCs enhance the progression of pancreatic cancer cells. CD10(+) PSCs may be a candidate for selective therapeutic targeting in the treatment of pancreatic cancer. 10.1053/j.gastro.2010.05.084
    p75 Neurotrophin receptor is a marker for precursors of stellate cells and portal fibroblasts in mouse fetal liver. Suzuki Kaori,Tanaka Minoru,Watanabe Natsumi,Saito Shigeru,Nonaka Hidenori,Miyajima Atsushi Gastroenterology BACKGROUND & AIMS:Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are 2 distinct mesenchymal cells in adult liver. HSCs in sinusoids accumulate lipids and express p75 neurotrophin receptor (p75NTR). HSCs and PFs play pivotal roles in liver regeneration and fibrosis. However, the roles of mesenchymal cells in fetal liver remain poorly understood. In this study, we aimed to characterize mesenchymal cells in mouse fetal liver. METHODS:We prepared an anti-p75NTR monoclonal antibody applicable for flow cytometry and immunohistochemistry. p75NTR(+) cells isolated from fetal liver by flow cytometry were characterized by reverse-transcription polymerase chain reaction, immunohistochemistry, and cell cultivation. Lipid-containing cells were visualized by Oil-red O staining. RESULTS:p75NTR(+) cells in fetal liver were clearly distinct from endothelial cells and showed characteristics of mesenchymal cells. At embryonic day (E) 10.5, p75NTR(+) cells were present at the periphery of the liver bud in close contact with endothelial cells, and spread over the liver at E11.5. With the formation of the liver architecture, they began to localize to 2 distinct areas, parenchymal and portal areas, and lipid-containing p75NTR(+) cells increased accordingly. p75NTR(+) cells around portal veins were adjacent to cholangiocytes and expressed Jagged1, a crucial factor for the commitment of hepatoblasts to cholangiocytes. By cultivation, p75NTR(+) cells showed features of adult HSCs with markedly increased expression of glial fibrillary acidic protein and alpha-smooth muscle actin. CONCLUSIONS:p75NTR(+) mesenchymal cells in fetal liver include progenitors for HSCs and PFs, and the anti-p75NTR monoclonal antibody is useful for their isolation. 10.1053/j.gastro.2008.03.075
    Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis. Weber Georg F,Chousterman Benjamin G,Hilgendorf Ingo,Robbins Clinton S,Theurl Igor,Gerhardt Louisa M S,Iwamoto Yoshiko,Quach Tam D,Ali Muhammad,Chen John W,Rothstein Thomas L,Nahrendorf Matthias,Weissleder Ralph,Swirski Filip K The Journal of experimental medicine Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF-dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity. 10.1084/jem.20131471
    Lymph node stromal cells acquire peptide-MHCII complexes from dendritic cells and induce antigen-specific CD4⁺ T cell tolerance. Dubrot Juan,Duraes Fernanda V,Potin Lambert,Capotosti Francesca,Brighouse Dale,Suter Tobias,LeibundGut-Landmann Salomé,Garbi Natalio,Reith Walter,Swartz Melody A,Hugues Stéphanie The Journal of experimental medicine Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8(+) T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4(+) T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide-MHCII complexes from DCs and induce CD4(+) T cell dysfunction by presenting transferred complexes to naive CD4(+) T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4(+) T cells through the presentation of self-antigens via transferred peptide-MHCII complexes of DC origin. 10.1084/jem.20132000
    MicroRNA-15a/16-1 Prevents Hepatocellular Carcinoma by Disrupting the Communication Between Kupffer Cells and Regulatory T Cells. Liu Ningning,Chang Ching Wen,Steer Clifford J,Wang Xin Wei,Song Guisheng Gastroenterology BACKGROUND & AIMS:Hepatocellular carcinoma (HCC) is characterized by intratumoral accumulation of regulatory T cells (Tregs), which suppresses antitumor immunity. This study was designed to investigate how microRNAs regulate immunosuppression in HCC. METHODS:FVB/NJ mice were hydrodynamically injected with AKT/Ras or c-Myc and Sleeping Beauty transposon to induce HCC. The Sleeping Beauty system was used to deliver microRNA-15a/16-1 into livers of mice. Flow cytometry and immunostaining were used to determine changes in the immune system. RESULTS:Hydrodynamic injection of AKT/Ras or c-Myc into mice resulted in hepatic enrichment of Tregs and reduced cytotoxic T cells (CTLs) and HCC development. HCC impaired microRNA-15a/16-1 biogenesis in Kupffer cells (KCs) of AKT/Ras and c-Myc mice. Hydrodynamic injection of microRNA-15a/16-1 fully prevented HCC in AKT/Ras and c-Myc mice, while 100% of control mice died of HCC. Therapeutically, microRNA-15a/16-1 promoted a regression of HCC in both mouse models, impaired hepatic enrichment of Tregs, and increased hepatic CTLs. Mechanistically, a significant increase was observed in serum C-C motif chemokine 22 (CCL22) and transcription of Ccl22 in KCs of AKT/Ras and c-Myc mice. MicroRNA-15a/16-1 prevented KCs from overproducing CCL22 by inhibiting nuclear factor-κB that activates transcription of Ccl22. By reducing CCL22 binding to C-C chemokine receptor type 4 on Tregs, microRNA-15a/16-1 impaired Treg chemotaxis. Disrupting the interaction between microRNA-15a/16-1 and nuclear factor-κB impaired the ability of microRNA-15a/16-1 to prevent hepatic Treg accumulation and HCC. Depletion of cluster of differentiation 8 T cells and additional treatment of CCL22 recovered growth of HCC that was fully prevented by microRNA-15a/16. CONCLUSIONS:MicroRNA-15a/16-1 attenuates immunosuppression by disrupting CCL22-mediated communication between KCs and Tregs. MicroRNA-15a/16-1 represents a potential immunotherapy against HCC. 10.1053/j.gastro.2021.10.015
    Reduced expression of FOXP3 and regulatory T-cell function in severe forms of early-onset autoimmune enteropathy. Moes Nicolette,Rieux-Laucat Frédéric,Begue Bernadette,Verdier Julien,Neven Bénédicte,Patey Natacha,Torgerson Troy T,Picard Capucine,Stolzenberg Marie-Claude,Ruemmele Corinne,Rings Edmond Hhm,Casanova Jean-Laurent,Piloquet Hugues,Biver Armand,Breton Anne,Ochs Hans D,Hermine Olivier,Fischer Alain,Goulet Olivier,Cerf-Bensussan Nadine,Ruemmele Frank M Gastroenterology BACKGROUND & AIMS:Little is known about the pathophysiology of early onset forms of autoimmune enteropathy (AIE). AIE has been associated with mutations in FOXP3-a transcription factor that controls regulatory T-cell development and function. We analyzed the molecular basis of neonatal or early postnatal AIE using clinical, genetic, and functional immunological studies. METHODS:Gastroenterological and immunological features were analyzed in 9 boys and 2 girls with AIE that began within the first 5 months of life. FOXP3 and IL2RA were genotyped in peripheral blood monocytes. FOXP3 messenger RNA and protein expression were analyzed using reverse-transcription polymerase chain reaction, flow cytometry, and confocal immunofluorescence of CD4(+) T cells. Regulatory T-cell function (CD4(+)CD25(+)) was assayed in coculture systems. RESULTS:AIE associated with extraintestinal autoimmunity was severe and life-threatening; all patients required total parenteral nutrition. Regulatory T cells from 7 patients had altered function and FOXP3 mutations that resulted in lost or reduced FOXP3 protein expression; 2 infants had reduced regulatory T-cell activity and reduced levels of FOXP3 protein, although we did not detect mutations in FOXP3 coding region, poly-A site, or promoter region (called FOXP3-dependent AIE). Two patients had a normal number of regulatory T cells that expressed normal levels of FOXP3 protein and normal regulatory activity in in vitro coculture assays (called FOXP3-independent AIE). No mutations in IL2RA were found. CONCLUSIONS:Most cases of AIE are associated with alterations in regulatory T-cell function; some, but not all, cases have mutations that affect FOXP3 expression levels. Further studies are needed to identify mechanisms of AIE pathogenesis. 10.1053/j.gastro.2010.06.006
    Divergent transcriptional programming of class-specific B cell memory by T-bet and RORα. Wang Nathaniel S,McHeyzer-Williams Louise J,Okitsu Shinji L,Burris Thomas P,Reiner Steven L,McHeyzer-Williams Michael G Nature immunology Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo. Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA(+) memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA(+) memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells. 10.1038/ni.2294
    The natural soluble form of IL-18 receptor beta exacerbates collagen-induced arthritis via modulation of T-cell immune responses. Veenbergen S,Smeets R L,Bennink M B,Arntz O J,Joosten L A B,van den Berg W B,van de Loo F A J Annals of the rheumatic diseases OBJECTIVE:IL-18 is a pluripotent cytokine that has been implicated in the development of rheumatoid arthritis. A soluble form of the IL-18 receptor accessory protein (sIL-18Rbeta) with unknown function has recently been identified. This study examined the ability of sIL-18Rbeta to inhibit IL-18 biological activities and to modulate immune responses during collagen-induced arthritis (CIA). METHODS:Adenoviruses encoding sIL-18Rbeta were administered intravenously in type II collagen-immunised DBA/1 mice. Humoral responses were analysed by determining anti-bovine collagen type II (BCII) antibody levels by ELISA. Cytokine production by splenic T cells and cytokine levels in serum were measured by Luminex multi-analyte technology. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) were measured by flow cytometry. RESULTS:Intravenous delivery of Ad5.sIL-18Rbeta in collagen-immunised mice led to enhanced transgene expression in splenic antigen-presenting cells (APC). A co-culture of these sIL-18Rbeta-transduced APC with purified splenic CD3(+) T cells led to a marked inhibition of IL-18-induced IFNgamma, IL-4 and IL-17 production by CD3(+) T cells. Remarkably, systemic treatment with Ad5.sIL-18Rbeta caused an exacerbation of arthritis, and histological evaluation of knee joints showed increased cartilage and bone erosion. No significant differences were observed in anti-BCII antibodies, but the aggravation was accompanied by decreased IFNgamma (-30%) and IL-4 (-44%) and increased IL-17 (+84%) production by splenic CD3(+) T cells. In addition, reduced circulating levels of CD4(+)CD25(+)Foxp3(+) Treg and anti-inflammatory IL-10 were shown. CONCLUSION:This study identifies sIL-18Rbeta as a novel IL-18 inhibitor, which promotes CIA after intravenous overexpression by affecting Treg levels and supporting a T helper type 17 response. 10.1136/ard.2008.100867
    One-pot Synthesis of Metal-Organic Frameworks with Encapsulated Target Molecules and Their Applications for Controlled Drug Delivery. Zheng Haoquan,Zhang Yuning,Liu Leifeng,Wan Wei,Guo Peng,Nyström Andreas M,Zou Xiaodong Journal of the American Chemical Society Many medical and chemical applications require target molecules to be delivered in a controlled manner at precise locations. Metal-organic frameworks (MOFs) have high porosity, large surface area, and tunable functionality and are promising carriers for such purposes. Current approaches for incorporating target molecules are based on multistep postfunctionalization. Here, we report a novel approach that combines MOF synthesis and molecule encapsulation in a one-pot process. We demonstrate that large drug and dye molecules can be encapsulated in zeolitic imidazolate framework (ZIF) crystals. The molecules are homogeneously distributed within the crystals, and their loadings can be tuned. We show that ZIF-8 crystals loaded with the anticancer drug doxorubicin (DOX) are efficient drug delivery vehicles in cancer therapy using pH-responsive release. Their efficacy on breast cancer cell lines is higher than that of free DOX. Our one-pot process opens new possibilities to construct multifunctional delivery systems for a wide range of applications. 10.1021/jacs.5b11720
    Comparative characterization of B cells specific for HBV nucleocapsid and envelope proteins in patients with chronic hepatitis B. Le Bert Nina,Salimzadeh Loghman,Gill Upkar Singh,Dutertre Charles-Antoine,Facchetti Floriana,Tan Anthony,Hung Magdeleine,Novikov Nikolai,Lampertico Pietro,Fletcher Simon Paul,Kennedy Patrick Thomas Francis,Bertoletti Antonio Journal of hepatology BACKGROUND & AIMS:Knowledge about the regulation of anti-HBV humoral immunity during natural HBV infection is limited. We recently utilized dual fluorochrome-conjugated HBsAg to demonstrate, in patients with chronic HBV (CHB) infection, the functional impairment of their HBsAg-specific B cells. However, the features of their HBcAg-specific B cells are unknown. Here we developed a method to directly visualize, select and characterize HBcAg-specific B cells in parallel with HBsAg-specific B cells. METHODS:Fluorochrome-conjugated HBcAg reagents were synthesized and utilized to directly detect ex vivo HBcAg-specific B cells in 36 patients with CHB. The frequency, phenotype, functional maturation and transcriptomic profile of HBcAg-specific B cells was studied by flow cytometry, in vitro maturation assays and NanoString-based detection of expression of immune genes, which we compared with HBsAg-specific B cells and total B cells. RESULTS:HBcAg-specific B cells are present at a higher frequency than HBsAg-specific B cells in patients with CHB and, unlike HBsAg-specific B cells, they mature efficiently into antibody-secreting cells in vitro. Their phenotypic and transcriptomic profiles show that HBcAg-specific B cells are preferentially IgG+ memory B cells. However, despite their phenotypic and functional differences, HBcAg- and HBsAg-specific B cells from patients with CHB share an mRNA expression pattern that differs from global memory B cells and is characterized by high expression of genes indicative of cross-presentation and innate immune activity. CONCLUSIONS:During chronic HBV infection, a direct relation exists between serological detection of anti-HBs and anti-HBc antibodies, and the quantity and function of their respective specific B cells. However, the transcriptomic analysis performed in HBsAg- and HBcAg-specific B cells suggests additional roles of HBV-specific B cells beyond the production of antibodies. LAY SUMMARY:Protection of viral infection necessitates the production of antibodies that are generated by specialized cells of the immune system called B cells. During chronic HBV infection, antibodies against the internal part of the virus (core or HBcAg) are detectable while the antibodies directed against the virus envelope (surface or HBsAg) are not present. Here we developed a method that allows us to directly visualize ex vivo the B cells specific for these 2 viral components, highlighting their differences and similarities, and showing how 2 components of the same virus can have different impacts on the function of antiviral B cells. 10.1016/j.jhep.2019.07.015
    Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma. Toh Tan Boon,Lim Jhin Jieh,Hooi Lissa,Rashid Masturah Bte Mohd Abdul,Chow Edward Kai-Hua Journal of hepatology BACKGROUND & AIMS:Hepatic resection and liver transplantation with adjuvant chemo- and radiotherapy are the mainstay of hepatocellular carcinoma (HCC) treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Only sorafenib and lenvatinib are currently approved for the first-line treatment of advanced, unresected HCC, but they yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modalities. METHODS:The HCC tumor model was generated by hydrodynamic transfection of AKT1 and β-catenin (CTNNB1) oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors was analyzed in vitro by using tumorsphere culture and in vivo using an allograft mouse model. RESULTS:Co-activation of both Wnt/β-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/β-catenin or Akt/mTOR pathway activation alone, demonstrating the clinical relevance of our study. In addition, we observed that Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor self-renewal and drug resistance. Consequently, we identified small molecule inhibitors of the Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/β-catenin-driven HCC. CONCLUSIONS:In conclusion, we have shown that Akt/β-catenin tumors contain a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics which can be effectively targeted with inhibitors of the Jak/Stat pathway, demonstrating that inhibition of the Jak/Stat pathway could be an alternative method to overcome drug resistance and effectively treat Akt/β-catenin-driven HCC tumors. LAY SUMMARY:The prognosis for patients with hepatocellular carcinoma is poor, partly because of the lack of effective treatment options for those with more advanced disease. In this study, we identified a subpopulation of cancer cells with stem cell-like properties that were critical for tumor maintenance and growth in a mouse model of hepatocellular carcinoma. Through further experiments, we demonstrated that the Jak/Stat pathway is a promising therapeutic target in hepatocellular carcinoma. 10.1016/j.jhep.2019.08.035
    Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis. Sauter Reinhard J,Sauter Manuela,Reis Edimara S,Emschermann Frederic N,Nording Henry,Ebenhöch Sonja,Kraft Peter,Münzer Patrick,Mauler Maximilian,Rheinlaender Johannes,Madlung Johannes,Edlich Frank,Schäffer Tilman E,Meuth Sven G,Duerschmied Daniel,Geisler Tobias,Borst Oliver,Gawaz Meinrad,Kleinschnitz Christoph,Lambris John D,Langer Harald F Circulation BACKGROUND:Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant. METHODS:We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry. RESULTS:We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca influx. Using C3aR mice or C3 mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR mice with C3aR platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation. CONCLUSIONS:Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases. 10.1161/CIRCULATIONAHA.118.034600
    Genome-wide whole blood transcriptome profiling in a large European cohort of systemic sclerosis patients. Beretta Lorenzo,Barturen Guillermo,Vigone Barbara,Bellocchi Chiara,Hunzelmann Nicolas,De Langhe Ellen,Cervera Ricard,Gerosa Maria,Kovács László,Ortega Castro Rafaela,Almeida Isabel,Cornec Divi,Chizzolini Carlo,Pers Jacques-Olivier,Makowska Zuzanna,Lesche Ralf,Kerick Martin,Alarcón-Riquelme Marta Eugenia,Martin Javier, , Annals of the rheumatic diseases OBJECTIVES:The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. METHODS:Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. RESULTS:Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. CONCLUSIONS:We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc. 10.1136/annrheumdis-2020-217116
    TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Pols Thijs W H,Nomura Mitsunori,Harach Taoufiq,Lo Sasso Giuseppe,Oosterveer Maaike H,Thomas Charles,Rizzo Giovanni,Gioiello Antimo,Adorini Luciano,Pellicciari Roberto,Auwerx Johan,Schoonjans Kristina Cell metabolism The G protein-coupled receptor TGR5 has been identified as an important component of the bile acid signaling network, and its activation has been linked to enhanced energy expenditure and improved glycemic control. Here, we demonstrate that activation of TGR5 in macrophages by 6α-ethyl-23(S)-methylcholic acid (6-EMCA, INT-777), a semisynthetic BA, inhibits proinflammatory cytokine production, an effect mediated by TGR5-induced cAMP signaling and subsequent NF-κB inhibition. TGR5 activation attenuated atherosclerosis in Ldlr(-/-)Tgr5(+/+) mice but not in Ldlr(-/-)Tgr5(-/-) double-knockout mice. The inhibition of lesion formation was associated with decreased intraplaque inflammation and less plaque macrophage content. Furthermore, Ldlr(-/-) animals transplanted with Tgr5(-/-) bone marrow did not show an inhibition of atherosclerosis by INT-777, further establishing an important role of leukocytes in INT-777-mediated inhibition of vascular lesion formation. Taken together, these data attribute a significant immune modulating function to TGR5 activation in the prevention of atherosclerosis, an important facet of the metabolic syndrome. 10.1016/j.cmet.2011.11.006
    Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion. Luis Tiago C,Naber Brigitta A E,Roozen Paul P C,Brugman Martijn H,de Haas Edwin F E,Ghazvini Mehrnaz,Fibbe Willem E,van Dongen Jacques J M,Fodde Riccardo,Staal Frank J T Cell stem cell Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool. 10.1016/j.stem.2011.07.017
    RNA:DNA hybrids are a novel molecular pattern sensed by TLR9. Rigby Rachel E,Webb Lauren M,Mackenzie Karen J,Li Yue,Leitch Andrea,Reijns Martin A M,Lundie Rachel J,Revuelta Ailsa,Davidson Donald J,Diebold Sandra,Modis Yorgo,MacDonald Andrew S,Jackson Andrew P The EMBO journal The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease. 10.1002/embj.201386117
    Effects of Hepatitis B Surface Antigen on Virus-Specific and Global T Cells in Patients With Chronic Hepatitis B Virus infection. Le Bert Nina,Gill Upkar S,Hong Michelle,Kunasegaran Kamini,Tan Damien Z M,Ahmad Raidah,Cheng Yang,Dutertre Charles-A,Heinecke Andreas,Rivino Laura,Tan Anthony,Hansi Navjyot K,Zhang Min,Xi Sujuan,Chong Yutian,Pflanz Stefan,Newell Evan W,Kennedy Patrick T F,Bertoletti Antonio Gastroenterology BACKGROUND & AIMS:Chronic hepatitis B virus (HBV) infection is characterized by the presence of defective viral envelope proteins (hepatitis B surface antigen [HBsAg]) and the duration of infection-most patients acquire the infection at birth or during the first years of life. We investigated the effects of these factors on patients' lymphocyte and HBV-specific T-cell populations. METHODS:We collected blood samples and clinical data from 243 patients with HBV infection (3-75 years old) in the United Kingdom and China. We measured levels of HBV DNA, HBsAg, hepatitis B e antigen, and alanine aminotransferase; analyzed HBV genotypes; and isolated peripheral blood mononuclear cells (PBMCs). In PBMCs from 48 patients with varying levels of serum HBsAg, we measured 40 markers on nature killer and T cells by mass cytometry. PBMCs from 189 patients with chronic infection and 38 patients with resolved infections were incubated with HBV peptide libraries, and HBV-specific T cells were identified by interferon gamma enzyme-linked immune absorbent spot (ELISpot) assays or flow cytometry. We used multivariate linear regression and performed variable selection using the Akaike information criterion to identify covariates associated with HBV-specific responses of T cells. RESULTS:Although T- and natural killer cell phenotypes and functions did not change with level of serum HBsAg, numbers of HBs-specific T cells correlated with serum levels of HBsAg (r = 0.3367; P < .00001). After we performed the variable selection, the multivariate linear regression model identified patient age as the only factor significantly associated with numbers of HBs-specific T cells (P = .000115). In patients younger than 30 years, HBs-specific T cells constituted 28.26% of the total HBV-specific T cells; this value decreased to 7.14% in patients older than 30 years. CONCLUSIONS:In an analysis of immune cells from patients with chronic HBV infection, we found that the duration of HBsAg exposure, rather than the quantity of HBsAg, was associated with the level of anti-HBV immune response. Although the presence of HBs-specific T cells might not be required for the clearance of HBV infection in all patients, strategies to restore anti-HBV immune responses should be considered in patients younger than 30 years. 10.1053/j.gastro.2020.04.019
    Proteomics of a fuzzy organelle: interphase chromatin. Kustatscher Georg,Hégarat Nadia,Wills Karen L H,Furlan Cristina,Bukowski-Wills Jimi-Carlo,Hochegger Helfrid,Rappsilber Juri The EMBO journal Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. 10.1002/embj.201387614
    In vitro measurement of cell death with the annexin A5 affinity assay. van Genderen Hugo,Kenis Heidi,Lux Petra,Ungeth Lisette,Maassen Cecile,Deckers Niko,Narula Jagat,Hofstra Leo,Reutelingsperger Chris Nature protocols One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here. 10.1038/nprot.2006.55
    Identification of human regulatory T cells in the setting of T-cell activation and anti-CTLA-4 immunotherapy on the basis of expression of latency-associated peptide. Sun Jingjing,Tang Derek Ng,Fu Tihui,Sharma Padmanee Cancer discovery UNLABELLED:Effector and regulatory T cells (Treg) share multiple markers that make it difficult to discern differences in these populations in humans. The transcription factor FoxP3 has been shown to identify Tregs. However, the detection of FoxP3 requires cell permeabilization, thereby preventing isolation of viable Tregs. Subsequently, the extracellular marker CD127 was established for the identification of Tregs. However, these studies were not conducted in the setting of immunotherapy. Here, we conducted studies to analyze CD127 and FoxP3 expression on T cells before and after in vitro activation as well as in the setting of patients treated with antibodies directed against cytotoxic T-lymphocyte antigen-4 (CTLA-4). We show that latency-associated peptide (LAP), as opposed to CD127, was capable of identifying Tregs after in vitro activation as well as after treatment with anti-CTLA-4. Therefore, we propose that LAP should be used as a marker of Tregs for immune monitoring studies in patients treated with active immunotherapy such as anti-CTLA-4. SIGNIFICANCE:Tregs play an important role in human diseases, including cancer and autoimmunity; however, it has been difficult to study these cells because of a lack of an appropriate marker. Here, we propose LAP as a marker that can be used to identify Tregs in patients treated with immunotherapy, thereby permitting isolation of these cells for functional studies and for ex vivo expansion. 10.1158/2159-8290.CD-11-0236
    Mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load. Oshansky Christine M,Gartland Andrew J,Wong Sook-San,Jeevan Trushar,Wang David,Roddam Philippa L,Caniza Miguela A,Hertz Tomer,Devincenzo John P,Webby Richard J,Thomas Paul G American journal of respiratory and critical care medicine RATIONALE:Children are an at-risk population for developing complications following influenza infection, but immunologic correlates of disease severity are not understood. We hypothesized that innate cellular immune responses at the site of infection would correlate with disease outcome. OBJECTIVES:To test the immunologic basis of severe illness during natural influenza virus infection of children and adults at the site of infection. METHODS:An observational cohort study with longitudinal sampling of peripheral and mucosal sites in 84 naturally influenza-infected individuals, including infants. Cellular responses, viral loads, and cytokines were quantified from nasal lavages and blood, and correlated to clinical severity. MEASUREMENTS AND MAIN RESULTS:We show for the first time that although viral loads in children and adults were similar, innate responses in the airways were stronger in children and varied considerably between plasma and site of infection. Adjusting for age and viral load, an innate immune profile characterized by increased nasal lavage monocyte chemotactic protein-3, IFN-α2, and plasma IL-10 levels at enrollment predicted progression to severe disease. Increased plasma IL-10, monocyte chemotactic protein-3, and IL-6 levels predicted hospitalization. This inflammatory cytokine production correlated significantly with monocyte localization from the blood to the site of infection, with conventional monocytes positively correlating with inflammation. Increased frequencies of CD14(lo) monocytes were in the airways of participants with lower inflammatory cytokine levels. CONCLUSIONS:An innate profile was identified that correlated with disease progression independent of viral dynamics and age. The airways and blood displayed dramatically different immune profiles emphasizing the importance of cellular migration and localized immune phenotypes. 10.1164/rccm.201309-1616OC
    Deficiency of melanoma differentiation-associated protein 5 results in exacerbated chronic postviral lung inflammation. Kim Won-Keun,Jain Deepika,Sánchez Melissa D,Koziol-White Cynthia J,Matthews Krystal,Ge Moyar Q,Haczku Angela,Panettieri Reynold A,Frieman Matthew B,López Carolina B American journal of respiratory and critical care medicine RATIONALE:Respiratory viral infections can result in the establishment of chronic lung diseases. Understanding the early innate immune mechanisms that participate in the development of chronic postviral lung disease may reveal new targets for therapeutic intervention. The intracellular viral sensor protein melanoma differentiation-associated protein 5 (MDA5) sustains the acute immune response to Sendai virus, a mouse pathogen that causes chronic lung inflammation, but its role in the development of postviral chronic lung disease is unknown. OBJECTIVES:To establish the role of MDA5 in the development of chronic lung disease. METHODS:MDA5-deficient or control mice were infected with Sendai virus. The acute inflammatory response was evaluated by profiling chemokine and cytokine expression and by characterizing the composition of the cellular infiltrate. The impact of MDA5 on chronic lung pathology and function was evaluated through histological studies, degree of oxygen saturation, and responsiveness to carbachol. MEASUREMENTS AND MAIN RESULTS:MDA5 deficiency resulted in normal virus replication and in a distinct profile of chemokines and cytokines that associated with acute lung neutropenia and enhanced accumulation of alternatively activated macrophages. Diminished expression of neutrophil-recruiting chemokines was also observed in cells infected with influenza virus, suggesting a key role of MDA5 in driving the early accumulation of neutrophils at the infection site. The biased acute inflammatory response of MDA5-deficient mice led to an enhanced chronic lung inflammation, epithelial cell hyperplasia, airway hyperreactivity, and diminished blood oxygen saturation. CONCLUSIONS:MDA5 modulates the development of chronic lung inflammation by regulating the early inflammatory response in the lung. 10.1164/rccm.201307-1338OC
    Prospective identification of myogenic endothelial cells in human skeletal muscle. Zheng Bo,Cao Baohong,Crisan Mihaela,Sun Bin,Li Guangheng,Logar Alison,Yap Solomon,Pollett Jonathan B,Drowley Lauren,Cassino Theresa,Gharaibeh Burhan,Deasy Bridget M,Huard Johnny,Péault Bruno Nature biotechnology We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease. 10.1038/nbt1334
    Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages. Cell Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target. 10.1016/j.cell.2021.04.038
    Tumor Necrosis Factor-producing T-regulatory Cells Are Associated With Severe Liver Injury in Patients With Acute Hepatitis A. Choi Yoon Seok,Jung Min Kyung,Lee Jeewon,Choi Seong Jin,Choi Sung Hoon,Lee Hyun Woong,Lee Jong-Joo,Kim Hyung Joon,Ahn Sang Hoon,Lee Dong Hyeon,Kim Won,Park Su-Hyung,Huh Jun R,Kim Hyoung-Pyo,Park Jun Yong,Shin Eui-Cheol Gastroenterology BACKGROUND AND AIMS:CD4CD25Foxp3 T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. METHODS:We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4CD25Foxp3) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. RESULTS:A higher proportion of CD4CD25Foxp3 Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. CONCLUSIONS:Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA. 10.1053/j.gastro.2017.11.277
    Toll-like Receptor 9 Pathway Mediates Schlafen-MDSC Polarization During Helicobacter-induced Gastric Metaplasias. Gastroenterology BACKGROUND & AIMS:A subset of myeloid-derived suppressor cells (MDSCs) that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia. We propose that individuals with a more robust response to damage-activated molecular patterns and increased Toll-like receptor 9 (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection. METHODS:A mouse or human Transwell co-culture system composed of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers, and Helicobacter were used to dissect the cellular source of interferon-α (IFNα) in the stomach by flow cytometry. Conditioned media from the co-cultures polarized primary myeloid cells. MDSC activity was determined by T-cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9T>C variant was genotyped and linked to TLR9, IFNα, and SLFN12L expression by immunohistochemistry. Nuclear factor-κB binding to the TLR9 C allele was determined by electrophoretic mobility shift assays. RESULTS:Helicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFNα. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFNα secretion, and SLFN-MDSC polarization. Neutralizing IFNα in vivo mitigated Helicobacter-induced spasmolytic polypeptide-expressing metaplasia. The TLR9 minor C allele creates a nuclear factor-κB binding site associated with higher levels of TLR9, IFNα, and SLFN12L in Helicobacter-infected stomachs that correlated with a greater incidence of metaplasias and cancer. CONCLUSIONS:TLR9 plays an essential role in the production of IFNα and polarization of SLFN MDSCs on Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected. 10.1053/j.gastro.2022.04.031
    Adrenergic nerves govern circadian leukocyte recruitment to tissues. Scheiermann Christoph,Kunisaki Yuya,Lucas Daniel,Chow Andrew,Jang Jung-Eun,Zhang Dachuan,Hashimoto Daigo,Merad Miriam,Frenette Paul S Immunity The multistep sequence leading to leukocyte migration is thought to be locally regulated at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves regulate rhythmic recruitment of leukocytes in tissues. Constitutive leukocyte adhesion and migration in murine bone marrow (BM) and skeletal-muscle microvasculature fluctuated with circadian peak values at night. Migratory oscillations, altered by experimental jet lag, were implemented by perivascular SNS fibers acting on β-adrenoreceptors expressed on nonhematopoietic cells and leading to tissue-specific, differential circadian oscillations in the expression of endothelial cell adhesion molecules and chemokines. We showed that these rhythms have physiological consequences through alteration of hematopoietic cell recruitment and overall survival in models of septic shock, sickle cell vaso-occlusion, and BM transplantation. These data provide unique insights in the leukocyte adhesion cascade and the potential for time-based therapeutics for transplantation and inflammatory diseases. 10.1016/j.immuni.2012.05.021
    Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Acton Sophie E,Astarita Jillian L,Malhotra Deepali,Lukacs-Kornek Veronika,Franz Bettina,Hess Paul R,Jakus Zoltan,Kuligowski Michael,Fletcher Anne L,Elpek Kutlu G,Bellemare-Pelletier Angelique,Sceats Lindsay,Reynoso Erika D,Gonzalez Santiago F,Graham Daniel B,Chang Jonathan,Peters Anneli,Woodruff Matthew,Kim Young-A,Swat Wojciech,Morita Takashi,Kuchroo Vijay,Carroll Michael C,Kahn Mark L,Wucherpfennig Kai W,Turley Shannon J Immunity To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces. 10.1016/j.immuni.2012.05.022
    STAT3 transcription factor promotes instability of nTreg cells and limits generation of iTreg cells during acute murine graft-versus-host disease. Laurence Arian,Amarnath Shoba,Mariotti Jacopo,Kim Yong Chan,Foley Jason,Eckhaus Michael,O'Shea John J,Fowler Daniel H Immunity Acute graft-versus-host disease (GvHD) is a major cause of mortality in allogeneic bone marrow transplantation (BMT), for which administration of FoxP3(+) regulatory T (Treg) cells has been proposed as a therapy. However, the phenotypic stability of Treg cells is controversial, and STAT3-dependent cytokines can inhibit FoxP3 expression. We assessed whether the elimination of STAT3 in T cells could limit the severity of GvHD. We found STAT3 limited FoxP3(+) Treg cell numbers following allogeneic BMT by two pathways: instability of natural Treg (nTreg) cells and inhibition of induced Treg (iTreg) cell polarization from naive CD4(+) T cells. Deletion of STAT3 within only the nTreg cell population was not sufficient to protect against lethal GvHD. In contrast, transfer of STAT3-deficient naive CD4(+) T cells increased FoxP3(+) Treg cells post-BMT and prevented lethality, suggesting that the consequence of STAT3 signaling may be greater for iTreg rather than nTreg cells during GvHD. 10.1016/j.immuni.2012.05.027
    Interleukin-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft versus host disease. Hanash Alan M,Dudakov Jarrod A,Hua Guoqiang,O'Connor Margaret H,Young Lauren F,Singer Natalie V,West Mallory L,Jenq Robert R,Holland Amanda M,Kappel Lucy W,Ghosh Arnab,Tsai Jennifer J,Rao Uttam K,Yim Nury L,Smith Odette M,Velardi Enrico,Hawryluk Elena B,Murphy George F,Liu Chen,Fouser Lynette A,Kolesnick Richard,Blazar Bruce R,van den Brink Marcel R M Immunity Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage. 10.1016/j.immuni.2012.05.028
    FLT3-ITDs instruct a myeloid differentiation and transformation bias in lymphomyeloid multipotent progenitors. Mead Adam J,Kharazi Shabnam,Atkinson Deborah,Macaulay Iain,Pecquet Christian,Loughran Stephen,Lutteropp Michael,Woll Petter,Chowdhury Onima,Luc Sidinh,Buza-Vidas Natalija,Ferry Helen,Clark Sally-Ann,Goardon Nicolas,Vyas Paresh,Constantinescu Stefan N,Sitnicka Ewa,Nerlov Claus,Jacobsen Sten Eirik W Cell reports Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies. 10.1016/j.celrep.2013.04.031
    Uncontrolled Innate and Impaired Adaptive Immune Responses in Patients with COVID-19 Acute Respiratory Distress Syndrome. Hue Sophie,Beldi-Ferchiou Asma,Bendib Inés,Surenaud Mathieu,Fourati Slim,Frapard Thomas,Rivoal Simon,Razazi Keyvan,Carteaux Guillaume,Delfau-Larue Marie-Héléne,Mekontso-Dessap Armand,Audureau Etienne,de Prost Nicolas American journal of respiratory and critical care medicine Uncontrolled inflammatory innate response and impaired adaptive immune response are associated with clinical severity in patients with coronavirus disease (COVID-19). To compare the immunopathology of COVID-19 acute respiratory distress syndrome (ARDS) with that of non-COVID-19 ARDS, and to identify biomarkers associated with mortality in patients with COVID-19 ARDS. Prospective observational monocenter study. Immunocompetent patients diagnosed with RT-PCR-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and ARDS admitted between March 8 and March 30, 2020, were included and compared with patients with non-COVID-19 ARDS. The primary clinical endpoint of the study was mortality at Day 28. Flow cytometry analyses and serum cytokine measurements were performed at Days 1-2 and 4-6 of ICU admission. As compared with patients with non-COVID-19 ARDS ( = 36), those with COVID-19 ( = 38) were not significantly different regarding age, sex, and Sequential Organ Failure Assessment and Simplified Acute Physiology Score II scores but exhibited a higher Day-28 mortality (34% vs. 11%,  = 0.030). Patients with COVID-19 showed profound and sustained T CD4 ( = 0.002), CD8 ( < 0.0001), and B ( < 0.0001) lymphopenia, higher HLA-DR expression on monocytes ( < 0.001) and higher serum concentrations of EGF (epithelial growth factor), GM-CSF, IL-10, CCL2/MCP-1, CCL3/MIP-1a, CXCL10/IP-10, CCL5/RANTES, and CCL20/MIP-3a. After adjusting on age and Sequential Organ Failure Assessment, serum CXCL10/IP-10 ( = 0.047) and GM-CSF ( = 0.050) were higher and nasopharyngeal RT-PCR cycle threshold values lower ( = 0.010) in patients with COVID-19 who were dead at Day 28. Profound global lymphopenia and a "chemokine signature" were observed in COVID-19 ARDS. Increased serum concentrations of CXCL10/IP-10 and GM-CSF, together with higher nasopharyngeal SARS-CoV-2 viral load, were associated with Day-28 mortality. 10.1164/rccm.202005-1885OC
    The paradox of NKp46+ natural killer cells: drivers of severe hepatitis C virus-induced pathology but in-vivo resistance to interferon α treatment. Pembroke Tom,Christian Adam,Jones Emma,Hills Robert K,Wang Eddie C Y,Gallimore Awen M,Godkin Andrew Gut OBJECTIVE:There is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). The phenotype and function of blood and intrahepatic NK cells, in steady state and after interferon (IFN) α treatment has not been fully elucidated. DESIGN:We performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from patients with a variety of chronic liver diseases. Furthermore, we obtained serial paired samples from an average of five time points in HCV patients treated with IFNα. RESULTS:Liver NK cells demonstrate a distinct activated phenotype compared to blood manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30; with increased spontaneous degranulation and IFN production. In contrast, NKp46 expression was not downregulated. Indeed, NKp46-rich NK populations were the most activated, correlating closely with the severity of liver inflammation. Following initiation of IFNα treatment there was a significant increase in the proportion of intrahepatic NK cells at days 1 and 3. NKp46-rich NK populations demonstrated no reserve activation capacity with IFNα treatment and were associated with poor viral control on treatment and treatment failure. CONCLUSIONS:NKp46 marks out pathologically activated NK cells, which may result from a loss of homeostatic control of activating receptor expression in HCV. Paradoxically these pathological NK cells do not appear to be involved in viral control in IFNα-treated individuals and, indeed, predict slower rates of viral clearance. 10.1136/gutjnl-2013-304472
    Obesity accelerates Helicobacter felis-induced gastric carcinogenesis by enhancing immature myeloid cell trafficking and TH17 response. Ericksen Russell E,Rose Shannon,Westphalen Christoph Benedikt,Shibata Wataru,Muthupalani Sureshkumar,Tailor Yagnesh,Friedman Richard A,Han Weiping,Fox James G,Ferrante Anthony W,Wang Timothy C Gut OBJECTIVE:To investigate the role of obesity-associated inflammation and immune modulation in gastric carcinogenesis during Helicobacter-induced chronic gastric inflammation. DESIGN:C57BL/6 male mice were infected with H felis and placed on a high-fat diet (45% calories from fat). Study animals were analysed for gastric and adipose pathology, inflammatory markers in serum, stomach and adipose tissue, and immune responses in blood, spleen, stomach and adipose tissue. RESULTS:H felis-induced gastric carcinogenesis was accelerated in diet-induced obese mice compared with lean controls. Obesity increased bone marrow-derived immature myeloid cells in blood and gastric tissue of H felis-infected mice. Obesity also led to elevations in CD4 T cells, IL-17A, granulocyte macrophage colony-stimulating factor, phosphorylated STAT3 and prosurvival gene expression in gastric tissue of H felis-infected mice. Conversely, in adipose tissue of obese mice, H felis infection increased macrophage accumulation and expression of IL-6, C-C motif ligand 7 (CCL7) and leptin. Finally, the combination of obesity and gastric inflammation synergistically increased serum proinflammatory cytokines, including IL-6. CONCLUSIONS:Here, we have established a model to study the molecular mechanism by which obesity predisposes individuals to gastric cancer. In H felis-infected mice, obesity increased proinflammatory immune responses and accelerated gastric carcinogenesis. Interestingly, gastric inflammation augmented obesity-induced adipose inflammation and production of adipose-derived factors in obese, but not lean, mice. Our findings suggest that obesity accelerates Helicobacter-associated gastric cancer through cytokine-mediated cross-talk between inflamed gastric and adipose tissues, augmenting immune responses at both tissue sites, and thereby contributing to a protumorigenic gastric microenvironment. 10.1136/gutjnl-2013-305092
    IL-10 production differentially influences the magnitude, quality, and protective capacity of Th1 responses depending on the vaccine platform. Darrah Patricia A,Hegde Sonia T,Patel Dipti T,Lindsay Ross W B,Chen Linda,Roederer Mario,Seder Robert A The Journal of experimental medicine The quality of a Th1 response can be a prospective correlate of vaccine-mediated protection against certain intracellular pathogens. Using two distinct vaccine platforms, we evaluate the influence of interleukin (IL) 10 production on the magnitude, quality, and protective capacity of CD4(+) T cell responses in the mouse model of Leishmania major infection. Multiparameter flow cytometry was used to delineate the CD4(+) T cell production of interferon (IFN) gamma, IL-2, tumor necrosis factor (TNF), and IL-10 (or combinations thereof) after vaccination. Immunization with a high dose of adenovirus (ADV) expressing leishmanial proteins (MML-ADV) elicited a limited proportion of multifunctional IFN-gamma(+)IL-2(+)TNF(+) Th1 cells, a high frequency of IL-10-producing CD4(+) T cells, and did not protect against subsequent challenge. Surprisingly, in the absence of IL-10, there was no change in the magnitude, quality, or protective capacity of the Th1 response elicited by high-dose MML-ADV. In contrast, after immunization with MML protein and CpG (MML + CpG), IL-10 limited the production of IL-12 by DCs in vivo, thereby decreasing the generation of multifunctional Th1 cells. Consequently, three immunizations with MML + CpG were required for full protection. However, inhibiting IL-10 at the time of immunization enhanced the magnitude and quality of the Th1 response sufficiently to mediate protection after only a single immunization. Overall, we delineate distinct mechanisms by which vaccines elicit protective Th1 responses and underscore the importance of multifunctional CD4(+) T cells. 10.1084/jem.20092532
    DNA flow cytometry in sperm cells from testicular cancer patients. Impact of different treatment modalities on spermatogenesis. Fosså S D,Melvik J E,Juul N O,Pettersen E O,Amellem O,Theodorsen L European urology In 77 patients with testicular cancer, sperm cell density was evaluated before and after treatment together with the following parameters as recorded by DNA flow cytometry: the percentage of condensed and non-condensed haploid sperm cells, and the number of non-haploid cells. The patients received either abdominal radiotherapy, cisplatin-based combination chemotherapy (+/- surgery) or did not undergo antiproliferative treatment at all. Ejaculates with a low sperm cell density had high numbers of non-condensed haploid and non-haploid cells, whereas the percentage of condensed haploid cells was decreased. However, even 'azoospermic' semen samples (by light microscopy) contained haploid cells indicating ongoing sperm cell production. One year after radiotherapy there was a significant decrease in the sperm cell density and the number of condensed haploid cells. Chemotherapy led to a significant reduction of sperm cell density evaluated 1 year after treatment. Three years after the diagnosis of testicular cancer, sperm cell production had recovered in most patients. In patients who did not have radiotherapy or chemotherapy the median density of sperm cells 3 years after treatment significantly exceeded the corresponding figure from the pretreatment situation. A low pretreatment percentage of condensed haploid cells (less than or equal to 90%) was correlated with the lack of 1-year recovery of sperm cell production. It is too early to state whether longitudinal flow cytometric data from ejaculates in testicular cancer patients yield clinically valuable information, in addition to that obtained by light microscopy. From this preliminary observation it seems that DNA flow cytometry in ejaculates from testicular cancer patients may give valuable clinical information about sperm cell production following treatment.
    Autoimmune pancreatitis in MRL/Mp mice is a T cell-mediated disease responsive to cyclosporine A and rapamycin treatment. Schwaiger Theresa,van den Brandt Cindy,Fitzner Brit,Zaatreh Sarah,Kraatz Franziska,Dummer Annegret,Nizze Horst,Evert Matthias,Bröker Barbara M,Brunner-Weinzierl Monika C,Wartmann Thomas,Salem Tareq,Lerch Markus M,Jaster Robert,Mayerle Julia Gut BACKGROUND:Autoimmune pancreatitis (AIP) in humans invariably responds to steroid treatment, but little is known about the underlying pathogenesis and the benefits of alternative treatments. OBJECTIVE:To study the pathogenesis, and the efficacy of alternative immunosuppressant agents in the MRL/Mp mouse model of AIP. DESIGN:MRL/Mp mice were pretreated for 4 weeks with polyinosinic:polycytidylic acid to induce AIP. Pancreatic sections of mice genetically deleted for CTLA-4 were analysed. Blockage of CTLA-4 was achieved by intraperitoneal antibody treatment with 2 μg/g anti-mouse-CD152. Subsequent therapeutic studies were performed for a period of 4 weeks using cyclosporine A (40 μg/g), rapamycin (1 μg/g) or azathioprine (15 μg/g). RESULTS:Blockage of CTLA-4 in MRL/Mp mice suppressed regulatory T cell (Treg) function and raised the effector T cell (Teff) response with subsequent histomorphological organ destruction, indicating that AIP is a T cell-driven disease. Using an established histopathological score, we found that dexamethasone, cyclosporine A and rapamycin, but less so azathioprine, reduced pancreatic damage. However, the beneficial effects of cyclosporine A and rapamycin were achieved via different mechanisms: cyclosporine A inhibited Teff activation and proliferation whereas rapamycin led to selective expansion of Tregs which subsequently suppressed the Teff response. CONCLUSIONS:The calcineurin inhibitor cyclosporine A and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, improve the course of AIP in MRL/Mp mice via different mechanisms. These findings further support the concept of autoreactive T cells as key players in the pathogenesis of AIP and suggest that cyclosporine A and rapamycin should be considered for treatment of AIP in humans. 10.1136/gutjnl-2012-303635
    Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation. The Journal of clinical investigation Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines. 10.1172/JCI140685
    Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis. Ramdzan Yasmin M,Trubetskov Mikhail M,Ormsby Angelique R,Newcombe Estella A,Sui Xiaojing,Tobin Mark J,Bongiovanni Marie N,Gras Sally L,Dewson Grant,Miller Jason M L,Finkbeiner Steven,Moily Nagaraj S,Niclis Jonathan,Parish Clare L,Purcell Anthony W,Baker Michael J,Wilce Jacqueline A,Waris Saboora,Stojanovski Diana,Böcking Till,Ang Ching-Seng,Ascher David B,Reid Gavin E,Hatters Danny M Cell reports Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis. 10.1016/j.celrep.2017.04.029
    A model for personalized in vivo analysis of human immune responsiveness. Kalscheuer Hannes,Danzl Nichole,Onoe Takashi,Faust Ted,Winchester Robert,Goland Robin,Greenberg Ellen,Spitzer Thomas R,Savage David G,Tahara Hiroyuki,Choi Goda,Yang Yong-Guang,Sykes Megan Science translational medicine Studies of human immune diseases are generally limited to the analysis of peripheral blood lymphocytes of heterogeneous patient populations. Improved models are needed to allow analysis of fundamental immunologic abnormalities predisposing to disease and in which to assess immunotherapies. Immunodeficient mice receiving human fetal thymus grafts and fetal CD34(+) cells intravenously produce robust human immune systems, allowing analysis of human T cell development and function. However, to use humanized mice to study human immune-mediated disorders, immune systems must be generated from adult hematopoietic cells. Here, we demonstrated robust immune reconstitution in mice with hematopoietic stem cells (HSCs) aspirated from bone marrow of adults with type 1 diabetes (T1D) and healthy control volunteers. In these humanized mice, cryopreservation of human leukocyte antigen allele-matched fetal thymic tissue prevented allogeneic adult HSC rejection. Newly generated T cells, which included regulatory T cells (T(regs)), were functional and self-tolerant and had a diverse repertoire. The immune recognition of these mice mimicked that of the adult CD34(+) cell donor, but the T cell phenotypes were more predominantly "naïve" than those of the adult donors. HSCs from T1D and control donors generated similar numbers of natural T(regs) intrathymically; however, peripheral T cells from T1D subjects showed increased proportions of activated or memory cells compared to controls, suggesting possible HSC-intrinsic differences in T cell homeostasis that might underlie immune pathology in T1D. This "personalized immune" mouse provides a new model for individualized analysis of human immune responses that may provide new insights into not only T1D but also other forms of immune function and dysfunction as well. 10.1126/scitranslmed.3003481
    Tuning Cellular Uptake of Molecular Probes by Rational Design of Their Assembly into Supramolecular Nanoprobes. Lock Lye Lin,Reyes Claudia D,Zhang Pengcheng,Cui Honggang Journal of the American Chemical Society Intracellular sensing of pathologically relevant biomolecules could provide essential information for accurate evaluation of disease staging and progression, yet the poor cellular uptake of water-soluble molecular probes limits their use as protease sensors. In other cases such as extracellular sensing, cellular uptake should be effectively inhibited. Self-assembly of molecular probes into supramolecular nanoprobes presents a potential strategy to alter their interaction mechanisms with cells to promote or reduce their cellular uptake. Here, we report on the design, synthesis, and assembly of peptide-based molecular beacons into supramolecular protease sensors of either spherical or filamentous shapes. We found that positively charged spherical nanobeacons demonstrate much higher cellular uptake efficiency than its monomeric form, thus making them most suitable for intracellular sensing of the lysosomal protease cathepsin B. Our results also suggest that assembly into filamentous nanobeacons significantly reduces their internalization by cancer cells, an important property that can be utilized for probing extracellular protease activities. These studies provide important guiding principles for rational design of supramolecular nanoprobes with tunable cellular uptake characteristics. 10.1021/jacs.6b00073
    Genomes of uncultured eukaryotes: sorting FACS from fiction. Worden Alexandra Z,Dupont Christopher,Allen Andrew E Genome biology A recent study explores the genome content of uncultured unicellular marine eukaryotes and provides insights about interactions between uncultured eukaryotes and other biological entities. 10.1186/gb-2011-12-6-117
    Postacute COVID-19 is Characterized by Gut Viral Antigen Persistence in Inflammatory Bowel Diseases. Gastroenterology BACKGROUND & AIMS:The coronavirus disease 2019 (COVID-19) pandemic has affected populations, societies, and lives for more than 2 years. Long-term sequelae of COVID-19, collectively termed the postacute COVID-19 syndrome, are rapidly emerging across the globe. Here, we investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen persistence underlies the postacute COVID-19 syndrome. METHODS:We performed an endoscopy study with 46 patients with inflammatory bowel disease (IBD) 219 days (range, 94-257) after a confirmed COVID-19 infection. SARS-CoV-2 antigen persistence was assessed in the small and large intestine using quantitative polymerase chain reaction of 4 viral transcripts, immunofluorescence of viral nucleocapsid, and virus cultivation from biopsy tissue. Postacute COVID-19 was assessed using a standardized questionnaire, and a systemic SARS-CoV-2 immune response was evaluated using flow cytometry and enzyme-linked immunosorbent assay at endoscopy. IBD activity was evaluated using clinical, biochemical, and endoscopic means. RESULTS:We report expression of SARS-CoV-2 RNA in the gut mucosa ∼7 months after mild acute COVID-19 in 32 of 46 patients with IBD. Viral nucleocapsid protein persisted in 24 of 46 patients in gut epithelium and CD8 T cells. Expression of SARS-CoV-2 antigens was not detectable in stool and viral antigen persistence was unrelated to severity of acute COVID-19, immunosuppressive therapy, and gut inflammation. We were unable to culture SARS-CoV-2 from gut tissue of patients with viral antigen persistence. Postacute sequelae of COVID-19 were reported from the majority of patients with viral antigen persistence, but not from patients without viral antigen persistence. CONCLUSION:Our results indicate that SARS-CoV-2 antigen persistence in infected tissues serves as a basis for postacute COVID-19. The concept that viral antigen persistence instigates immune perturbation and postacute COVID-19 requires validation in controlled clinical trials. 10.1053/j.gastro.2022.04.037
    Induction of Fibroblast Growth Factor Receptor 4 by Helicobacter pylori via Signal Transducer and Activator of Transcription 3 With a Feedforward Activation Loop Involving Steroid Receptor Coactivator Signaling in Gastric Cancer. Gastroenterology BACKGROUND & AIMS:Helicobacter pylori (H pylori) infection is the main risk factor for gastric cancer. The role of fibroblast growth factor receptors (FGRFs) in H pylori-mediated gastric tumorigenesis remains largely unknown. This study investigated the molecular and mechanistic links between H pylori, inflammation, and FGFR4 in gastric cancer. METHODS:Cell lines, human and mouse gastric tissue samples, and gastric organoids models were implemented. Infection with H pylori was performed using in vitro and in vivo models. Western blot, quantitative real-time reverse-transcription polymerase chain reaction, flow cytometry, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and luciferase reporter assays were used for molecular, mechanistic, and functional studies. RESULTS:Analysis of FGFR family members using The Cancer Genome Atlas data, followed by validation, indicated that FGFR4 messenger (m)RNA was the most significantly overexpressed member in human gastric cancer tissue samples (P < .001). We also detected high levels of Fgfr4 mRNA and protein in gastric dysplasia and adenocarcinoma lesions in mouse models. Infection with J166, 7.13, and PMSS1 cytotoxin-associated gene A (CagA)+ H pylori strains induced FGFR4 mRNA and protein expression in in vitro and in vivo models. This was associated with a concordant activation of signal transducer and activator of transcription 3 (STAT3). Analysis of the FGFR4 promoter suggested several putative binding sites for STAT3. Using chromatin immunoprecipitation assay and an FGFR-promoter luciferase reporter containing putative STAT3 binding sites and their mutants, we confirmed a direct functional binding of STAT3 on the FGFR4 promoter. Mechanistically, we also discovered a feedforward activation loop between FGFR4 and STAT3 where the fibroblast growth factor 19-FGFR4 axis played an essential role in activating STAT3 in a steroid receptor coactivator-dependent manner. Functionally, we found that FGFR4 protected against H pylori-induced DNA damage and cell death. CONCLUSIONS:Our findings demonstrated a link between infection, inflammation, and FGFR4 activation, where a feedforward activation loop between FGFR4 and STAT3 is established via steroid receptor coactivator in response to H pylori infection. Given the relevance of FGFR4 to the etiology and biology of gastric cancer, we propose FGFR4 as a druggable molecular vulnerability that can be tested in patients with gastric cancer. 10.1053/j.gastro.2022.05.016
    Affinity peptide for targeted detection of dysplasia in Barrett's esophagus. Li Meng,Anastassiades Constantinos P,Joshi Bishnu,Komarck Chris M,Piraka Cyrus,Elmunzer Badih J,Turgeon Danielle K,Johnson Timothy D,Appelman Henry,Beer David G,Wang Thomas D Gastroenterology BACKGROUND & AIMS:Dysplasia is a premalignant condition in Barrett's esophagus that is difficult to detect on endoscopy because of its flat architecture and patchy distribution. Peptides are promising for use as novel molecular probes that identify cell surface targets unique to disease and can be fluorescence-labeled for detection. We aim to select and validate an affinity peptide that binds to esophageal dysplasia for future clinical studies. METHODS:Peptide selection was performed using phage display by removing nonspecific binders using Q-hTERT (intestinal metaplasia) cells and achieving specific binding against OE33 (esophageal adenocarcinoma) cells. Selective binding was confirmed on bound phage counts, enzyme-linked immunosorbent assay (ELISA), flow cytometry, competitive inhibition, and fluorescence microscopy. On stereomicroscopy, specific peptide binding to dysplasia on endoscopically resected specimens was assessed by rigorous registration of fluorescence intensity to histology in 1-mm intervals. RESULTS:The peptide sequence SNFYMPL was selected and showed preferential binding to target cells. Reduced binding was observed on competition with unlabeled peptide in a dose-dependent manner, an affinity of K(d) = 164 nmol/L was measured, and peptide binding to the surface of OE33 cells was validated on fluorescence microscopy. On esophageal specimens (n = 12), the fluorescence intensity (mean ± SEM) in 1-mm intervals classified histologically as squamous (n = 145), intestinal metaplasia (n = 83), dysplasia (n = 61), and gastric mucosa (n = 69) was 46.5 ± 1.6, 62.3 ± 5.8, 100.0 ± 9.0, and 42.4 ± 3.0 arb units, respectively. CONCLUSIONS:The peptide sequence SNFYMPL binds specifically to dysplasia in Barrett's esophagus and can be fluorescence labeled to target premalignant mucosa on imaging. 10.1053/j.gastro.2010.07.007
    Interplay of Th1 and Th17 cells in murine models of malignant pleural effusion. Lin Hua,Tong Zhao-Hui,Xu Qian-Qian,Wu Xiu-Zhi,Wang Xiao-Juan,Jin Xiao-Guang,Ma Wan-Li,Cheng Xiang,Zhou Qiong,Shi Huan-Zhong American journal of respiratory and critical care medicine RATIONALE:IFN-γ-producing CD4(+) T (Th1) cells and IL-17-producing CD4(+) T (Th17) cells have been found to be involved in multiple malignancies; however, the reciprocal relationship between Th1 and Th17 cells in malignant pleural effusion (MPE) remains to be elucidated. OBJECTIVES:To explore the differentiation and immune regulation of Th1 and Th17 cells in the development of MPE in murine models. METHODS:The distribution and differentiation of Th1 and Th17 cells in MPE were investigated in IFN-γ(-/-), IL-17(-/-), and wild-type mice. The effects of Th1 and Th17 cells on the development of MPE and the survival of mice bearing MPE were also investigated. MEASUREMENTS AND MAIN RESULTS:We have demonstrated that increased Th1 and Th17 cells could be found in MPE as compared with blood and spleen. Compared with wild-type mice, Th17 cells were markedly augmented in MPE from IFN-γ(-/-) mice, and improved survival could be seen in IFN-γ(-/-) mice. Th1 cell numbers were elevated in MPE from IL-17(-/-) mice, and decreased survival could be seen in IL-17(-/-) mice. The in vitro experiments showed that IFN-γ deficiency promoted Th17-cell differentiation by suppressing the STAT3 pathway and that IL-17 deficiency promoted Th1-cell differentiation by suppressing the STAT1 pathway. CONCLUSIONS:In mouse models of MPE, IFN-γ inhibited Th17-cell differentiation, whereas IL-17 inhibited Th1-cell differentiation. IL-17 inhibited the formation of MPE and improved the survival of mice bearing MPE; in contrast, IFN-γ promoted MPE formation and mouse death. 10.1164/rccm.201310-1776OC
    Intermittent hypoxia-induced changes in tumor-associated macrophages and tumor malignancy in a mouse model of sleep apnea. Almendros Isaac,Wang Yang,Becker Lev,Lennon Frances E,Zheng Jiamao,Coats Brittney R,Schoenfelt Kelly S,Carreras Alba,Hakim Fahed,Zhang Shelley X,Farré Ramon,Gozal David American journal of respiratory and critical care medicine RATIONALE:An increased cancer aggressiveness and mortality have been recently reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, enhances melanoma growth and metastasis in mice. OBJECTIVES:To assess whether OSA-related adverse cancer outcomes occur via IH-induced changes in host immune responses, namely tumor-associated macrophages (TAMs). MEASUREMENTS AND MAIN RESULTS:Lung epithelial TC1 cell tumors were 84% greater in mice subjected to IH for 28 days compared with room air (RA). In addition, TAMs in IH-exposed tumors exhibited reductions in M1 polarity with a shift toward M2 protumoral phenotype. Although TAMs from tumors harvested from RA-exposed mice increased TC1 migration and extravasation, TAMs from IH-exposed mice markedly enhanced such effects and also promoted proliferative rates and invasiveness of TC1 cells. Proliferative rates of melanoma (B16F10) and TC1 cells exposed to IH either in single culture or in coculture with macrophages (RAW 264.7) increased only when RAW 264.7 macrophages were concurrently present. CONCLUSIONS:Our findings support the notion that IH-induced alterations in TAMs participate in the adverse cancer outcomes reported in OSA. 10.1164/rccm.201310-1830OC
    Consolidation and Maintenance in Newly Diagnosed Multiple Myeloma. Sonneveld Pieter,Dimopoulos Meletios A,Beksac Meral,van der Holt Bronno,Aquino Sara,Ludwig Heinz,Zweegman Sonja,Zander Thilo,Zamagni Elena,Wester Ruth,Hajek Roman,Pantani Lucia,Dozza Luca,Gay Francesca,Cafro AnneMaria,De Rosa Luca,Morelli Annamaria,Gregersen Henrik,Gulbrandsen Nina,Cornelisse Petra,Troia Rosella,Oliva Stefania,van de Velden Vincent,Wu KaLung,Ypma Paula F,Bos Gerard,Levin Mark-David,Pour Luca,Driessen Christoph,Broijl Annemiek,Croockewit Alexandra,Minnema Monique C,Waage Anders,Hveding Cecilie,van de Donk Niels W C J,Offidani Massimo,Palumbo Giuseppe A,Spencer Andrew,Boccadoro Mario,Cavo Michele Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:To address the role of consolidation treatment for newly diagnosed, transplant eligible patients with multiple myeloma in a controlled clinical trial. PATIENTS AND METHODS:The EMN02/HOVON95 trial compared consolidation treatment with two cycles of bortezomib, lenalidomide, and dexamethasone (VRD) or no consolidation after induction and intensification therapy, followed by continuous lenalidomide maintenance. Primary study end point was progression-free survival (PFS). RESULTS:Eight hundred seventy-eight eligible patients were randomly assigned to receive VRD consolidation (451 patients) or no consolidation (427 patients). At a median follow-up of 74.8 months, median PFS with adjustment for pretreatment was prolonged in patients randomly assigned to VRD consolidation (59.3 42.9 months, hazard ratio [HR] = 0.81; 95% CI, 0.68 to 0.96; = .016). The PFS benefit was observed across most predefined subgroups, including revised International Staging System (ISS) stage, cytogenetics, and prior treatment. Revised ISS3 stage (HR, 2.00; 95% CI, 1.41 to 2.86) and ampl1q (HR, 1.67; 95% CI, 1.37 to 2.04) were significant adverse prognostic factors. The median duration of maintenance was 33 months (interquartile range 13-86 months). Response ≥ complete response (CR) after consolidation versus no consolidation before start of maintenance was 34% versus 18%, respectively ( < .001). Response ≥ CR on protocol including maintenance was 59% with consolidation and 46% without ( < .001). Minimal residual disease analysis by flow cytometry in a subgroup of 226 patients with CR or stringent complete response or very good partial response before start of maintenance demonstrated a 74% minimal residual disease-negativity rate in VRD-treated patients. Toxicity from VRD was acceptable and manageable. CONCLUSION:Consolidation treatment with VRD followed by lenalidomide maintenance improves PFS and depth of response in newly diagnosed patients with multiple myeloma as compared to maintenance alone. 10.1200/JCO.21.01045
    Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions. Bao Xiaoping,Lian Xiaojun,Qian Tongcheng,Bhute Vijesh J,Han Tianxiao,Palecek Sean P Nature protocols Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-β inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications. 10.1038/nprot.2017.080
    The early bird catches the worm: new technologies for the Caenorhabditis elegans toolkit. Xu Xiao,Kim Stuart K Nature reviews. Genetics The inherent simplicity of Caenorhabditis elegans and its extensive genetic toolkit make it ideal for studying complex biological processes. Recent developments further increase the usefulness of the worm, including new methods for: altering gene expression, altering physiology using optogenetics, manipulating large numbers of worms, automating laborious processes and processing high-resolution images. These developments both enhance the worm as a model for studying processes such as development and ageing and make it an attractive model in areas such as neurobiology and behaviour. 10.1038/nrg3050
    Increased DNA and/or RNA content of synovial fluid cells in rheumatoid arthritis: a flow-cytometry study. Bonvoisin B,Cordier G,Revillard J P,Lejeune E,Bouvier M Annals of the rheumatic diseases Flow-cytometry studies of DNA and RNA content were carried out in acridine orange-stained synovial fluid lymphocytes from 11 patients presenting with classical or definite rheumatoid arthritis. Monoclonal antibodies were used to detect specific T cell surface antigens (OKT3, OKT4, OKT8) and antigens associated with lymphocyte activation (OKIa 1, OKT10). T3 positive cell percentages were comparable to those of normal blood, although T4/T8 ratios were decreased in 4 out of 5 cases, and HLA-DR positive cells increased. Six out of 11 patients showed percentages of dividing cells varying from 2.2 to 7.2% as compared with less than 1% in the other patients and in normal blood. Nondividing cells were characterised by an increase in their RNA content compared with normal blood. A greater increase of RNA content was observed in patients with lower percentages of dividing cells, suggesting a G1/S block. Changes in cellular DNA and/or RNA contents provide a valuable parameter of lymphocyte activation, not necessarily linked to the expression of differentiation antigens by activated cells. 10.1136/ard.43.2.222
    Inflammatory monocytes activate memory CD8(+) T and innate NK lymphocytes independent of cognate antigen during microbial pathogen invasion. Soudja Saïdi M'Homa,Ruiz Anne L,Marie Julien C,Lauvau Grégoire Immunity Memory CD8(+) T cells induced upon immunization exhibit improved functional features that contribute to protection of immunized hosts. Although both cognate antigen recognition and inflammation are important for memory CD8(+) T cell reactivation, the relative contribution of these factors and the cell types providing these signals in vivo are poorly defined. Here, we show that Ly6C(+)CCR2(+) inflammatory monocytes, a subset of monocytes, largely orchestrate memory CD8(+) T and NK lymphocytes activation by differentiating into interleukin-18 (IL-18)- and IL-15-producing cells in an inflammasome and type I interferon-IRF3-dependent manner. Memory CD8(+) T cells became potent effector cells by sensing inflammation from monocytes independently of their cognate antigen. Like NK cells, they underwent rapid mobilization, upregulated intense and sustained effector functions during bacterial, viral, and parasitic infections, and contributed to innate responses and protection in vivo. Thus, inflammatory monocyte-derived IL-18 and IL-15 are critical to initiate memory CD8(+) T and NK lymphocytes differentiation into antimicrobial effector cells. 10.1016/j.immuni.2012.05.029
    γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response. Zeng Xun,Wei Yu-Ling,Huang Jun,Newell Evan W,Yu Hongxiang,Kidd Brian A,Kuhns Michael S,Waters Ray W,Davis Mark M,Weaver Casey T,Chien Yueh-hsiu Immunity γδ T cells contribute uniquely to immune competence. Nevertheless, how they function remains an enigma. It is unclear what most γδ T cells recognize, what is required for them to mount an immune response, and how the γδ T cell response is integrated into host immune defense. Here, we report that a noted B cell antigen, the algae protein phycoerythrin (PE), is a murine and human γδ T cell antigen. Employing this specificity, we demonstrated that antigen recognition activated naive γδ T cells to make interleukin-17 and respond to cytokine signals that perpetuate the response. High frequencies of antigen-specific γδ T cells in naive animals and their ability to mount effector response without extensive clonal expansion allow γδ T cells to initiate a swift, substantial response. These results underscore the adaptability of lymphocyte antigen receptors and suggest an antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. 10.1016/j.immuni.2012.06.011
    Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production. Lynch Lydia,Nowak Michael,Varghese Bindu,Clark Justice,Hogan Andrew E,Toxavidis Vasillis,Balk Steven P,O'Shea Donal,O'Farrelly Cliona,Exley Mark A Immunity Invariant natural killer T (iNKT) cells are evolutionarily conserved innate T cells that influence inflammatory responses. We have shown that iNKT cells, previously thought to be rare in humans, were highly enriched in human and murine adipose tissue, and that as adipose tissue expanded in obesity, iNKT cells were depleted, correlating with proinflammatory macrophage infiltration. iNKT cell numbers were restored in mice and humans after weight loss. Mice lacking iNKT cells had enhanced weight gain, larger adipocytes, fatty livers, and insulin resistance on a high-fat diet. Adoptive transfer of iNKT cells into obese mice or in vivo activation of iNKT cells via their lipid ligand, alpha-galactocylceramide, decreased body fat, triglyceride levels, leptin, and fatty liver and improved insulin sensitivity through anti-inflammatory cytokine production by adipose-derived iNKT cells. This finding highlights the potential of iNKT cell-targeted therapies, previously proven to be safe in humans, in the management of obesity and its consequences. 10.1016/j.immuni.2012.06.016
    Oxysterol gradient generation by lymphoid stromal cells guides activated B cell movement during humoral responses. Yi Tangsheng,Wang Xiaoming,Kelly Lisa M,An Jinping,Xu Ying,Sailer Andreas W,Gustafsson Jan-Ake,Russell David W,Cyster Jason G Immunity 7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G protein-coupled receptor EBI2; however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We showed that all three enzymes control EBI2 ligand concentration in lymphoid tissues. Lymphoid stromal cells were the main CH25H- and CYP7B1-expressing cells required for positioning of B cells, and they also mediated 7α,25-OHC inactivation. CH25H and CYP7B1 were abundant at the follicle perimeter, whereas CH25H expression by follicular dendritic cells was repressed. CYP7B1, CH25H, and HSD3B7 deficiencies each resulted in defective T cell-dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues, and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses. 10.1016/j.immuni.2012.06.015
    A broad range of self-reactivity drives thymic regulatory T cell selection to limit responses to self. Lee Hyang-Mi,Bautista Jhoanne L,Scott-Browne James,Mohan James F,Hsieh Chyi-Song Immunity The degree of T cell self-reactivity considered dangerous by the immune system, thereby requiring thymic selection processes to prevent autoimmunity, is unknown. Here, we analyzed a panel of T cell receptors (TCRs) with a broad range of reactivity to ovalbumin (OVA(323-339)) in the rat insulin promoter (RIP)-mOVA self-antigen model for their ability to trigger thymic self-tolerance mechanisms. Thymic regulatory T (Treg) cell generation in vivo was directly correlated with in vitro TCR reactivity to OVA-peptide in a broad ~1,000-fold range. Interestingly, higher TCR affinity was associated with a larger Treg cell developmental "niche" size, even though the amount of antigen should remain constant. The TCR-reactivity threshold to elicit thymic negative selection and peripheral T cell responses was ~100-fold higher than that of Treg cell differentiation. Thus, these data suggest that the broad range of self-reactivity that elicits thymic Treg cell generation is tuned to secure peripheral tolerance to self. 10.1016/j.immuni.2012.07.009
    T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor β2. Koch Meghan A,Thomas Kerri R,Perdue Nikole R,Smigiel Kate S,Srivastava Shivani,Campbell Daniel J Immunity Foxp3(+) regulatory T (Treg) cells limit inflammatory responses and maintain immune homeostasis. Although comprised of several phenotypically and functionally distinct subsets, the differentiation of specialized Treg cell populations within the periphery is poorly characterized. We demonstrate that the development of T-bet(+) Treg cells that potently inhibit T helper 1 (Th1) cell responses was dependent on the transcription factor STAT1 and occurred directly in response to interferon-γ produced by effector T cells. Additionally, delayed induction of the IL-12Rβ2 receptor component after STAT1 activation helped ensure that Treg cells do not readily complete STAT4-dependent Th1 cell development and lose their ability to suppress effector T cell proliferation. Thus, we define a pathway of abortive Th1 cell development that results in the specialization of peripheral Treg cells and demonstrate that impaired expression of a single cytokine receptor helps maintain Treg cell-suppressive function in the context of inflammatory Th1 cell responses. 10.1016/j.immuni.2012.05.031
    The BH3-only proteins Bim and Puma cooperate to impose deletional tolerance of organ-specific antigens. Gray Daniel H D,Kupresanin Fiona,Berzins Stuart P,Herold Marco J,O'Reilly Lorraine A,Bouillet Philippe,Strasser Andreas Immunity Although the proapoptotic BH3-only protein, Bim, is required for deletion of autoreactive thymocytes, Bim-deficient mice do not succumb to extensive organ-specific autoimmune disease. To determine whether other BH3-only proteins safeguard tolerance in the absence of Bim, we screened mice lacking Bim as well as other BH3-only proteins. Most strains showed no additional defects; however, mice deficient for both Puma and Bim spontaneously developed autoimmunity in multiple organs, and their T cells could transfer organ-specific autoimmunity. Puma- and Bim-double-deficient mice had a striking accumulation of mature, single-positive thymocytes, suggesting an additional defect in thymic deletion was the basis for disease. Transgenic mouse models of thymocyte deletion by peripheral neoantigens confirmed that the loss of Bim and Puma allowed increased numbers of autoreactive thymocytes to escape deletion. Our data show that Puma cooperates with Bim to impose a thymic-deletion checkpoint to peripheral self-antigens and cement the notion that defects in apoptosis alone are sufficient to cause autoimmune disease. 10.1016/j.immuni.2012.05.030
    Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation. Halim Timotheus Y F,MacLaren Aric,Romanish Mark T,Gold Matthew J,McNagny Kelly M,Takei Fumio Immunity Natural helper (NH) cells are innate lymphoid cells (ILCs) that produce T helper-2 (Th2)-cell-type cytokines in the lung- and gut-associated lymphoid tissues. Currently, the lineage relationship between NH cells in different tissues and between NH cells and interleukin-22 (IL-22)-producing retinoic-acid-receptor-related orphan receptor (ROR)γt-positive ILCs is unclear. Here, we report that NH cells express RORα, but not RORγt. RORα-deficient, but not RORγt-deficient, mice lacked NH cells in all tissues, whereas all other lymphocytes, including RORγt(+) ILCs, were unaffected. NH-cell-deficient mice generated by RORα-deficient bone-marrow transplantation had normal Th2 cell responses but failed to develop acute lung inflammation in response to protease allergen, thus confirming the essential role of NH cells in allergic lung inflammation. We have also identified RORα-dependent NH cell progenitors in the bone marrow. Thus, all NH cells belong to a unique RORα-dependent cell lineage separate from other lymphoid cell lineages. 10.1016/j.immuni.2012.06.012
    The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology. Hall Aisling O'Hara,Beiting Daniel P,Tato Cristina,John Beena,Oldenhove Guillaume,Lombana Claudia Gonzalez,Pritchard Gretchen Harms,Silver Jonathan S,Bouladoux Nicolas,Stumhofer Jason S,Harris Tajie H,Grainger John,Wojno Elia D Tait,Wagage Sagie,Roos David S,Scott Philip,Turka Laurence A,Cherry Sara,Reiner Steven L,Cua Daniel,Belkaid Yasmine,Elloso M Merle,Hunter Christopher A Immunity Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation. 10.1016/j.immuni.2012.06.014
    Compromised intestinal epithelial barrier induces adaptive immune compensation that protects from colitis. Khounlotham Manirath,Kim Wooki,Peatman Eric,Nava Porfirio,Medina-Contreras Oscar,Addis Caroline,Koch Stefan,Fournier Benedicte,Nusrat Asma,Denning Timothy L,Parkos Charles A Immunity Mice lacking junctional adhesion molecule A (JAM-A, encoded by F11r) exhibit enhanced intestinal epithelial permeability, bacterial translocation, and elevated colonic lymphocyte numbers, yet do not develop colitis. To investigate the contribution of adaptive immune compensation in response to increased intestinal epithelial permeability, we examined the susceptibility of F11r(-/-)Rag1(-/-) mice to acute colitis. Although negligible contributions of adaptive immunity in F11r(+/+)Rag1(-/-) mice were observed, F11r(-/-)Rag1(-/-) mice exhibited increased microflora-dependent colitis. Elimination of T cell subsets and cytokine analyses revealed a protective role for TGF-β-producing CD4(+) T cells in F11r(-/-) mice. Additionally, loss of JAM-A resulted in elevated mucosal and serum IgA that was dependent upon CD4(+) T cells and TGF-β. Absence of IgA in F11r(+/+)Igha(-/-) mice did not affect disease, whereas F11r(-/-)Igha(-/-) mice displayed markedly increased susceptibility to acute injury-induced colitis. These data establish a role for adaptive immune-mediated protection from acute colitis under conditions of intestinal epithelial barrier compromise. 10.1016/j.immuni.2012.06.017
    Characterization of human FDCs reveals regulation of T cells and antigen presentation to B cells. Heesters Balthasar A,van Megesen Kyah,Tomris Ilhan,de Vries Robert P,Magri Giuliana,Spits Hergen The Journal of experimental medicine Stromal-derived follicular dendritic cells (FDCs) are essential for germinal centers (GCs), the site where B cells maturate their antibodies. FDCs present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDCs remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRCs) rather than FDCs expressed B cell activating factor (BAFF). Furthermore, we found that human FDCs expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDCs activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DRA, CD40, and others. These data suggest intimate contact between human FDCs and T cells. 10.1084/jem.20210790
    Genetic evidence for the role of plasmacytoid dendritic cells in systemic lupus erythematosus. Sisirak Vanja,Ganguly Dipyaman,Lewis Kanako L,Couillault Coline,Tanaka Lena,Bolland Silvia,D'Agati Vivette,Elkon Keith B,Reizis Boris The Journal of experimental medicine Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of antibodies to self-nucleic acids, immune complex deposition, and tissue inflammation such as glomerulonephritis. Innate recognition of self-DNA and -RNA and the ensuing production of cytokines such as type I interferons (IFNs) contribute to SLE development. Plasmacytoid dendritic cells (pDCs) have been proposed as a source of pathogenic IFN in SLE; however, their net contribution to the disease remains unclear. We addressed this question by reducing gene dosage of the pDC-specific transcription factor E2-2 (Tcf4), which causes a specific impairment of pDC function in otherwise normal animals. We report that global or DC-specific Tcf4 haplodeficiency ameliorated SLE-like disease caused by the overexpression of the endosomal RNA sensor Tlr7. Furthermore, Tcf4 haplodeficiency in the B6.Sle1.Sle3 multigenic model of SLE nearly abolished key disease manifestations including anti-DNA antibody production and glomerulonephritis. Tcf4-haplodeficient SLE-prone animals showed a reduction of the spontaneous germinal center reaction and its associated gene expression signature. These results provide genetic evidence that pDCs are critically involved in SLE pathogenesis and autoantibody production, confirming their potential utility as therapeutic targets in the disease. 10.1084/jem.20132522
    Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits. Heise Nicole,De Silva Nilushi S,Silva Kathryn,Carette Amanda,Simonetti Giorgia,Pasparakis Manolis,Klein Ulf The Journal of experimental medicine Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis. 10.1084/jem.20132613
    Early, transient depletion of plasmacytoid dendritic cells ameliorates autoimmunity in a lupus model. Rowland Sarah L,Riggs Jeffrey M,Gilfillan Susan,Bugatti Mattia,Vermi William,Kolbeck Roland,Unanue Emil R,Sanjuan Miguel A,Colonna Marco The Journal of experimental medicine Plasmacytoid dendritic cells (pDCs) have long been implicated in the pathogenesis of lupus. However, this conclusion has been largely based on a correlative link between the copious production of IFN-α/β by pDCs and the IFN-α/β "signature" often seen in human lupus patients. The specific contribution of pDCs to disease in vivo has not been investigated in detail. For this reason, we generated a strain of BXSB lupus-prone mice in which pDCs can be selectively depleted in vivo. Early, transient ablation of pDCs before disease initiation resulted in reduced splenomegaly and lymphadenopathy, impaired expansion and activation of T and B cells, reduced antibodies against nuclear autoantigens and improved kidney pathology. Amelioration of pathology coincided with decreased transcription of IFN-α/β-induced genes in tissues. PDC depletion had an immediate impact on the activation of immune cells, and importantly, the beneficial effects on pathology were sustained even though pDCs later recovered, indicating an early pDC contribution to disease. Together, our findings demonstrate a critical function for pDCs during the IFN-α/β-dependent initiation of autoimmune lupus and point to pDCs as an attractive therapeutic target for the treatment of SLE. 10.1084/jem.20132620
    Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections. Conti Heather R,Peterson Alanna C,Brane Lucas,Huppler Anna R,Hernández-Santos Nydiaris,Whibley Natasha,Garg Abhishek V,Simpson-Abelson Michelle R,Gibson Gregory A,Mamo Anna J,Osborne Lisa C,Bishu Shrinivas,Ghilardi Nico,Siebenlist Ulrich,Watkins Simon C,Artis David,McGeachy Mandy J,Gaffen Sarah L The Journal of experimental medicine Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα(-/-), and Rag1(-/-) mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1-2 d by tongue-resident populations of γδ T cells and CD3(+)CD4(+)CD44(hi)TCRβ(+)CCR6(+) natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β(-/-) and TCR-δ(-/-) mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1(-/-), IL-7Rα(-/-), and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens. 10.1084/jem.20130877
    Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction. Kurktschiev Peter D,Raziorrouh Bijan,Schraut Winfried,Backmund Markus,Wächtler Martin,Wendtner Clemens-Martin,Bengsch Bertram,Thimme Robert,Denk Gerald,Zachoval Reinhart,Dick Andrea,Spannagl Michael,Haas Jürgen,Diepolder Helmut M,Jung Maria-Christina,Gruener Norbert H The Journal of experimental medicine The transcription factor T-bet regulates the production of interferon-γ and cytotoxic molecules in effector CD8 T cells, and its expression correlates with improved control of chronic viral infections. However, the role of T-bet in infections with differential outcome remains poorly defined. Here, we report that high expression of T-bet in virus-specific CD8 T cells during acute hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was associated with spontaneous resolution, whereas T-bet deficiency was more characteristic of chronic evolving infection. T-bet strongly correlated with interferon-γ production and proliferation of virus-specific CD8 T cells, and its induction by antigen and IL-2 stimulation partially restored functionality in previously dysfunctional T-bet-deficient CD8 T cells. However, restoration of a strong interferon-γ response required additional stimulation with IL-12, which selectively induced the phosphorylation of STAT4 in T-bet(+) CD8 T cells. The observation that T-bet expression rendered CD8 T cells responsive to IL-12 suggests a stepwise mechanism of T cell activation in which T-bet facilitates the recruitment of additional transcription factors in the presence of key cytokines. These findings support a critical role of T-bet for viral clearance and suggest T-bet deficiency as an important mechanism behind chronic infection. 10.1084/jem.20131333
    Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6. Ma Ji Su,Sasai Miwa,Ohshima Jun,Lee Youngae,Bando Hironori,Takeda Kiyoshi,Yamamoto Masahiro The Journal of experimental medicine Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b(+) Ly6G(+) cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner. 10.1084/jem.20131272
    Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication. Giugliano Silvia,Kriss Michael,Golden-Mason Lucy,Dobrinskikh Evgenia,Stone Amy E L,Soto-Gutierrez Alejandro,Mitchell Angela,Khetani Salman R,Yamane Daisuke,Stoddard Mark,Li Hui,Shaw George M,Edwards Michael G,Lemon Stanley M,Gale Michael,Shah Vijay H,Rosen Hugo R Gastroenterology BACKGROUND & AIMS:Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. METHODS:Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays. RESULTS:HLSECs internalized HCV, independent of cell-cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid-inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN λ3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8(+) T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. CONCLUSIONS:Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity. 10.1053/j.gastro.2014.10.040
    Bi-allelic LoF NRROS Variants Impairing Active TGF-β1 Delivery Cause a Severe Infantile-Onset Neurodegenerative Condition with Intracranial Calcification. Dong Xiaomin,Tan Natalie B,Howell Katherine B,Barresi Sabina,Freeman Jeremy L,Vecchio Davide,Piccione Maria,Radio Francesca Clementina,Calame Daniel,Zong Shan,Eggers Stefanie,Scheffer Ingrid E,Tan Tiong Y,Van Bergen Nicole J,Tartaglia Marco,Christodoulou John,White Susan M American journal of human genetics Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat-containing protein that uniquely associates with latent transforming growth factor beta-1 (TGF- β1) and anchors it on the cell surface; this anchoring is required for activation of TGF-β1 in macrophages and microglia. We report six individuals from four families with bi-allelic variants in NRROS. All affected individuals had neurodegenerative disease with refractory epilepsy, developmental regression, and reduced white matter volume with delayed myelination. The clinical course in affected individuals began with normal development or mild developmental delay, and the onset of seizures occurred within the first year of life, followed by developmental regression. Intracranial calcification was detected in three individuals. The phenotypic features in affected individuals are consistent with those observed in the Nrros knockout mouse, and they overlap with those seen in the human condition associated with TGF-β1 deficiency. The disease-causing NRROS variants involve two significant functional NRROS domains. These variants result in aberrant NRROS proteins with impaired ability to anchor latent TGF-β1 on the cell surface. Using confocal microscopy in HEK293T cells, we demonstrate that wild-type and mutant NRROS proteins co-localize with latent TGF-β1 intracellularly. However, using flow cytometry, we show that our mutant NRROS proteins fail to anchor latent TGF-β1 at the cell surface in comparison to wild-type NRROS. Moreover, wild-type NRROS rescues the defect of our disease-associated mutants in presenting latent TGF-β1 to the cell surface. Taken together, our findings suggest that loss of NRROS function causes a severe childhood-onset neurodegenerative condition with features suggestive of a disordered response to inflammation. 10.1016/j.ajhg.2020.02.014
    Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP-1 and p38. Escolano Amelia,Martínez-Martínez Sara,Alfranca Arántzazu,Urso Katia,Izquierdo Helena M,Delgado Mario,Martín Francisco,Sabio Guadalupe,Sancho David,Gómez-del Arco Pablo,Redondo Juan Miguel The EMBO journal Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status. 10.1002/embj.201386369
    Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation in podocytes and endothelial cells. Wang Wenjian,Wang Yin,Long Jianyin,Wang Jinrong,Haudek Sandra B,Overbeek Paul,Chang Benny H J,Schumacker Paul T,Danesh Farhad R Cell metabolism Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled coil-containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1 and an inducible podocyte-specific knockin mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics. 10.1016/j.cmet.2012.01.009
    Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells. Gupta Rana K,Mepani Rina J,Kleiner Sandra,Lo James C,Khandekar Melin J,Cohen Paul,Frontini Andrea,Bhowmick Diti Chatterjee,Ye Li,Cinti Saverio,Spiegelman Bruce M Cell metabolism Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots. 10.1016/j.cmet.2012.01.010
    Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Mayer Günter,Ahmed Marie-Sophie L,Dolf Andreas,Endl Elmar,Knolle Percy A,Famulok Michael Nature protocols Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19(+) Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library. 10.1038/nprot.2010.163
    Regulation of proximal T cell receptor signaling and tolerance induction by deubiquitinase Usp9X. Naik Edwina,Webster Joshua D,DeVoss Jason,Liu Jinfeng,Suriben Rowena,Dixit Vishva M The Journal of experimental medicine The T cell hyperproliferation and autoimmune phenotypes that manifest in mice lacking E3 ubiquitin ligases such as Cbl, ITCH, or GRAIL highlight the importance of ubiquitination for the maintenance of peripheral T cell tolerance. Less is known, however, about the deubiquitinating enzymes that regulate T cell proliferation and effector function. Here, we define a cell intrinsic role for the deubiquitinase Usp9X during proximal TCR signaling. Usp9X-deficient T cells were hypoproliferative, yet mice with T cell-specific Usp9x deletion had elevated numbers of antigen-experienced T cells and expanded PD-1 and OX40-expressing populations consistent with immune hyperactivity. Aged Usp9x KO mice developed lupus-like autoimmunity and lymphoproliferative disease, indicating that ubiquitin ligases and deubiquitinases maintain the delicate balance between effective immunity and self-tolerance. 10.1084/jem.20140860
    IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell-dependent antigen. Jacque Emilie,Schweighoffer Edina,Visekruna Alexander,Papoutsopoulou Stamatia,Janzen Julia,Zillwood Rachel,Tarlinton David M,Tybulewicz Victor L J,Ley Steven C The Journal of experimental medicine The importance of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in B cells was investigated using Nfkb1(SSAA/SSAA) mice, in which this NF-κB signaling pathway is blocked. Nfkb1(SSAA) mutation had no effect on the development and homeostasis of follicular mature (FM) B cells. However, analysis of mixed bone marrow chimeras revealed that Nfkb1(SSAA/SSAA) FM B cells were completely unable to mediate T cell-dependent antibody responses. Nfkb1(SSAA) mutation decreased B cell antigen receptor (BCR) activation of NF-κB in FM B cells, which selectively blocked BCR stimulation of cell survival and antigen-induced differentiation into plasmablasts and germinal center B cells due to reduced expression of Bcl-2 family proteins and IRF4, respectively. In contrast, the antigen-presenting function of FM B cells and their BCR-induced migration to the follicle T cell zone border, as well as their growth and proliferation after BCR stimulation, were not affected. All of the inhibitory effects of Nfkb1(SSAA) mutation on B cell functions were rescued by normalizing NF-κB activation genetically. Our study identifies critical B cell-intrinsic functions for IKK-induced NF-κB1 p105 proteolysis in the antigen-induced survival and differentiation of FM B cells, which are essential for T-dependent antibody responses. 10.1084/jem.20132019
    WASH is required for the differentiation commitment of hematopoietic stem cells in a c-Myc-dependent manner. Xia Pengyan,Wang Shuo,Huang Guanling,Zhu Pingping,Li Man,Ye Buqing,Du Ying,Fan Zusen The Journal of experimental medicine Hematopoiesis is fully dependent on hematopoietic stem cells (HSCs) that possess the capacity to self-renew and differentiate into all blood cell lineages. WASH, Wiskott-Aldrich syndrome protein (WASP) and SCAR homologue (WASH) is involved in endosomal sorting as an actin-nucleating protein. Here, we show that conditional WASH deletion in the hematopoietic system causes defective blood production of the host, leading to severe cytopenia and rapid anemia. WASH deficiency causes the accumulation of long-term (LT)-HSCs in bone marrow and perturbs their differentiation potential to mature blood lineages. Importantly, WASH is located in the nucleus of LT-HSCs and associates with the nucleosome remodeling factor (NURF) complex. WASH assists the NURF complex to the promoter of c-Myc gene through its VCA domain-dependent nuclear actin nucleation. WASH deletion suppresses the transcriptional activation of c-Myc gene and impairs the differentiation of LT-HSCs. WASH acts as an upstream regulator to modulate c-Myc transcription for hematopoietic regulation. 10.1084/jem.20140169
    Exhaustion of bacteria-specific CD4 T cells and microbial translocation in common variable immunodeficiency disorders. Perreau Matthieu,Vigano Selena,Bellanger Florence,Pellaton Céline,Buss Guillaume,Comte Denis,Roger Thierry,Lacabaratz Christine,Bart Pierre-Alexandre,Levy Yves,Pantaleo Giuseppe The Journal of experimental medicine In the present study, we have investigated the functional profile of CD4 T cells from patients with common variable immunodeficiency (CVID), including production of cytokines and proliferation in response to bacteria and virus-derived antigens. We show that the functional impairment of CD4 T cells, including the reduced capacity to proliferate and to produce IFN-γ and IL-2, was restricted to bacteria-specific and not virus-specific CD4 T cells. High levels of endotoxins were found in the plasma of patients with CVID, suggesting that CD4 T cell dysfunction might be caused by bacterial translocation. Of note, endotoxemia was associated with significantly higher expression of programmed death 1 (PD-1) on CD4 T cells. The blockade of the PD-1-PD-L1/2 axis in vitro restored CD4 T cell proliferation capacity, thus indicating that PD-1 signaling negatively regulates CD4 T cell functions. Finally, we showed that intravenous immunoglobulin G (IVIG) treatment significantly reduced endotoxemia and the percentage of PD-1(+) CD4 T cells, and restored bacteria-specific CD4 T cell cytokine production and proliferation. In conclusion, the present study demonstrates that the CD4 T cell exhaustion and functional impairment observed in CVID patients is associated with bacterial translocation and that IVIG treatment resolves bacterial translocation and restores CD4 T cell functions. 10.1084/jem.20140039
    Mucin 5AC-Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity. Gastroenterology BACKGROUND & AIMS:Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes. METHODS:Autochthonous murine models of PC (Kras, Pdx1-Cre [KC] and Kras, Pdx1-Cre, Muc5ac [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay. RESULTS:The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-β1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors. CONCLUSION:MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity. 10.1053/j.gastro.2022.02.032
    METTL3 Inhibits Antitumor Immunity by Targeting mA-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer. Gastroenterology BACKGROUND & AIMS:N-Methyladenosine (mA) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic mA modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS:METTL3 knockout mice, CD34 humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. MA sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS:We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4 and CD8 T cells, and eventually suppressed CRC in ApcMettl3 mice, CD34 humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the mA-BHLHE41-CXCL1 axis by analysis of mA sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an mA-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 treatment. CONCLUSIONS:Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the mA-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-programmed cell death protein 1 treatment shows promising antitumor efficacy against CRC. 10.1053/j.gastro.2022.06.024
    Dectin-1 Contributes to Myocardial Ischemia/Reperfusion Injury by Regulating Macrophage Polarization and Neutrophil Infiltration. Fan Qin,Tao Rong,Zhang Hang,Xie Hongyang,Lu Lin,Wang Ting,Su Min,Hu Jian,Zhang Qi,Chen Qiujing,Iwakura Yoichiro,Shen Weifeng,Zhang Ruiyan,Yan Xiaoxiang Circulation BACKGROUND:Macrophage-associated immune response plays an important role in myocardial ischemia/reperfusion (IR) injury. Dectin-1, expressed mainly on activated myeloid cells, is crucial for the regulation of immune homeostasis as a pattern recognition receptor. However, its effects and roles during the myocardial IR injury remain unknown. METHODS:Genetic ablation, antibody blockade, or Dectin-1 activation, along with the adoptive bone marrow transfer chimeric model, was used to determine the functional significance of Dectin-1 in myocardial IR injury. Immune cell filtration and inflammation were examined by flow cytometry, quantitative real-time polymerase chain reaction, and immunohistochemistry. Moreover, Dectin-1 cells were analyzed by flow cytometry in the blood of patients with ST-segment-elevation myocardial infarction and stable patients with normal coronary artery (control). RESULTS:We demonstrated that Dectin-1 expression observed on the bone marrow-derived macrophages is increased in the heart during the early phase after IR injury. Dectin-1 deficiency and antibody-mediated Dectin-1 inhibition led to a considerable improvement in cardiac function, accompanied by a reduction in cardiomyocyte apoptosis, which was associated with a decrease in M1 macrophage polarization and Ly-6C monocyte and neutrophil infiltration. Activation of Dectin-1 with its agonist had the opposite effects. Furthermore, Dectin-1 contributed to neutrophil recruitment through the regulation of Cxcl1 and granulocyte colony-stimulating factor expression. In addition, Dectin-1-dependent interleukin-23/interleukin-1β production was shown to be essential for interleukin-17A expression by γδT cells, leading to neutrophil recruitment and myocardial IR injury. Furthermore, we demonstrated that circulating Dectin-1CD14CD16 and Dectin-1CD14CD16 monocyte levels were significantly higher in patients with ST-segment-elevation myocardial infarction than in controls and positively correlated with the severity of cardiac dysfunction. CONCLUSIONS:Our results reveal a crucial role of Dectin-1 in the process of mouse myocardial IR injury and provide a new, clinically significant therapeutic target. 10.1161/CIRCULATIONAHA.118.036044
    The vascular endothelium of the adipose tissue gives rise to both white and brown fat cells. Tran Khanh-Van,Gealekman Olga,Frontini Andrea,Zingaretti Maria Cristina,Morroni Manrico,Giordano Antonio,Smorlesi Arianna,Perugini Jessica,De Matteis Rita,Sbarbati Andrea,Corvera Silvia,Cinti Saverio Cell metabolism Adipose tissue expansion involves the enlargement of existing adipocytes, the formation of new cells from committed preadipocytes, and the coordinated development of the tissue vascular network. Here we find that murine endothelial cells (ECs) of classic white and brown fat depots share ultrastructural characteristics with pericytes, which are pluripotent and can potentially give rise to preadipocytes. Lineage tracing experiments using the VE-cadherin promoter reveal localization of reporter genes in ECs and also in preadipocytes and adipocytes of white and brown fat depots. Furthermore, capillary sprouts from human adipose tissue, which have predominantly EC characteristics, are found to express Zfp423, a recently identified marker of preadipocyte determination. In response to PPARγ activation, endothelial characteristics of sprouting cells are progressively lost, and cells form structurally and biochemically defined adipocytes. Together these data support an endothelial origin of murine and human adipocytes, suggesting a model for how adipogenesis and angiogenesis are coordinated during adipose tissue expansion. 10.1016/j.cmet.2012.01.008
    Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. Coan Kristin E D,Shoichet Brian K Journal of the American Chemical Society Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a "critical aggregation concentration" (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10,000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 x 10(11) and 4.7 x 10(10) A(3), respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate. 10.1021/ja802977h
    Normalization of low-density lipoprotein receptor expression in receptor defective homozygous familial hypercholesterolemia by inhibition of PCSK9 with alirocumab. Lambert Gilles,Chatelais Mathias,Petrides Francine,Passard Maxime,Thedrez Aurélie,Rye Kerry-Anne,Schwahn Uwe,Gusarova Viktoria,Blom Dirk J,Sasiela William,Marais A David Journal of the American College of Cardiology 10.1016/j.jacc.2014.07.995
    Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells. Lee Jonghyeob,Sugiyama Takuya,Liu Yinghua,Wang Jing,Gu Xueying,Lei Ji,Markmann James F,Miyazaki Satsuki,Miyazaki Jun-Ichi,Szot Gregory L,Bottino Rita,Kim Seung K eLife Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001. 10.7554/eLife.00940
    Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Pinder Jordan,Salsman Jayme,Dellaire Graham Nucleic acids research CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains such as the nuclear lamina and promyelocytic leukemia nuclear bodies (PML NBs). Using this approach, we compared strategies for enhancing CRISPR-mediated HDR, focusing on known genes and small molecules that impact non-homologous end joining (NHEJ) and homologous recombination (HR). Ultimately, we identified the small molecule RS-1 as a potent enhancer of CRISPR-based genome editing, enhancing HDR 3- to 6-fold depending on the locus and transfection method. We also characterized U2OS human osteosarcoma cells expressing Clover-tagged PML and demonstrate that this strategy generates cell lines with PML NBs that are structurally and functionally similar to bodies in the parental cell line. Thus, the nuclear domain knock-in screen that we describe provides a simple means of rapidly evaluating methods and small molecules that have the potential to enhance Cas9-mediated HDR. 10.1093/nar/gkv993
    Depletion of circulating regulatory T cells during severe respiratory syncytial virus infection in young children. Raiden Silvina,Pandolfi Julieta,Payasliàn Florencia,Anderson Mariana,Rivarola Norma,Ferrero Fernando,Urtasun Marcela,Fainboim Leonardo,Geffner Jorge,Arruvito Lourdes American journal of respiratory and critical care medicine 10.1164/rccm.201311-1977LE
    Metabolic differentiation in the embryonic retina. Agathocleous Michalis,Love Nicola K,Randlett Owen,Harris Julia J,Liu Jinyue,Murray Andrew J,Harris William A Nature cell biology Unlike healthy adult tissues, cancers produce energy mainly by aerobic glycolysis instead of oxidative phosphorylation. This adaptation, called the Warburg effect, may be a feature of all dividing cells, both normal and cancerous, or it may be specific to cancers. It is not known whether, in a normally growing tissue during development, proliferating and postmitotic cells produce energy in fundamentally different ways. Here we show in the embryonic Xenopus retina in vivo, that dividing progenitor cells depend less on oxidative phosphorylation for ATP production than non-dividing differentiated cells, and instead use glycogen to fuel aerobic glycolysis. The transition from glycolysis to oxidative phosphorylation is connected to the cell differentiation process. Glycolysis is indispensable for progenitor proliferation and biosynthesis, even when it is not used for ATP production. These results suggest that the Warburg effect can be a feature of normal proliferation in vivo, and that the regulation of glycolysis and oxidative phosphorylation is critical for normal development. 10.1038/ncb2531
    Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells. Cao Nan,Liu Zumei,Chen Zhongyan,Wang Jia,Chen Taotao,Zhao Xiaoyang,Ma Yu,Qin Lianju,Kang Jiuhong,Wei Bin,Wang Liu,Jin Ying,Yang Huang-Tian Cell research Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells. 10.1038/cr.2011.195
    TGFβ inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy. Wang Chengyan,Tang Xuming,Sun Xiaomeng,Miao Zhenchuan,Lv Yaxin,Yang Yanlei,Zhang Huidan,Zhang Pengbo,Liu Yang,Du Liying,Gao Yang,Yin Ming,Ding Mingxiao,Deng Hongkui Cell research Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGFβ as a novel signal to regulate hematopoietic development, as the TGFβ inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development. 10.1038/cr.2011.138
    Genomic Risk Score impact on susceptibility to systemic sclerosis. Bossini-Castillo Lara,Villanueva-Martin Gonzalo,Kerick Martin,Acosta-Herrera Marialbert,López-Isac Elena,Simeón Carmen P,Ortego-Centeno Norberto,Assassi Shervin, , , , ,Hunzelmann Nicolas,Gabrielli Armando,de Vries-Bouwstra J K,Allanore Yannick,Fonseca Carmen,Denton Christopher P,Radstake Timothy Rdj,Alarcón-Riquelme Marta Eugenia,Beretta Lorenzo,Mayes Maureen D,Martin Javier Annals of the rheumatic diseases OBJECTIVES:Genomic Risk Scores (GRS) successfully demonstrated the ability of genetics to identify those individuals at high risk for complex traits including immune-mediated inflammatory diseases (IMIDs). We aimed to test the performance of GRS in the prediction of risk for systemic sclerosis (SSc) for the first time. METHODS:Allelic effects were obtained from the largest SSc Genome-Wide Association Study (GWAS) to date (9 095 SSc and 17 584 healthy controls with European ancestry). The best-fitting GRS was identified under the additive model in an independent cohort that comprised 400 patients with SSc and 571 controls. Additionally, GRS for clinical subtypes (limited cutaneous SSc and diffuse cutaneous SSc) and serological subtypes (anti-topoisomerase positive (ATA+) and anti-centromere positive (ACA+)) were generated. We combined the estimated GRS with demographic and immunological parameters in a multivariate generalised linear model. RESULTS:The best-fitting SSc GRS included 33 single nucleotide polymorphisms (SNPs) and discriminated between patients with SSc and controls (area under the receiver operating characteristic (ROC) curve (AUC)=0.673). Moreover, the GRS differentiated between SSc and other IMIDs, such as rheumatoid arthritis and Sjögren's syndrome. Finally, the combination of GRS with age and immune cell counts significantly increased the performance of the model (AUC=0.787). While the SSc GRS was not able to discriminate between ATA+ and ACA+ patients (AUC<0.5), the serological subtype GRS, which was based on the allelic effects observed for the comparison between ACA+ and ATA+ patients, reached an AUC=0.693. CONCLUSIONS:GRS was successfully implemented in SSc. The model discriminated between patients with SSc and controls or other IMIDs, confirming the potential of GRS to support early and differential diagnosis for SSc. 10.1136/annrheumdis-2020-218558
    Dihydropyrimidine accumulation is required for the epithelial-mesenchymal transition. Shaul Yoav D,Freinkman Elizaveta,Comb William C,Cantor Jason R,Tam Wai Leong,Thiru Prathapan,Kim Dohoon,Kanarek Naama,Pacold Michael E,Chen Walter W,Bierie Brian,Possemato Richard,Reinhardt Ferenc,Weinberg Robert A,Yaffe Michael B,Sabatini David M Cell It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits. 10.1016/j.cell.2014.07.032
    Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis. Li Haisen,Yue Rui,Wei Bin,Gao Ge,Du Jiulin,Pei Gang The EMBO journal Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX-LPA signaling was mediated by PI3K/Akt-Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient-derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation. 10.15252/embj.201387594
    Multiplexed single-cell morphometry for hematopathology diagnostics. Tsai Albert G,Glass David R,Juntilla Marisa,Hartmann Felix J,Oak Jean S,Fernandez-Pol Sebastian,Ohgami Robert S,Bendall Sean C Nature medicine The diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to 'see' like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of 'morphometric' markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases. 10.1038/s41591-020-0783-x
    Cord-Blood Transplantation in Patients with Minimal Residual Disease. Milano Filippo,Gooley Ted,Wood Brent,Woolfrey Ann,Flowers Mary E,Doney Kristine,Witherspoon Robert,Mielcarek Marco,Deeg Joachim H,Sorror Mohamed,Dahlberg Ann,Sandmaier Brenda M,Salit Rachel,Petersdorf Effie,Appelbaum Frederick R,Delaney Colleen The New England journal of medicine BACKGROUND:The majority of patients in need of a hematopoietic-cell transplant do not have a matched related donor. Data are needed to inform the choice among various alternative donor-cell sources. METHODS:In this retrospective analysis, we compared outcomes in 582 consecutive patients with acute leukemia or the myelodysplastic syndrome who received a first myeloablative hematopoietic-cell transplant from an unrelated cord-blood donor (140 patients), an HLA-matched unrelated donor (344), or an HLA-mismatched unrelated donor (98). RESULTS:The relative risks of death and relapse between the cord-blood group and the two other unrelated-donor groups appeared to vary according to the presence of minimal residual disease status before transplantation. Among patients with minimal residual disease, the risk of death was higher in the HLA-mismatched group than in the cord-blood group (hazard ratio, 2.92; 95% confidence interval [CI], 1.52 to 5.63; P=0.001); the risk was also higher in the HLA-matched group than in the cord-blood group but not significantly so (hazard ratio, 1.69; 95% CI, 0.94 to 3.02; P=0.08). Among patients without minimal residual disease, the hazard ratios were lower (hazard ratio in the HLA-mismatched group, 1.36; 95% CI, 0.76 to 2.46; P=0.30; hazard ratio in the HLA-matched group, 0.78; 95% CI, 0.48 to 1.28; P=0.33). The risk of relapse among patients with minimal residual disease was significantly higher in the two unrelated-donor groups than in the cord-blood group (hazard ratio in the HLA-mismatched group, 3.01; 95% CI, 1.22 to 7.38; P=0.02; hazard ratio in the HLA-matched group, 2.92; 95% CI, 1.34 to 6.35; P=0.007). Among patients without minimal residual disease, the magnitude of these associations was lower (hazard ratio in the HLA-mismatched group, 1.28; 95% CI, 0.51 to 3.25; P=0.60; hazard ratio in the HLA-matched group, 1.30; 95% CI, 0.65 to 2.58; P=0.46). CONCLUSIONS:Our data suggest that among patients with pretransplantation minimal residual disease, the probability of overall survival after receipt of a transplant from a cord-blood donor was at least as favorable as that after receipt of a transplant from an HLA-matched unrelated donor and was significantly higher than the probability after receipt of a transplant from an HLA-mismatched unrelated donor. Furthermore, the probability of relapse was lower in the cord-blood group than in either of the other groups. 10.1056/NEJMoa1602074
    Homozygosity for a null allele of SMIM1 defines the Vel-negative blood group phenotype. Storry Jill R,Jöud Magnus,Christophersen Mikael Kronborg,Thuresson Britt,Åkerström Bo,Sojka Birgitta Nilsson,Nilsson Björn,Olsson Martin L Nature genetics The Vel antigen is present on red blood cells (RBCs) from all humans except rare Vel-negative individuals who can form antibodies to Vel in response to transfusion or pregnancy. These antibodies may cause severe hemolytic reactions in blood recipients. We combined SNP profiling and transcriptional network modeling to link the Vel-negative phenotype to SMIM1, located in a 97-kb haplotype block on chromosome 1p36. This gene encodes a previously undiscovered, evolutionarily conserved transmembrane protein expressed on RBCs. Notably, 35 of 35 Vel-negative individuals were homozygous for a frameshift deletion of 17 bp in exon 3. Functional studies using antibodies raised against SMIM1 peptides confirmed a null phenotype in RBC membranes, and SMIM1 overexpression induced Vel expression. Genotype screening estimated that ~1 of 17 Swedish blood donors is a heterozygous deletion carrier and ~1 of 1,200 is a homozygous deletion knockout and enabled identification of Vel-negative donors. Our results establish SMIM1 as a new erythroid gene and Vel as a new blood group system. 10.1038/ng.2600
    Covalent binding of nanoliposomes to the surface of magnetotactic bacteria for the synthesis of self-propelled therapeutic agents. Taherkhani Samira,Mohammadi Mahmood,Daoud Jamal,Martel Sylvain,Tabrizian Maryam ACS nano The targeted and effective delivery of therapeutic agents remains an unmet goal in the field of controlled release systems. Magnetococcus marinus MC-1 magnetotactic bacteria (MTB) are investigated as potential therapeutic carriers. By combining directional magnetotaxis-microaerophilic control of these self-propelled agents, a larger amount of therapeutics can be delivered surpassing the diffusion limits of large drug molecules toward hard-to-treat hypoxic regions in solid tumors. The potential benefits of these carriers emphasize the need to develop an adequate method to attach therapeutic cargos, such as drug-loaded nanoliposomes, without substantially affecting the cell's ability to act as delivery agents. In this study, we report on a strategy for the attachment of liposomes to MTB (MTB-LP) through carbodiimide chemistry. The attachment efficacy, motility, and magnetic response of the MTB-LP were investigated. Results confirm that a substantial number of nanoliposomes (∼70) are efficiently linked with MTB without compromising functionality and motility. Cytotoxicity assays using three different cell types (J774, NIH/3T3, and Colo205) reveal that liposomal attachments to MTB formulation improve the biocompatibility of MTB, whereas attachment does not interfere with liposomal uptake. 10.1021/nn5011304
    Prognostic role of flow cytometry in superficial bladder cancer. Di Silverio F,von Heland M,De Berardinis E,Izzi R,Buscarini M,De Vita R,Forte R,Seccareccia F,Menotti A European urology Prognostic factors in superficial transitional cell carcinoma of the bladder were assessed with histopathological and flow cytometry analysis in a series of 61 patients operated transurethrally between 1988 and 1990. In particular, we evaluated the usefulness of flow cytometry in order to identify patients who require a more accurate clinical follow-up or a more aggressive therapy. A multivariate analysis was done in 61 cases, considering patient age and sex, stage, grade and number of lesions (unifocal or multifocal), post-TUR therapy (therapy or not), and DNA ploidy (diploid/aneuploid). DNA pattern and number of recurrences were determinant for selecting risk cases for progression.
    A voltage-gated sodium channel is essential for the positive selection of CD4(+) T cells. Lo Wan-Lin,Donermeyer David L,Allen Paul M Nature immunology The sustained entry of Ca(2+) into CD4(+)CD8(+) double-positive thymocytes is required for positive selection. Here we identified a voltage-gated Na(+) channel (VGSC) that was essential for positive selection of CD4(+) T cells. Pharmacological inhibition of VGSC activity inhibited the sustained Ca(2+) influx induced by positively selecting ligands and the in vitro positive selection of CD4(+) but not CD8(+) T cells. In vivo short hairpin RNA (shRNA)-mediated knockdown of the gene encoding a regulatory β-subunit of a VGSC specifically inhibited the positive selection of CD4(+) T cells. Ectopic expression of VGSC in peripheral AND CD4(+) T cells bestowed the ability to respond to a positively selecting ligand, which directly demonstrated that VGSC expression was responsible for the enhanced sensitivity. Thus, active VGSCs in thymocytes provide a mechanism by which a weak positive selection signal can induce the sustained Ca(2+) signals required for CD4(+) T cell development. 10.1038/ni.2379
    TGF-β is responsible for NK cell immaturity during ontogeny and increased susceptibility to infection during mouse infancy. Marcoe Jeffrey P,Lim James R,Schaubert Keri L,Fodil-Cornu Nassima,Matka Marsel,McCubbrey Alexandra L,Farr Alexander R,Vidal Silvia M,Laouar Yasmina Nature immunology A large gap in our understanding of infant immunity is why natural killer (NK) cell responses are deficient, which makes infants more prone to viral infection. Here we demonstrate that transforming growth factor-β (TGF-β) was responsible for NK cell immaturity during infancy. We found more fully mature NK cells in CD11c(dnR) mice, whose NK cells lack TGF-β receptor (TGF-βR) signaling. Ontogenic maturation of NK cells progressed faster in the absence of TGF-β signaling, which results in the formation of a mature NK cell pool early in life. As a consequence, infant CD11c(dnR) mice efficiently controlled viral infections. These data thus demonstrate an unprecedented role for TGF-β in ontogeny that can explain why NK cell responses are deficient early in life. 10.1038/ni.2388
    The effect of rosuvastatin on platelet-leukocyte interactions in the setting of acute coronary syndrome. Sexton Travis R,Wallace Eric L,Macaulay Tracy E,Charnigo Richard J,Evangelista Virgilio,Campbell Charles L,Bailey Alison L,Smyth Susan S Journal of the American College of Cardiology 10.1016/j.jacc.2014.10.047
    Flow cytometry, cellular DNA content, and prognosis in human malignancy. Merkel D E,Dressler L G,McGuire W L Journal of clinical oncology : official journal of the American Society of Clinical Oncology The use of flow cytometry to analyze the cellular DNA content of human malignancies has become increasingly commonplace. The relationship between abnormalities in DNA content or proliferative characteristics and prognosis is becoming clear for a variety of malignancies in part through new techniques that permit analysis of archival material. High- and low-risk groups of patients with early breast and bladder carcinomas, non-small-cell lung cancer, and colorectal, ovarian, and cervical carcinoma can be distinguished on the basis of abnormal stemline DNA content. In several hematologic and common pediatric malignancies, the prognostic relevance of DNA content flow cytometry has been similarly established. Though the interpretation of tumor cell cycle analyses is less certain, this characteristic may also be prognostically important. However, generalizations cannot be made when applying flow cytometric DNA analysis to clinical decision making. The prognostic importance of an abnormal DNA histogram for an individual patient must be assessed on the basis of the relevant data base for that particular tumor type. The current extent of this data base for various malignancies is reviewed. 10.1200/JCO.1987.5.10.1690
    Endocytic pH-triggered degradation of nanoengineered multilayer capsules. Liang Kang,Such Georgina K,Johnston Angus P R,Zhu Zhiyuan,Ejima Hirotaka,Richardson Joseph J,Cui Jiwei,Caruso Frank Advanced materials (Deerfield Beach, Fla.) The synthesis of cross-linker free layer-by-layer (LbL) capsules that solely utilize cellular pH variations as a trigger to specifically deconstruct and subsequently release cargo in cells is reported. These capsules demonstrate retention of water-soluble therapeutic molecules as small as 500 Da at extracellular pH. Triggered capsule degradation and release of cargo is observed within 30 min of cell uptake. 10.1002/adma.201305144
    Evidence for Faster X Chromosome Evolution in Spiders. Bechsgaard Jesper,Schou Mads Fristrup,Vanthournout Bram,Hendrickx Frederik,Knudsen Bjarne,Settepani Virginia,Schierup Mikkel Heide,Bilde Trine Molecular biology and evolution In species with chromosomal sex determination, X chromosomes are predicted to evolve faster than autosomes because of positive selection on recessive alleles or weak purifying selection. We investigated X chromosome evolution in Stegodyphus spiders that differ in mating system, sex ratio, and population dynamics. We assigned scaffolds to X chromosomes and autosomes using a novel method based on flow cytometry of sperm cells and reduced representation sequencing. We estimated coding substitution patterns (dN/dS) in a subsocial outcrossing species (S. africanus) and its social inbreeding and female-biased sister species (S. mimosarum), and found evidence for faster-X evolution in both species. X chromosome-to-autosome diversity (piX/piA) ratios were estimated in multiple populations. The average piX/piA estimates of S. africanus (0.57 [95% CI: 0.55-0.60]) was lower than the neutral expectation of 0.75, consistent with more hitchhiking events on X-linked loci and/or a lower X chromosome mutation rate, and we provide evidence in support of both. The social species S. mimosarum has a significantly higher piX/piA ratio (0.72 [95% CI: 0.65-0.79]) in agreement with its female-biased sex ratio. Stegodyphus mimosarum also have different piX/piA estimates among populations, which we interpret as evidence for recurrent founder events. Simulations show that recurrent founder events are expected to decrease the piX/piA estimates in S. mimosarum, thus underestimating the true effect of female-biased sex ratios. Finally, we found lower synonymous divergence on X chromosomes in both species, and the male-to-female substitution ratio to be higher than 1, indicating a higher mutation rate in males. 10.1093/molbev/msz074
    Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1. Chiba Shigeki,Baghdadi Muhammad,Akiba Hisaya,Yoshiyama Hironori,Kinoshita Ichiro,Dosaka-Akita Hirotoshi,Fujioka Yoichiro,Ohba Yusuke,Gorman Jacob V,Colgan John D,Hirashima Mitsuomi,Uede Toshimitsu,Takaoka Akinori,Yagita Hideo,Jinushi Masahisa Nature immunology The mechanisms by which tumor microenvironments modulate nucleic acid-mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids. 10.1038/ni.2376
    Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation. van Gisbergen Klaas P J M,Kragten Natasja A M,Hertoghs Kirsten M L,Wensveen Felix M,Jonjic Stipan,Hamann Jörg,Nolte Martijn A,van Lier Rene A W Nature immunology The transcriptional repressor Blimp-1 mediates the terminal differentiation of many cell types, including T cells. Here we identified Hobit (Znf683) as a previously unrecognized homolog of Blimp-1 that was specifically expressed in mouse natural killer T cells (NKT cells). Through studies of Hobit-deficient mice, we found that Hobit was essential for the formation of mature thymic NKT cells. In the periphery, Hobit repressed the accumulation of interferon-γ (IFN-γ)-producing NK1.1(lo) NKT cells at steady state. After antigenic stimulation, Hobit repressed IFN-γ expression, whereas after innate stimulation, Hobit induced granzyme B expression. Thus, reminiscent of the function of Blimp-1 in other lymphocytes, Hobit controlled the maintenance of quiescent, fully differentiated NKT cells and regulated their immediate effector functions. 10.1038/ni.2393
    Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity. Rego E Hesper,Audette Rebecca E,Rubin Eric J Nature Microorganisms are often studied as populations but the behaviour of single, individual cells can have important consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population dies rapidly. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed. In fact, mycobacteria create variability each time a cell divides, producing daughter cells with different sizes and growth rates. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a fluorescence-activated cell sorting (FACS)-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologues in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (the 'divisome'). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure. 10.1038/nature22361
    Methylome analysis using MeDIP-seq with low DNA concentrations. Taiwo Oluwatosin,Wilson Gareth A,Morris Tiffany,Seisenberger Stefanie,Reik Wolf,Pearce Daniel,Beck Stephan,Butcher Lee M Nature protocols DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d. 10.1038/nprot.2012.012
    Functions of human liver CD69CD103CD8 T cells depend on HIF-2α activity in healthy and pathologic livers. Kim Jong Hoon,Han Ji Won,Choi Young Joon,Rha Min-Seok,Koh June Young,Kim Kyung Hwan,Kim Chang Gon,Lee Yong Joon,Kim A Reum,Park Junsik,Kim Hong Kwan,Min Byung Soh,Seo Seong Il,Kang Minyong,Park Hye Jung,Han Dai Hoon,Kim Soon Il,Kim Myoung Soo,Lee Jae Geun,Lee Dong Hyeon,Kim Won,Park Jun Yong,Park Su-Hyung,Joo Dong Jin,Shin Eui-Cheol Journal of hepatology BACKGROUND & AIMS:Human liver CD69CD8 T cells are ~95% CD103 and ~5% CD103. Although CD69CD103CD8 T cells show tissue residency and robustly respond to antigens, CD69CD103CD8 T cells are not yet well understood. METHODS:Liver perfusate and paired peripheral blood were collected from healthy living donors and recipients with cirrhosis during liver transplantation. Liver tissues were obtained from patients with acute hepatitis A. Phenotypic and functional analyses were performed by flow cytometry. Gene expression profiles were determined by microarray and quantitative reverse transcription PCR. PT-2385 was used to inhibit hypoxia-inducible factor (HIF)-2α. RESULTS:Human liver CD69CD103CD8 T cells exhibited HIF-2α upregulation with a phenotype of tissue residency and terminal differentiation. CD103 cells comprised non-hepatotropic virus-specific T cells as well as hepatotropic virus-specific T cells, but CD103 cells exhibited only hepatotropic virus specificity. Although CD103 cells were weaker effectors on a per cell basis than CD103 cells, following T cell receptor or interleukin-15 stimulation, they remained the major CD69CD8 effector population in the liver, surviving with less cell death. An HIF-2α inhibitor suppressed the effector functions and survival of CD69CD103CD8 T cells. In addition, HIF-2α expression in liver CD69CD103CD8 T cells was significantly increased in patients with acute hepatitis A or cirrhosis. CONCLUSIONS:Liver CD69CD103CD8 T cells are tissue resident and terminally differentiated, and their effector functions depend on HIF-2α. Furthermore, activation of liver CD69CD103CD8 T cells with HIF-2α upregulation is observed during liver pathology. LAY SUMMARY:The immunologic characteristics and the role of CD69CD103CD8 T cells, which are a major population of human liver CD8 T cells, remain unknown. Our study shows that these T cells have a terminally differentiated tissue-resident phenotype, and their effector functions depend on a transcription factor, HIF-2α. Furthermore, these T cells were activated and expressed higher levels of HIF-2α in liver pathologies, suggesting that they play an important role in immune responses in liver tissues and the pathogenesis of human liver disease. 10.1016/j.jhep.2020.01.010
    Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis. Beldman Thijs J,Senders Max L,Alaarg Amr,Pérez-Medina Carlos,Tang Jun,Zhao Yiming,Fay Francois,Deichmöller Jacqueline,Born Benjamin,Desclos Emilie,van der Wel Nicole N,Hoebe Ron A,Kohen Fortune,Kartvelishvily Elena,Neeman Michal,Reiner Thomas,Calcagno Claudia,Fayad Zahi A,de Winther Menno P J,Lutgens Esther,Mulder Willem J M,Kluza Ewelina ACS nano Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis. 10.1021/acsnano.7b01385
    BRAF(V600E) mutation in a histiocytic sarcoma arising from hairy cell leukemia. Michonneau David,Kaltenbach Sophie,Derrieux Coralie,Trinquand Amelie,Brouzes Chantal,Gibault Laure,North Marie-Odile,Delarue Richard,Varet Bruno,Emile Jean-François,Brousse Nicole,Hermine Olivier Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10.1200/JCO.2013.49.0078
    Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. Velardi Enrico,Tsai Jennifer J,Holland Amanda M,Wertheimer Tobias,Yu Vionnie W C,Zakrzewski Johannes L,Tuckett Andrea Z,Singer Natalie V,West Mallory L,Smith Odette M,Young Lauren F,Kreines Fabiana M,Levy Emily R,Boyd Richard L,Scadden David T,Dudakov Jarrod A,van den Brink Marcel R M The Journal of experimental medicine Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function. 10.1084/jem.20131289
    Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice. Mirsoian Annie,Bouchlaka Myriam N,Sckisel Gail D,Chen Mingyi,Pai Chien-Chun Steven,Maverakis Emanuel,Spencer Richard G,Fishbein Kenneth W,Siddiqui Sana,Monjazeb Arta M,Martin Bronwen,Maudsley Stuart,Hesdorffer Charles,Ferrucci Luigi,Longo Dan L,Blazar Bruce R,Wiltrout Robert H,Taub Dennis D,Murphy William J The Journal of experimental medicine Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF(+) macrophages in response to αCD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with αCD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon αCD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT. 10.1084/jem.20140116
    Antigen-specific expansion and differentiation of natural killer cells by alloantigen stimulation. Nabekura Tsukasa,Lanier Lewis L The Journal of experimental medicine Natural killer (NK) cells provide important host defense against microbial pathogens and can generate a population of long-lived memory NK cells after infection or immunization. Here, we addressed whether NK cells can expand and differentiate after alloantigen stimulation, which may be important in hematopoietic stem cell and solid tissue transplantation. A subset of NK cell in C57BL/6 mice expresses the activating Ly49D receptor that is specific for H-2D(d). These Ly49D(+) NK cells can preferentially expand and differentiate when challenged with allogeneic H-2D(d) cells in the context of an inflammatory environment. H-2D(d) is also recognized by the inhibitory Ly49A receptor, which, when coexpressed on Ly49D(+) NK cells, suppresses the expansion of Ly49D(+) NK cells. Specificity of the secondary response of alloantigen-primed NK cells was defined by the expression of activating Ly49 receptors and regulated by the inhibitory receptors for MHC class I. Thus, the summation of signals through a repertoire of Ly49 receptors controls the adaptive immune features of NK cells responding to allogeneic cells. 10.1084/jem.20140798
    MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures. Muraoka Naoto,Yamakawa Hiroyuki,Miyamoto Kazutaka,Sadahiro Taketaro,Umei Tomohiko,Isomi Mari,Nakashima Hanae,Akiyama Mizuha,Wada Rie,Inagawa Kohei,Nishiyama Takahiko,Kaneda Ruri,Fukuda Toru,Takeda Shu,Tohyama Shugo,Hashimoto Hisayuki,Kawamura Yoshifumi,Goshima Naoki,Aeba Ryo,Yamagishi Hiroyuki,Fukuda Keiichi,Ieda Masaki The EMBO journal Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. 10.15252/embj.201387605
    Identification of a gene regulatory network associated with prion replication. Marbiah Masue M,Harvey Anna,West Billy T,Louzolo Anais,Banerjee Priya,Alden Jack,Grigoriadis Anita,Hummerich Holger,Kan Ho-Man,Cai Ying,Bloom George S,Jat Parmjit,Collinge John,Klöhn Peter-Christian The EMBO journal Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrP(C)). They propagate by recruiting host-encoded PrP(C) although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrP(C) expression differences, to identify such factors. Transcriptome analysis of prion-resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease-related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrP(C) deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)-2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state. 10.15252/embj.201387150
    Staphylococcus aureus infection induces protein A-mediated immune evasion in humans. Pauli Noel T,Kim Hwan Keun,Falugi Fabiana,Huang Min,Dulac John,Henry Dunand Carole,Zheng Nai-Ying,Kaur Kaval,Andrews Sarah F,Huang Yunping,DeDent Andrea,Frank Karen M,Charnot-Katsikas Angella,Schneewind Olaf,Wilson Patrick C The Journal of experimental medicine Staphylococcus aureus bacterial infection commonly results in chronic or recurrent disease, suggesting that humoral memory responses are hampered. Understanding how S. aureus subverts the immune response is critical for the rescue of host natural humoral immunity and vaccine development. S. aureus expresses the virulence factor Protein A (SpA) on all clinical isolates, and SpA has been shown in mice to expand and ablate variable heavy 3 (VH3) idiotype B cells. The effects of SpA during natural infection, however, have not been addressed. Acutely activated B cells, or plasmablasts (PBs), were analyzed to dissect the ongoing immune response to infection through the production of monoclonal antibodies (mAbs). The B cells that were activated by infection had a highly limited response. When screened against multiple S. aureus antigens, only high-affinity binding to SpA was observed. Consistently, PBs underwent affinity maturation, but their B cell receptors demonstrated significant bias toward the VH3 idiotype. These data suggest that the superantigenic activity of SpA leads to immunodominance, limiting host responses to other S. aureus virulence factors that would be necessary for protection and memory formation. 10.1084/jem.20141404
    Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated Th17 cell-dependent inflammation. Wang Jian,Li Fengqi,Wei Haiming,Lian Zhe-Xiong,Sun Rui,Tian Zhigang The Journal of experimental medicine Influenza in humans is often accompanied by gastroenteritis-like symptoms such as diarrhea, but the underlying mechanism is not yet understood. We explored the occurrence of gastroenteritis-like symptoms using a mouse model of respiratory influenza infection. We found that respiratory influenza infection caused intestinal injury when lung injury occurred, which was not due to direct intestinal viral infection. Influenza infection altered the intestinal microbiota composition, which was mediated by IFN-γ produced by lung-derived CCR9(+)CD4(+) T cells recruited into the small intestine. Th17 cells markedly increased in the small intestine after PR8 infection, and neutralizing IL-17A reduced intestinal injury. Moreover, antibiotic depletion of intestinal microbiota reduced IL-17A production and attenuated influenza-caused intestinal injury. Further study showed that the alteration of intestinal microbiota significantly stimulated IL-15 production from intestinal epithelial cells, which subsequently promoted Th17 cell polarization in the small intestine in situ. Thus, our findings provide new insights into an undescribed mechanism by which respiratory influenza infection causes intestinal disease. 10.1084/jem.20140625
    An inherited mutation in NLRC4 causes autoinflammation in human and mice. Kitamura Akiko,Sasaki Yuki,Abe Takaya,Kano Hirotsugu,Yasutomo Koji The Journal of experimental medicine Autoinflammatory syndromes cause sterile inflammation in the absence of any signs of autoimmune responses. Familial cold autoinflammatory syndrome (FCAS) is characterized by intermittent episodes of rash, arthralgia, and fever after exposure to cold stimuli. We have identified a missense mutation in the NLRC4 gene in patients with FCAS. NLRC4 has been known as a crucial sensor for several Gram-negative intracellular bacteria. The mutation in NLRC4 in FCAS patients promoted the formation of NLRC4-containing inflammasomes that cleave procaspase-1 and increase production of IL-1β. Transgenic mice that expressed mutant Nlrc4 under the invariant chain promoter developed dermatitis and arthritis. Inflammation within tissues depended on IL-1β-mediated production of IL-17A from neutrophils but not from T cells. Our findings reveal a previously unrecognized link between NLRC4 and a hereditary autoinflammatory disease and highlight the importance of NLRC4 not only in the innate immune response to bacterial infections but also in the genesis of inflammatory diseases. 10.1084/jem.20141091
    Mechanisms of nuclear content loading to exosomes. Yokoi Akira,Villar-Prados Alejandro,Oliphint Paul Allen,Zhang Jianhua,Song Xingzhi,De Hoff Peter,Morey Robert,Liu Jinsong,Roszik Jason,Clise-Dwyer Karen,Burks Jared K,O'Halloran Theresa J,Laurent Louise C,Sood Anil K Science advances Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development. 10.1126/sciadv.aax8849
    Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling. Wang Jigang,Zhang Jianbin,Lee Yew Mun,Ng Shukie,Shi Yin,Hua Zi-Chun,Lin Qingsong,Shen Han-Ming Nature protocols At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli. Cells then undergo a 'click' reaction between the azide group of AHA and a fluorescently tagged alkyne probe. The AHA-containing proteins can then be detected by flow cytometry. This protocol is nonradioactive, sensitive and quantitative, and it is easy to perform. It is also applicable to various cell culture systems. The whole protocol is estimated to take 4-5 d to complete. 10.1038/nprot.2016.160
    Genomic analysis at the single-cell level. Kalisky Tomer,Blainey Paul,Quake Stephen R Annual review of genetics Studying complex biological systems such as a developing embryo, a tumor, or a microbial ecosystem often involves understanding the behavior and heterogeneity of the individual cells that constitute the system and their interactions. In this review, we discuss a variety of approaches to single-cell genomic analysis. 10.1146/annurev-genet-102209-163607
    Loss of hepatic Mboat7 leads to liver fibrosis. Thangapandi Veera Raghavan,Knittelfelder Oskar,Brosch Mario,Patsenker Eleonora,Vvedenskaya Olga,Buch Stephan,Hinz Sebastian,Hendricks Alexander,Nati Marina,Herrmann Alexander,Rekhade Devavrat Ravindra,Berg Thomas,Matz-Soja Madlen,Huse Klaus,Klipp Edda,Pauling Josch K,Wodke Judith Ah,Miranda Ackerman Jacobo,Bonin Malte von,Aigner Elmar,Datz Christian,von Schönfels Witigo,Nehring Sophie,Zeissig Sebastian,Röcken Christoph,Dahl Andreas,Chavakis Triantafyllos,Stickel Felix,Shevchenko Andrej,Schafmayer Clemens,Hampe Jochen,Subramanian Pallavi Gut OBJECTIVE:The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN:Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS:Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7 mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p0.05), hydroxyproline content (p0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7 mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7 livers and human rs641738TT carriers were similar. CONCLUSION:Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD. 10.1136/gutjnl-2020-320853
    Highly sensitive single domain antibody-quantum dot conjugates for detection of HER2 biomarker in lung and breast cancer cells. Rakovich Tatsiana Y,Mahfoud Omar K,Mohamed Bashir M,Prina-Mello Adriele,Crosbie-Staunton Kieran,Van Den Broeck Tina,De Kimpe Line,Sukhanova Alyona,Baty Daniel,Rakovich Aliaksandra,Maier Stefan A,Alves Frauke,Nauwelaers Frans,Nabiev Igor,Chames Patrick,Volkov Yuri ACS nano Despite the widespread availability of immunohistochemical and other methodologies for screening and early detection of lung and breast cancer biomarkers, diagnosis of the early stage of cancers can be difficult and prone to error. The identification and validation of early biomarkers specific to lung and breast cancers, which would permit the development of more sensitive methods for detection of early disease onset, is urgently needed. In this paper, ultra-small and bright nanoprobes based on quantum dots (QDs) conjugated to single domain anti-HER2 (human epidermal growth factor receptor 2) antibodies (sdAbs) were applied for immunolabeling of breast and lung cancer cell lines, and their performance was compared to that of anti-HER2 monoclonal antibodies conjugated to conventional organic dyes Alexa Fluor 488 and Alexa Fluor 568. The sdAbs-QD conjugates achieved superior staining in a panel of lung cancer cell lines with differential HER2 expression. This shows their outstanding potential for the development of more sensitive assays for early detection of cancer biomarkers. 10.1021/nn500212h
    IFNL3-adjuvanted HCV DNA vaccine reduces regulatory T cell frequency and increases virus-specific T cell responses. Han Ji Won,Sung Pil Soo,Hong Seon-Hui,Lee Hoyoung,Koh June Young,Lee Hyojin,White Scott,Maslow Joel N,Weiner David B,Park Su-Hyung,Jeong Moonsup,Heo Jeong,Ahn Sang Hoon,Shin Eui-Cheol Journal of hepatology BACKGROUND & AIMS:Although direct-acting antiviral (DAA) treatment results in a sustained virologic response (SVR) in most patients with chronic HCV infection, they are at risk of re-infection. Moreover, the immune system is not completely normalized even after SVR (e.g. increased regulatory T [Treg] cell frequency). We developed a DNA vaccine, GLS-6150, to prevent re-infection of patients with DAA-induced SVR and evaluated its safety and immunogenicity in individuals with chronic HCV infection. METHODS:GLS-6150 consists of plasmids encoding HCV non-structural proteins (NS3-NS5A) and adjuvant IFNL3. The vaccine was administered 4 times at 4-weekly intervals to 3 groups (1, 3, or 6 mg/vaccination; n = 6 per group), followed by a 6 mg boost at 24 weeks (n = 14). Peripheral blood T cell responses were evaluated by interferon (IFN)-γ enzyme-linked immunospot assays, intracellular cytokine staining, and major histocompatibility complex class-I (MHC-I) dextramer staining. Treg cell frequency was assessed by flow cytometry. RESULTS:Severe adverse events or vaccine discontinuation were not reported. The IFN-γ spot-forming cells specific to NS3-NS5A were increased by GLS-6150. Both CD4 and CD8 T cells produced multiple cytokines. However, the frequency and phenotype of HCV-specific MHC-I dextramerCD8 T cells were not changed. Interestingly, the frequency of Treg cells, particularly activated Treg cells, was decreased by GLS-6150, as expected from previous reports that IFNL3 adjuvants decrease Treg cell frequency. Ex vivo IFN-λ3 treatment reduced Treg frequency in pre-vaccination peripheral blood mononuclear cells. Finally, Treg cell frequency inversely correlated with HCV-specific, IFN-γ-producing T cell responses in the study participants. CONCLUSIONS:We demonstrate that GLS-6150 decreases Treg cell frequency and enhances HCV-specific T cell responses without significant side effects. A phase I clinical trial of GLS-6150 is currently underway in patients with DAA-induced SVR. CLINICAL TRIAL NUMBER:NCT02027116. LAY SUMMARY:Although direct-acting antivirals (DAAs) are successfully used for the treatment of chronic hepatitis C virus (HCV) infection, a prophylactic HCV vaccine needs to be developed, especially for patients who achieve a sustained virologic response. In the current study, we show that a DNA vaccine (GLS-6150) was safe and increased HCV-specific T cell responses. A clinical trial is underway to test this vaccine in patients with a sustained virologic response following DAA therapy. 10.1016/j.jhep.2020.02.009
    In vivo identification of bipotential adipocyte progenitors recruited by β3-adrenoceptor activation and high-fat feeding. Lee Yun-Hee,Petkova Anelia P,Mottillo Emilio P,Granneman James G Cell metabolism Nutritional and pharmacological stimuli can dramatically alter the cellular phenotypes in white adipose tissue (WAT). Utilizing genetic lineage tracing techniques, we demonstrate that brown adipocytes (BA) that are induced by β3-adrenergic receptor activation in abdominal WAT arise from the proliferation and differentiation of cells expressing platelet-derived growth factor receptor alpha (PDGFRα), CD34, and Sca-1 (PDGFRα(+) cells). PDGFRα(+) cells have a unique morphology in which extended processes contact multiple cells in the tissue microenvironment. Surprisingly, these cells also give rise to white adipocytes (WA) that can comprise up to 25% of total fat cells in abdominal fat pads following 8 weeks of high-fat feeding. Isolated PDGFRα(+) cells differentiated into both BA and WA in vitro and generated WA after transplantation in vivo. The identification of PDGFRα(+) cells as bipotential adipocyte progenitors will enable further investigation of mechanisms that promote therapeutic cellular remodeling in adult WAT. 10.1016/j.cmet.2012.03.009
    Identification of naturally occurring fatty acids of the myelin sheath that resolve neuroinflammation. Ho Peggy P,Kanter Jennifer L,Johnson Amanda M,Srinagesh Hrishikesh K,Chang Eun-Ju,Purdy Timothy M,van Haren Keith,Wikoff William R,Kind Tobias,Khademi Mohsen,Matloff Laura Y,Narayana Sirisha,Hur Eun Mi,Lindstrom Tamsin M,He Zhigang,Fiehn Oliver,Olsson Tomas,Han Xianlin,Han May H,Steinman Lawrence,Robinson William H Science translational medicine Lipids constitute 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain, and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as therapeutics for MS. 10.1126/scitranslmed.3003831
    Measurable Residual Disease Response and Prognosis in Treatment-Naïve Acute Myeloid Leukemia With Venetoclax and Azacitidine. Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:There is limited evidence on the clinical utility of monitoring measurable residual disease (MRD) in patients with acute myeloid leukemia treated with lower-intensity therapy. Herein, we explored the outcomes of patients treated with venetoclax and azacitidine who achieved composite complete remission (CRc; complete remission + complete remission with incomplete hematologic recovery) and MRD < 10 in the VIALE-A trial. METHODS:The patients included in this report were treated with venetoclax and azacitidine. Bone marrow aspirate samples for multiparametric flow cytometry assessments were collected for central analysis at baseline, end of cycle 1, and every three cycles thereafter. MRD-negative response was defined as < 1 residual blast per 1,000 leukocytes (< 10 or 0.1%) with an estimated analytic sensitivity of 0.0037%-0.0027%. CRc, duration of remission (DoR), event-free survival (EFS), and overall survival (OS) were assessed. A multivariate Cox regression analysis identified prognostic factors associated with OS. RESULTS:One hundred sixty-four of one hundred ninety (86%) patients with CRc were evaluable for MRD. MRD < 10 was achieved by 67 of 164 (41%), and 97 of 164 (59%) had MRD ≥ 10. The median DoR, EFS, and OS were not reached in patients with CRc and MRD < 10, and the 12-month estimates for DoR, EFS, and OS in this group were 81.2%, 83.2%, and 94.0%. Among patients with CRc and MRD ≥ 10, the median DoR, EFS, and OS were 9.7, 10.6, and 18.7 months. Multivariate analysis showed that CRc with MRD < 10 was a strong predictor of OS (adjusted hazard ratio = 0.285; 95% CI, 0.159 to 0.510; < .001). CONCLUSION:Patients who achieved CRc and MRD < 10 with venetoclax and azacitidine had longer DoR, EFS, and OS, than responding patients with MRD ≥ 10. 10.1200/JCO.21.01546
    Quantifying the Ligand-Coated Nanoparticle Delivery to Cancer Cells in Solid Tumors. Dai Qin,Wilhelm Stefan,Ding Ding,Syed Abdullah Muhammad,Sindhwani Shrey,Zhang Yuwei,Chen Yih Yang,MacMillan Presley,Chan Warren C W ACS nano Coating the nanoparticle surface with cancer cell recognizing ligands is expected to facilitate specific delivery of nanoparticles to diseased cells in vivo. While this targeting strategy is appealing, no nanoparticle-based active targeting formulation for solid tumor treatment had made it past phase III clinical trials. Here, we quantified the cancer cell-targeting efficiencies of Trastuzumab (Herceptin) and folic acid coated gold and silica nanoparticles in multiple mouse tumor models. Surprisingly, we showed that less than 14 out of 1 million (0.0014% injected dose) intravenously administrated nanoparticles were delivered to targeted cancer cells, and that only 2 out of 100 cancer cells interacted with the nanoparticles. The majority of the intratumoral nanoparticles were either trapped in the extracellular matrix or taken up by perivascular tumor associated macrophages. The low cancer cell targeting efficiency and significant uptake by noncancer cells suggest the need to re-evaluate the active targeting process and therapeutic mechanisms using quantitative methods. This will be important for developing strategies to deliver emerging therapeutics such as genome editing, nucleic acid therapy, and immunotherapy for cancer treatment using nanocarriers. 10.1021/acsnano.8b03900
    Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to . American journal of respiratory and critical care medicine Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype. In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model. Aspiration with a mixture of human oral commensals (MOC; , , and ) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds. 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with led to a decrease in hosts' susceptibility to this pathogen. Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to . Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens. 10.1164/rccm.202005-1596OC
    Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns. Sanchez-Freire Veronica,Ebert Antje D,Kalisky Tomer,Quake Stephen R,Wu Joseph C Nature protocols Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation. 10.1038/nprot.2012.021
    Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP. Bonn Stefan,Zinzen Robert P,Perez-Gonzalez Alexis,Riddell Andrew,Gavin Anne-Claude,Furlong Eileen E M Nature protocols This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d. 10.1038/nprot.2012.049
    Highly multiplexed and strand-specific single-cell RNA 5' end sequencing. Islam Saiful,Kjällquist Una,Moliner Annalena,Zajac Pawel,Fan Jian-Bing,Lönnerberg Peter,Linnarsson Sten Nature protocols Single-cell analysis of gene expression is increasingly important for the analysis of complex tissues, including cancer, developing organs and adult stem cell niches. Here we present a detailed protocol for quantitative gene expression analysis in single cells, by the sequencing of mRNA 5' ends. In all, 96 cells are lysed, and their mRNA is converted to cDNA. By using a template-switching mechanism, a bar code and an upstream primer-binding sequence are introduced simultaneously with reverse transcription. All cDNA is pooled and then prepared for 5' end sequencing, including fragmentation, adapter ligation and PCR amplification. The chief advantage of this approach is the great reduction in cost and time, afforded by the early bar-coding strategy. Compared with previous methods, it is more suitable for large-scale quantitative analysis, as well as for the characterization of transcription start sites, but it is unsuitable for the detection of alternatively spliced transcripts. Sample preparation takes 3 d, and two sets of 96 cells can be prepared in parallel. Finally, the sequencing and data analysis can take an additional 4 d altogether. 10.1038/nprot.2012.022
    Preexisting BCG-specific T cells improve intravesical immunotherapy for bladder cancer. Biot Claire,Rentsch Cyrill A,Gsponer Joel R,Birkhäuser Frédéric D,Jusforgues-Saklani Hélène,Lemaître Fabrice,Auriau Charlotte,Bachmann Alexander,Bousso Philippe,Demangel Caroline,Peduto Lucie,Thalmann George N,Albert Matthew L Science translational medicine Therapeutic intravesical instillation of bacillus Calmette-Guérin (BCG) is effective at triggering inflammation and eliciting successful tumor immunity in patients with non-muscle invasive bladder cancer, with 50 to 70% clinical response. Therapeutic success relies on repeated instillations of live BCG administered as adjuvant therapy shortly after tumor resection; however, the precise mechanisms remain unclear. Using an experimental model, we demonstrate that after a single instillation, BCG could disseminate to bladder draining lymph nodes and prime interferon-γ-producing T cells. Nonetheless, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Parenteral exposure to BCG before instillation overcame this requirement; after the first intravesical instillation, BCG triggered a more robust acute inflammatory process and accelerated T cell entry into the bladder, as compared to the standard protocol. Moreover, parenteral exposure to BCG before intravesical treatment of an orthotopic tumor markedly improved response to therapy. Indeed, patients with sustained preexisting immunity to BCG showed a significant improvement in recurrence-free survival. Together, these data suggest that monitoring patients' response to purified protein derivative, and, in their absence, boosting BCG responses by parenteral exposure before intravesical treatment initiation, may be a safe and effective means of improving intravesical BCG-induced clinical responses. 10.1126/scitranslmed.3003586
    Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1 Tissue-Resident Memory T Cells. Snyder Mark E,Sembrat John,Noda Kentaro,Myerburg Michael M,Craig Andrew,Mitash Nilay,Harano Takashi,Furukawa Masashi,Pilewski Joseph,McDyer John,Rojas Mauricio,Sanchez Pablo American journal of respiratory and critical care medicine Tissue-resident memory T cells (T) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (M) are limited by experimental constraints. To characterize the spatial and functional relationship between M and human lung tissue-resident memory T cells using lung perfusion (EVLP). T and M were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3 and CD68 cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung T and M colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8 T are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that M provide costimulatory signaling to PD1 CD4 and CD8 lung T, augmenting the effector cytokine production and degranulation of T. EVLP provides an innovative technique to study resident immune populations in humans. Human M colocalize with and provide costimulation signaling to T, augmenting their effector function. 10.1164/rccm.202006-2403OC
    Effects of acute insulin-induced hypoglycemia on indices of inflammation: putative mechanism for aggravating vascular disease in diabetes. Diabetes care OBJECTIVE:To examine the effects of acute insulin-induced hypoglycemia on inflammation, endothelial dysfunction, and platelet activation in adults with and without type 1 diabetes. RESEARCH DESIGN AND METHODS:We studied 16 nondiabetic adults and 16 subjects with type 1 diabetes during euglycemia (blood glucose 4.5 mmol/l) and hypoglycemia (blood glucose 2.5 mmol/l). Markers of inflammation, thrombosis, and endothelial dysfunction (soluble P-selectin, interleukin-6, von Willebrand factor [vWF], tissue plasminogen activator [tPA], high-sensitivity C-reactive protein [hsCRP], and soluble CD40 ligand [sCD40L]) were measured; platelet-monocyte aggregation and CD40 expression on monocytes were determined using flow cytometry. RESULTS:In nondiabetic participants, platelet activation occurred after hypoglycemia, with increments in platelet-monocyte aggregation and P-selectin (P <or= 0.02). Inflammation was triggered with CD40 expression increasing maximally at 24 h (3.13 +/- 2.3% vs. 2.06 +/- 1.0%) after hypoglycemia (P = 0.009). Both sCD40L and hsCRP (P = 0.02) increased with a nonsignificant rise in vWF and tPA, indicating a possible endothelial effect. A reduction in sCD40L, tPA, and P-selectin occurred during euglycemia (P = 0.03, P <or= 0.006, and P = 0.006, respectively). In type 1 diabetes, both CD40 expression (5.54 +/- 4.4% vs. 3.65 +/- 1.8%; P = 0.006) and plasma sCD40L concentrations increased during hypoglycemia (peak 3.41 +/- 3.2 vs. 2.85 +/- 2.8 ng/ml; P = 0.03). Platelet-monocyte aggregation also increased significantly at 24 h after hypoglycemia (P = 0.03). A decline in vWF and P-selectin occurred during euglycemia (P <or= 0.04). CONCLUSIONS:Acute hypoglycemia may provoke upregulation and release of vasoactive substances in adults with and without type 1 diabetes. This may be a putative mechanism for hypoglycemia-induced vascular injury. 10.2337/dc10-0013
    T-cell epitope mapping by flow cytometry. Kern F,Surel I P,Brock C,Freistedt B,Radtke H,Scheffold A,Blasczyk R,Reinke P,Schneider-Mergener J,Radbruch A,Walden P,Volk H D Nature medicine
    The telomere deprotection response is functionally distinct from the genomic DNA damage response. Cesare Anthony J,Hayashi Makoto T,Crabbe Laure,Karlseder Jan Molecular cell Loss of chromosome end protection through telomere erosion is a hallmark of aging and senescence. Here we developed an experimental system that mimics physiological telomere deprotection in human cells and discovered that the telomere deprotection response is functionally distinct from the genomic DNA damage response. We found that, unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion-resistant intermediate state activate differential ataxia telangiectasia mutated (ATM) signaling where CHK2 is not phosphorylated. Also unlike genomic breaks, we found that deprotected telomeres do not contribute to the G2/M checkpoint and are instead passed through cell division to induce p53-dependent G1 arrest in the daughter cells. Telomere deprotection is therefore an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest occurs in stable diploid G1 phase cells before genome instability can occur. 10.1016/j.molcel.2013.06.006
    Inhibition of PU.1 ameliorates metabolic dysfunction and non-alcoholic steatohepatitis. Liu Qiongming,Yu Junjie,Wang Liheng,Tang Yuliang,Zhou Quan,Ji Shuhui,Wang Yi,Santos Luis,Haeusler Rebecca A,Que Jianwen,Rajbhandari Prashant,Lei Xiaoguang,Valenti Luca,Pajvani Utpal B,Qin Jun,Qiang Li Journal of hepatology BACKGROUND & AIMS:Obesity is a well-established risk factor for type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH), but the underlying mechanisms remain incompletely understood. Herein, we aimed to identify novel pathogenic factors (and possible therapeutic targets) underlying metabolic dysfunction in the liver. METHODS:We applied a tandem quantitative proteomics strategy to enrich and identify transcription factors (TFs) induced in the obese liver. We used flow cytometry of liver cells to analyze the source of the induced TFs. We employed conditional knockout mice, shRNA, and small-molecule inhibitors to test the metabolic consequences of the induction of identified TFs. Finally, we validated mouse data in patient liver biopsies. RESULTS:We identified PU.1/SPI1, the master hematopoietic regulator, as one of the most upregulated TFs in livers from diet-induced obese (DIO) and genetically obese (db/db) mice. Targeting PU.1 in the whole liver, but not hepatocytes alone, significantly improved glucose homeostasis and suppressed liver inflammation. Consistently, treatment with the PU.1 inhibitor DB1976 markedly reduced inflammation and improved glucose homeostasis and dyslipidemia in DIO mice, and strongly suppressed glucose intolerance, liver steatosis, inflammation, and fibrosis in a dietary NASH mouse model. Furthermore, hepatic PU.1 expression was positively correlated with insulin resistance and inflammation in liver biopsies from patients. CONCLUSIONS:These data suggest that the elevated hematopoietic factor PU.1 promotes liver metabolic dysfunction, and may be a useful therapeutic target for obesity, insulin resistance/T2D, and NASH. LAY SUMMARY:Expression of the immune regulator PU.1 is increased in livers of obese mice and people. Blocking PU.1 improved glucose homeostasis, and reduced liver steatosis, inflammation and fibrosis in mouse models of non-alcoholic steatohepatitis. Inhibition of PU.1 is thus a potential therapeutic strategy for treating obesity-associated liver dysfunction and metabolic diseases. 10.1016/j.jhep.2020.02.025
    A protocol for phenotypic detection and characterization of vascular cells of different origins in a lung neovascularization model in rodents. Jones Rosemary C,Capen Diane E,Cohen Kenneth S,Munn Lance L,Jain Rakesh K,Duda Dan G Nature protocols The goal of many current studies of neovascularization is to define the phenotype of vascular cell populations of different origins and to determine how such cells promote assembly of vascular channel. Here, we describe a protocol to immunophenotype vascular cells by high-resolution imaging and by fluorescence-activated flow cytometry in an in vivo rodent model of pulmonary microvascular remodeling. Analysis of cells by this combined approach will characterize their phenotype, quantify their number and identify their role in the assembly of vascular channels. 10.1038/nprot.2007.537
    A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis. Davis Richard P,Costa Magdaline,Grandela Catarina,Holland Andrew M,Hatzistavrou Tanya,Micallef Suzanne J,Li Xueling,Goulburn Adam L,Azzola Lisa,Elefanty Andrew G,Stanley Edouard G Nature protocols The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month. 10.1038/nprot.2008.146
    Minimal Residual Disease After Autologous Stem-Cell Transplant for Patients With Myeloma: Prognostic Significance and the Impact of Lenalidomide Maintenance and Molecular Risk. de Tute Ruth M,Pawlyn Charlotte,Cairns David A,Davies Faith E,Menzies Tom,Rawstron Andy,Jones John R,Hockaday Anna,Henderson Rowena,Cook Gordon,Drayson Mark T,Jenner Matthew W,Kaiser Martin F,Gregory Walter M,Morgan Gareth J,Jackson Graham H,Owen Roger G Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Minimal residual disease (MRD) can predict outcomes in patients with multiple myeloma, but limited data are available on the prognostic impact of MRD when assessed at serial time points in the context of maintenance therapy after autologous stem-cell transplant (ASCT) and the interaction between MRD and molecular risk. METHODS:Data from a large phase III trial (Myeloma XI) were examined to determine the relationship between MRD status, progression-free survival (PFS), and overall survival (OS) in post-ASCT patients randomly assigned to lenalidomide maintenance or no maintenance at 3 months after ASCT. MRD status was assessed by flow cytometry (median sensitivity 0.004%) before maintenance random assignment (ASCT + 3) and 6 months later (ASCT + 9). RESULTS:At ASCT + 3, 475 of 750 (63.3%) patients were MRD-negative and 275 (36.7%) were MRD-positive. MRD-negative status was associated with improved PFS (hazard ratio [HR] = 0.47; 95% CI, 0.37 to 0.58 < .001) and OS (HR = 0.59; 95% CI, 0.40 to 0.85; = .0046). At ASCT + 9, 214 of 326 (65.6%) were MRD-negative and 112 (34.4%) were MRD-positive. MRD-negative status was associated with improved PFS (HR = 0.20; 95% CI, 0.13 to 0.31; < .0001) and OS (HR = 0.33; 95% CI, 0.15 to 0.75; = .0077). The findings were very similar when restricted to patients with complete response/near complete response. Sustained MRD negativity from ASCT + 3 to ASCT + 9 or the conversion to MRD negativity by ASCT + 9 was associated with the longest PFS/OS. Patients randomly assigned to lenalidomide maintenance were more likely to convert from being MRD-positive before maintenance random assignment to MRD-negative 6 months later (lenalidomide 30%, observation 17%). High-risk molecular features had an adverse effect on PFS and OS even for those patients achieving MRD-negative status. On multivariable analysis of MRD status, maintenance therapy and molecular risk maintained prognostic impact at both ASCT + 3 and ASCT + 9. CONCLUSION:In patients with multiple myeloma, MRD status at both ASCT + 3 and ASCT + 9 is a powerful predictor of PFS and OS. 10.1200/JCO.21.02228
    Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs. Benz Claudia,Copley Michael R,Kent David G,Wohrer Stefan,Cortes Adrian,Aghaeepour Nima,Ma Elaine,Mader Heidi,Rowe Keegan,Day Christopher,Treloar David,Brinkman Ryan R,Eaves Connie J Cell stem cell Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues. 10.1016/j.stem.2012.02.007
    Neuronal Ig/Caspr recognition promotes the formation of axoaxonic synapses in mouse spinal cord. Ashrafi Soha,Betley J Nicholas,Comer John D,Brenner-Morton Susan,Bar Vered,Shimoda Yasushi,Watanabe Kazutada,Peles Elior,Jessell Thomas M,Kaltschmidt Julia A Neuron Inhibitory microcircuits are wired with a precision that underlies their complex regulatory roles in neural information processing. In the spinal cord, one specialized class of GABAergic interneurons (GABApre) mediates presynaptic inhibitory control of sensory-motor synapses. The synaptic targeting of these GABAergic neurons exhibits an absolute dependence on proprioceptive sensory terminals, yet the molecular underpinnings of this specialized axoaxonic organization remain unclear. Here, we show that sensory expression of an NB2 (Contactin5)/Caspr4 coreceptor complex, together with spinal interneuron expression of NrCAM/CHL1, directs the high-density accumulation of GABAergic boutons on sensory terminals. Moreover, genetic elimination of NB2 results in a disproportionate stripping of inhibitory boutons from high-density GABApre-sensory synapses, suggesting that the preterminal axons of GABApre neurons compete for access to individual sensory terminals. Our findings define a recognition complex that contributes to the assembly and organization of a specialized GABAergic microcircuit. 10.1016/j.neuron.2013.10.060
    CD4 and CD8 T-cell responses to mycobacterial antigens in African children. Tena-Coki Nontobeko G,Scriba Thomas J,Peteni Nomathemba,Eley Brian,Wilkinson Robert J,Andersen Peter,Hanekom Willem A,Kampmann Beate American journal of respiratory and critical care medicine RATIONALE:The current tuberculosis (TB) vaccine, bacille Calmette-Guérin (BCG), does not provide adequate protection against TB disease in children. Furthermore, more efficacious TB vaccines are needed for children with immunodeficiencies such as HIV infection, who are at highest risk of disease. OBJECTIVES:To characterize mycobacteria-specific T cells in children who might benefit from vaccination against TB, focusing on responses to antigens contained in novel TB vaccines. METHODS:Whole blood was collected from three groups of BCG-vaccinated children: HIV-seronegative children receiving TB treatment (n = 30), HIV-infected children (n = 30), and HIV-unexposed healthy children (n = 30). Blood was stimulated with Ag85B and TB10.4, or purified protein derivative, and T-cell cytokine production by CD4 and CD8 was determined by flow cytometry. The memory phenotype of antigen-specific CD4 and CD8 T cells was also determined. MEASUREMENTS AND MAIN RESULTS:Mycobacteria-specific CD4 and CD8 T-cell responses were detectable in all three groups of children. Children receiving TB treatment had significantly higher frequencies of antigen-specific CD4 T cells compared with HIV-infected children (P = 0.0176). No significant differences in magnitude, function, or phenotype of specific T cells were observed in HIV-infected children compared with healthy control subjects. CD4 T cells expressing IFN-gamma, IL-2, or both expressed a CD45RA(-)CCR7(-)CD27(+/-) effector memory phenotype. Mycobacteria-specific CD8 T cells expressed mostly IFN-gamma in all groups of children; these cells expressed CD45RA(-)CCR7(-)CD27(+/-) or CD45RA(+)CCR7(-)CD27(+/-) effector memory phenotypes. CONCLUSIONS:Mycobacteria-specific T-cell responses could be demonstrated in all groups of children, suggesting that the responses could be boosted by new TB vaccines currently in clinical trials. 10.1164/rccm.200912-1862OC
    Circulating microparticles from pulmonary hypertensive rats induce endothelial dysfunction. Tual-Chalot Simon,Guibert Christelle,Muller Bernard,Savineau Jean-Pierre,Andriantsitohaina Ramaroson,Martinez M Carmen American journal of respiratory and critical care medicine RATIONALE:Pulmonary arterial hypertension (PAH) is a severe disease characterized by an increase of pulmonary vascular resistance, which is accompanied by functional and structural changes in pulmonary arteries. Microparticles (MPs) have been described as biological vector of endothelial dysfunction in other pathologies. OBJECTIVES:The purpose of this work was to characterize circulating MPs during hypoxic PAH and to study their effects on endothelial function. METHODS:Male Wistar rats were exposed or not to chronic hypoxia, and normoxic or hypoxic MPs from blood were characterized by flow cytometry. Endothelial cells (ECs) from rat aorta or pulmonary arteries were incubated with MPs, and then expression and phosphorylation of enzymes involved in nitric oxide (NO) and reactive oxygen species productions were analyzed. Hypoxic MPs were injected into rats, and endothelium-dependent relaxation was assessed. MEASUREMENTS AND MAIN RESULTS:Circulating levels of MPs from hypoxic rats were twofold higher than those present in normoxic rats. In vitro treatment of ECs with hypoxic MPs reduced NO production in aortas and pulmonary arteries by enhancing phosphorylation of endothelial NO synthase at the inhibitory site. Hypoxic MPs increased oxidative stress only in pulmonary ECs via xanthine oxidase and mitochondrial implication. In vivo injection of hypoxic MPs into rat impaired endothelium-dependent relaxation both in aorta and pulmonary arteries. CONCLUSIONS:These data provide evidence that hypoxic circulating MPs induce endothelial dysfunction in rat aorta and pulmonary arteries by decreasing NO production. Moreover, MPs display tissue specificity with respect to increased oxidative stress, which occurs only in pulmonary ECs. 10.1164/rccm.200909-1347OC
    The HIF-1α hypoxia response in tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immunotherapy. Palazón Asís,Martínez-Forero Iván,Teijeira Alvaro,Morales-Kastresana Aizea,Alfaro Carlos,Sanmamed Miguel F,Perez-Gracia Jose Luis,Peñuelas Iván,Hervás-Stubbs Sandra,Rouzaut Ana,de Landázuri Manuel Ortiz,Jure-Kunkel Maria,Aragonés Julian,Melero Ignacio Cancer discovery UNLABELLED:The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by positron emission tomographic (PET) imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) of transplanted colon carcinomas, melanomas, and spontaneous breast adenocarcinomas are CD137 (4-1BB)-positive, as opposed to their counterparts in tumor-draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Importantly, hypoxia does not upregulate CD137 in hypoxia-inducible factor (HIF)-1α-knockout T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating and co-transferred HIF-1α-sufficient T lymphocytes. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies to the tumor tissue. As a result, low-dose intratumoral injections avoid liver inflammation, achieve antitumor systemic effects, and permit synergistic therapeutic effects with PD-L1/B7-H1 blockade. SIGNIFICANCE:CD137 (4-1BB) is an important molecular target to augment antitumor immunity. Hypoxia in the tumor microenvironment as sensed by the HIF-1α system increases expression of CD137 on tumor-infiltrating lymphocytes that thereby become selectively responsive to the immunotherapeutic effects of anti-CD137 agonist monoclonal antibodies as those used in ongoing clinical trials. 10.1158/2159-8290.CD-11-0314
    Functional antagonism between Sall4 and Plzf defines germline progenitors. Hobbs Robin M,Fagoonee Sharmila,Papa Antonella,Webster Kaitlyn,Altruda Fiorella,Nishinakamura Ryuichi,Chai Li,Pandolfi Pier Paolo Cell stem cell Transcription factors required for formation of embryonic tissues often maintain their expression in adult stem cell populations, but whether their function remains equivalent is not clear. Here we demonstrate critical and distinct roles for Sall4 in development of embryonic germ cells and differentiation of postnatal spermatogonial progenitor cells (SPCs). In differentiating SPCs, Sall4 levels transiently increase and Sall4 physically interacts with Plzf, a transcription factor exclusively required for adult stem cell maintenance. Mechanistically, Sall4 sequesters Plzf to noncognate chromatin domains to induce expression of Kit, a target of Plzf-mediated repression required for differentiation. Plzf in turn antagonizes Sall4 function by displacing Sall4 from cognate chromatin to induce Sall1 expression. Taken together, these data suggest that transcription factors required for embryonic tissue development postnatally take on distinct roles through interaction with opposing factors, which hence define properties of the adult stem cell compartment. 10.1016/j.stem.2012.02.004
    Antigen-Specific T-Cell Activation Distinguishes between Recent and Remote Tuberculosis Infection. Mpande Cheleka A M,Musvosvi Munyaradzi,Rozot Virginie,Mosito Boitumelo,Reid Timothy D,Schreuder Constance,Lloyd Tessa,Bilek Nicole,Huang Huang,Obermoser Gerlinde,Davis Mark M,Ruhwald Morten,Hatherill Mark,Scriba Thomas J,Nemes Elisa, American journal of respiratory and critical care medicine Current diagnostic tests fail to identify individuals at higher risk of progression to tuberculosis disease, such as those with recent infection, who should be prioritized for targeted preventive treatment. To define a blood-based biomarker, measured with a simple flow cytometry assay, that can stratify different stages of tuberculosis infection to infer risk of disease. South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 mo) and persistent (QuantiFERON-TB+ for >1 yr) infection. We defined the ΔHLA-DR median fluorescence intensity biomarker as the difference in HLA-DR expression between IFN-γ TNF -specific T cells and total CD3 T cells. Biomarker performance was assessed by blinded prediction in untouched test cohorts with recent versus persistent infection or tuberculosis disease and by unblinded analysis of asymptomatic adolescents with tuberculosis infection who remained healthy (nonprogressors) or who progressed to microbiologically confirmed disease (progressors). In the test cohorts, frequencies of -specific T cells differentiated between QuantiFERON-TB- ( = 25) and QuantiFERON-TB+ ( = 47) individuals (area under the receiver operating characteristic curve, 0.94; 95% confidence interval, 0.87-1.00). ΔHLA-DR significantly discriminated between recent ( = 20) and persistent ( = 22) QuantiFERON-TB+ (0.91; 0.83-1.00); persistent QuantiFERON-TB+ and newly diagnosed tuberculosis ( = 19; 0.99; 0.96-1.00); and tuberculosis progressors ( = 22) and nonprogressors ( = 34; 0.75; 0.63-0.87). However, ΔHLA-DR median fluorescent intensity could not discriminate between recent QuantiFERON-TB+ and tuberculosis (0.67; 0.50-0.84). The ΔHLA-DR biomarker can identify individuals with recent QuantiFERON-TB conversion and those with disease progression, allowing targeted provision of preventive treatment to those at highest risk of tuberculosis. Further validation studies of this novel immune biomarker in various settings and populations at risk are warranted. 10.1164/rccm.202007-2686OC
    Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity. MacArthur Ben D,Sevilla Ana,Lenz Michel,Müller Franz-Josef,Schuldt Berhard M,Schuppert Andreas A,Ridden Sonya J,Stumpf Patrick S,Fidalgo Miguel,Ma'ayan Avi,Wang Jianlong,Lemischka Ihor R Nature cell biology A number of key regulators of mouse embryonic stem (ES) cell identity, including the transcription factor Nanog, show strong expression fluctuations at the single-cell level. The molecular basis for these fluctuations is unknown. Here we used a genetic complementation strategy to investigate expression changes during transient periods of Nanog downregulation. Employing an integrated approach that includes high-throughput single-cell transcriptional profiling and mathematical modelling, we found that early molecular changes subsequent to Nanog loss are stochastic and reversible. However, analysis also revealed that Nanog loss severely compromises the self-sustaining feedback structure of the ES cell regulatory network. Consequently, these nascent changes soon become consolidated to committed fate decisions in the prolonged absence of Nanog. Consistent with this, we found that exogenous regulation of Nanog-dependent feedback control mechanisms produced a more homogeneous ES cell population. Taken together our results indicate that Nanog-dependent feedback loops have a role in controlling both ES cell fate decisions and population variability. 10.1038/ncb2603
    Vascular endothelial growth factor drives autocrine epithelial cell proliferation and survival in chronic rhinosinusitis with nasal polyposis. Lee Hyun Sil,Myers Allen,Kim Jean American journal of respiratory and critical care medicine RATIONALE:The pathogenesis of nasal polyps in chronic rhinosinusitis is poorly understood. OBJECTIVES:These studies seek to implicate a functional role for vascular endothelial growth factor (VEGF) in perpetuating primary nasal epithelial cell overgrowth, a key feature of hyperplastic polyps. METHODS:Comparison of VEGF and receptor expression was assessed by ELISA of nasal lavage, immunohistochemistry of sinus tissue, flow cytometry of nasal epithelial cells, and ELISA of supernatants. VEGF-dependent cell growth and apoptosis were assessed with blocking antibodies to VEGF, their receptors, or small interfering RNA knockdown of neuropilin-1 by cell proliferation assays and flow cytometric binding of annexin V. MEASUREMENTS AND MAIN RESULTS:VEGF protein was sevenfold higher in nasal lavage from patients with polyposis compared with control subjects (P < 0.001). We also report elevated expression of VEGF (P < 0.012), receptors VEGFR2 and phospho-VEGFR2 (both P < 0.04), and identification of VEGF coreceptor neuropilin-1 in these tissues. Nasal epithelial cells from patients with polyps demonstrated faster growth rates (P < 0.005). Exposure of cells to blocking antibodies against VEGF resulted in inhibition of cell growth (P < 0.05). VEGF receptor blockade required blockade of neuropilin-1 (P < 0.05) and resulted in increased apoptosis (P < 0.001) and inhibition of autocrine epithelial VEGF production (P < 0.05). CONCLUSIONS:These data demonstrate that VEGF is a novel biomarker for chronic rhinosinusitis with hyperplastic sinonasal polyposis that functions in an autocrine feed-forward manner to promote nasal epithelial cell growth and to inhibit apoptosis. These findings implicate a previously unrecognized and novel role of VEGF functioning through neuropilin-1 on nonneoplastic primary human airway epithelial cells, to amplify cell growth, contributing to exuberant hyperplastic polyposis. 10.1164/rccm.200905-0740OC
    Optical Microfluidic Waveguides and Solution Lasers of Colloidal Semiconductor Quantum Wells. Maskoun Joudi,Gheshlaghi Negar,Isik Furkan,Delikanli Savas,Erdem Onur,Erdem Emine Yegan,Demir Hilmi Volkan Advanced materials (Deerfield Beach, Fla.) The realization of high-quality lasers in microfluidic devices is crucial for numerous applications, including biological and chemical sensors and flow cytometry, and the development of advanced lab-on-chip (LOC) devices. Herein, an ultralow-threshold microfluidic single-mode laser is proposed and demonstrated using an on-chip cavity. CdSe/CdS@Cd Zn S core/crown@gradient-alloyed shell colloidal semiconductor quantum wells (CQWs) dispersed in toluene are employed in the cavity created inside a poly(dimethylsiloxane) (PDMS) microfluidic device using SiO -protected Ag mirrors to achieve in-solution lasing. Lasing from such a microfluidic device having CQWs solution as a microfluidic gain medium is shown for the first time with a record-low optical gain threshold of 17.1 µJ cm ² and lasing threshold of 68.4 µJ cm ² among all solution-based lasing demonstrations. In addition, air-stable SiO protected Ag films are used and designed to form highly tunable and reflective mirrors required to attain a high-quality Fabry-Pérot cavity. These realized record-low thresholds emanate from the high-quality on-chip cavity together with the core/crown@gradient-alloyed shell CQWs having giant gain cross-section and slow Auger rates. This microfabricated CQW laser provides a compact and inexpensive coherent light source for microfluidics and integrated optics covering the visible spectral region. 10.1002/adma.202007131
    Canonical and atypical E2Fs regulate the mammalian endocycle. Chen Hui-Zi,Ouseph Madhu M,Li Jing,Pécot Thierry,Chokshi Veda,Kent Lindsey,Bae Sooin,Byrne Morgan,Duran Camille,Comstock Grant,Trikha Prashant,Mair Markus,Senapati Shantibhusan,Martin Chelsea K,Gandhi Sagar,Wilson Nicholas,Liu Bin,Huang Yi-Wen,Thompson John C,Raman Sundaresan,Singh Shantanu,Leone Marcelo,Machiraju Raghu,Huang Kun,Mo Xiaokui,Fernandez Soledad,Kalaszczynska Ilona,Wolgemuth Debra J,Sicinski Piotr,Huang Tim,Jin Victor,Leone Gustavo Nature cell biology The endocycle is a variant cell cycle consisting of successive DNA synthesis and gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals. 10.1038/ncb2595
    2018 Ebola virus disease outbreak in Équateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation. Mbala-Kingebeni Placide,Pratt Catherine B,Wiley Michael R,Diagne Moussa M,Makiala-Mandanda Sheila,Aziza Amuri,Di Paola Nicholas,Chitty Joseph A,Diop Mamadou,Ayouba Ahidjo,Vidal Nicole,Faye Ousmane,Faye Oumar,Karhemere Stormy,Aruna Aaron,Nsio Justus,Mulangu Felix,Mukadi Daniel,Mukadi Patrick,Kombe John,Mulumba Anastasie,Duraffour Sophie,Likofata Jacques,Pukuta Elisabeth,Caviness Katie,Bartlett Maggie L,Gonzalez Jeanette,Minogue Timothy,Sozhamannan Shanmuga,Gross Stephen M,Schroth Gary P,Kuhn Jens H,Donaldson Eric F,Delaporte Eric,Sanchez-Lockhart Mariano,Peeters Martine,Muyembe-Tamfum Jean-Jacques,Alpha Sall Amadou,Palacios Gustavo,Ahuka-Mundeke Steve The Lancet. Infectious diseases BACKGROUND:The 2018 Ebola virus disease (EVD) outbreak in Équateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures). METHODS:We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Équateur Province outbreak. Combining these genomes with genomes associated with known outbreaks from GenBank, we constructed a maximum-likelihood phylogenetic tree. In-silico analyses were used to assess potential mismatches between the outbreak strain and the probes and primers of diagnostic assays and the antigenic sites of the experimental rVSVΔG-ZEBOV-GP vaccine and therapeutics. An in-vitro flow cytometry assay was used to assess the binding capability of the individual components of the monoclonal antibody cocktail ZMapp. FINDINGS:A targeted sequencing approach produced 16 near-complete genomes. Phylogenetic analysis of these genomes and 1011 genomes from GenBank revealed a distinct cluster, confirming a new Ebola virus variant, for which we propose the name "Tumba". This new variant appears to have evolved at a slower rate than other Ebola virus variants (0·69 × 10 substitutions per site per year with "Tumba" vs 1·06 × 10 substitutions per site per year without "Tumba"). We found few sequence mismatches in the assessed assay target regions and antigenic sites. We identified nine amino acid changes in the Ebola virus surface glycoprotein, of which one resulted in reduced binding of the 13C6 antibody within the ZMapp cocktail. INTERPRETATION:Retrospectively, we show the feasibility of using genomics to rapidly characterise a new Ebola virus variant within the timeframe of an outbreak. Phylogenetic analysis provides further indications that these variants are evolving at differing rates. Rapid in-silico analyses can direct in-vitro experiments to quickly assess medical countermeasures. FUNDING:Defense Biological Product Assurance Office. 10.1016/S1473-3099(19)30124-0
    IgA-producing plasma cells originate from germinal centers that are induced by B-cell receptor engagement in humans. Barone Francesca,Vossenkamper Anna,Boursier Laurent,Su Wen,Watson Alan,John Susan,Dunn-Walters Deborah K,Fields Paul,Wijetilleka Sonali,Edgeworth Jonathan D,Spencer Jo Gastroenterology BACKGROUND & AIMS:IgA contributes to homeostatic balance between host and intestinal microbiota. Mechanisms that initiate the IgA response are unclear and likely to differ between humans and animal models. We used multiple experimental approaches to investigate the origin of human intestinal plasma cells that produce IgA in the gastrointestinal tract. METHODS:Complexity of IgA-producing plasma cell populations in human gastrointestinal mucosa and bone marrow and the specific response to oral cholera vaccine were compared by analysis of immunoglobulin genes. Flow cytometry, gene expression analysis, and immunohistochemistry were used to analyze signaling pathways induced by B-cell receptor engagement in human gut-associated lymphoid tissue (GALT) and involvement of innate immunity in B-cell activation in GALT compared with nonintestinal sites. RESULTS:Human intestinal IgA-producing plasma cells appeared to be of germinal center origin; there was no evidence for the population complexity that accompanies multiple pathways of derivation observed in bone marrow. In germinal center B cells of human GALT, Btk and Erk are phosphorylated, CD22 is down-regulated, Lyn is translocated to the cell membrane, and Fos and Jun are up-regulated; these features indicate B-cell receptor ligation during germinal center evolution. No differences in innate activation of B cells were observed in GALT, compared with peripheral immune compartments. CONCLUSIONS:IgA-producing plasma cells appear to be derived from GALT germinal centers in humans. B-cell receptor engagement promotes formation of germinal centers of GALT, with no more evidence for innate immune receptor activation in the mucosa than nonintestinal immune compartments. Germinal centers in GALT should be targets of mucosal vaccinations because they are the source of human intestinal IgA response. 10.1053/j.gastro.2010.12.005
    Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity. Weber Felix C,Németh Tamás,Csepregi Janka Z,Dudeck Anne,Roers Axel,Ozsvári Béla,Oswald Eva,Puskás László G,Jakob Thilo,Mócsai Attila,Martin Stefan F The Journal of experimental medicine Allergic contact dermatitis and its animal model, contact hypersensitivity (CHS), are T cell-mediated inflammatory skin diseases induced by contact allergens. Though numerous cellular and molecular players are known, the mechanism of chemical-induced sensitization remains poorly understood. Here, we identify neutrophils as crucial players in the sensitization phase of CHS. Genetic deficiency of neutrophils caused by myeloid-specific deletion of Mcl-1 or antibody-mediated depletion of neutrophils before sensitization abrogated the CHS response. Neutrophil deficiency reduced contact allergen-induced cytokine production, gelatinase release, and reactive oxygen species production in naive mice. Mast cell deficiency inhibited neutrophil accumulation at the site of sensitization. In turn, neutrophils were required for contact allergen-induced release of further neutrophil-attracting chemokines, migration of DCs to the draining lymph nodes, and priming of allergen-specific T cells. Lymph node cells from mice sensitized in the absence of neutrophils failed to transfer sensitization to naive recipients. Furthermore, no CHS response could be induced when neutrophils were depleted before elicitation or when normally sensitized lymph node cells were transferred to neutrophil-deficient recipients, indicating an additional role for neutrophils in the elicitation phase. Collectively, our data identify neutrophils to be critically involved in both the sensitization and elicitation phase of CHS. 10.1084/jem.20130062
    T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. Tran Eric,Robbins Paul F,Lu Yong-Chen,Prickett Todd D,Gartner Jared J,Jia Li,Pasetto Anna,Zheng Zhili,Ray Satyajit,Groh Eric M,Kriley Isaac R,Rosenberg Steven A The New England journal of medicine We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×10 HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02. 10.1056/NEJMoa1609279
    mTOR inhibition rescues osteopenia in mice with systemic sclerosis. Chen Chider,Akiyama Kentaro,Wang Dandan,Xu Xingtian,Li Bei,Moshaverinia Alireza,Brombacher Frank,Sun Lingyun,Shi Songtao The Journal of experimental medicine Fibrillin-1 (FBN1) deficiency-induced systemic sclerosis is attributed to elevation of interleukin-4 (IL4) and TGF-β, but the mechanism underlying FBN1 deficiency-associated osteopenia is not fully understood. We show that bone marrow mesenchymal stem cells (BMMSCs) from FBN1-deficient (Fbn1(+/-)) mice exhibit decreased osteogenic differentiation and increased adipogenic differentiation. Mechanistically, this lineage alteration is regulated by IL4/IL4Rα-mediated activation of mTOR signaling to down-regulate RUNX2 and up-regulate PPARγ2, respectively, via P70 ribosomal S6 protein kinase (P70S6K). Additionally, we reveal that activation of TGF-β/SMAD3/SP1 signaling results in enhancement of SP1 binding to the IL4Rα promoter to synergistically activate mTOR pathway in Fbn1(+/-) BMMSCs. Blockage of mTOR signaling by osteoblastic-specific knockout or rapamycin treatment rescues osteopenia phenotype in Fbn1(+/-) mice by improving osteogenic differentiation of BMMSCs. Collectively, this study identifies a previously unrecognized role of the FBN1/TGF-β/IL4Rα/mTOR cascade in BMMSC lineage selection and provides experimental evidence that rapamycin treatment may provide an anabolic therapy for osteopenia in Fbn1(+/-) mice. 10.1084/jem.20140643
    Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity. Burbage Marianne,Keppler Selina J,Gasparrini Francesca,Martínez-Martín Nuria,Gaya Mauro,Feest Christoph,Domart Marie-Charlotte,Brakebusch Cord,Collinson Lucy,Bruckbauer Andreas,Batista Facundo D The Journal of experimental medicine The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology. 10.1084/jem.20141143
    Neutrophil-related factors as biomarkers in EAE and MS. Rumble Julie M,Huber Amanda K,Krishnamoorthy Gurumoorthy,Srinivasan Ashok,Giles David A,Zhang Xu,Wang Lu,Segal Benjamin M The Journal of experimental medicine A major function of T helper (Th) 17 cells is to induce the production of factors that activate and mobilize neutrophils. Although Th17 cells have been implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE), little attention has been focused on the role of granulocytes in those disorders. We show that neutrophils, as well as monocytes, expand in the bone marrow and accumulate in the circulation before the clinical onset of EAE, in response to systemic up-regulation of granulocyte colony-stimulating factor (G-CSF) and the ELR(+) CXC chemokine CXCL1. Neutrophils comprised a relatively high percentage of leukocytes infiltrating the central nervous system (CNS) early in disease development. G-CSF receptor deficiency and CXCL1 blockade suppressed myeloid cell accumulation in the blood and ameliorated the clinical course of mice that were injected with myelin-reactive Th17 cells. In relapsing MS patients, plasma levels of CXCL5, another ELR(+) CXC chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS. 10.1084/jem.20141015
    A lipid E-MAP identifies Ubx2 as a critical regulator of lipid saturation and lipid bilayer stress. Surma Michal A,Klose Christian,Peng Debby,Shales Michael,Mrejen Caroline,Stefanko Adam,Braberg Hannes,Gordon David E,Vorkel Daniela,Ejsing Christer S,Farese Robert,Simons Kai,Krogan Nevan J,Ernst Robert Molecular cell Biological membranes are complex, and the mechanisms underlying their homeostasis are incompletely understood. Here, we present a quantitative genetic interaction map (E-MAP) focused on various aspects of lipid biology, including lipid metabolism, sorting, and trafficking. This E-MAP contains ∼250,000 negative and positive genetic interaction scores and identifies a molecular crosstalk of protein quality control pathways with lipid bilayer homeostasis. Ubx2p, a component of the endoplasmic-reticulum-associated degradation pathway, surfaces as a key upstream regulator of the essential fatty acid (FA) desaturase Ole1p. Loss of Ubx2p affects the transcriptional control of OLE1, resulting in impaired FA desaturation and a severe shift toward more saturated membrane lipids. Both the induction of the unfolded protein response and aberrant nuclear membrane morphologies observed in cells lacking UBX2 are suppressed by the supplementation of unsaturated FAs. Our results point toward the existence of dedicated bilayer stress responses for membrane homeostasis. 10.1016/j.molcel.2013.06.014
    The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase. Dohrmann Paul R,Correa Raul,Frisch Ryan L,Rosenberg Susan M,McHenry Charles S Nucleic acids research There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ'χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C(tag)) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-C(tag) per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ'χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F' episome. 10.1093/nar/gkv1510
    Circulating endothelial progenitor cells and depression: a possible novel link between heart and soul. Dome P,Teleki Z,Rihmer Z,Peter L,Dobos J,Kenessey I,Tovari J,Timar J,Paku S,Kovacs G,Dome B Molecular psychiatry Although depression is known to be an independent risk factor for cardiovascular disorders, the mechanisms behind this connection are not well understood. However, the reduction in the number of endothelial progenitor cells (EPCs) in patients with cardiovascular risk factors has led us to hypothesize that depression influences the number of EPCs. EPCs labeled with CD34, CD133 and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood (PB) of 33 patients with a current episode of major depression and of 16 control subjects. Mature (CD34+/VEGFR2+) and immature (CD133+/VEGFR2+) EPC counts were decreased in patients (vs controls; P<0.01 for both comparisons), and there was a significant inverse relationship between EPC levels and the severity of depressive symptoms (P<0.01 for both EPC phenotypes). Additionally, we assayed the plasma levels of VEGF, C-reactive protein (CRP) and tumor necrosis factor (TNF)-alpha and observed significantly elevated TNF-alpha concentrations in patients (vs controls; P<0.05) and, moreover, a significant inverse correlation between TNF-alpha and EPC levels (P<0.05). Moreover, by means of a quantitative RT-PCR approach, we measured CD34, CD133 and VEGFR2 mRNA levels of PB samples and found a net trend toward a decrease in all the investigated EPC-specific mRNA levels in patients as compared with controls. However, statistical significance was reached only for VEGFR2 and CD133 levels (P<0.01 for both markers). This is the first paper that demonstrates evidence of decreased numbers of circulating EPCs in patients with a current episode of major depression. 10.1038/sj.mp.4002138
    Blood dendritic cells in systemic lupus erythematosus exhibit altered activation state and chemokine receptor function. Gerl Velia,Lischka Alexandra,Panne Daniel,Grossmann Patrick,Berthold Rita,Hoyer Bimba Franziska,Biesen Robert,Bruns Anne,Alexander Tobias,Jacobi Annett,Dörner Thomas,Burmester Gerd-Rüdiger,Radbruch Andreas,Hiepe Falk Annals of the rheumatic diseases BACKGROUND:Dendritic cells (DCs) have a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Reduced numbers of blood DCs and the accumulation of DCs at inflammatory sites have been observed in SLE. One crucial feature of DCs is their ability to migrate. OBJECTIVE:To analyse the maturation/activation state and the migratory capacity of different DC precursor subsets in SLE to further elucidate their role in autoimmunity. METHODS:Plasmacytoid DCs (pDCs), myeloid DCs (mDCs) and monocytes from patients with SLE, healthy volunteers and healthy volunteers immunised with tetanus/diphtheria were examined by flow cytometry for expression of subset-specific antigens (BDCA-2, CD11c, CD14, HLA-DR), activation/maturation markers (CD83, CD86, CD40, BLyS) and chemokine receptors (CCR1, CCR5, CCR7, ChemR23). Additionally, migratory capacity to chemokine receptors was investigated in vitro using the chemokines RANTES, CCL19 and chemerin. RESULTS:SLE monocytes and mDCs had higher CD86 and B-lymphocyte stimulatory factor (BLyS) expression levels. ChemR23 expression was lower in SLE pDCs and mDCs. Basal and CCL19-specific migration levels were higher in SLE pDCs. Altered DC function in SLE had no correlative changes in chemokine receptor expression, whereas immunisation-induced blood DC migration patterns in healthy donors were accompanied by changes in chemokine receptor expression. CONCLUSIONS:The phenotypic and migratory disturbances observed in SLE blood DCs could result in altered distribution of DCs in peripheral tissues, contributing to dysregulated immune responses and autoimmunity. 10.1136/ard.2009.111021
    Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1-E2F pathway activity. Henriques Rossana,Magyar Zoltán,Monardes Antonia,Khan Safina,Zalejski Christine,Orellana Juan,Szabados László,de la Torre Consuelo,Koncz Csaba,Bögre László The EMBO journal The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single- and double-knockout mutations and RNA-interference (RNAi)-silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non-viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma-related 1 (RBR1)-E2FB complex and this is partly mediated by its N-terminal LVxCxE motif. Moreover, the S6K1-RBR1 association regulates RBR1 nuclear localization, as well as E2F-dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient-limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels. 10.1038/emboj.2010.164
    A role for dendritic cells in bleomycin-induced pulmonary fibrosis in mice? Bantsimba-Malanda Claudie,Marchal-Sommé Joëlle,Goven Delphine,Freynet Olivia,Michel Laurence,Crestani Bruno,Soler Paul American journal of respiratory and critical care medicine RATIONALE:Lung dendritic cells (DCs) have been shown to accumulate in human fibrotic lung disease, but little is known concerning a role for DCs in the pathogenesis of fibrotic lung. OBJECTIVES:To characterize lung DCs in an in vivo model of bleomycin-induced pulmonary fibrosis in mice. METHODS:We characterized the kinetics and activation of pulmonary DCs during the course of bleomycin-induced lung injury by flow cytometry on lung single-cell suspensions. We also characterized the lymphocytes accumulating in bleomycin lung and the chemokines susceptible to favor the recruitment of immune cells. MEASUREMENTS AND MAIN RESULTS:We show, for the first time, that increased numbers of CD11c(+)/major histocompatibility complex class II(+) DCs, including CD11b(hi) monocyte-derived inflammatory DCs, infiltrate the lung of treated animals during the fibrotic phase of the response to bleomycin. These DCs are mature DCs expressing CD40, CD86, and CD83. They are associated with increased numbers of recently activated memory T cells expressing CD44, CD40L, and CD28, suggesting that fully mature DCs and Ag-experienced T cells can drive an efficient effector immune response within bleomycin lung. Most importantly, when DCs are inactivated with VAG539, a recently described new immunomodulator, VAG539 treatment attenuates the hallmarks of bleomycin lung injury. CONCLUSIONS:These findings identify lung DCs as key proinflammatory cells potentially able to sustain pulmonary inflammation and fibrosis in the bleomycin model. 10.1164/rccm.200907-1164OC
    Resolution of allergic airway inflammation and airway hyperreactivity is mediated by IL-17-producing {gamma}{delta}T cells. Murdoch Jenna R,Lloyd Clare M American journal of respiratory and critical care medicine RATIONALE:gammadeltaT lymphocytes are enriched within the epithelial microenvironment, where they are thought to maintain homeostasis and limit immunopathology. gammadeltaT cells are postulated to exert a regulatory influence during acute allergic airway disease, but the mechanism is unknown. Although regulation of allergic airway disease has been attributed to IL-17-producing T helper (Th) 17 cells, we have found that gammadeltaT cells represent the major source of IL-17 in the allergic lung. OBJECTIVES:The aim of this study was to determine the contribution of these IL-17-producing gammadeltaT cells to regulation of allergic airway inflammation. METHODS:Flow cytometry revealed that IL-17-producing gammadeltaT cells are more prevalent than IL-17(+)alphabetaT cells (Th17) in a murine model of ovalbumin-induced allergic inflammation. MEASUREMENTS AND MAIN RESULTS:Transfer of gammadeltaT cells at the peak of acute allergic responses ameliorated airway hyperresponsiveness with a corresponding acceleration in the resolution of eosinophilic and Th2-driven inflammation. Conversely, functional blockade of gammadeltaT cells led to exacerbation of injury. Neither treatment changed pulmonary Th17 cell numbers. Moreover, transfer of Th17 cells had no effect on disease outcome. Importantly, IL-17-deficient gammadeltaT cells were unable to promote resolution of injury. These data identify IL-17-producing gammadeltaT cells as key regulators of the allergic response in vivo. CONCLUSIONS:This unfolds a new perspective for the understanding of gammadeltaT cell function with regard to innate regulation of the adaptive immune responses, emphasizing that resolution of responses are important in determining the outcome of acute inflammatory episodes as well as for maintenance of tissue integrity and homeostasis. 10.1164/rccm.200911-1775OC
    Inhibition of LTi cell development by CD25 blockade is associated with decreased intrathecal inflammation in multiple sclerosis. Perry Justin S A,Han Sungpil,Xu Quangang,Herman Matthew L,Kennedy Lucy B,Csako Gyorgy,Bielekova Bibiana Science translational medicine Genetic polymorphisms in the interleukin-2 receptor α (IL-2Rα) chain (CD25) locus are associated with several human autoimmune diseases, including multiple sclerosis (MS). Blockade of CD25 by the humanized monoclonal antibody daclizumab decreases MS-associated inflammation but has surprisingly limited direct inhibitory effects on activated T cells. The present study describes unexpected effects of daclizumab therapy on innate lymphoid cells (ILCs). The number of circulating retinoic acid receptor-related orphan receptor γt-positive ILCs, which include lymphoid tissue inducer (LTi) cells, was found to be elevated in untreated MS patients compared to healthy subjects. Daclizumab therapy not only decreased numbers of ILCs but also modified their phenotype away from LTi cells and toward a natural killer (NK) cell lineage. Mechanistic studies indicated that daclizumab inhibited differentiation of LTi cells from CD34⁺ hematopoietic progenitor cells or c-kit⁺ ILCs indirectly, steering their differentiation toward immunoregulatory CD56(bright) NK cells through enhanced intermediate-affinity IL-2 signaling. Because adult LTi cells may retain lymphoid tissue-inducing capacity or stimulate adaptive immune responses, we indirectly measured intrathecal inflammation in daclizumab-treated MS patients by quantifying the cerebrospinal fluid chemokine (C-X-C motif) ligand 13 and immunoglobulin G index. Both of these inflammatory biomarkers were inhibited by daclizumab treatment. Our study indicates that ILCs are involved in the regulation of adaptive immune responses, and their role in human autoimmunity should be investigated further, including their potential as therapeutic targets. 10.1126/scitranslmed.3004140
    Adult interfollicular tumour-initiating cells are reprogrammed into an embryonic hair follicle progenitor-like fate during basal cell carcinoma initiation. Youssef Khalil Kass,Lapouge Gaëlle,Bouvrée Karine,Rorive Sandrine,Brohée Sylvain,Appelstein Ornella,Larsimont Jean-Christophe,Sukumaran Vijayakumar,Van de Sande Bram,Pucci Doriana,Dekoninck Sophie,Berthe Jean-Valery,Aerts Stein,Salmon Isabelle,del Marmol Véronique,Blanpain Cédric Nature cell biology Basal cell carcinoma, the most frequent human skin cancer, arises from activating hedgehog (HH) pathway mutations; however, little is known about the temporal changes that occur in tumour-initiating cells from the first oncogenic hit to the development of invasive cancer. Using an inducible mouse model enabling the expression of a constitutively active Smoothened mutant (SmoM2) in the adult epidermis, we carried out transcriptional profiling of SmoM2-expressing cells at different times during cancer initiation. We found that tumour-initiating cells are massively reprogrammed into a fate resembling that of embryonic hair follicle progenitors (EHFPs). Wnt/ β-catenin signalling was very rapidly activated following SmoM2 expression in adult epidermis and coincided with the expression of EHFP markers. Deletion of β-catenin in adult SmoM2-expressing cells prevents EHFP reprogramming and tumour initiation. Finally, human basal cell carcinomas also express genes of the Wnt signalling and EHFP signatures. 10.1038/ncb2628
    Telomere-driven tetraploidization occurs in human cells undergoing crisis and promotes transformation of mouse cells. Davoli Teresa,de Lange Titia Cancer cell Human cancers with a subtetraploid karyotype are thought to originate from tetraploid precursors, but the cause of tetraploidization is unknown. We previously documented endoreduplication in mouse cells with persistent telomere dysfunction or genome-wide DNA damage. We now report that endoreduplication and mitotic failure occur during telomere crisis in human fibroblasts and mammary epithelial cells and document the role of p53 and Rb in repressing tetraploidization. Using an inducible system to generate transient telomere damage, we show that telomere-driven tetraploidization enhances the tumorigenic transformation of mouse cells. Similar to human solid cancers, the resulting tumors evolved subtetraploid karyotypes. These data establish that telomere-driven tetraploidization is induced by critically short telomeres and has the potential to promote tumorigenesis in early cancerous lesions. 10.1016/j.ccr.2012.03.044
    Proliferation and tumorigenesis of a murine sarcoma cell line in the absence of DICER1. Ravi Arvind,Gurtan Allan M,Kumar Madhu S,Bhutkar Arjun,Chin Christine,Lu Victoria,Lees Jacqueline A,Jacks Tyler,Sharp Phillip A Cancer cell MicroRNAs are a class of short ~22 nucleotide RNAs predicted to regulate nearly half of all protein coding genes, including many involved in basal cellular processes and organismal development. Although a global reduction in miRNAs is commonly observed in various human tumors, complete loss has not been documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding that transformed or immortalized Dicer1 null somatic cells can be isolated readily in vitro, maintain the characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1 null cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence, miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral environment. 10.1016/j.ccr.2012.04.037
    Ex Vivo Model of Human Penile Transplantation and Rejection: Implications for Erectile Tissue Physiology. Sopko Nikolai A,Matsui Hotaka,Lough Denver M,Miller Devin,Harris Kelly,Kates Max,Liu Xiaopu,Billups Kevin,Redett Richard,Burnett Arthur L,Brandacher Gerald,Bivalacqua Trinity J European urology BACKGROUND:Penile transplantation is a potential treatment option for severe penile tissue loss. Models of human penile rejection are lacking. OBJECTIVE:Evaluate effects of rejection and immunosuppression on cavernous tissue using a novel ex vivo mixed lymphocyte reaction (MLR) model. DESIGN, SETTING, AND PARTICIPANTS:Cavernous tissue and peripheral blood mononuclear cells (PBMCs) from 10 patients undergoing penile prosthesis operations and PBMCs from a healthy volunteer were obtained. Ex vivo MLRs were prepared by culturing cavernous tissue for 48h in media alone, in media with autologous PBMCs, or in media with allogenic PBMCs to simulate control, autotransplant, and allogenic transplant conditions with or without 1μM cyclosporine A (CsA) or 20nM tacrolimus (FK506) treatment. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:Rejection was characterized by PBMC flow cytometry and gene expression transplant array. Cavernous tissues were evaluated by histomorphology and myography to assess contraction and relaxation. Data were analyzed using two-way analysis of variance and unpaired Student t test. RESULTS AND LIMITATIONS:Flow cytometry and tissue array demonstrated allogenic PBMC activation consistent with rejection. Rejection impaired cavernous tissue physiology and was associated with cellular infiltration and apoptosis. CsA prevented rejection but did not improve tissue relaxation. CsA treatment impaired relaxation in tissues cultured without PBMCs compared with media and FK506. Study limitations included the use of penile tissue with erectile dysfunction and lack of cross-matching data. CONCLUSIONS:This model could be used to investigate the effects of penile rejection and immunosuppression. Additional studies are needed to optimize immunosuppression to prevent rejection and maximize corporal tissue physiology. PATIENT SUMMARY:This report describes a novel ex vivo model of human penile transplantation rejection. Tissue rejection impaired erectile tissue physiology. This report suggests that cyclosporin A might hinder corporal physiology and that other immunosuppressant agents, such as FK506, might be better suited to penile transplantation. 10.1016/j.eururo.2016.07.006
    Barrett's esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Reid B J,Haggitt R C,Rubin C E,Rabinovitch P S Gastroenterology The value of endoscopic surveillance biopsy for dysplasia and carcinoma in patients with Barrett's esophagus is controversial. One reason is that the available histologic criteria are not adequate to separate patients with lesser degrees of dysplasia or predysplastic changes who are at increased risk for carcinoma and therefore require more frequent surveillance from those patients who are not at increased risk. We used flow cytometry and histology to evaluate 317 biopsy specimens from 64 consecutive patients who were in a cancer surveillance program for Barrett's esophagus and 3 additional patients with adenocarcinoma in Barrett's esophagus. Specimens from 10 patients had aneuploid cells; 9 of these had dysplasia or carcinoma, or both, but 1 patient had only specialized metaplastic epithelium. Twenty specimens ahd G2/tetraploid fractions greater than 6%; all 20 came from patients who had cancer or dysplasia, or were indefinite for dysplasia. All patients with dysplasia or adenocarcinoma had evidence of genomic instability (aneuploidy) or abnormalities of mucosal proliferation by flow cytometry, even when the dysplasia was focal or difficult to recognize histologically. In a small subset of patients with specialized metaplastic epithelium whose specimens were histologically negative or indefinite for dysplasia, the mucosa had aneuploid cell populations or proliferative abnormalities that were otherwise found only in dysplasia or carcinoma. Additional study may prove that this subset of patients merits more frequent endoscopic biopsy surveillance because of an increased risk for developing carcinoma. Because the abnormalities we have detected by flow cytometry correlate well with the conventional histologic diagnoses of dysplasia and carcinoma, they may prove to be a valuable objective adjunct in the diagnosis of dysplasia and carcinoma in Barrett's esophagus.
    YAP mediates crosstalk between the Hippo and PI(3)K–TOR pathways by suppressing PTEN via miR-29. Tumaneng Karen,Schlegelmilch Karin,Russell Ryan C,Yimlamai Dean,Basnet Harihar,Mahadevan Navin,Fitamant Julien,Bardeesy Nabeel,Camargo Fernando D,Guan Kun-Liang Nature cell biology Organ development is a complex process governed by the interplay of several signalling pathways that have critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear. Here we provide evidence that YAP activates the mammalian target of rapamycin (mTOR), a major regulator of cell growth. We have identified the tumour suppressor PTEN, an upstream negative regulator of mTOR, as a critical mediator of YAP in mTOR regulation. We demonstrate that YAP downregulates PTEN by inducing miR-29 to inhibit PTEN translation. Last, we show that PI(3)K–mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia. Our studies reveal a functional link between Hippo and PI(3)K–mTOR, providing a molecular basis for the coordination of these two pathways in organ size regulation. 10.1038/ncb2615
    Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature. Rinkevich Yuval,Mori Taisuke,Sahoo Debashis,Xu Pin-Xian,Bermingham John R,Weissman Irving L Nature cell biology Fibroblasts and smooth muscle cells (FSMCs) are principal cell types of connective and adventitial tissues that participate in the development, physiology and pathology of internal organs, with incompletely defined cellular origins. Here, we identify and prospectively isolate from the mesothelium a mouse cell lineage that is committed to FSMCs. The mesothelium is an epithelial monolayer covering the vertebrate thoracic and abdominal cavities and internal organs. Time-lapse imaging and transplantation experiments reveal robust generation of FSMCs from the mesothelium. By targeting mesothelin (MSLN), a surface marker expressed on mesothelial cells, we identify and isolate precursors capable of clonally generating FSMCs. Using a genetic lineage tracing approach, we show that embryonic and adult mesothelium represents a common lineage to trunk FSMCs, and trunk vasculature, with minimal contributions from neural crest, or circulating cells. The isolation of FSMC precursors enables the examination of multiple aspects of smooth muscle and fibroblast biology as well as the prospective isolation of these precursors for potential regenerative medicine purposes. 10.1038/ncb2610
    Self-renewal as a therapeutic target in human colorectal cancer. Kreso Antonija,van Galen Peter,Pedley Nicholas M,Lima-Fernandes Evelyne,Frelin Catherine,Davis Thomas,Cao Liangxian,Baiazitov Ramil,Du Wu,Sydorenko Nadiya,Moon Young-Choon,Gibson Lianne,Wang Yadong,Leung Cherry,Iscove Norman N,Arrowsmith Cheryl H,Szentgyorgyi Eva,Gallinger Steven,Dick John E,O'Brien Catherine A Nature medicine Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (CICs) in tumors that survive therapy. Together with recent reports showing that CIC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key 'stemness' property unique to CICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation and, more specifically, human colorectal CIC function are dependent on the canonical self-renewal regulator BMI-1. Downregulation of BMI-1 inhibits the ability of colorectal CICs to self-renew, resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with a small-molecule BMI-1 inhibitor resulted in colorectal CIC loss with long-term and irreversible impairment of tumor growth. Targeting the BMI-1-related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. 10.1038/nm.3418
    Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity. Kim Hye Young,Lee Hyun Jun,Chang Ya-Jen,Pichavant Muriel,Shore Stephanie A,Fitzgerald Katherine A,Iwakura Yoichiro,Israel Elliot,Bolger Kenneth,Faul John,DeKruyff Rosemarie H,Umetsu Dale T Nature medicine Obesity is associated with the development of asthma, which is often difficult to control. To understand the immunological pathways that lead to obesity-associated asthma, we fed mice a high-fat diet for 12 weeks, which resulted in obesity and the development of airway hyperreactivity (AHR), a cardinal feature of asthma. This AHR was independent of adaptive immunity, as it occurred in obese Rag1(-/-) mice, which lack B and T cells, and was dependent on interleukin-17A (IL-17A) and the NLRP3 inflammasome, as it did not develop in obese Il17a(-/-) or Nlrp3(-/-) mice. AHR was also associated with the expansion of CCR6(+) type 3 innate lymphoid cells (ILCs) producing IL-17A (ILC3 cells) in the lung, which could by themselves mediate AHR when adoptively transferred into Rag2(-/-); Il2rg(-/-) mice treated with recombinant IL-1β. Macrophage-derived IL-1β production was induced by HFD and expanded the number of lung ILC3 cells. Blockade of IL-1β with an IL-1 receptor antagonist abolished obesity-induced AHR and reduced the number of ILC3 cells. As we found ILC3-like cells in the bronchoalveolar lavage fluid of individuals with asthma, we suggest that obesity-associated asthma is facilitated by inflammation mediated by NLRP3, IL-1β and ILC3 cells. 10.1038/nm.3423
    Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection. Liehl Peter,Zuzarte-Luís Vanessa,Chan Jennie,Zillinger Thomas,Baptista Fernanda,Carapau Daniel,Konert Madlen,Hanson Kirsten K,Carret Céline,Lassnig Caroline,Müller Mathias,Kalinke Ulrich,Saeed Mohsan,Chora Angelo Ferreira,Golenbock Douglas T,Strobl Birgit,Prudêncio Miguel,Coelho Luis P,Kappe Stefan H,Superti-Furga Giulio,Pichlmair Andreas,Vigário Ana M,Rice Charles M,Fitzgerald Katherine A,Barchet Winfried,Mota Maria M Nature medicine Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN. 10.1038/nm.3424
    Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Jan Max,Snyder Thomas M,Corces-Zimmerman M Ryan,Vyas Paresh,Weissman Irving L,Quake Stephen R,Majeti Ravindra Science translational medicine Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions. 10.1126/scitranslmed.3004315
    Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis. Komatsu Noriko,Okamoto Kazuo,Sawa Shinichiro,Nakashima Tomoki,Oh-hora Masatsugu,Kodama Tatsuhiko,Tanaka Sakae,Bluestone Jeffrey A,Takayanagi Hiroshi Nature medicine Autoimmune diseases often result from an imbalance between regulatory T (Treg) cells and interleukin-17 (IL-17)-producing T helper (TH17) cells; the origin of the latter cells remains largely unknown. Foxp3 is indispensable for the suppressive function of Treg cells, but the stability of Foxp3 has been under debate. Here we show that TH17 cells originating from Foxp3(+) T cells have a key role in the pathogenesis of autoimmune arthritis. Under arthritic conditions, CD25(lo)Foxp3(+)CD4(+) T cells lose Foxp3 expression (herein called exFoxp3 cells) and undergo transdifferentiation into TH17 cells. Fate mapping analysis showed that IL-17-expressing exFoxp3 T (exFoxp3 TH17) cells accumulated in inflamed joints. The conversion of Foxp3(+)CD4(+) T cells to TH17 cells was mediated by synovial fibroblast-derived IL-6. These exFoxp3 TH17 cells were more potent osteoclastogenic T cells than were naive CD4(+) T cell-derived TH17 cells. Notably, exFoxp3 TH17 cells were characterized by the expression of Sox4, chemokine (C-C motif) receptor 6 (CCR6), chemokine (C-C motif) ligand 20 (CCL20), IL-23 receptor (IL-23R) and receptor activator of NF-κB ligand (RANKL, also called TNFSF11). Adoptive transfer of autoreactive, antigen-experienced CD25(lo)Foxp3(+)CD4(+) T cells into mice followed by secondary immunization with collagen accelerated the onset and increased the severity of arthritis and was associated with the loss of Foxp3 expression in the majority of transferred T cells. We observed IL-17(+)Foxp3(+) T cells in the synovium of subjects with active rheumatoid arthritis (RA), which suggests that plastic Foxp3(+) T cells contribute to the pathogenesis of RA. These findings establish the pathological importance of Foxp3 instability in the generation of pathogenic TH17 cells in autoimmunity. 10.1038/nm.3432
    Dopamine Receptor-Mediated Binding and Cellular Uptake of Polydopamine-Coated Nanoparticles. Liu Yao,Choi Chun Kit K,Hong Huiling,Xiao Yu,Kwok Man Long,Liu Hanzhuang,Tian Xiao Yu,Choi Chung Hang Jonathan ACS nano Polydopamine (PDA)-coated nanoparticles (NPs) are emerging carriers of therapeutic agents for nanomedicine applications due to their biocompatibility and abundant entry to various cell types, yet it remains unknown whether their cellular entry engages cell-surface receptors. As monomeric dopamine (DA) is an endogenous ligand of dopamine receptor and raw ingredient of PDA, we elucidate the interaction between polyethylene glycol-stabilized, PDA-coated gold NPs (Au@PDA@PEG NPs) and dopamine receptors, particularly D2 (D2DR). After proving the binding of Au@PDA@PEG NPs to recombinant and cellular D2DR, we employ antibody blocking, gene knockdown, and gene overexpression to establish the role of D2DR in the cellular uptake of Au@PDA@PEG NPs . By preparing a series of PEG-coated AuNPs that contain different structural analogues of DA (Au@PEG-X NPs), we demonstrate that catechol and amine groups collectively enhance the binding of NPs to D2DR and their cellular uptake. By intravenously injecting Au@PDA@PEG NPs to Balb/c mice, we reveal their binding to D2DR in the liver by competitive inhibition and immunohistochemistry together with their preferential association to D2DR-rich resident Kupffer cells by flow cytometry, a result consistent with the profuse expression of D2DR by resident Kupffer cells. Catechol and amine groups jointly contribute to the preferential association of NPs to D2DR-rich Kupffer cells. Our data highlight the importance of D2DR expression and DA-related functional groups in mediating the cell-nano interactions of PDA-based nanomedicines. 10.1021/acsnano.1c06081
    Mutations in GPAA1, Encoding a GPI Transamidase Complex Protein, Cause Developmental Delay, Epilepsy, Cerebellar Atrophy, and Osteopenia. Nguyen Thi Tuyet Mai,Murakami Yoshiko,Sheridan Eamonn,Ehresmann Sophie,Rousseau Justine,St-Denis Anik,Chai Guoliang,Ajeawung Norbert F,Fairbrother Laura,Reimschisel Tyler,Bateman Alexandra,Berry-Kravis Elizabeth,Xia Fan,Tardif Jessica,Parry David A,Logan Clare V,Diggle Christine,Bennett Christopher P,Hattingh Louise,Rosenfeld Jill A,Perry Michael Scott,Parker Michael J,Le Deist Françoise,Zaki Maha S,Ignatius Erika,Isohanni Pirjo,Lönnqvist Tuula,Carroll Christopher J,Johnson Colin A,Gleeson Joseph G,Kinoshita Taroh,Campeau Philippe M American journal of human genetics Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs102] and c.920delG [p.Gly307Alafs11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system. 10.1016/j.ajhg.2017.09.020
    The role of IFN-gamma Elispot assay in HIV vaccine research. Streeck Hendrik,Frahm Nicole,Walker Bruce D Nature protocols The interferon (IFN)-gamma Elispot assay has been widely used as a general screening method for the quantification and characterization of the human immunodeficiency virus (HIV)-specific CD8+ T cell responses. However, the predictive power of this assay has been challenged due to the lack of efficacy of a recently conducted HIV vaccine phase IIb trial, despite induction of robust Elispot responses. This finding plus improvements in multiparameter flow cytometry, which has the potential advantage of simultaneously quantifying numerous parameters, raises questions regarding the future role of IFN-gamma Elispot as a gateway to moving forward with clinical trials of candidate vaccines. However, the IFN-gamma Elispot assay has been, unlike other techniques, evaluated and validated in several proficiency panels and is advantageous in cost-effectively detecting and mapping T-cell responses. Here we present a detailed protocol for a state-of-the-art 3-d IFN-gamma Elispot assay and review further advantages and disadvantages of this method for the characterization of HIV-specific CD8+ T cell responses. 10.1038/nprot.2009.7
    Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars. Bond Michelle R,Zhang Haochi,Vu Peter D,Kohler Jennifer J Nature protocols Transient interactions among glycoconjugates underlie developmental, immunological and metastatic recognition. Glycan-mediated interactions have low binding affinities and rapid dissociation rates. As a result, these complexes dissociate when removed from their cellular context, complicating characterization. Photocrosslinkers introduce a covalent bond between glycoconjugates and their binding partners, allowing physiologically relevant complexes to be isolated. This protocol describes metabolic incorporation of a diazirine photocrosslinker into sialic acids in cellular glycoconjugates. Subsequent irradiation results in photocrosslinking of sialic acid to neighboring macromolecules, providing a photochemical 'snapshot' of binding events. As photocrosslinking sugars are light activated, these reagents have the potential to be used for temporally and/or spatially restricted crosslinking. We provide instructions for the synthesis of photocrosslinking sugar precursors, cell culture for metabolic incorporation, flow cytometry to evaluate metabolic incorporation, photoirradiation and analysis of the crosslinked complexes. Synthesis of photocrosslinking sugars requires 4-6 d, and photocrosslinking experiments can be completed in an additional 6 d. 10.1038/nprot.2009.85
    Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Logue Susan E,Elgendy Mohamed,Martin Seamus J Nature protocols Apoptosis is a mode of programmed cell death that is widely used to eliminate cells during development, tissue homeostasis, infection or in response to injury. Alterations to the plasma membranes of apoptotic cells trigger recognition and engulfment of such cells by phagocytes. Measurement of plasma membrane phosphatidylserine externalization, using fluorescently labeled annexin V, is widely used for the detection of apoptotic cells. Here we describe protocols for bacterial expression, purification and FITC labeling of recombinant annexin V. By following the method outlined in this protocol, it is possible to produce milligram amounts of recombinant annexin V within 3 d. We also describe a method for the assessment of annexin V binding to cell populations by flow cytometry or fluorescence microscopy. 10.1038/nprot.2009.143
    Bladder wash cytology and flow cytometry for the diagnosis of transitional cell carcinoma of the urinary bladder. Romero J,Alos L,Mallofre C,Sole M,Gutierrez R,Alcover J,Carretero P European urology The role of bladder wash (BW) cytology and flow cytometry in the diagnosis of low-grade transitional cell carcinoma (TCC) of the bladder is yet to be demonstrated. We have studied a series of BW specimens by both conventional cytology and flow cytometry: there were 16 BW from patients with histologically proven TCC and 14 BW from patients with clinical suspicion of tumor or under follow-up for previous TCC in which no evidence of tumor was found by cystoscopy and multiple biopsies. As control group, 21 BW were studied from patients undergoing cystoscopy for causes other than TCC. In conclusion, the conventional cytologic study of BW specimens was highly sensitive for grade II-III TCC, but missed most grade I TCC; flow cytometric analysis did not improve significantly the detection rate in low-grade TCC.
    Detection of circulating endothelial cells: CD146-based magnetic separation enrichment or flow cytometric assay? Dignat-George Françoise,Sabatier Florence,Blann Andrew,Woywodt Alexander Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10.1200/JCO.2006.07.7677
    Defining patient-specific risk in acute myeloid leukemia. Ho Tzu-Chieh,Becker Michael W Journal of clinical oncology : official journal of the American Society of Clinical Oncology 10.1200/JCO.2013.51.4307