Chikungunya virus-induced autophagy delays caspase-dependent cell death.
Joubert Pierre-Emmanuel,Werneke Scott W,de la Calle Claire,Guivel-Benhassine Florence,Giodini Alessandra,Peduto Lucie,Levine Beth,Schwartz Olivier,Lenschow Deborah J,Albert Matthew L
The Journal of experimental medicine
Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α-XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16L(HM) mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease.
Association of Adipose Tissue Inflammation With Histologic Severity of Nonalcoholic Fatty Liver Disease.
du Plessis Johannie,van Pelt Jos,Korf Hannelie,Mathieu Chantal,van der Schueren Bart,Lannoo Matthias,Oyen Tom,Topal Baki,Fetter Gary,Nayler Simon,van der Merwe Tessa,Windmolders Petra,Van Gaal Luc,Verrijken An,Hubens Guy,Gericke Martin,Cassiman David,Francque Sven,Nevens Frederik,van der Merwe Schalk
BACKGROUND & AIMS:The prevalence of nonalcoholic fatty liver disease (NAFLD) has increased with the obesity pandemic. We analyzed the transcriptional profiles of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT), and phenotypes and functional characteristics of adipocyte tissue macrophages (ATMs), in obese patients undergoing bariatric surgery. METHODS:We collected anthropometric data; plasma samples; and SAT, VAT, and liver tissues from 113 obese patients undergoing bariatric surgery at academic hospitals in Europe (Antwerp and Leuven) and South Africa. Based on clinical and histologic features, patients were assigned to the following groups: obese, NAFLD, nonalcoholic steatohepatitis (NASH), or NASH with fibrosis. Microarray analyses were performed to identify genes expressed differentially among groups. We measured levels of cytokines and chemokines in plasma samples and levels of RNAs in adipose tissues by quantitative reverse-transcription polymerase chain reaction. ATMs were isolated from patients and 13 lean individuals undergoing cholecystectomy (controls), analyzed by flow cytometry, and cultured; immunophenotypes and levels of cytokines and chemokines in supernatants were determined. RESULTS:We observed increased expression of genes that regulate inflammation in adipose tissues from patients with NAFLD and NASH; expression of these genes increased as disease progressed from NAFLD to NASH. We found 111 genes associated with inflammation that were expressed differentially between VAT and SAT. Serum levels of interleukin 8, chemokine (C-C motif) ligand 3, and tumor necrosis factor-α correlated with liver inflammation and NAFLD activity score. We developed 2 models that could be used to determine patients' liver histology based on gene expression in VAT and SAT. Flow cytometry showed increased proportions of CD11c+CD206+ and CCR2+ macrophages in VAT from patients with NASH, and supernatants of cultured macrophages had increased levels of cytokines and chemokines compared with controls. CONCLUSIONS:VAT and SAT from patients with NAFLD and NASH have an increased expression of genes that regulate inflammation, and ATM produce increased levels of inflammatory cytokines, compared with adipose tissues from controls. We identified an expression profile of 5 genes in SAT that accurately predict liver histology in these patients. Transcript profiling: accession numbers: GSE58979 and GSE59045.
Immunosuppression in patients who die of sepsis and multiple organ failure.
Boomer Jonathan S,To Kathleen,Chang Kathy C,Takasu Osamu,Osborne Dale F,Walton Andrew H,Bricker Traci L,Jarman Stephen D,Kreisel Daniel,Krupnick Alexander S,Srivastava Anil,Swanson Paul E,Green Jonathan M,Hotchkiss Richard S
CONTEXT:Severe sepsis is typically characterized by initial cytokine-mediated hyperinflammation. Whether this hyperinflammatory phase is followed by immunosuppression is controversial. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting. OBJECTIVES:To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression. DESIGN, SETTING, AND PARTICIPANTS:Rapid postmortem spleen and lung tissue harvest was performed at the bedsides of 40 patients who died in intensive care units (ICUs) of academic medical centers with active severe sepsis to characterize their immune status at the time of death (2009-2011). Control spleens (n = 29) were obtained from patients who were declared brain-dead or had emergent splenectomy due to trauma; control lungs (n = 20) were obtained from transplant donors or from lung cancer resections. MAIN OUTCOME MEASURES:Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. RESULTS:The mean ages of patients with sepsis and controls were 71.7 (SD, 15.9) and 52.7 (SD, 15.0) years, respectively. The median number of ICU days for patients with sepsis was 8 (range, 1-195 days), while control patients were in ICUs for 4 or fewer days. The median duration of sepsis was 4 days (range, 1-40 days). Compared with controls, anti-CD3/anti-CD28-stimulated splenocytes from sepsis patients had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5361 (95% CI, 3327-7485) pg/mL vs 418 (95% CI, 98-738) pg/mL; interferon γ, 1374 (95% CI, 550-2197) pg/mL vs 37.5 (95% CI, -5 to 80) pg/mL; interleukin 6, 3691 (95% CI, 2313-5070) vs 365 (95% CI, 87-642) pg/mL; and interleukin 10, 633 (95% CI, -269 to 1534) vs 58 (95% CI, -39 to 156) pg/mL; (P < .001 for all). There were similar reductions in 5-hour lipopolysaccharide-stimulated cytokine secretion. Cytokine secretion in sepsis patients was generally less than 10% that in controls, independent of age, duration of sepsis, corticosteroid use, and nutritional status. Although differences existed between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of sepsis patients vs cancer patients and vs transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells. CONCLUSIONS:Patients who die in the ICU following sepsis compared with patients who die of nonsepsis etiologies have biochemical, flow cytometric, and immunohistochemical findings consistent with immunosuppression. Targeted immune-enhancing therapy may be a valid approach in selected patients with sepsis.
Expansion of somatically reverted memory CD8+ T cells in patients with X-linked lymphoproliferative disease caused by selective pressure from Epstein-Barr virus.
Palendira Umaimainthan,Low Carol,Bell Andrew I,Ma Cindy S,Abbott Rachel J M,Phan Tri Giang,Riminton D Sean,Choo Sharon,Smart Joanne M,Lougaris Vassilios,Giliani Silvia,Buckley Rebecca H,Grimbacher Bodo,Alvaro Frank,Klion Amy D,Nichols Kim E,Adelstein Stephen,Rickinson Alan B,Tangye Stuart G
The Journal of experimental medicine
Patients with the primary immunodeficiency X-linked lymphoproliferative disease (XLP), which is caused by mutations in SH2D1A, are highly susceptible to Epstein-Barr virus (EBV) infection. Nonetheless, some XLP patients demonstrate less severe clinical manifestations after primary infection. SH2D1A encodes the adaptor molecule SLAM-associated protein (SAP), which is expressed in T and natural killer cells and is required for cytotoxicity against B cells, the reservoir for EBV. It is not known why the clinical presentation of XLP is so variable. In this study, we report for the first time the occurrence of somatic reversion in XLP. Reverted SAP-expressing cells resided exclusively within the CD8(+) T cell subset, displayed a CD45RA(-)CCR7(-) effector memory phenotype, and were maintained at a stable level over time. Importantly, revertant CD8(+) SAP(+) T cells, but not SAP(-) cells, proliferated in response to EBV and killed EBV-infected B cells. As somatic reversion correlated with EBV infection, we propose that the virus exerts a selective pressure on the reverted cells, resulting in their expansion in vivo and host protection against ongoing infection.
Human invariant natural killer T cells acquire transient innate responsiveness via histone H4 acetylation induced by weak TCR stimulation.
Wang Xiaohua,Bishop Kathleen A,Hegde Subramanya,Rodenkirch Lance A,Pike J Wesley,Gumperz Jenny E
The Journal of experimental medicine
Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent T(H1) differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.
A role for GPx3 in activity of normal and leukemia stem cells.
Herault Olivier,Hope Kristin J,Deneault Eric,Mayotte Nadine,Chagraoui Jalila,Wilhelm Brian T,Cellot Sonia,Sauvageau Martin,Andrade-Navarro Miguel A,Hébert Josée,Sauvageau Guy
The Journal of experimental medicine
The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.
Dll4-Notch signaling in Flt3-independent dendritic cell development and autoimmunity in mice.
Billiard Fabienne,Lobry Camille,Darrasse-Jèze Guillaume,Waite Janelle,Liu Xia,Mouquet Hugo,DaNave Amanda,Tait Michelle,Idoyaga Juliana,Leboeuf Marylène,Kyratsous Christos A,Burton Jacquelynn,Kalter Julie,Klinakis Apostolos,Zhang Wen,Thurston Gavin,Merad Miriam,Steinman Ralph M,Murphy Andrew J,Yancopoulos George D,Aifantis Iannis,Skokos Dimitris
The Journal of experimental medicine
Delta-like ligand 4 (Dll4)-Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4-Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg) cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural T(reg) cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4(-)CD8(-)c-kit(+)CD44(+)CD25(-) (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4(+) T cells into T(reg) cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of T(reg) cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a T(reg) cell-mediated mechanism and inhibits CD8(+) T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased T(reg) cell numbers in the pancreas-draining lymph nodes. These results identify Dll4-Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated T(reg) cell homeostasis and autoimmunity.
B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells.
Barr Tom A,Shen Ping,Brown Sheila,Lampropoulou Vicky,Roch Toralf,Lawrie Sarah,Fan Boli,O'Connor Richard A,Anderton Stephen M,Bar-Or Amit,Fillatreau Simon,Gray David
The Journal of experimental medicine
B cells have paradoxical roles in autoimmunity, exerting both pathogenic and protective effects. Pathogenesis may be antibody independent, as B cell depletion therapy (BCDT) leads to amelioration of disease irrespective of autoantibody ablation. However, the mechanisms of pathogenesis are poorly understood. We demonstrate that BCDT alleviates central nervous system autoimmunity through ablation of IL-6-secreting pathogenic B cells. B cells from mice with experimental autoimmune encephalomyelitis (EAE) secreted elevated levels of IL-6 compared with B cells from naive controls, and mice with a B cell-specific IL-6 deficiency showed less severe disease than mice with wild-type B cells. Moreover, BCDT ameliorated EAE only in mice with IL-6-sufficient B cells. This mechanism of pathogenesis may also operate in multiple sclerosis (MS) because B cells from MS patients produced more IL-6 than B cells from healthy controls, and this abnormality was normalized with B cell reconstitution after Rituximab treatment. This suggests that BCDT improved disease progression, at least partly, by eliminating IL-6-producing B cells in MS patients. Taking these data together, we conclude that IL-6 secretion is a major mechanism of B cell-driven pathogenesis in T cell-mediated autoimmune disease such as EAE and MS.
Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays.
Gribble Susan M,Ng Bee Ling,Prigmore Elena,Fitzgerald Tomas,Carter Nigel P
Array painting is a technique that uses microarray technology to rapidly map chromosome translocation breakpoints. Previous methods to map translocation breakpoints have used fluorescence in situ hybridization (FISH) and have consequently been labor-intensive, time-consuming and restricted to the low breakpoint resolution imposed by the use of metaphase chromosomes. Array painting combines the isolation of derivative chromosomes (chromosomes with translocations) and high-resolution microarray analysis to refine the genomic location of translocation breakpoints in a single experiment. In this protocol, we describe array painting by isolation of derivative chromosomes using a MoFlo flow sorter, amplification of these derivatives using whole-genome amplification and hybridization onto commercially available oligonucleotide microarrays. Although the sorting of derivative chromosomes is a specialized procedure requiring sophisticated equipment, the amplification, labeling and hybridization of DNA is straightforward, robust and can be completed within 1 week. The protocol described produces good quality data; however, array painting is equally achievable using any combination of the available alternative methodologies for chromosome isolation, amplification and hybridization.
A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood.
Duda Dan G,Cohen Kenneth S,Scadden David T,Jain Rakesh K
Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2-2.5 h, and is expected to detect 0.1-6.0% viable CECs and 0.01-0.20% CPCs within blood mononuclear cell population.
Kinetics of adult hematopoietic stem cell differentiation in vivo.
Upadhaya Samik,Sawai Catherine M,Papalexi Efthymia,Rashidfarrokhi Ali,Jang Geunhyo,Chattopadhyay Pratip,Satija Rahul,Reizis Boris
The Journal of experimental medicine
Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.
Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis.
Gössl Mario,Mödder Ulrike I,Atkinson Elizabeth J,Lerman Amir,Khosla Sundeep
Journal of the American College of Cardiology
OBJECTIVES:This study was designed to test whether patients with coronary atherosclerosis have increases in circulating endothelial progenitor cells (EPCs) expressing an osteogenic phenotype. BACKGROUND:Increasing evidence indicates a link between bone and the vasculature, and bone marrow and circulating osteogenic cells have been identified by staining for the osteoblastic marker, osteocalcin (OCN). Endothelial progenitor cells contribute to vascular repair, but repair of vascular injury may result in calcification. Using cell surface markers (CD34, CD133, kinase insert domain receptor [KDR]) to identify EPCs, we examined whether patients with coronary atherosclerosis had increases in the percentage of EPCs expressing OCN. METHODS:We studied 72 patients undergoing invasive coronary assessment: control patients (normal coronary arteries and no endothelial dysfunction, n = 21) versus 2 groups with coronary atherosclerosis-early coronary atherosclerosis (normal coronary arteries but with endothelial dysfunction, n = 22) and late coronary atherosclerosis (severe, multivessel coronary artery disease, n = 29). Peripheral blood mononuclear cells were analyzed using flow cytometry. RESULTS:Compared with control patients, patients with early or late coronary atherosclerosis had significant increases (approximately 2-fold) in the percentage of CD34+/KDR+ and CD34+/CD133+/KDR+ cells costaining for OCN. Even larger increases were noted in the early and late coronary atherosclerosis patients in the percentage of CD34+/CD133-/KDR+ cells costaining for OCN (5- and 2-fold, p < 0.001 and 0.05, respectively). CONCLUSIONS:A higher percentage of EPCs express OCN in patients with coronary atherosclerosis compared with subjects with normal endothelial function and no structural coronary artery disease. These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary atherosclerosis.
Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction.
Chen Guo-Yun,Chen Xi,King Samantha,Cavassani Karen A,Cheng Jiansong,Zheng Xincheng,Cao Hongzhi,Yu Hai,Qu Jingyao,Fang Dexing,Wu Wei,Bai Xue-Feng,Liu Jin-Qing,Woodiga Shireen A,Chen Chong,Sun Lei,Hogaboam Cory M,Kunkel Steven L,Zheng Pan,Liu Yang
Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction [corrected] of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid-based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid-based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.
Deficiency of Prebiotic Fiber and Insufficient Signaling Through Gut Metabolite-Sensing Receptors Leads to Cardiovascular Disease.
Kaye David M,Shihata Waled A,Jama Hamdi A,Tsyganov Kirill,Ziemann Mark,Kiriazis Helen,Horlock Duncan,Vijay Amrita,Giam Beverly,Vinh Antony,Johnson Chad,Fiedler April,Donner Daniel,Snelson Matthew,Coughlan Melinda T,Phillips Sarah,Du Xiao-Jun,El-Osta Assam,Drummond Grant,Lambert Gavin W,Spector Tim D,Valdes Ana M,Mackay Charles R,Marques Francine Z
BACKGROUND:High blood pressure (BP) continues to be a major, poorly controlled but modifiable risk factor for cardiovascular death. Among key Western lifestyle factors, a diet poor in fiber is associated with prevalence of high BP. The impact of lack of prebiotic fiber and the associated mechanisms that lead to higher BP are unknown. Here we show that lack of prebiotic dietary fiber leads to the development of a hypertensinogenic gut microbiota, hypertension and its complications, and demonstrate a role for G-protein coupled-receptors (GPCRs) that sense gut metabolites. METHODS:One hundred seventy-nine mice including C57BL/6J, gnotobiotic C57BL/6J, and knockout strains for GPR41, GPR43, GPR109A, and GPR43/109A were included. C57BL/6J mice were implanted with minipumps containing saline or a slow-pressor dose of angiotensin II (0.25 mg·kg·d). Mice were fed diets lacking prebiotic fiber with or without addition of gut metabolites called short-chain fatty acids ([SCFA)] produced during fermentation of prebiotic fiber in the large intestine), or high prebiotic fiber diets. Cardiac histology and function, BP, sodium and potassium excretion, gut microbiome, flow cytometry, catecholamines and methylation-wide changes were determined. RESULTS:Lack of prebiotic fiber predisposed mice to hypertension in the presence of a mild hypertensive stimulus, with resultant pathological cardiac remodeling. Transfer of a hypertensinogenic microbiota to gnotobiotic mice recapitulated the prebiotic-deprived hypertensive phenotype, including cardiac manifestations. Reintroduction of SCFAs to fiber-depleted mice had protective effects on the development of hypertension, cardiac hypertrophy, and fibrosis. The cardioprotective effect of SCFAs were mediated via the cognate SCFA receptors GPR43/GPR109A, and modulated L-3,4-dihydroxyphenylalanine levels and the abundance of T regulatory cells regulated by DNA methylation. CONCLUSIONS:The detrimental effects of low fiber Westernized diets may underlie hypertension, through deficient SCFA production and GPR43/109A signaling. Maintaining a healthy, SCFA-producing microbiota is important for cardiovascular health.
Noise in gene expression is coupled to growth rate.
Keren Leeat,van Dijk David,Weingarten-Gabbay Shira,Davidi Dan,Jona Ghil,Weinberger Adina,Milo Ron,Segal Eran
Genetically identical cells exposed to the same environment display variability in gene expression (noise), with important consequences for the fidelity of cellular regulation and biological function. Although population average gene expression is tightly coupled to growth rate, the effects of changes in environmental conditions on expression variability are not known. Here, we measure the single-cell expression distributions of approximately 900 Saccharomyces cerevisiae promoters across four environmental conditions using flow cytometry, and find that gene expression noise is tightly coupled to the environment and is generally higher at lower growth rates. Nutrient-poor conditions, which support lower growth rates, display elevated levels of noise for most promoters, regardless of their specific expression values. We present a simple model of noise in expression that results from having an asynchronous population, with cells at different cell-cycle stages, and with different partitioning of the cells between the stages at different growth rates. This model predicts non-monotonic global changes in noise at different growth rates as well as overall higher variability in expression for cell-cycle-regulated genes in all conditions. The consistency between this model and our data, as well as with noise measurements of cells growing in a chemostat at well-defined growth rates, suggests that cell-cycle heterogeneity is a major contributor to gene expression noise. Finally, we identify gene and promoter features that play a role in gene expression noise across conditions. Our results show the existence of growth-related global changes in gene expression noise and suggest their potential phenotypic implications.
Blockade of transforming growth factor beta upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa.
Di Sabatino A,Pickard K M,Rampton D,Kruidenier L,Rovedatti L,Leakey N A B,Corazza G R,Monteleone G,MacDonald T T
BACKGROUND AND AIMS:The role of transforming growth factor beta (TGFbeta) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFbeta inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFbeta blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon gamma (IFN gamma) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. METHODS:TGFbeta transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFbeta neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFN gamma, tumour necrosis factor alpha (TNFalpha), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. RESULTS:A higher number of TGFbeta transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFbeta antibody than in those cultured with control antibody. TGFbeta blockade downregulated T cell apoptosis, and induced a significant increase in IFN gamma, TNFalpha, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFbeta antibody. CONCLUSIONS:The findings support a crucial role for TGFbeta in dampening T cell-mediated tissue-damaging responses in the human gut.
Endocytosis promotes rapid dopaminergic signaling.
Kotowski Sarah J,Hopf F Woodward,Seif Taban,Bonci Antonello,von Zastrow Mark
D(1) dopamine receptors are primary mediators of dopaminergic signaling in the CNS. These receptors internalize rapidly following agonist-induced activation, but the functional significance of this process is unknown. We investigated D(1) receptor endocytosis and signaling in HEK293 cells and cultured striatal neurons using real-time fluorescence imaging and cAMP biosensor technology. Agonist-induced activation of D(1) receptors promoted endocytosis of receptors with a time course overlapping that of acute cAMP accumulation. Inhibiting receptor endocytosis blunted acute D(1) receptor-mediated signaling in both dissociated cells and striatal slice preparations. Although endocytic inhibition markedly attenuated acute cAMP accumulation, inhibiting the subsequent recycling of receptors had no effect. Further, D(1) receptors localized in close proximity to endomembrane-associated trimeric G protein and adenylyl cyclase immediately after endocytosis. Together, these results suggest a previously unanticipated role of endocytosis, and the early endocytic pathway, in supporting rapid dopaminergic neurotransmission.
The alveolar immune cell landscape is dysregulated in checkpoint inhibitor pneumonitis.
Suresh Karthik,Naidoo Jarushka,Zhong Qiong,Xiong Ye,Mammen Jennifer,de Flores Marcia Villegas,Cappelli Laura,Balaji Aanika,Palmer Tsvi,Forde Patrick M,Anagnostou Valsamo,Ettinger David S,Marrone Kristen A,Kelly Ronan J,Hann Christine L,Levy Benjamin,Feliciano Josephine L,Lin Cheng-Ting,Feller-Kopman David,Lerner Andrew D,Lee Hans,Shafiq Majid,Yarmus Lonny,Lipson Evan J,Soloski Mark,Brahmer Julie R,Danoff Sonye K,D'Alessio Franco
The Journal of clinical investigation
BACKGROUND:Checkpoint inhibitor pneumonitis (CIP) is a highly morbid complication of immune checkpoint immunotherapy (ICI), one which precludes the continuation of ICI. Yet, the mechanistic underpinnings of CIP are unknown. METHODS:To better understand the mechanism of lung injury in CIP, we prospectively collected bronchoalveolar lavage (BAL) samples in ICI-treated patients with (n=12) and without CIP (n=6), prior to initiation of first-line therapy for CIP (high dose corticosteroids. We analyzed BAL immune cell populations using a combination of traditional multicolor flow cytometry gating, unsupervised clustering analysis and BAL supernatant cytokine measurements. RESULTS:We found increased BAL lymphocytosis, predominantly CD4+ T cells, in CIP. Specifically, we observed increased numbers of BAL central memory T-cells (Tcm), evidence of Type I polarization, and decreased expression of CTLA-4 and PD-1 in BAL Tregs, suggesting both activation of pro-inflammatory subsets and an attenuated suppressive phenotype. CIP BAL myeloid immune populations displayed enhanced expression of IL-1β and decreased expression of counter-regulatory IL-1RA. We observed increased levels of T cell chemoattractants in the BAL supernatant, consistent with our pro-inflammatory, lymphocytic cellular landscape. CONCLUSION:We observe several immune cell subpopulations that are dysregulated in CIP, which may represent possible targets that could lead to therapeutics for this morbid immune related adverse event.
Treatment of Relapsing HPV Diseases by Restored Function of Natural Killer Cells.
The New England journal of medicine
Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene (), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.).
Antisense therapy against CCR3 and the common beta chain attenuates allergen-induced eosinophilic responses.
Gauvreau Gail M,Boulet Louis Philippe,Cockcroft Donald W,Baatjes Adrian,Cote Johanne,Deschesnes Francine,Davis Beth,Strinich Tara,Howie Karen,Duong Mylinh,Watson Richard M,Renzi Paolo M,O'Byrne Paul M
American journal of respiratory and critical care medicine
RATIONALE:The drug product TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides designed to inhibit allergic inflammation by down-regulating human CCR3 and the common beta chain (beta(c)) of IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors. OBJECTIVES:This study examined the effects of inhaled TPI ASM8 on sputum cellular influx, CCR3 and beta(c) mRNA and protein levels, and the airway physiologic response after inhaled allergen. METHODS:Seventeen subjects with mild atopic asthma were randomized in a crossover study to inhale 1,500 microg TPI ASM8 or placebo by nebulizer, once daily for 4 days. On Day 3, subjects underwent allergen inhalation challenge. Sputum samples were collected before and after allergen. CCR3 and beta(c) protein levels were measured by flow cytometry, mRNA was measured using real-time quantitative polymerase chain reaction, and the FEV1 was measured over 7 hours after challenge. MEASUREMENTS AND MAIN RESULTS:Compared with placebo, TPI ASM8 inhibited sputum eosinophil influx by 46% (P = 0.02) and blunted the increase in total cells (63%) after allergen challenge. TPI ASM8 significantly reduced the early asthmatic response (P = 0.04) with a trend for the late asthmatic response (P = 0.08). The allergen-induced (Day 2 to Day 3) levels of beta(c) mRNA and CCR3 mRNA in sputum-derived cells were inhibited by TPI ASM8 (P = 0.039 and P = 0.054, respectively), with no significant effects on the cell surface protein expression of CCR3 and beta(c) (P > 0.05). No serious adverse events were reported. CONCLUSIONS:TPI ASM8 attenuates the allergen-induced increase in target gene mRNA and airway responses in subjects with mild asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 00264966).
Ontogeny-recapitulating generation and tissue integration of ES cell-derived Purkinje cells.
Muguruma Keiko,Nishiyama Ayaka,Ono Yuichi,Miyawaki Hiroyuki,Mizuhara Eri,Hori Seiji,Kakizuka Akira,Obata Kunihiko,Yanagawa Yuchio,Hirano Tomoo,Sasai Yoshiki
Purkinje cells are the sole output neurons of the cerebellar cortex and their dysfunction causes severe ataxia. We found that Purkinje cells could be robustly generated from mouse embryonic stem (ES) cells by recapitulating the self-inductive signaling microenvironments of the isthmic organizer. The cell-surface marker Neph3 enabled us to carry out timed prospective selection of Purkinje cell progenitors, which generated morphologically characteristic neurons with highly arborized dendrites that expressed mature Purkinje cell-specific markers such as the glutamate receptor subunit GluRδ2. Similar to mature Purkinje cells, these neurons also showed characteristic spontaneous and repeated action potentials and their postsynaptic excitatory potentials were generated exclusively through nonNMDA glutamate receptors. Fetal transplantation of precursors isolated by fluorescence-activated cell sorting showed orthotopic integration of the grafted neurons into the Purkinje cell layer with their axons extending to the deep cerebellar nuclei and dendrites receiving climbing and parallel fibers. This selective preparation of bona fide Purkinje cells should aid future investigation of this important neuron.
Transcription and histone methylation changes correlate with imprint acquisition in male germ cells.
Henckel Amandine,Chebli Karim,Kota Satya K,Arnaud Philippe,Feil Robert
The EMBO journal
Genomic imprinting in mammals is controlled by DNA methylation imprints that are acquired in the gametes, at essential sequence elements called 'imprinting control regions' (ICRs). What signals paternal imprint acquisition in male germ cells remains unknown. To address this question, we explored histone methylation at ICRs in mouse primordial germ cells (PGCs). By 13.5 days post coitum (d.p.c.), H3 lysine-9 and H4 lysine-20 trimethylation are depleted from ICRs in male (and female) PGCs, indicating that these modifications do not signal subsequent imprint acquisition, which initiates at ∼15.5 d.p.c. Furthermore, during male PGC development, H3 lysine-4 trimethylation becomes biallelically enriched at 'maternal' ICRs, which are protected against DNA methylation, and whose promoters are active in the male germ cells. Remarkably, high transcriptional read-through is detected at the paternal ICRs H19-DMR and Ig-DMR at the time of imprint establishment, from one of the strands predominantly. Combined, our data evoke a model in which differential histone modification states linked to transcriptional events may signal the specificity of imprint acquisition during spermatogenesis.
CD44 deficiency attenuates chronic murine ileitis.
Collins Colm B,Ho Johnson,Wilson Theodore E,Wermers Joshua D,Tlaxca José L,Lawrence Michael B,Solga Michael,Lannigan Joanne,Rivera-Nieves Jesús
BACKGROUND & AIMS:Lymphocyte recruitment to sites of inflammation requires the sequential engagement of adhesion molecules and chemokine receptors. In the current studies we analyzed the role of CD44 for the development of chronic small-intestinal inflammatory infiltrates in vivo. METHODS:By using a tumor necrosis factor (TNF)-driven model of chronic ileitis (ie, B6.129P-TNF(DeltaAU-rich element [ARE])) that recapitulates many features of Crohn's disease, we noticed dynamic changes in the expression and functional state of CD44 and its ligand hyaluronan via enzyme-linked immunosorbent assay, real-time reverse-transcription polymerase chain reaction, immunohistochemistry, and flow cytometry. In addition, we assessed the role of lymphocyte populations during induction of ileitis through adoptive transfer studies, and generated CD44-deficient TNFDeltaARE mice to assess the role of CD44 for development of ileitis. RESULTS:Soluble hyaluronan levels and expression of hyaluronan synthase-1 were increased in TNFDeltaARE mice. This coincided with increased expression of CD44 (including variant 7) and reactivity towards hyaluronan on CD4(+) T cells. CD44 was spatially colocalized with the gut-homing integrin alpha(4)beta(7), spatially linking lymphocyte rolling with arrest. These cells had an effector phenotype because they lacked L-selectin and a higher proportion in diseased mice produced TNF and interleukin-2 compared with wild-type littermates. Lastly, CD4(+) but not CD8(+) T cells conferred ileitis to RAG(-/-) recipients and deficiency of one or both alleles of the CD44 gene resulted in attenuation of the severity of ileitis in TNFDeltaARE mice. CONCLUSIONS:Our findings support an important role of CD44 expressed by CD4(+) and CD8(+) for development of ileitis mediated by TNF overproduction.
Control and signal processing by transcriptional interference.
Buetti-Dinh Antoine,Ungricht Rosemarie,Kelemen János Z,Shetty Chetak,Ratna Prasuna,Becskei Attila
Molecular systems biology
A transcriptional activator can suppress gene expression by interfering with transcription initiated by another activator. Transcriptional interference has been increasingly recognized as a regulatory mechanism of gene expression. The signals received by the two antagonistically acting activators are combined by the polymerase trafficking along the DNA. We have designed a dual-control genetic system in yeast to explore this antagonism systematically. Antagonism by an upstream activator bears the hallmarks of competitive inhibition, whereas a downstream activator inhibits gene expression non-competitively. When gene expression is induced weakly, the antagonistic activator can have a positive effect and can even trigger paradoxical activation. Equilibrium and non-equilibrium models of transcription shed light on the mechanism by which interference converts signals, and reveals that self-antagonism of activators imitates the behavior of feed-forward loops. Indeed, a synthetic circuit generates a bell-shaped response, so that the induction of expression is limited to a narrow range of the input signal. The identification of conserved regulatory principles of interference will help to predict the transcriptional response of genes in their genomic context.
Myeloid dendritic cells of patients with chronic HCV infection induce proliferation of regulatory T lymphocytes.
Dolganiuc Angela,Paek Edward,Kodys Karen,Thomas Joanne,Szabo Gyongyi
BACKGROUND & AIMS:Dendritic cells (DCs) initiate and sustain an efficient T-lymphocyte response. Chronic hepatitis C virus (HCV) infection is associated with inefficient T-cell functions that fail to eradicate the virus, so defects in DC function might be involved in HCV pathogenesis. This study analyzed the activities of myeloid DCs and distinct CD4(+) T-cell populations in samples collected from patients with HCV. METHODS:The abilities of primary BDCA1(+) or monocyte-derived DCs from HCV patients (HCV-DC) to stimulate CD4(+), CD4(+)CD25(-), or different ratios of CD4(+)CD25(+)/CD4(+)CD25(-) T cells were evaluated in mixed lymphocyte reactions. T-cell proliferation and phenotype were evaluated by flow cytometry; cytokine production was evaluated by enzyme-linked immunosorbent assay and marker expression by polymerase chain reaction analyses. RESULTS:HCV-DCs were poor activators of CD4(+) T cells; this defect was reversed by addition of interleukin-2, neutralization of interleukin-10, or elimination of CD4(+)CD25(+) T cells. HCV-DC stimulated proliferation of regulatory T cells (Tregs; CD4(+)CD25(+)FoxP3(+)), which limit proliferation of HCV-specific T lymphocytes. We observed an increased frequency of CD4(+)CD25(+) T cells in peripheral blood of HCV patients and that HCV-DC overexpressed a number of alternative costimulatory molecules, including PD-L1. Finally, HCV-DC stimulated expansion rather than de novo induction of FoxP3(+) Tregs. CONCLUSIONS:Our results indicate a role for myeloid DC in expansion of Tregs to promote chronic infection of patients with HCV.
Helicobacter pylori lipopolysaccharide interacts with TFF1 in a pH-dependent manner.
Reeves Emer P,Ali Tehmeena,Leonard Paul,Hearty Stephen,O'Kennedy Richard,May Felicity E B,Westley Bruce R,Josenhans Christine,Rust Melanie,Suerbaum Sebastian,Smith Angeline,Drumm Brendan,Clyne Marguerite
BACKGROUND & AIMS:Little is known about how bacteria establish chronic infections of mucosal surfaces. Helicobacter pylori (H. pylori), a chronic pathogen that lives in the gastric mucosa of humans, interacts with the trefoil factor family (TFF) protein TFF1, which is found in gastric mucus. We aimed to characterize the interaction of H. pylori with TFF1 and to assess the role of this interaction in mediating colonization. METHODS:Subcellular fractions of H. pylori were immobilized and then probed with TFF1, TFF2, or TFF3. The effect of glycosidases and preincubation with monosaccharides on the interaction and binding of TFF1 to a H. pylori adhesin was assessed. The interaction between H. pylori adhesin and TFF1 was characterized using surface plasmon resonance, flow cytometry, nondenaturing polyacrylamide gel electrophoresis, coimmunofluoresence, and incubation with tissue sections. RESULTS:The H. pylori core oligosaccharide portion (rough form) of lipopolysaccharide (RF-LPS) bound to TFF1 and to a lesser extent TFF3; this interaction was inhibited by incubation of RF-LPS with mannosidase, glucosidase, or mixed monosaccharides. TFF1 also bound to human serum albumin-conjugated mannose and glucose. The optimum pH for binding was 5.0-6.0 for TFF1 and 7.0 for TFF3. H. pylori bound TFF1 in gastric mucus ex vivo; binding of LPS-coated latex beads to human antral gastric tissue was inhibited by TFF1. CONCLUSIONS:TFF1 interacts specifically with H. pylori RF-LPS. The pH dependence of this interaction indicates that binding of H. pylori to TFF1 in the stomach could promote colonization of the mucus layer adjacent to the gastric epithelial surface.
SARAF inactivates the store operated calcium entry machinery to prevent excess calcium refilling.
Palty Raz,Raveh Adi,Kaminsky Ido,Meller Ruth,Reuveny Eitan
Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Using heterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.
Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.
Jeselsohn Rinath,Brown Nelson E,Arendt Lisa,Klebba Ina,Hu Miaofen G,Kuperwasser Charlotte,Hinds Philip W
Transplantation studies have demonstrated the existence of mammary progenitor cells with the ability to self-renew and regenerate a functional mammary gland. Although these progenitors are the likely targets for oncogenic transformation, correlating progenitor populations with certain oncogenic stimuli has been difficult. Cyclin D1 is required for lobuloalveolar development during pregnancy and lactation as well as MMTV-ErbB2- but not MMTV-Wnt1-mediated tumorigenesis. Using a kinase-deficient cyclin D1 mouse, we identified two functional mammary progenitor cell populations, one of which is the target of MMTV-ErbB2. Moreover, cyclin D1 activity is required for the self-renewal and differentiation of mammary progenitors because its abrogation leads to a failure to maintain the mammary epithelial regenerative potential and also results in defects in luminal lineage differentiation.
CD28 Signaling Controls Metabolic Fitness of Pathogenic T Cells in Medium and Large Vessel Vasculitis.
Zhang Hui,Watanabe Ryu,Berry Gerald J,Nadler Steven G,Goronzy Jörg J,Weyand Cornelia M
Journal of the American College of Cardiology
BACKGROUND:In giant cell arteritis, vessel-wall infiltrating CD4 T cells and macrophages form tissue-destructive granulomatous infiltrates, and the artery responds with a maladaptive response to injury, leading to intramural neoangiogenesis, intimal hyperplasia, and luminal occlusion. Lesion-residing T cells receive local signals, which represent potential therapeutic targets. OBJECTIVES:The authors examined how CD28 signaling affects vasculitis induction and maintenance, and which pathogenic processes rely on CD28-mediated T-cell activation. METHODS:Vasculitis was induced by transferring peripheral blood mononuclear cells from giant cell arteritis patients into immunodeficient NSG mice engrafted with human arteries. Human artery-NSG chimeras were treated with anti-CD28 domain antibody or control antibody. Treatment effects and immunosuppressive mechanisms were examined in vivo and in vitro applying tissue transcriptome analysis, immunohistochemistry, flow cytometry, and immunometabolic analysis. RESULTS:Blocking CD28-dependent signaling markedly reduced tissue-infiltrating T cells and effectively suppressed vasculitis. Mechanistic studies implicated CD28 in activating AKT signaling, T-cell proliferation and differentiation of IFN-γ and IL-21-producing effector T cells. Blocking CD28 was immunosuppressive by disrupting T-cell metabolic fitness; specifically, the ability to utilize glucose. Expression of the glucose transporter Glut1 and of glycolytic enzymes as well as mitochondrial oxygen consumption were all highly sensitive to CD28 blockade. Also, induction and maintenance of CD4CD103 tissue-resident memory T cells, needed to replenish the vasculitic infiltrates, depended on CD28 signaling. CD28 blockade effectively suppressed vasculitis-associated remodeling of the vessel wall. CONCLUSIONS:CD28 stimulation provides a metabolic signal required for pathogenic effector functions in medium and large vessel vasculitis. Disease-associated glycolytic activity in wall-residing T-cell populations can be therapeutically targeted by blocking CD28 signaling.
Proliferation assay of human gastric remnant by bromodeoxyuridine and flow cytometry.
Ohyama S,Yonemura Y,Miwa K,Miyazaki I,Sasaki T
The cell kinetics of gastric epithelium were studied by bromodeoxyuridine (BrdU) and flow cytometry in seven patients with remnant stomachs reconstructed with a Billroth type II procedure and in 25 patients with whole stomachs. Each patient received an intravenous injection of BrdU (200 mg/m2) 6 hours before surgery. Fresh specimens obtained from the lesser curvature, greater curvature, and stomal areas in the cases of remnant stomachs and from the antrum and fundus in the case of whole stomachs were studied. The BrdU labeling index was higher in the stomal area of the gastric remnant than in other areas, and DNA synthesis time was shortened in the stomal area of the gastric remnant (P less than 0.01) but not in other areas. The turnover time of the mucosa in the stomal area was 4.1 +/- 1.2 days, significantly shorter (P less than 0.01) than in other areas (7.6 +/- 2.3 to 8.2 +/- 1.2 days). The present study showed that the cell proliferation was extremely rapid in the stomal area of the gastric remnant, suggesting that this enhanced turnover of epithelial cells may assist in promoting carcinogenesis in the stomal area of the gastric remnant.
Plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes.
Koyama Shohei,Aoshi Taiki,Tanimoto Takeshi,Kumagai Yutaro,Kobiyama Kouji,Tougan Takahiro,Sakurai Kazuo,Coban Cevayir,Horii Toshihiro,Akira Shizuo,Ishii Ken J
Science translational medicine
A variety of different vaccine types are available for H1N1 influenza A virus infections; however, their immunological mechanisms of action remain unclear. Here, we show that plasmacytoid dendritic cells (pDCs) and type I interferon (IFN)-mediated signaling delineate the immunogenicity of live attenuated virus, inactivated whole-virus (WV), and split-virus vaccines. Although Toll-like receptor 7 acted as the adjuvant receptor for the immunogenicity of both live virus and WV vaccines, the requirement for type I IFN production by pDCs for the immunogenicity of the vaccines was restricted to WV. A split vaccine commonly used in humans failed to immunize naïve mice, but a pDC-activating adjuvant could restore immunogenicity. In blood from human adults, however, split vaccine alone could recall memory T cell responses, underscoring the importance of this adjuvant pathway for primary, but not secondary, vaccination.
Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.
Baladi Tom,Nilsson Jesper R,Gallud Audrey,Celauro Emanuele,Gasse Cécile,Levi-Acobas Fabienne,Sarac Ivo,Hollenstein Marcel R,Dahlén Anders,Esbjörner Elin K,Wilhelmsson L Marcus
Journal of the American Chemical Society
Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tC, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tC in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tC is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tC-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tC-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.
Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6-blockade-refractory idiopathic multicentric Castleman disease.
Fajgenbaum David C,Langan Ruth-Anne,Japp Alberto Sada,Partridge Helen L,Pierson Sheila K,Singh Amrit,Arenas Daniel J,Ruth Jason R,Nabel Christopher S,Stone Katie,Okumura Mariko,Schwarer Anthony,Jose Fábio Freire,Hamerschlak Nelson,Wertheim Gerald B,Jordan Michael B,Cohen Adam D,Krymskaya Vera,Rubenstein Arthur,Betts Michael R,Kambayashi Taku,van Rhee Frits,Uldrick Thomas S
The Journal of clinical investigation
BACKGROUND:Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6(IL-6)-blockade refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6-blockade refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype. METHODS:We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus. RESULTS:Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months. CONCLUSION:This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904). FUNDING:Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.
Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development.
Doulatov Sergei,Notta Faiyaz,Eppert Kolja,Nguyen Linh T,Ohashi Pamela S,Dick John E
The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. Here we provide a comprehensive analysis of the human hematopoietic hierarchy by clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow. Human multilymphoid progenitors, identified as a distinct population of Thy-1(neg-lo)CD45RA(+) cells in the CD34(+)CD38(-) stem cell compartment, gave rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells, which indicated that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation.
Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions.
Connelly John T,Gautrot Julien E,Trappmann Britta,Tan David Wei-Min,Donati Giacomo,Huck Wilhelm T S,Watt Fiona M
Nature cell biology
Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM). A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled. Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20 microm diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 microm diameter) islands. Differentiation did not depend on ECM composition or density. Rather, the actin cytoskeleton mediated shape-induced differentiation by regulating serum response factor (SRF) transcriptional activity. Knockdown of SRF or its co-factor MAL inhibited differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues are transduced into transcriptional responses that determine epidermal cell fate.
Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells.
Nucleic acids research
Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.
Bright Surface-Enhanced Raman Scattering with Fluorescence Quenching from Silica Encapsulated J-Aggregate Coated Gold Nanoparticles.
Walters Christopher M,Pao Caroline,Gagnon Brandon P,Zamecnik Colin R,Walker Gilbert C
Advanced materials (Deerfield Beach, Fla.)
Plexitonic nanoparticles offer variable optical properties through tunable excitations, in addition to electric field enhancements that far exceed molecular resonators. This study demonstrates a way to design an ultrabright surface-enhanced Raman spectroscopy (SERS) signal while simultaneously quenching the fluorescence background through silica encapsulation of the semiconductor-metal composite nanoparticles. Using a multistep approach, a J-aggregate-forming organic dye is assembled on the surface of gold nanoparticles using a cationic linker. Excitonic resonance of the J-aggregate-metal system shows an enhanced SERS signal at an appropriate excitation wavelength. Further encapsulation of the decorated particles in silica shows a significant reduction in the fluorescence signal of the Raman spectra (5× reduction) and an increase in Raman scattering (7× enhancement) when compared to phospholipid encapsulation. This reduction in fluorescence is important for maximizing the useful SERS enhancement from the particle, which shows a signal increase on the order of 10 times greater than J-aggregated dye in solution and 24 times greater than Oxonica S421 SERS tag. The silica layer also serves to promote colloidal stability. The combination of reduced fluorescence background, enhanced SERS intensity, and temporal stability makes these particles highly distinguishable with potential to enable high-throughput applications such as SERS flow cytometry.
Foxp3 expression in CD4+ T cells of patients with systemic lupus erythematosus: a comparative phenotypic analysis.
Bonelli M,von Dalwigk K,Savitskaya A,Smolen J S,Scheinecker C
Annals of the rheumatic diseases
OBJECTIVES:The forkhead family transcription factor Foxp3 currently represents the most specific marker molecule for CD4(+)CD25(+) T cells with suppressive/regulatory capacity (Treg) in the mouse. Recent studies in the human system, however, indicate that the expression of Foxp3 can be T cell activation dependent. This tempted us to evaluate the significance of Foxp3 expression under autoimmune conditions with chronic T cell activation in patients with systemic lupus erythematosus (SLE) as compared with healthy controls (HCs). METHODS:Proportions of peripheral blood CD4(+)Foxp3(+) T cells and CD4(+)CD25(high) T cells were determined in patients with active and inactive SLE as compared with HC by flow cytometry. Comparative analysis of the percentage of CD4(+)Foxp3(+) T cells and of percentage of CD4(+)CD25(high) T cells with clinical disease activity and T cell activation marker molecule expression were performed. Finally, the induction of Foxp3 expression was analysed upon T cell activation in vitro. RESULTS:Proportions of CD4(+)Foxp3(+) T cells were significantly increased in patients with SLE as compared with HC and a significant correlation was observed between clinical disease activity and proportions of CD4(+)Foxp3(+) T cells. On the other hand, proportions of CD4(+)CD25(high) were decreased in SLE and no correlation with a T cell activation marker expression of was observed. In addition, in vitro activation of T cells induced Foxp3 expression. CONCLUSIONS:Our data suggest that the expression of Foxp3 on CD4(+) T cells in patients with SLE, at least to some extent, reflects the activation of CD4(+) T cells due to underlying disease activity and does not necessarily indicate a functional regulatory T cell capacity.
Interplay between Cdh1 and JNK activity during the cell cycle.
Gutierrez Gustavo J,Tsuji Toshiya,Chen Meifan,Jiang Wei,Ronai Ze'ev A
Nature cell biology
The ubiquitin ligase APC/C(Cdh1) coordinates degradation of key cell cycle regulators. We report here that a nuclear-localized portion of the stress-activated kinase JNK is degraded by the APC/C(Cdh1) during exit from mitosis and the G1 phase of the cell cycle. Expression of a non-degradable JNK induces prometaphase-like arrest and aberrant mitotic spindle dynamics. Moreover, JNK phosphorylates Cdh1 directly, during G2 and early mitosis, changing its subcellular localization and attenuating its ability to activate the APC/C during G2/M. This regulatory mechanism between JNK and Cdh1 reveals an important function for JNK during the cell cycle.
High-Fat Diet Accelerates Carcinogenesis in a Mouse Model of Barrett's Esophagus via Interleukin 8 and Alterations to the Gut Microbiome.
Münch Natasha Stephens,Fang Hsin-Yu,Ingermann Jonas,Maurer H Carlo,Anand Akanksha,Kellner Victoria,Sahm Vincenz,Wiethaler Maria,Baumeister Theresa,Wein Frederik,Einwächter Henrik,Bolze Florian,Klingenspor Martin,Haller Dirk,Kavanagh Maria,Lysaght Joanne,Friedman Richard,Dannenberg Andrew J,Pollak Michael,Holt Peter R,Muthupalani Sureshkumar,Fox James G,Whary Mark T,Lee Yoomi,Ren Tony Y,Elliot Rachael,Fitzgerald Rebecca,Steiger Katja,Schmid Roland M,Wang Timothy C,Quante Michael
BACKGROUND & AIMS:Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Progression from BE to cancer is associated with obesity, possibly due to increased abdominal pressure and gastroesophageal reflux disease, although this pathogenic mechanism has not been proven. We investigated whether environmental or dietary factors associated with obesity contribute to the progression of BE to EAC in mice. METHODS:Tg(ED-L2-IL1RN/IL1B)#Tcw mice (a model of BE, called L2-IL1B mice) were fed a chow (control) or high-fat diet (HFD) or were crossbred with mice that express human interleukin (IL) 8 (L2-IL1B/IL8 mice). Esophageal tissues were collected and analyzed for gene expression profiles and by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. Organoids were established from BE tissue of mice and cultured with serum from lean or obese individuals or with neutrophils from L2-IL1B mice. Feces from mice were analyzed by 16s ribosomal RNA sequencing and compared to 16s sequencing data from patients with dysplasia or BE. L2-IL1B were mice raised in germ-free conditions. RESULTS:L2-IL1B mice fed an HFD developed esophageal dysplasia and tumors more rapidly than mice fed the control diet; the speed of tumor development was independent of body weight. The acceleration of dysplasia by the HFD in the L2-IL1B mice was associated with a shift in the gut microbiota and an increased ratio of neutrophils to natural killer cells in esophageal tissues compared with mice fed a control diet. We observed similar differences in the microbiomes from patients with BE that progressed to EAC vs patients with BE that did not develop into cancer. Tissues from dysplasias of L2-IL1B mice fed the HFD contained increased levels of cytokines that are produced in response to CXCL1 (the functional mouse homolog of IL8, also called KC). Serum from obese patients caused organoids from L2-IL1B/IL8 mice to produce IL8. BE tissues from L2-IL1B mice fed the HFD and from L2-IL1B/IL8 mice contained increased numbers of myeloid cells and cells expressing Cxcr2 and Lgr5 messenger RNAs (epithelial progenitors) compared with mice fed control diets. BE tissues from L2-IL1B mice raised in germ-free housing had fewer progenitor cells and developed less dysplasia than in L2-IL1 mice raised under standard conditions; exposure of fecal microbiota from L2-IL1B mice fed the HFD to L2-IL1B mice fed the control diet accelerated tumor development. CONCLUSIONS:In a mouse model of BE, we found that an HFD promoted dysplasia by altering the esophageal microenvironment and gut microbiome, thereby inducing inflammation and stem cell expansion, independent of obesity.
Protective HIV-specific CD8+ T cells evade Treg cell suppression.
Elahi Shokrollah,Dinges Warren L,Lejarcegui Nicholas,Laing Kerry J,Collier Ann C,Koelle David M,McElrath M Juliana,Horton Helen
Specific human leukocyte antigens (HLAs), notably HLA-B*27 and HLA-B*57 allele groups, have long been associated with control of HIV-1. Although the majority of HIV-specific CD8(+) T cells lose proliferative capacity during chronic infection, T cells restricted by HLA-B*27 or HLA-B*57 allele groups do not. Here we show that CD8(+) T cells restricted by 'protective' HLA allele groups are not suppressed by T(reg) cells, whereas, within the same individual, T cells restricted by 'nonprotective' alleles are highly suppressed ex vivo. This differential sensitivity of HIV-specific CD8(+) T cells to T(reg) cell-mediated suppression correlates with their expression of the inhibitory receptor T cell immunoglobulin domain and mucin domain 3 (Tim-3) after stimulation with their cognate epitopes. Furthermore, we show that HLA-B*27- and HLA-B*57-restricted effectors also evade T(reg) cell-mediated suppression by directly killing T(reg) cells they encounter in a granzyme B (GzmB)-dependent manner. This study uncovers a previously unknown explanation for why HLA-B*27 and HLA-B*57 allele groups are associated with delayed HIV-1 disease progression.
Foxp3+ follicular regulatory T cells control the germinal center response.
Linterman Michelle A,Pierson Wim,Lee Sau K,Kallies Axel,Kawamoto Shimpei,Rayner Tim F,Srivastava Monika,Divekar Devina P,Beaton Laura,Hogan Jennifer J,Fagarasan Sidonia,Liston Adrian,Smith Kenneth G C,Vinuesa Carola G
Follicular helper (T(FH)) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T(FH) numbers maintains self tolerance. We describe a population of Foxp3(+)Blimp-1(+)CD4(+) T cells constituting 10-25% of the CXCR5(high)PD-1(high)CD4(+) T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T(FR)) cells share phenotypic characteristics with T(FH) and conventional Foxp3(+) regulatory T (T(reg)) cells yet are distinct from both. Similar to T(FH) cells, T(FR) cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T(FR) cells originate from thymic-derived Foxp3(+) precursors, not naive or T(FH) cells. T(FR) cells are suppressive in vitro and limit T(FH) cell and germinal center B cell numbers in vivo. In the absence of T(FR) cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T(FH) differentiation pathway is co-opted by T(reg) cells to control the germinal center response.
Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis.
Wei Changli,El Hindi Shafic,Li Jing,Fornoni Alessia,Goes Nelson,Sageshima Junichiro,Maiguel Dony,Karumanchi S Ananth,Yap Hui-Kim,Saleem Moin,Zhang Qingyin,Nikolic Boris,Chaudhuri Abanti,Daftarian Pirouz,Salido Eduardo,Torres Armando,Salifu Moro,Sarwal Minnie M,Schaefer Franz,Morath Christian,Schwenger Vedat,Zeier Martin,Gupta Vineet,Roth David,Rastaldi Maria Pia,Burke George,Ruiz Phillip,Reiser Jochen
Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte β(3) integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte β(3) integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR-β(3) integrin interaction through antibodies and small molecules targeting either uPAR or β(3) integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS.
The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche.
Omatsu Yoshiki,Sugiyama Tatsuki,Kohara Hiroshi,Kondoh Gen,Fujii Nobutaka,Kohno Kenji,Nagasawa Takashi
Hematopoietic stem cells (HSCs) and their lympho-hematopoietic progeny are supported by microenvironmental niches within bone marrow; however, the identity, nature, and function of these niches remain unclear. Short-term ablation of CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells in vivo did not affect the candidate niches, bone-lining osteoblasts, or endothelial cells but severely impaired the adipogenic and osteogenic differentiation potential of marrow cells and production of the cytokines SCF and CXCL12 and led to a marked reduction in cycling lymphoid and erythroid progenitors. HSCs from CAR cell-depleted mice were reduced in number and cell size, were more quiescent, and had increased expression of early myeloid selector genes, similar to the phenotype of wild-type HSCs cultured without a niche. Thus, the niche composed of adipo-osteogenic progenitors is required for proliferation of HSCs and lymphoid and erythroid progenitors, as well as maintenance of HSCs in an undifferentiated state.
Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.
Chaudhary Neelkamal,Datta Kausik,Askin Frederic B,Staab Janet F,Marr Kieren A
American journal of respiratory and critical care medicine
RATIONALE:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. OBJECTIVES:To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. METHODS:A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. MEASUREMENTS AND MAIN RESULTS:Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. CONCLUSIONS:Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.
Runx1-Cbfβ facilitates early B lymphocyte development by regulating expression of Ebf1.
Seo Wooseok,Ikawa Tomokatsu,Kawamoto Hiroshi,Taniuchi Ichiro
The Journal of experimental medicine
Although Runx and Cbfβ transcription factor complexes are involved in the development of multiple hematopoietic lineages, their precise roles in early mouse B lymphocyte differentiation remain elusive. In this study, we examined mouse strains in which Runx1, Runx3, or Cbfβ were deleted in early B lineage progenitors by an mb1-cre transgene. Loss of Runx1, but not Runx3, caused a developmental block during early B lymphopoiesis, resulting in the lack of IgM(+) B cells and reduced V(H) to DJ(H) recombination. Expression of core transcription factors regulating early B cell development, such as E2A, Ebf1, and Pax5, was reduced in B cell precursors lacking Runx1. We detected binding of Runx1-Cbfβ complexes to the Ebf1 proximal promoter, and these Runx-binding motifs were essential to drive reporter gene expression. Runx1-deficient pro-B cells harbored excessive amounts of the repressive histone mark H3K27 trimethylation in the Ebf1 proximal promoter. Interestingly, retroviral transduction of Ebf1, but not Pax5, into Runx1-deficient progenitors restored not only development of B220(+) cells that underwent V(H) to DJ(H) rearrangement but also expression of B lineage signature genes. Collectively, these results demonstrate that Runx1-Cbfβ complexes are essential to facilitate B lineage specification, in part via epigenetic activation of the Ebf1 gene.
Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice.
Han May H,Lundgren Deborah H,Jaiswal Siddhartha,Chao Mark,Graham Kareem L,Garris Christopher S,Axtell Robert C,Ho Peggy P,Lock Christopher B,Woodard Joslyn I,Brownell Sara E,Zoudilova Maria,Hunt Jack F V,Baranzini Sergio E,Butcher Eugene C,Raine Cedric S,Sobel Raymond A,Han David K,Weissman Irving,Steinman Lawrence
The Journal of experimental medicine
Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.
DNA released from dying host cells mediates aluminum adjuvant activity.
Marichal Thomas,Ohata Keiichi,Bedoret Denis,Mesnil Claire,Sabatel Catherine,Kobiyama Kouji,Lekeux Pierre,Coban Cevayir,Akira Shizuo,Ishii Ken J,Bureau Fabrice,Desmet Christophe J
Aluminum-based adjuvants (aluminum salts or alum) are widely used in human vaccination, although their mechanisms of action are poorly understood. Here we report that, in mice, alum causes cell death and the subsequent release of host cell DNA, which acts as a potent endogenous immunostimulatory signal mediating alum adjuvant activity. Furthermore, we propose that host DNA signaling differentially regulates IgE and IgG1 production after alum-adjuvanted immunization. We suggest that, on the one hand, host DNA induces primary B cell responses, including IgG1 production, through interferon response factor 3 (Irf3)-independent mechanisms. On the other hand, we suggest that host DNA also stimulates 'canonical' T helper type 2 (T(H)2) responses, associated with IgE isotype switching and peripheral effector responses, through Irf3-dependent mechanisms. The finding that host DNA released from dying cells acts as a damage-associated molecular pattern that mediates alum adjuvant activity may increase our understanding of the mechanisms of action of current vaccines and help in the design of new adjuvants.
Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11.
Sasaki Masato,Kawahara Kohichi,Nishio Miki,Mimori Koshi,Kogo Ryunosuke,Hamada Koichi,Itoh Bunsho,Wang Jia,Komatsu Yukako,Yang Yong Ryoul,Hikasa Hiroki,Horie Yasuo,Yamashita Takayuki,Kamijo Takehiko,Zhang Yanping,Zhu Yan,Prives Carol,Nakano Toru,Mak Tak Wah,Sasaki Takehiko,Maehama Tomohiko,Mori Masaki,Suzuki Akira
PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G(1) and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.
Increased nicotinamide adenine dinucleotide phosphate oxidase 4 expression mediates intrinsic airway smooth muscle hypercontractility in asthma.
Sutcliffe Amanda,Hollins Fay,Gomez Edith,Saunders Ruth,Doe Camille,Cooke Marcus,Challiss R A John,Brightling Chris E
American journal of respiratory and critical care medicine
RATIONALE:Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood. OBJECTIVES:We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility. METHODS:We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility. MEASUREMENTS AND MAIN RESULTS:We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress-induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin. CONCLUSIONS:Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.
UNG shapes the specificity of AID-induced somatic hypermutation.
Pérez-Durán Pablo,Belver Laura,de Yébenes Virginia G,Delgado Pilar,Pisano David G,Ramiro Almudena R
The Journal of experimental medicine
Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.
Serum from patients with SLE instructs monocytes to promote IgG and IgA plasmablast differentiation.
Joo Hyemee,Coquery Christine,Xue Yaming,Gayet Ingrid,Dillon Stacey R,Punaro Marilynn,Zurawski Gerard,Banchereau Jacques,Pascual Virginia,Oh Sangkon
The Journal of experimental medicine
The development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). SLE serum can induce monocyte differentiation into dendritic cells (DCs) in a type I IFN-dependent manner. Such SLE-DCs activate T cells, but whether they promote B cell responses is not known. In this study, we demonstrate that SLE-DCs can efficiently stimulate naive and memory B cells to differentiate into IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. SLE-DC-mediated IgG-PB differentiation is dependent on B cell-activating factor (BAFF) and IL-10, whereas IgA-PB differentiation is dependent on a proliferation-inducing ligand (APRIL). Importantly, SLE-DCs express CD138 and trans-present CD138-bound APRIL to B cells, leading to the induction of IgA switching and PB differentiation in an IFN-α-independent manner. We further found that this mechanism of providing B cell help is relevant in vivo, as CD138-bound APRIL is expressed on blood monocytes from active SLE patients. Collectively, our study suggests that a direct myeloid DC-B cell interplay might contribute to the pathogenesis of SLE.
Visualising the interaction of CD4 T cells and DCs in the evolution of inflammatory arthritis.
Prendergast Catriona T,Patakas Agapitos,Al-Khabouri Shaima,McIntyre Claire L,McInnes Iain B,Brewer James M,Garside Paul,Benson Robert A
Annals of the rheumatic diseases
OBJECTIVES:Successful early intervention in rheumatoid arthritis (RA) with the aim of resetting immunological tolerance requires a clearer understanding of how specificity, cellular kinetics and spatial behaviour shape the evolution of articular T cell responses. We aimed to define initial seeding of articular CD4 T cell responses in early experimental arthritis, evaluating their dynamic behaviour and interactions with dendritic cells (DCs) in the inflamed articular environment. METHODS:Antigen-induced arthritis was used to model articular inflammation. Flow cytometry and PCR of T cell receptor (TCR) diversity genes allowed phenotypic analysis of infiltrating T cells. The dynamic interactions of T cells with joint residing DCs were visualised using intravital multiphoton microscopy. RESULTS:Initial recruitment of antigen-specific T cells into the joint was paralleled by accumulation of CD4 T cells with diverse antigen-receptor expression and ability to produce tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) on mitogenic restimulation. A proportion of this infiltrate demonstrated slower motility speeds and engaged for longer periods with articular DCs in vivo. Abatacept treatment did not disrupt these interactions but did reduce T cell expression of inducible costimulatory (ICOS) molecule. We also demonstrated that non-specific CD4 T cells could be recruited during these early articular events. CONCLUSIONS:We demonstrate that CD4 T cells engage with articular DCs supporting antigen specific T cell reactivation. This cellular dialogue can be targeted therapeutically to reduce local T cell activation.
Engineered biological nanofactories trigger quorum sensing response in targeted bacteria.
Fernandes Rohan,Roy Varnika,Wu Hsuan-Chen,Bentley William E
Biological nanofactories, which are engineered to contain modules that can target, sense and synthesize molecules, can trigger communication between different bacterial populations. These communications influence biofilm formation, virulence, bioluminescence and many other bacterial functions in a process called quorum sensing. Here, we show the assembly of a nanofactory that can trigger a bacterial quorum sensing response in the absence of native quorum molecules. The nanofactory comprises an antibody (for targeting) and a fusion protein that produces quorum molecules when bound to the targeted bacterium. Our nanofactory selectively targets the appropriate bacteria and triggers a quorum sensing response when added to two populations of bacteria. The nanofactories also trigger communication between two bacterial populations that are otherwise non-communicating. We envision the use of these nanofactories in generating new antimicrobial treatments that target the communication networks of bacteria rather than their viability.
Shrink-film configurable multiscale wrinkles for functional alignment of human embryonic stem cells and their cardiac derivatives.
Chen Aaron,Lieu Deborah K,Freschauf Lauren,Lew Valerie,Sharma Himanshu,Wang Jiaxian,Nguyen Diep,Karakikes Ioannis,Hajjar Roger J,Gopinathan Ajay,Botvinick Elliot,Fowlkes Charless C,Li Ronald A,Khine Michelle
Advanced materials (Deerfield Beach, Fla.)
A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.
Bromoisobutyramide as an intermolecular surface binder for the preparation of free-standing biopolymer assemblies.
Mertz Damien,Tan Pramudana,Wang Yajun,Goh Tor Kit,Blencowe Anton,Caruso Frank
Advanced materials (Deerfield Beach, Fla.)
Bromoisobutyramide (BrIBAM)-modified silica templates facilitate the formation of bio-functional thin films made of a range of biopolymers (e.g., polypeptides, nucleic acids or polysaccharides). Upon template removal, non-covalent free-standing biopolymeric assemblies (e.g., hollow capsules or replicated spheres and fibers) are formed without the need for covalent cross-linking.
HLA-Bw4 80(T) and multiple HLA-Bw4 copies combined with KIR3DL1 associate with spontaneous clearance of HCV infection in people who inject drugs.
Thöns Christine,Senff Tina,Hydes Theresa J,Manser Angela R,Heinemann Falko M,Heinold Andreas,Heilmann Martin,Kim Arthur Y,Uhrberg Markus,Scherbaum Norbert,Lauer Georg M,Khakoo Salim I,Timm Jörg
Journal of hepatology
BACKGROUND & AIMS:Natural killer (NK) cell function is regulated by inhibitory and activating receptors including killer cell immunoglobulin-like receptors (KIRs). Here, we analyzed the impact of different KIR/KIR-ligand genotypes on the outcome of hepatitis C virus (HCV) infection in people who inject drugs (PWID). METHODS:KIR/KIR-ligand genotypes associated with spontaneous clearance of HCV infection were identified in a cohort of PWID from Germany (n=266) and further validated in a second anti-HCV positive cohort of PWID recruited in North America (n=342). NK cells of PWID and healthy donors were functionally characterized according to their KIR/KIR-ligand genotype by flow cytometry. RESULTS:Multivariate logistic regression analysis revealed that KIR3DL1/HLA-Bw4 80(T) was associated with spontaneous clearance of HCV infection in PWID, which was confirmed in the PWID cohort from North America. Compared with PWID with detectable HCV RNA, the frequency of individuals with multiple HLA-Bw4 alleles was significantly higher in anti-HCV positive PWID with resolved HCV infection (29.7% vs. 15.2%; p=0.0229) and in anti-HCV seronegative PWID (39.2%; p=0.0006). KIR3DL1 NK cells from HLA-Bw4 80(T)-positive PWID showed superior functionality compared to HLA-Bw4 80(I)-positive PWID. This differential impact was not observed in healthy donors; however, the HLA-Bw4 copy number strongly correlated with the functionality of KIR3DL1 NK cells. CONCLUSIONS:HLA-Bw4-80(T) and multiple HLA-Bw4 copies in combination with KIR3DL1 are associated with protection against chronic hepatitis C in PWID by distinct mechanisms. Better licensing of KIR3DL1 NK cells in the presence of multiple HLA-Bw4 copies is beneficial prior to seroconversion whereas HLA-Bw4 80(T) may be beneficial during acute hepatitis C. Lay summary: Natural killer (NK) cells are part of the innate immune system and are regulated by a complex network of activating and inhibiting receptors. The regulating receptor-ligand pairs of an individual are genetically determined. Here, we identified a particular set of ligand and receptor genes that are associated with better functionality of NK cells and better outcome upon exposure to HCV in a high-risk group.
Eukaryotic life without tQCUG: the role of Elongator-dependent tRNA modifications in Dictyostelium discoideum.
Schäck Manfred A,Jablonski Kim Philipp,Gräf Stefan,Klassen Roland,Schaffrath Raffael,Kellner Stefanie,Hammann Christian
Nucleic acids research
In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only are previously established modifications observable by mass spectrometry in strains with the most conserved genes of each step deleted, but also additional modifications are detected, indicating a certain plasticity of the pathway in the amoeba. Unlike described for yeast, D. discoideum allows for an unconditional deletion of the single tQCUG gene, as long as the Elongator-dependent modification pathway is intact. In gene deletion strains of the modification pathway, protein amounts are significantly reduced as shown by flow cytometry and Western blotting, using strains expressing different glutamine leader constructs fused to GFP. Most dramatic are these effects, when the tQCUG gene is deleted, or Elp3, the catalytic component of the Elongator complex is missing. In addition, Elp3 is the most strongly conserved protein of the modification pathway, as our phylogenetic analysis reveals. The implications of this observation are discussed with respect to the evolutionary age of the components acting in the Elongator-dependent modification pathway.
Bright and stable near-infrared fluorescent protein for in vivo imaging.
Filonov Grigory S,Piatkevich Kiryl D,Ting Li-Min,Zhang Jinghang,Kim Kami,Verkhusha Vladislav V
Imaging biological processes in mammalian tissues will be facilitated by fluorescent probes with excitation and emission bands within the near-infrared optical window of high transparency. Here we report a phytochrome-based near-infrared fluorescent protein (iRFP) with excitation and emission maxima at 690 nm and 713 nm, respectively. iRFP does not require an exogenous supply of the chromophore biliverdin and has higher effective brightness, intracellular stability and photostability than earlier phytochrome-derived fluorescent probes. Compared with far-red GFP-like proteins, iRFP has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra.
Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing.
Holmqvist Erik,Reimegård Johan,Wagner E Gerhart H
Nucleic acids research
We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.
Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
Martinez Paula,Thanasoula Maria,Carlos Ana R,Gómez-López Gonzalo,Tejera Agueda M,Schoeftner Stefan,Dominguez Orlando,Pisano David G,Tarsounas Madalena,Blasco Maria A
Nature cell biology
Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.
Trichostatin A sensitises rheumatoid arthritis synovial fibroblasts for TRAIL-induced apoptosis.
Jüngel A,Baresova V,Ospelt C,Simmen B R,Michel B A,Gay R E,Gay S,Seemayer C A,Neidhart M
Annals of the rheumatic diseases
BACKGROUND:Histone acetylation/deacetylation has a critical role in the regulation of transcription by altering the chromatin structure. OBJECTIVE:To analyse the effect of trichostatin A (TSA), a streptomyces metabolite which specifically inhibits mammalian histone deacetylases, on TRAIL-induced apoptosis of rheumatoid arthritis synovial fibroblasts (RASF). METHODS:Apoptotic cells were detected after co-treatment of RASF with TRAIL (200 ng/ml) and TSA (0.5, 1, and 2 micromol/l) by flow cytometry using propidium iodide/annexin-V-FITC staining. Cell proliferation was assessed using the MTS proliferation test. Induction of the cell cycle inhibitor p21Waf/Cip1 by TSA was analysed by western blot. Expression of the TRAIL receptor-2 (DR5) on the cell surface of RASF was analysed by flow cytometry. Levels of soluble TRAIL were measured in synovial fluid of patients with RA and osteoarthritis (OA) by ELISA. RESULTS:Co-treatment of the cells with TSA and TRAIL induced cell death in a synergistic and dose dependent manner, whereas TRAIL and TSA alone had no effect or only a modest effect. RASF express DR5 (TRAIL receptor 2), but treatment of the cells with TSA for 24 hours did not change the expression level of DR5, as it is shown for cancer cells. TSA induced cell cycle arrest in RASF through up regulation of p21Waf1/Cip1. Levels of soluble TRAIL were significantly higher in RA than in OA synovial fluids. CONCLUSION:Because TSA sensitises RASF for TRAIL-induced apoptosis, it is concluded that TSA discloses sensitive sites in the cascade of TRAIL signalling and may represent a new principle for the treatment of RA.
Translation initiator EIF4G1 mutations in familial Parkinson disease.
Chartier-Harlin Marie-Christine,Dachsel Justus C,Vilariño-Güell Carles,Lincoln Sarah J,Leprêtre Frédéric,Hulihan Mary M,Kachergus Jennifer,Milnerwood Austen J,Tapia Lucia,Song Mee-Sook,Le Rhun Emilie,Mutez Eugénie,Larvor Lydie,Duflot Aurélie,Vanbesien-Mailliot Christel,Kreisler Alexandre,Ross Owen A,Nishioka Kenya,Soto-Ortolaza Alexandra I,Cobb Stephanie A,Melrose Heather L,Behrouz Bahareh,Keeling Brett H,Bacon Justin A,Hentati Emna,Williams Lindsey,Yanagiya Akiko,Sonenberg Nahum,Lockhart Paul J,Zubair Abba C,Uitti Ryan J,Aasly Jan O,Krygowska-Wajs Anna,Opala Grzegorz,Wszolek Zbigniew K,Frigerio Roberta,Maraganore Demetrius M,Gosal David,Lynch Tim,Hutchinson Michael,Bentivoglio Anna Rita,Valente Enza Maria,Nichols William C,Pankratz Nathan,Foroud Tatiana,Gibson Rachel A,Hentati Faycal,Dickson Dennis W,Destée Alain,Farrer Matthew J
American journal of human genetics
Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.
Bone Marrow Endothelial Cells Regulate Myelopoiesis in Diabetes Mellitus.
Hoyer Friedrich Felix,Zhang Xinyi,Coppin Emilie,Vasamsetti Sathish Babu,Modugu Ganesh,Schloss Maximilian J,Rohde David,McAlpine Cameron S,Iwamoto Yoshiko,Libby Peter,Naxerova Kamila,Swirski Filip K,Dutta Partha,Nahrendorf Matthias
BACKGROUND:Diabetes mellitus is a prevalent public health problem that affects about one-third of the US population and leads to serious vascular complications with increased risk for coronary artery disease. How bone marrow hematopoiesis contributes to diabetes mellitus complications is incompletely understood. We investigated the role of bone marrow endothelial cells in diabetic regulation of inflammatory myeloid cell production. METHODS:In 3 types of mouse diabetes mellitus, including streptozotocin, high-fat diet, and genetic induction using leptin-receptor-deficient db/db mice, we assayed leukocytes, hematopoietic stem and progenitor cells (HSPC). In addition, we investigated bone marrow endothelial cells with flow cytometry and expression profiling. RESULTS:In diabetes mellitus, we observed enhanced proliferation of HSPC leading to augmented circulating myeloid cell numbers. Analysis of bone marrow niche cells revealed that endothelial cells in diabetic mice expressed less , a retention factor promoting HSPC quiescence. Transcriptome-wide analysis of bone marrow endothelial cells demonstrated enrichment of genes involved in epithelial growth factor receptor (Egfr) signaling in mice with diet-induced diabetes mellitus. To explore whether endothelial Egfr plays a functional role in myelopoiesis, we generated mice with endothelial-specific deletion of Egfr ( ). We found enhanced HSPC proliferation and increased myeloid cell production in mice compared with wild-type mice with diabetes mellitus. Disrupted Egfr signaling in endothelial cells decreased their expression of the HSPC retention factor angiopoietin-1. We tested the functional relevance of these findings for wound healing and atherosclerosis, both implicated in complications of diabetes mellitus. Inflammatory myeloid cells accumulated more in skin wounds of diabetic mice, significantly delaying wound closure. Atherosclerosis was accelerated in mice, leading to larger and more inflamed atherosclerotic lesions in the aorta. CONCLUSIONS:In diabetes mellitus, bone marrow endothelial cells participate in the dysregulation of bone marrow hematopoiesis. Diabetes mellitus reduces endothelial production of Cxcl12, a quiescence-promoting niche factor that reduces stem cell proliferation. We describe a previously unknown counterregulatory pathway, in which protective endothelial Egfr signaling curbs HSPC proliferation and myeloid cell production.
Abnormal T cell differentiation persists in patients with rheumatoid arthritis in clinical remission and predicts relapse.
Burgoyne C H,Field S L,Brown A K,Hensor E M,English A,Bingham S L,Verburg R,Fearon U,Lawson C A,Hamlin P J,Straszynski L,Veale D,Conaghan P,Hull M A,van Laar J M,Tennant A,Emery P,Isaacs J D,Ponchel F
Annals of the rheumatic diseases
OBJECTIVES:An abnormal CD4+ T cell subset related to inflammation exposure (inflammation-related cells, IRC) has been identified in rheumatoid arthritis (RA). Patients with inflammatory and non-inflammatory diseases were used to examine the relationship between inflammation and this T cell subset in vivo. METHODS:Blood was collected from healthy controls and patients with RA (active disease or in clinical remission), Crohn's disease and osteoarthritis. IRC and chemokine receptors were quantified by flow cytometry. Thymic activity and apoptotic factors were measured by real-time polymerase chain reaction. Circulating cytokines were measured by enzyme-linked immunosorbent assay. CXCR4 and SDF1 in synovial biopsies were measured using immunohistochemistry. RESULTS:IRC were identified in patients with RA (p<0.0001) and Crohn's disease (p = 0.005), but not in those with osteoarthritis. In RA in remission, IRC persisted (p<0.001). In remission, hyperproliferation of IRC was lost, chemokine receptor expression was significantly lowered (p<0.007), Bax expression dropped significantly (p<0.001) and was inversely correlated with IRC (rho = -0.755, p = 0.03). High IRC frequency in remission was associated with relapse within 18 months (OR = 6.4, p<0.001) and a regression model predicted 72% of relapse. CONCLUSIONS:These results suggest a model in which, despite the lack of systemic inflammation, IRC persist in remission, indicating that IRC are an acquired feature of RA. They have, however, lost their hyper-responsiveness, acquired a potential for survival, and no longer express chemokine receptors. IRC persistence in remission confirms their important role in chronic inflammation as circulating precursors of pathogenic cells. This was further demonstrated by much higher incidence of relapse in patients with high IRC frequency in remission.
Hematopoietic Stem-Cell Transplantation Does Not Improve the Poor Outcome of Children With Hypodiploid Acute Lymphoblastic Leukemia: A Report From Children's Oncology Group.
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:Children and young adults with hypodiploid B-lymphoblastic leukemia (B-ALL) fare poorly and hematopoietic stem-cell transplantation (HSCT) is often pursued in first complete remission (CR1). We retrospectively reviewed the outcomes of children and young adults with hypodiploid B-ALL who were enrolled in recent Children's Oncology Group (COG) trials to evaluate the impact of HSCT on outcome. PATIENTS AND METHODS:Cytogenetic analyses and DNA index were performed at COG-approved laboratories, and hypodiploidy was defined as modal chromosome number less than 44 and/or DNA index less than 0.81. Minimal residual disease (MRD) was determined centrally using flow cytometry at two reference laboratories. Patients with hypodiploid ALL came off protocol therapy postinduction and we retrospectively collected details on their subsequent therapy and outcomes. Event-free survival (EFS) and overall survival (OS) were estimated for the cohort. RESULTS:Between 2003 and 2011, 8,522 patients with National Cancer Institute standard-risk and high-risk B-ALL were enrolled in COG AALL03B1 ( ClinicalTrials.gov identifier: NCT00482352). Hypodiploidy occurred in 1.5% of patients (n = 131), 98.3% of whom achieved CR after induction therapy. Five-year EFS and OS were 52.2% ± 4.9% and 58.9% ± 4.8%, respectively. Outcomes for patients undergoing CR1 HSCT were not significantly improved: 5-year EFS and OS were 57.4% ± 7.0% and 66.2% ± 6.6% compared with 47.8% ± 7.5% and 53.8% ± 7.6%, respectively ( P = .49 and .34, respectively) for those who did not undergo transplantation. Patients with MRD of 0.01% or greater at the end of induction had 5-year EFS and OS of 26.7% ± 9.3% and 29.3% ± 10.1%, respectively, and HSCT had no significant impact on outcomes. CONCLUSION:Children and young adults with hypodiploid B-ALL continue to fare poorly and do not seem to benefit from CR1 HSCT. This is especially true for patients with MRD of 0.01% or greater at the end of induction. New treatment strategies are urgently needed for these patients.
Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido.
Balachandran Vinod P,Cavnar Michael J,Zeng Shan,Bamboat Zubin M,Ocuin Lee M,Obaid Hebroon,Sorenson Eric C,Popow Rachel,Ariyan Charlotte,Rossi Ferdinand,Besmer Peter,Guo Tianhua,Antonescu Cristina R,Taguchi Takahiro,Yuan Jianda,Wolchok Jedd D,Allison James P,DeMatteo Ronald P
Imatinib mesylate targets mutated KIT oncoproteins in gastrointestinal stromal tumor (GIST) and produces a clinical response in 80% of patients. The mechanism is believed to depend predominantly on the inhibition of KIT-driven signals for tumor-cell survival and proliferation. Using a mouse model of spontaneous GIST, we found that the immune system contributes substantially to the antitumor effects of imatinib. Imatinib therapy activated CD8(+) T cells and induced regulatory T cell (T(reg) cell) apoptosis within the tumor by reducing tumor-cell expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (Ido). Concurrent immunotherapy augmented the efficacy of imatinib in mouse GIST. In freshly obtained human GIST specimens, the T cell profile correlated with imatinib sensitivity and IDO expression. Thus, T cells are crucial to the antitumor effects of imatinib in GIST, and concomitant immunotherapy may further improve outcomes in human cancers treated with targeted agents.
Murine and Non-Human Primate Dendritic Cell Targeting Nanoparticles for in Vivo Generation of Regulatory T-Cells.
Stead Sebastian O,Kireta Svjetlana,McInnes Steve J P,Kette Francis D,Sivanathan Kisha N,Kim Juewan,Cueto-Diaz Eduardo J,Cunin Frederique,Durand Jean-Olivier,Drogemuller Christopher J,Carroll Robert P,Voelcker Nicolas H,Coates Patrick T
Porous silicon nanoparticles (pSiNP), modified to target dendritic cells (DC), provide an alternate strategy for the delivery of immunosuppressive drugs. Here, we aimed to develop a DC-targeting pSiNP displaying c-type lectin, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), and CD11c monoclonal antibodies. The in vivo tracking of these fluorescent DC-targeting nanoparticles was assessed in both C57BL/6 mice and common marmosets ( Callithrix jacchus) by intravenous injection (20 mg/kg). Rapamycin and ovalbumin (OVA) peptide loaded pSiNP were employed to evaluate their ability to generate murine CD4CD25FoxP3 regulatory T-cells in vivo within OVA sensitized mice. In vivo, pSiNP migrated to the liver, kidneys, lungs, and spleen in both mice and marmosets. Flow cytometry confirmed pSiNP uptake by splenic and peripheral blood DC when functionalized with targeting antibodies. C57BL/6 OVA sensitized mice injected with CD11c-pSiNP loaded with rapamycin + OVA produced a 5-fold higher number of splenic regulatory T-cells compared to control mice, at 40 days post-pSiNP injection. These results demonstrate the importance of the immobilized targeting antibodies to enhance cellular uptake and enable the in vivo generation of splenic regulatory T-cells.
A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus.
Liu Zhenzhen,Sun Mengmeng,Liu Jilin,Liu Tao,Ye Qing,Li Yingjun,Peng Nan
Nucleic acids research
CRISPR-Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.
Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis.
Whelan Kelly A,Merves Jamie F,Giroux Veronique,Tanaka Koji,Guo Andy,Chandramouleeswaran Prasanna M,Benitez Alain J,Dods Kara,Que Jianwen,Masterson Joanne C,Fernando Shahan D,Godwin Bridget C,Klein-Szanto Andres J,Chikwava Kudakwashe,Ruchelli Eduardo D,Hamilton Kathryn E,Muir Amanda B,Wang Mei-Lun,Furuta Glenn T,Falk Gary W,Spergel Jonathan M,Nakagawa Hiroshi
OBJECTIVE:The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu. DESIGN:Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry. RESULTS:EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies. CONCLUSIONS:Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.
Stem cell gene expression programs influence clinical outcome in human leukemia.
Eppert Kolja,Takenaka Katsuto,Lechman Eric R,Waldron Levi,Nilsson Björn,van Galen Peter,Metzeler Klaus H,Poeppl Armando,Ling Vicki,Beyene Joseph,Canty Angelo J,Danska Jayne S,Bohlander Stefan K,Buske Christian,Minden Mark D,Golub Todd R,Jurisica Igor,Ebert Benjamin L,Dick John E
Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.
Differentiation antigens identify subpopulations of rabbit T and B lymphocytes. Definition by flow cytometry.
Jackson S,Chused T M,Wilkinson J M,Leiserson W M,Kindt T J
The Journal of experimental medicine
A panel of six monoclonal antibodies produced against cell surface glycoproteins of a rabbit T lymphocyte line was used with flow cytometry to define rabbit lymphocyte subpopulations. Four thymocyte populations were characterized by size and expression of cell surface antigens and appear to represent stages in thymocyte differentiation. Rabbit spleen contained five subpopulations: two of T lineage, two of B, and a null cell subset. Bimodal distribution of staining of thymocytes and peripheral T cells was observed using an antibody (9AE10) directed against a Thy-1 analogue in the rabbit, suggesting two separate T cell lineages. One of the monoclonal reagents, L11/135, reacted strongly with peripheral rabbit T cells as shown by two-color immunofluorescence. In functional studies, only the L11/135-bearing cells responded to the T cell mitogens concanavalin A and phytohemagglutinin and to allogeneic splenocytes. The thymocyte subpopulations and the peripheral T and B cell subsets differ from those described in mouse and man.
Oligoclonal expansion of major histocompatibility complex class I-restricted cytolytic T lymphocytes during a primary immune response in vivo: direct monitoring by flow cytometry and polymerase chain reaction.
MacDonald H R,Casanova J L,Maryanski J L,Cerottini J C
The Journal of experimental medicine
Previous T cell receptor (TCR) sequence analysis of a panel of 23 H-2Kd restricted cytotoxic T lymphocyte (CTL) clones recognizing the decapeptide HLA-CW3 170-179 revealed a striking conservation of TCR structure, in that all clones examined used V beta 10 and J alpha pHDS58 segments. We show here that the primary response in vivo after intraperitoneal injection of DBA/2 mice with HLA-CW3 expressing transfectants of syngeneic P815 (H-2d) tumor cells is characterized by a dramatic expansion of CD8+ V beta 10+ CTL in the peritoneal cavity and draining (mesenteric) lymph node, as well as in peripheral blood. Additional analysis of TCR on HLA-CW3 immune populations by flow cytometry and polymerase chain reaction further indicates that the vast majority of responding CD8+ cells express restricted V alpha domains, a dominant J alpha segment (pHDS58), and a conserved CDR3 length for both alpha and beta chains. This novel system provides a unique opportunity to directly monitor an oligoclonal primary antigen specific immune response in vivo at the single cell level independently of functional assays.
Imbalance of Genes Encoding Natural Killer Immunoglobulin-Like Receptors and Human Leukocyte Antigen in Patients With Biliary Cancer.
Cornillet Martin,Jansson Hannes,Schaffer Marie,Hertwig Laura,Berglin Lena,Zimmer Christine L,Johansson Helene,Ellis Ewa,Isaksson Bengt,Gonzalez-Galarza Faviel F,Middleton Derek,Malmberg Karl-Johan,Sparrelid Ernesto,Björkström Niklas K
BACKGROUND & AIMS:Bile duct tumors are rare and have poor prognoses. Natural killer (NK) cells are frequent in human liver and infiltrate these tumors but do not control their progression. Responses of NK cells are regulated by NK immunoglobulin-like receptors (KIRs), which interact with HLA class I ligands. We aimed to characterize the features of the KIR gene loci and their ligands in patients with bile duct cancer (BDC). METHODS:We performed combined multidimensional characterization of genes that encode KIRs and their ligands in blood samples from patients with BDC from Sweden, followed for up to 8 years after diagnosis (n = 148), in 2 geographically matched cohorts of healthy individuals from Northern Europe (n = 204 and n = 900), and in healthy individuals from 6 geographically unrelated populations (n = 2917). We used real-time polymerase chain reaction, RNA sequencing, immunohistochemistry, and flow cytometry to evaluate NK-cell presence, as well as KIR and KIR-ligand expression in bile duct tumors and control tissues. RESULTS:Patients with bile duct tumors had multiple alterations at the KIR gene loci. KIR loci are grouped into genotypes that encode more inhibitory (group A) and more activating (group B) receptors, which can be subdivided into centromeric and telomeric fragments. Patients with BDC had a lower prevalence of KIR2DL3, which was linked to disequilibrium in centromeric A/B and B/B genotypes, compared with control individuals. The associations between KIRs and KIR ligands differed between patients with BDC and control individuals; patients had an altered balance between activating and inhibitory KIRs. KIR-positive NK cells infiltrated biliary tumors that expressed matched KIR ligands. CONCLUSIONS:In a multidimensional analysis of DNA from blood samples of patients with BDC in Europe, we found patients to have multiple alterations at the KIR and HLA gene loci compared with control individuals. These alterations might affect NK-cell tumor surveillance. NK cells from bile duct tumors expressed KIRs and were found in tumors that expressed cognate ligands. This should be considered in development of immune-based therapies for BDC.
Human colon organoids reveal distinct physiologic and oncogenic Wnt responses.
Michels Birgitta E,Mosa Mohammed H,Grebbin Britta M,Yepes Diego,Darvishi Tahmineh,Hausmann Johannes,Urlaub Henning,Zeuzem Stefan,Kvasnicka Hans M,Oellerich Thomas,Farin Henner F
The Journal of experimental medicine
Constitutive Wnt activation upon loss of () acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.
A kinase shRNA screen links LATS2 and the pRB tumor suppressor.
Tschöp Katrin,Conery Andrew R,Litovchick Larisa,Decaprio James A,Settleman Jeffrey,Harlow Ed,Dyson Nicholas
Genes & development
pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.
Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.
Juan Aster H,Derfoul Assia,Feng Xuesong,Ryall James G,Dell'Orso Stefania,Pasut Alessandra,Zare Hossein,Simone James M,Rudnicki Michael A,Sartorelli Vittorio
Genes & development
Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5⁻ stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.
Widespread genomic breaks generated by activation-induced cytidine deaminase are prevented by homologous recombination.
Hasham Muneer G,Donghia Nina M,Coffey Eliot,Maynard Jane,Snow Kathy J,Ames Jacquelyn,Wilpan Robert Y,He Yishu,King Benjamin L,Mills Kevin D
Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and immunoglobulin class switching in activated B cells. Because AID has no known target-site specificity, there have been efforts to identify non-immunoglobulin AID targets. We show here that AID acts promiscuously, generating widespread DNA double-strand breaks (DSBs), genomic instability and cytotoxicity in B cells with less homologous recombination ability. We demonstrate that the homologous-recombination factor XRCC2 suppressed AID-induced off-target DSBs, promoting B cell survival. Finally, we suggest that aberrations that affect human chromosome 7q36, including XRCC2, correlate with genomic instability in B cell cancers. Our findings demonstrate that AID has promiscuous genomic DSB-inducing activity, identify homologous recombination as a safeguard against off-target AID action, and have implications for genomic instability in B cell cancers.
Loss of Protease-Activated Receptor 4 Prevents Inflammation Resolution and Predisposes the Heart to Cardiac Rupture After Myocardial Infarction.
BACKGROUND:Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. METHODS:We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. RESULTS:Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4 mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4 mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4 neutrophils, but not of Par4 platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4 mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. CONCLUSIONS:These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.
NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.
Rolas Loïc,Boussif Abdelali,Weiss Emmanuel,Lettéron Philippe,Haddad Oualid,El-Benna Jamel,Rautou Pierre-Emmanuel,Moreau Richard,Périanin Axel
OBJECTIVE:Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients' susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients' neutrophils and blood. DESIGN:Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR. RESULTS:The impaired superoxide production by patients' neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 /NOX2 and p22 ), its cytosolic partner p47 but not p67 . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients' plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients' neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients' neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production. CONCLUSION:Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.
Interleukin 17, Produced by γδ T Cells, Contributes to Hepatic Inflammation in a Mouse Model of Biliary Atresia and Is Increased in Livers of Patients.
Klemann Christian,Schröder Arne,Dreier Anika,Möhn Nora,Dippel Stephanie,Winterberg Thomas,Wilde Anne,Yu Yi,Thorenz Anja,Gueler Faikah,Jörns Anne,Tolosa Eva,Leonhardt Johannes,Haas Jan D,Prinz Immo,Vieten Gertrud,Petersen Claus,Kuebler Joachim F
BACKGROUND & AIMS:Biliary atresia (BA) is a rare disease in infants, with unknown mechanisms of pathogenesis. It is characterized by hepatobiliary inflammatory, progressive destruction of the biliary system leading to liver fibrosis, and deterioration of liver function. Interleukin (IL) 17A promotes inflammatory and autoimmune processes. We studied the role of IL17A and cells that produce this cytokine in a mouse model of BA and in hepatic biopsy samples from infants with BA. METHODS:We obtained peripheral blood and liver tissue specimens from 20 patients with BA, collected at the time of Kasai portoenterostomy, along with liver biopsies from infants without BA (controls). The tissue samples were analyzed by reverse transcription quantitative polymerase chain reaction (PCR), in situ PCR, and flow cytometry analyses. BA was induced in balb/cAnNCrl mice by rhesus rotavirus infection; uninfected mice were used as controls. Liver tissues were collected from mice and analyzed histologically and by reverse transcriptase PCR; leukocytes were isolated, stimulated, and analyzed by flow cytometry and PCR analyses. Some mice were given 3 intraperitoneal injections of a monoclonal antibody against IL17 or an isotype antibody (control). RESULTS:Livers from rhesus rota virus-infected mice with BA had 7-fold more Il17a messenger RNA than control mice (P = .02). γδ T cells were the exclusive source of IL17; no T-helper 17 cells were detected in livers of mice with BA. The increased number of IL17a-positive γδ T cells liver tissues of mice with BA was associated with increased levels of IL17A, IL17F, retinoid-orphan-receptor C, C-C chemokine receptor 6, and the IL23 receptor. Mice that were developing BA and given antibodies against IL17 had lower levels of liver inflammation and mean serum levels of bilirubin than mice receiving control antibodies (191 μmol/L vs 78 μmol/L, P = .002). Liver tissues from patients with BA had 4.6-fold higher levels of IL17 messenger RNA than control liver tissues (P = .02). CONCLUSIONS:In livers of mice with BA, γδ T cells produce IL17, which is required for inflammation and destruction of the biliary system. IL17 is up-regulated in liver tissues from patients with BA, compared with controls, and might serve as a therapeutic target.
The human cytomegalovirus microRNA miR-UL112 acts synergistically with a cellular microRNA to escape immune elimination.
Nachmani Daphna,Lankry Dikla,Wolf Dana G,Mandelboim Ofer
Although approximately 200 viral microRNAs are known, only very few share similar targets with their host's microRNAs. A notable example of this is the stress-induced ligand MICB, which is targeted by several distinct viral and cellular microRNAs. Through the investigation of the microRNA-mediated immune-evasion strategies of herpesviruses, we initially identified two new cellular microRNAs that targeted MICB and were expressed differently both in healthy tissues and during melanocyte transformation. We show that coexpression of various pairs of cellular microRNAs interfered with the downregulation of MICB, whereas the viral microRNAs optimized their targeting ability to efficiently downregulate MICB. Moreover, we demonstrate that through site proximity and possibly inhibition of translation, a human cytomegalovirus (HCMV) microRNA acts synergistically with a cellular microRNA to suppress MICB expression during HCMV infection.
Oligo(ethylene glycol)-based thermosensitive dendrimers and their tumor accumulation and penetration.
Wu Wei,Driessen Wouter,Jiang Xiqun
Journal of the American Chemical Society
Dendrimers have several featured advantages over other nanomaterials as drug carriers, such as well-defined structure, specific low-nanometer size, and abundant peripheral derivable groups, etc. However, these advantages have not been fully exploited yet to optimize their biological performance, especially tumor penetration, which is a shortcoming of current nanomaterials. Here we show the syntheses of a new class of oligo(ethylene glycol) (OEG)-based thermosensitive dendrimers up to the fourth generation. Each dendrimer shows monodisperse structure. OEG/poly(ethylene glycol) (PEG) moieties with different precise lengths were introduced to the periphery of the fourth-generation dendrimer followed by an antitumor agent, gemcitabine (GEM). The biodistributions of the GEM-conjugated dendrimers were investigated by micro positron emission tomography and multispectral optoacoustic tomography imaging techniques and compared with that of GEM-conjugated poly(amidoamine) (PAMAM). The GEM-conjugated dendrimer with the longest peripheral PEG segments exhibited the most desirable tumor accumulation and penetration and thus had significantly higher antitumor activity than the GEM-conjugated PAMAM.
An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells.
Tang Chad,Lee Andrew S,Volkmer Jens-Peter,Sahoo Debashis,Nag Divya,Mosley Adriane R,Inlay Matthew A,Ardehali Reza,Chavez Shawn L,Pera Renee Reijo,Behr Barry,Wu Joseph C,Weissman Irving L,Drukker Micha
An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.
Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations.
Chen Xiaoli,Lingala Shilpa,Khoobyari Shiva,Nolta Jan,Zern Mark A,Wu Jian
Journal of hepatology
BACKGROUND & AIMS:Our previous studies showed that CD133, EpCAM, and aldehyde dehydrogenase (ALDH) are useful markers to identify cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) tissues. The present study aims to evaluate chemosensitivity and invasion capability of HCC based on CSC marker profiles, and to explore the underlying molecular mechanisms. METHODS:Hepatoma cell lines were separated into subpopulations according to CD133, EpCAM, and ALDH expression profiles. Epithelial mesenchymal transition (EMT) and hedgehog (Hh) signaling were examined to identify their links with chemoresistance and aggressive invasion. RESULTS:Well-differentiated cell lines were positive for CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) at 1.5-15% and 2.3-8.3%; whereas, poorly-differentiated cells were almost all negative for these markers. FACS-enriched CD133(+)/ALDH(high) and CD133(+)/EpCAM(+) Hep3B and Huh-7 cells formed more spheroids in vitro. CD133(-)/ALDH(low) HLE cells were more resistant to cisplatin, doxorubicin or sorafenib than their positive counterparts. CD133(-)/EpCAM(-) Huh-7 cells or CD133(-)/ALDH(-) HLE cells exhibited a higher invasion rate than their positive counterparts. HLE and HLF cells acquired EMT in double negative subpopulations. Hh activity in Huh-7 CD133(-)/EpCAM(-) cells was higher than in their positive counterparts, and the inhibition of Hh activity by cyclopamine resulted in reduced cell proliferation. CONCLUSIONS:Well-differentiated CD133(+)/ALDH(high) or CD133(+)/EpCAM(+) cells appear to be a CSC/initiating subpopulation; whereas, in poorly-differentiated hepatoma cells, EMT and enhanced hedgehog signaling activity may be responsible for their chemoresistance and invasion. These findings underscore the significance of EMT and enhanced Hh signaling in liver cancer stem or initiating cells.
TLR9 independent interferon α production by neutrophils on NETosis in response to circulating chromatin, a key lupus autoantigen.
Lindau Dennis,Mussard Julie,Rabsteyn Armin,Ribon Matthieu,Kötter Ina,Igney Annette,Adema Gosse J,Boissier Marie-Christophe,Rammensee Hans-Georg,Decker Patrice
Annals of the rheumatic diseases
OBJECTIVES:Interferon (IFN) α is a key immunoregulatory cytokine secreted by activated plasmacytoid dendritic cells (PDC) that constitute less than 1% of leucocytes. IFNα plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Nevertheless, the natural IFNα inducers in SLE as well as the different IFNα secreting cell types are only partially characterised. METHODS:Chromatin was purified from calf thymus. Human peripheral blood mononuclear cells (PBMC), neutrophils and mouse bone marrow neutrophils were purified and cultured with different stimuli. IFNα production was estimated by flow cytometry, ELISA and a bioassay, and gene expression by quantitative real time PCR. Neutrophil activation and NETosis were analysed by flow cytometry, ELISA and confocal microscopy. RESULTS:Neutrophils produced a bioactive IFNα on stimulation with purified chromatin. IFNα secretion was observed with steady state neutrophils purified from 56 independent healthy individuals and autoimmune patients in response to free chromatin and not chromatin containing immune complexes. Chromatin induced IFNα secretion occurred independently of Toll-like receptor 9 (TLR9). Neutrophil priming by granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor or IFNα was not necessary but PBMC sustained IFNα secretion by neutrophils. PDC were 27 times more efficient than neutrophils but blood neutrophils were 100 times more frequent than PDC. Finally, neutrophil activation by chromatin was associated with NETosis and DNA sensor upregulation. CONCLUSIONS:Neutrophils have the capability of producing IFNα on selective triggering, and we identified a natural lupus stimulus involved, unveiling a new mechanism involved in SLE. Neutrophils represent another important source of IFNα and important targets for future therapies aimed at influencing IFNα levels.
Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1.
Bothmer Anne,Robbiani Davide F,Di Virgilio Michela,Bunting Samuel F,Klein Isaac A,Feldhahn Niklas,Barlow Jacqueline,Chen Hua-Tang,Bosque David,Callen Elsa,Nussenzweig André,Nussenzweig Michel C
53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.
Micelle-Forming Dexamethasone Prodrug Attenuates Nephritis in Lupus-Prone Mice without Apparent Glucocorticoid Side Effects.
Jia Zhenshan,Wang Xiaobei,Wei Xin,Zhao Gang,Foster Kirk W,Qiu Fang,Gao Yangyang,Yuan Fang,Yu Fang,Thiele Geoffrey M,Bronich Tatiana K,O'Dell James R,Wang Dong
Nephritis is one of the major complications of systemic lupus erythematosus. While glucocorticoids (GCs) are frequently used as the first-line treatment for lupus nephritis (LN), long-term GC usage is often complicated by severe adverse effects. To address this challenge, we have developed a polyethylene glycol-based macromolecular prodrug (ZSJ-0228) of dexamethasone, which self-assembles into micelles in aqueous media. When compared to the dose equivalent daily dexamethasone 21-phosphate disodium (Dex) treatment, monthly intravenous administration of ZSJ-0228 for two months significantly improved the survival of lupus-prone NZB/W F1 mice and was much more effective in normalizing proteinuria, with clear histological evidence of nephritis resolution. Different from the dose equivalent daily Dex treatment, monthly ZSJ-0228 administration has no impact on the serum anti-double-stranded DNA (anti-dsDNA) antibody level but can significantly reduce renal immune complex deposition. No significant systemic toxicities of GCs ( e. g., total IgG reduction, adrenal gland atrophy, and osteopenia) were found to be associated with ZSJ-0228 treatment. In vivo imaging and flow cytometry studies revealed that the fluorescent-labeled ZSJ-0228 primarily distributed to the inflamed kidney after systemic administration, with renal myeloid cells and proximal tubular epithelial cells mainly responsible for its kidney retention. Collectively, these data suggest that the ZSJ-0228's potent local anti-inflammatory/immunosuppressive effects and improved safety may be attributed to its nephrotropicity and cellular sequestration at the inflamed kidney tissues. Pending further optimization, it may be developed into an effective and safe therapy for improved clinical management of LN.
High-throughput optical screening of cellular mechanotransduction.
Compton Jonathan L,Luo Justin C,Ma Huan,Botvinick Elliot,Venugopalan Vasan
We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated micro-cavitation bubbles (μCBs) without requiring flow chambers or microfluidics. These μCBs expose adherent cells to a microTsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1mm. We demonstrate microTsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation resulting in Ca release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microTsunami-induced Ca signalling by introducing a known inhibitor to this pathway. The imaging of Ca signalling, and its modulation by exogenous molecules, demonstrates the capacity to initiate and assess cellular mechanosignalling in real-time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry.
Isolation and in vitro expansion of human colonic stem cells.
Jung Peter,Sato Toshiro,Merlos-Suárez Anna,Barriga Francisco M,Iglesias Mar,Rossell David,Auer Herbert,Gallardo Mercedes,Blasco Maria A,Sancho Elena,Clevers Hans,Batlle Eduard
Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.
Evidence for osteocyte regulation of bone homeostasis through RANKL expression.
Nakashima Tomoki,Hayashi Mikihito,Fukunaga Takanobu,Kurata Kosaku,Oh-Hora Masatsugu,Feng Jian Q,Bonewald Lynda F,Kodama Tatsuhiko,Wutz Anton,Wagner Erwin F,Penninger Josef M,Takayanagi Hiroshi
Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.
A human memory T cell subset with stem cell-like properties.
Gattinoni Luca,Lugli Enrico,Ji Yun,Pos Zoltan,Paulos Chrystal M,Quigley Máire F,Almeida Jorge R,Gostick Emma,Yu Zhiya,Carpenito Carmine,Wang Ena,Douek Daniel C,Price David A,June Carl H,Marincola Francesco M,Roederer Mario,Restifo Nicholas P
Immunological memory is thought to depend on a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and IL-7Rα(+) T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell-like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.
Controlling the specificity of modularly assembled small molecules for RNA via ligand module spacing: targeting the RNAs that cause myotonic muscular dystrophy.
Lee Melissa M,Childs-Disney Jessica L,Pushechnikov Alexei,French Jonathan M,Sobczak Krzysztof,Thornton Charles A,Disney Matthew D
Journal of the American Chemical Society
Myotonic muscular dystrophy types 1 and 2 (DM1 and DM2, respectively) are caused by expansions of repeating nucleotides in noncoding regions of RNA. In DM1, the expansion is an rCUG triplet repeat, whereas the DM2 expansion is an rCCUG quadruplet repeat. Both RNAs fold into hairpin structures with periodically repeating internal loops separated by two 5'GC/3'CG base pairs. The sizes of the loops, however, are different: the DM1 repeat forms 1 x 1 nucleotide UU loops while the DM2 repeat forms 2 x 2 nucleotide 5'CU/3'UC loops. DM is caused when the expanded repeats bind the RNA splicing regulator Muscleblind-like 1 protein (MBNL1), thus compromising its function. Therefore, one potential therapeutic strategy for these diseases is to prevent MBNL1 from binding the toxic RNA repeats. Previously, we designed nanomolar inhibitors of the DM2-MBNL1 interaction by modularly assembling 6'-N-5-hexyonate kanamycin A (K) onto a peptoid backbone. The K ligand binds the 2 x 2 pyrimidine-rich internal loops found in the DM2 RNA with high affinity. The best compound identified from that study contains three K modules separated by four propylamine spacing modules and is 20-fold selective for the DM2 RNA over the DM1 RNA. Because the modularly assembled K-containing compounds also bound the DM1 RNA, albeit with lower affinity, and because the loop size is different, we hypothesized that the optimal DM1 RNA binder may display K modules separated by a shorter distance. Indeed, here the ideal DM1 RNA binder has only two propylamine spacing modules separating the K ligands. Peptoids displaying three and four K modules on a peptoid scaffold bind the DM1 RNA with K(d)'s of 20 nM (3-fold selective for DM1 over DM2) and 4 nM (6-fold selective) and inhibit the RNA-protein interaction with IC(50)'s of 40 and 7 nM, respectively. Importantly, by coupling the two studies together, we have determined that appropriate spacing can affect binding selectivity by 60-fold (20- x 3-fold). The trimer and tetramer also bind approximately 13- and approximately 63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and nontoxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and, (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.
An association between neutrophils and immunoglobulin free light chains in the pathogenesis of chronic obstructive pulmonary disease.
Braber Saskia,Thio Marco,Blokhuis Bart R,Henricks Paul A J,Koelink Pim J,Groot Kormelink Tom,Bezemer Gillina F G,Kerstjens Huib A M,Postma Dirkje S,Garssen Johan,Kraneveld Aletta D,Redegeld Frank A,Folkerts Gert
American journal of respiratory and critical care medicine
RATIONALE:Neutrophils are key players in chronic obstructive pulmonary disease (COPD), and increased numbers of neutrophils are present in sputum and lung tissue of patients with COPD. Interestingly, immunoglobulin free light chains (IgLC) are able to prolong the life of neutrophils; therefore, IgLC may contribute to the chronic state of inflammation. OBJECTIVES:In this study, the relation between IgLC and COPD has been investigated. METHODS:We investigated the presence of IgLC in different murine lung emphysema models. IgLC levels in serum from mice and patients with COPD were examined by Western blot analysis and ELISA, respectively. IgLC levels in lung tissue were determined by immunohistochemistry. Fluorescence-activated cell sorter and immunofluorescent analysis were used to detect binding between IgLC and human neutrophils. Interleukin-8 (CXCL8) release by neutrophils after IgLC incubation was measured by ELISA. The effect of F991, an IgLC antagonist, was examined on the neutrophil influx in murine lungs after 5 days of smoke exposure. MEASUREMENTS AND MAIN RESULTS:Increased levels of IgLC in serum of cigarette smoke-exposed and cigarette smoke extract-treated mice compared with control mice were observed. Patients with COPD showed increased serum IgLC and expression of IgLC in lung tissue compared with healthy volunteers. Interestingly, IgLC bound to neutrophils and activated neutrophils to release CXCL8. F991 inhibited the IgLC binding to neutrophils and reduced the smoke-induced neutrophil influx in murine lungs after smoke exposure. CONCLUSIONS:This study describes for the first time an association between neutrophils and IgLC in the pathophysiology of COPD, which could open new avenues to targeted treatment of this chronic disease.
TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.
Kirsch Ilan R,Watanabe Rei,O'Malley John T,Williamson David W,Scott Laura-Louise,Elco Christopher P,Teague Jessica E,Gehad Ahmed,Lowry Elizabeth L,LeBoeuf Nicole R,Krueger James G,Robins Harlan S,Kupper Thomas S,Clark Rachael A
Science translational medicine
Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin.
A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.
Sweeney Kari A,Dao Dee N,Goldberg Michael F,Hsu Tsungda,Venkataswamy Manjunatha M,Henao-Tamayo Marcela,Ordway Diane,Sellers Rani S,Jain Paras,Chen Bing,Chen Mei,Kim John,Lukose Regy,Chan John,Orme Ian M,Porcelli Steven A,Jacobs William R
We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.
Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis.
Bollée Guillaume,Flamant Martin,Schordan Sandra,Fligny Cécile,Rumpel Elisabeth,Milon Marine,Schordan Eric,Sabaa Nathalie,Vandermeersch Sophie,Galaup Ariane,Rodenas Anita,Casal Ibrahim,Sunnarborg Susan W,Salant David J,Kopp Jeffrey B,Threadgill David W,Quaggin Susan E,Dussaule Jean-Claude,Germain Stéphane,Mesnard Laurent,Endlich Karlhans,Boucheix Claude,Belenfant Xavier,Callard Patrice,Endlich Nicole,Tharaux Pierre-Louis
Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.
gp130 Controls Cardiomyocyte Proliferation and Heart Regeneration.
Li Yandong,Feng Jie,Song Shen,Li Haotong,Yang Huijun,Zhou Bin,Li Yan,Yue Zhang,Lian Hong,Liu Lihui,Hu Shengshou,Nie Yu
BACKGROUND:A key cause of the high mortality of cardiovascular diseases is the cardiomyocyte inability to renew after cardiac injury. As a promising strategy to supplement functional myocytes for cardiac repair, there is a pressing need to understand the cellular and molecular mechanisms of heart regeneration. METHODS:Seven genetic mouse lines were used: global OSM (oncostatin M) knockout, monocyte-/macrophage-specific OSM deletion, cardiomyocyte-specific lines, including OSM receptor deletion, gp130 (glycoprotein 130) deletion, gp130 activation, and Yap (yes-associated protein) ablation with gp130 activation mice. A series of molecular signaling experiments, including RNA sequencing, immunostaining, coimmunoprecipitation, and imaging flow cytometry, were conducted. Two models of cardiac injury, apical resection and myocardial infarction operation, were performed in neonatal, juvenile, and adult mice. Heart regeneration and cardiac function were evaluated by Masson staining and echocardiography, respectively. Gene recombinant adenovirus-associated virus was constructed and infected myocardial-infarcted mice as a gene therapy. RESULTS:OSM was identified by RNA sequencing as a key upstream regulator of cardiomyocyte proliferation during neonatal heart regeneration in mice. Cardiomyocyte proliferation and heart regeneration were suspended in neonatal mice after cardiac injury when OSM was conditionally knockout in macrophages. The cardiomyocyte-specific deficiency of the OSM receptor heterodimers, OSM receptor and gp130, individually in cardiomyocytes reduced myocyte proliferation and neonatal heart regeneration. Conditional activation of gp130 in cardiomyocytes promoted cardiomyocyte proliferation and heart regeneration in juvenile and adult mice. Using RNA sequencing and functional screening, we found that Src mediated gp130-triggered cardiomyocyte proliferation by activating Yap (yes-associated protein) with Y357 phosphorylation independently of the Hippo pathway. Cardiomyocyte-specific deletion of Yap in mice blocked the effect of gp130 activation-induced heart regeneration in juvenile mice. Gene therapy with adenovirus-associated virus encoding constitutively activated gp130 promoted cardiomyocyte proliferation and heart regeneration in adult mice after myocardial infarction. CONCLUSIONS:Macrophage recruitment is essential for heart regeneration through the secretion of OSM, which promotes cardiomyocyte proliferation. As the coreceptor of OSM, gp130 activation is sufficient to promote cardiomyocyte proliferation by activating Yap through Src during heart regeneration. gp130 is a potential therapeutic target to improve heart regeneration after cardiac injury.
TATA is a modular component of synthetic promoters.
Mogno Ilaria,Vallania Francesco,Mitra Robi D,Cohen Barak A
The expression of most genes is regulated by multiple transcription factors. The interactions between transcription factors produce complex patterns of gene expression that are not always obvious from the arrangement of cis-regulatory elements in a promoter. One critical element of promoters is the TATA box, the docking site for the RNA polymerase holoenzyme. Using a synthetic promoter system coupled to a thermodynamic model of combinatorial regulation, we analyze the effects of different strength TATA boxes on various aspects of combinatorial cis-regulation. The thermodynamic model explains 75% of the variance in gene expression in synthetic promoter libraries with different strength TATA boxes, suggesting that many of the salient aspects of cis-regulation are captured by the model. Our results demonstrate that the effect of changing the TATA box on gene expression is the same for all synthetic promoters regardless of the arrangement of cis-regulatory sites we studied. Our analysis also showed that in our synthetic system the strength of the RNA polymerase-TATA interaction does not alter the combinatorial interactions between transcription factors, or between transcription factors and RNA polymerase. Finally, we show that although stronger TATA boxes increase expression in a predictable fashion, stronger TATA boxes have very little effect on noise in our synthetic promoters, regardless of the arrangement of cis-regulatory sites. Our results support a modular model of promoter function, where cis-regulatory elements can be mixed and matched (programmed) with outcomes on expression that are predictable based on the rules of simple protein-protein and protein-DNA interactions.
Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B-Reactive CD4 T-Regulatory Cells.
Wolf Dennis,Gerhardt Teresa,Winkels Holger,Michel Nathaly Anto,Pramod Akula Bala,Ghosheh Yanal,Brunel Simon,Buscher Konrad,Miller Jacqueline,McArdle Sara,Baas Livia,Kobiyama Kouji,Vassallo Melanie,Ehinger Erik,Dileepan Thamotharampillai,Ali Amal,Schell Maximilian,Mikulski Zbigniew,Sidler Daniel,Kimura Takayuki,Sheng Xia,Horstmann Hauke,Hansen Sophie,Mitre Lucia Sol,Stachon Peter,Hilgendorf Ingo,Gaddis Dalia E,Hedrick Catherine,Benedict Chris A,Peters Bjoern,Zirlik Andreas,Sette Alessandro,Ley Klaus
BACKGROUND:Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 T cells with an atheroprotective, regulatory T cell (T) phenotype in healthy individuals. Yet, the function of apoB-reactive T and their relationship with pathogenic T1 cells remain unknown. METHODS:To interrogate the function of autoreactive CD4 T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B (apoB) at the single-cell level. RESULTS:We found that apoB T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T-like transcriptome, although only 21% of all apoB T cells expressed the T transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB T cells formed several clusters with mixed T signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T1, T helper cell type 2 (T2), and T helper cell type 17 (T17), and of follicular-helper T cells. ApoB T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T1/T17-like cells with proinflammatory properties and only a residual T transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T1/T17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB T in lineage tracing of hyperlipidemic mice. In adoptive transfer experiments, converting apoB T failed to protect from atherosclerosis. CONCLUSIONS:Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T as a novel cellular target in atherosclerosis.
A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.
Chen Ruihuan,Nishimura Merry C,Bumbaca Stephanie M,Kharbanda Samir,Forrest William F,Kasman Ian M,Greve Joan M,Soriano Robert H,Gilmour Laurie L,Rivers Celina Sanchez,Modrusan Zora,Nacu Serban,Guerrero Steve,Edgar Kyle A,Wallin Jeffrey J,Lamszus Katrin,Westphal Manfred,Heim Susanne,James C David,VandenBerg Scott R,Costello Joseph F,Moorefield Scott,Cowdrey Cynthia J,Prados Michael,Phillips Heidi S
The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.
Persistent Increase in Microglial RAGE Contributes to Chronic Stress-Induced Priming of Depressive-like Behavior.
Franklin Tina C,Wohleb Eric S,Zhang Yi,Fogaça Manoela,Hare Brendan,Duman Ronald S
BACKGROUND:Chronic stress-induced inflammatory responses occur in part via danger-associated molecular pattern (DAMP) molecules, such as high mobility group box 1 protein (HMGB1), but the receptor(s) underlying DAMP signaling have not been identified. METHODS:Microglia morphology and DAMP signaling in enriched rat hippocampal microglia were examined during the development and expression of chronic unpredictable stress (CUS)-induced behavioral deficits, including long-term, persistent changes after CUS. RESULTS:The results show that CUS promotes significant morphological changes and causes robust upregulation of HMGB1 messenger RNA in enriched hippocampal microglia, an effect that persists for up to 6 weeks after CUS exposure. This coincides with robust and persistent upregulation of receptor for advanced glycation end products (RAGE) messenger RNA, but not toll-like receptor 4 in hippocampal microglia. CUS also increased surface expression of RAGE protein on hippocampal microglia as determined by flow cytometry and returned to basal levels 5 weeks after CUS. Importantly, exposure to short-term stress was sufficient to increase RAGE surface expression as well as anhedonic behavior, reflecting a primed state that results from a persistent increase in RAGE messenger RNA expression. Further evidence for DAMP signaling in behavioral responses is provided by evidence that HMGB1 infusion into the hippocampus was sufficient to cause anhedonic behavior and by evidence that RAGE knockout mice were resilient to stress-induced anhedonia. CONCLUSIONS:Together, the results provide evidence of persistent microglial HMGB1-RAGE expression that increases vulnerability to depressive-like behaviors long after chronic stress exposure.
Bone marrow-derived pancreatic stellate cells in rats.
Sparmann Gisela,Kruse Marie-Luise,Hofmeister-Mielke Nicole,Koczan Dirk,Jaster Robert,Liebe Stefan,Wolff Daniel,Emmrich Jörg
Origin and fate of pancreatic stellate cells (PSCs) before, during and after pancreatic injury are a matter of debate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. However, the turnover of the cells remains obscure. The present study addressed the issue of a potential bone marrow (BM) origin of PSCs. We used a model of stable hematopoietic chimerism by grafting enhanced green fluorescence protein (eGFP)-expressing BM cells after irradiation of acceptor rats. Chimerism was detected by FACS analysis of eGFP-positive cells in the peripheral blood. Dibutyltin dichloride (DBTC) was used to induce acute pancreatic inflammation with subsequent recovery over 4 weeks. Investigations have been focused on isolated cells to detect the resting PSC population. The incidence of eGFP-positive PSC obtained from the pancreas of chimeric rats was approximately 7% in healthy pancreatic tissue and increased significantly to a mean of 18% in the restored pancreas 4 weeks after DBTC-induced acute inflammation. Our results suggest that BM-derived progenitor cells represent a source of renewable stellate cells in the pancreas. Increased numbers of resting PSCs after regeneration point toward enhanced recruitment of BM-derived cells to the pancreas and/or re-acquisition of a quiescent state after inflammation-induced activation.
A versatile microsatellite instability reporter system in human cells.
Koole Wouter,Schäfer Henning S,Agami Reuven,van Haaften Gijs,Tijsterman Marcel
Nucleic acids research
Here, we report the investigation of microsatellite instability (MSI) in human cells with a newly developed reporter system based on fluorescence. We composed a vector into which microsatellites of different lengths and nucleotide composition can be introduced between a functional copy of the fluorescent protein mCherry and an out-of-frame copy of EGFP; in vivo frameshifting will lead to EGFP expression, which can be quantified by fluorescence activated cell sorting (FACS). Via targeted recombineering, single copy reporters were introduced in HEK293 and MCF-7 cells. We found predominantly -1 and +1 base pair frameshifts, the levels of which are kept in tune by mismatch repair. We show that tract length and composition greatly influences MSI. In contrast, a tracts' potential to form a G-quadruplex structure, its strand orientation or its transcriptional status is not affecting MSI. We further validated the functionality of the reporter system for screening microsatellite mutagenicity of compounds and for identifying modifiers of MSI: using a retroviral miRNA expression library, we identified miR-21, which targets MSH2, as a miRNA that induces MSI when overexpressed. Our data also provide proof of principle for the strategy of combining fluorescent reporters with next-generation sequencing technology to identify genetic factors in specific pathways.
Factors that determine the antiviral efficacy of HCV-specific CD8(+) T cells ex vivo.
Seigel Bianca,Bengsch Bertram,Lohmann Volker,Bartenschlager Ralf,Blum Hubert E,Thimme Robert
BACKGROUND & AIMS:Dysfunctional CD8(+) T cells are believed to contribute to the ability of hepatitis C virus (HCV) to evade the immune response. Most studies have focused on the effector functions of HCV-specific CD8(+) T cells or their surface expression of inhibitory receptors. There is currently no information available about the ex vivo ability of HCV-specific CD8(+) T cells to inhibit viral replication (antiviral efficacy). METHODS:To analyze the antiviral efficacy of virus-specific CD8(+) T cells ex vivo, we used an immunologic model based on a cell line that expresses HLA-A*02 and contains a stably replicating HCV reporter replicon. We isolated HCV-specific CD8(+) T cells from 18 HLA-A*02-positive patients with chronic HCV infection and 15 subjects with resolved HCV infection (7 spontaneous, 8 after therapy). Replicon cells were labeled with virus-specific peptides; inhibition of HCV replication was determined by measuring luciferase activity after 72 hours of coculture with virus-specific CD8(+) T cells. RESULTS:HCV-specific CD8(+) T cells from patients with chronic HCV infection had a significantly lower antiviral efficacy than influenza-, Epstein-Barr virus-, and cytomegalovirus-specific CD8(+) T cells. Antiviral efficacy was associated with the ability of virus-specific CD8(+) T cells to secrete interferon gamma. The antiviral efficacy of HCV-specific CD8(+) T cells was linked to surface expression of CD127 and PD-1. The cytokines interleukin-2, interleukin-7, and interleukin-15 increased the antiviral efficacy of CD127-positive but not of CD127-negative, HCV-specific CD8(+) T cells. Spontaneous, but not antiviral therapy-induced, viral clearance was associated with increased antiviral efficacy. CONCLUSIONS:The ability of CD8(+) T cells to inhibit HCV replication ex vivo is associated with their ability to secrete interferon gamma and their surface expression of CD127 and PD-1.
Isolation, cultivation and characterization of adult murine prostate stem cells.
Lukacs Rita U,Goldstein Andrew S,Lawson Devon A,Cheng Donghui,Witte Owen N
The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we describe step-by-step procedures on the basis of previous work in our laboratory for the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; and the cultivation of prostate stem cells in vitro and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally, it will take approximately 8 h to harvest prostate cells, isolate the stem cell-enriched population and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo.
A new strategy for gene targeting and functional proteomics using the DT40 cell line.
Orlowska Kinga P,Klosowska Kamila,Szczesny Roman J,Cysewski Dominik,Krawczyk Pawel S,Dziembowski Andrzej
Nucleic acids research
DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry-based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination-based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.
Phenotypic states become increasingly sensitive to perturbations near a bifurcation in a synthetic gene network.
Axelrod Kevin,Sanchez Alvaro,Gore Jeff
Microorganisms often exhibit a history-dependent phenotypic response after exposure to a stimulus which can be imperative for proper function. However, cells frequently experience unexpected environmental perturbations that might induce phenotypic switching. How cells maintain phenotypic states in the face of environmental fluctuations remains an open question. Here, we use environmental perturbations to characterize the resilience of phenotypic states in a synthetic gene network near a critical transition. We find that far from the critical transition an environmental perturbation may induce little to no phenotypic switching, whereas close to the critical transition the same perturbation can cause many cells to switch phenotypic states. This loss of resilience was observed for perturbations that interact directly with the gene circuit as well as for a variety of generic perturbations-such as salt, ethanol, or temperature shocks-that alter the state of the cell more broadly. We obtain qualitatively similar findings in natural gene circuits, such as the yeast GAL network. Our findings illustrate how phenotypic memory can become destabilized by environmental variability near a critical transition.
Blocking the natural killer cell inhibitory receptor NKG2A increases activity of human natural killer cells and clears hepatitis B virus infection in mice.
Li Fenglei,Wei Hairong,Wei Haiming,Gao Yufeng,Xu Long,Yin Wenwei,Sun Rui,Tian Zhigang
BACKGROUND & AIMS:We studied the functions of natural killer (NK) cells and the role of the NK cell inhibitory receptor (NKG2A) during hepatitis B virus (HBV) infection in patients and mice. METHODS:We analyzed levels of NKG2A on peripheral blood NK cells from 42 patients with active chronic hepatitis B (CHB), 31 patients with inactive CHB, and 35 healthy volunteers (controls). Five patients with CHB treated with antiviral therapy were also included to evaluate changes in NK cells after HBV titers decreased. We examined the effects of blocking antibodies against NKG2A or its ligand Qa-1 (equivalent to HLA-E in humans) in immunocompetent mice that express HBV from a plasmid and are positive for serum hepatitis B surface antigen (a mouse model of HBV infection). RESULTS:A higher percentage of NK cells from patients with active CHB were positive for NKG2A (38.47%) than from patients with inactive CHB (19.33%; P < .01) or controls (27.96%; P < .05). The percentage of NKG2A(+) cells correlated with serum viral load (r = 0.5457; P < .001). The percentage of NKG2A(+) cells decreased along with HBV load in patients that received antiviral therapy (P < .05). Blocking NKG2A interaction with HLA-E in peripheral NK cells from patients with active CHB increased their cytotoxicity in vitro. NK cells of HBV carrier mice also had higher percentages of NK cells that expressed NKG2A compared with control mice; NKG2A was likely to be up-regulated by production of interleukin-10 by hepatic regulatory CD4(+)CD25(+) T cells. Blocking Qa-1 in these mice promoted viral clearance in an NK cell-dependent manner. CONCLUSIONS:Infection with HBV increases levels of the inhibitory receptor NKG2A on NK cells in mice and humans, and reduces their ability to clear HBV. Reagents designed to block the interaction between NKG2A and HLA-E might be developed to treat CHB infection.
Bacterial sensor triggering receptor expressed on myeloid cells-2 regulates the mucosal inflammatory response.
Correale Carmen,Genua Marco,Vetrano Stefania,Mazzini Elisa,Martinoli Chiara,Spinelli Antonino,Arena Vincenzo,Peyrin-Biroulet Laurent,Caprioli Flavio,Passini Nadia,Panina-Bordignon Paola,Repici Alessandro,Malesci Alberto,Rutella Sergio,Rescigno Maria,Danese Silvio
BACKGROUND AND AIMS:Triggering receptor expressed on myeloid cells (TREM)-2 is a surface receptor detected on macrophages, dendritic cells, and microglia that binds repeated anionic motifs on yeast and Gram-positive and Gram-negative bacteria. Little is known about TREM-2 expression and function in the intestine or its role in inflammatory bowel disease (IBD). We investigated the expression of TREM-2 in the intestinal lamina propria and its role in the development of colonic inflammation. METHODS:We measured levels of TREM-2 in lamina propria mononuclear cells from surgical specimens collected from patients with IBD or cancer (controls). We analyzed the development of colitis in TREM-2 knockout and wild-type mice. Colon samples were isolated from mice and analyzed for cytokine expression, phagocytosis of bacteria, proliferation in colonic crypts, lamina propria mononuclear cell function, and T-cell activation by ovalbumin. RESULTS:TREM-2 was virtually absent from colon samples of control patients, but levels were significantly higher in within the inflamed mucosa of patients with IBD; it was mainly expressed by CD11c(+) cells. Levels of TREM-2 increased as acute or chronic colitis was induced in mice. TREM-2 knockout mice developed less severe colitis than wild-type mice; the knockout mice lost less body weight, had a lower disease activity index, and had smaller mucosal lesions in endoscopic analysis. Colon dendritic cells from TREM-2 knockout mice produced lower levels of inflammatory cytokines and had reduced levels of bacterial killing and T-cell activation than cells from wild-type mice. CONCLUSIONS:TREM-2 contributes to mucosal inflammation during development of colitis in mice. Levels of TREM-2 are increased within the inflamed mucosa of patients with IBD, indicating its potential as a therapeutic target.
Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories.
Nucleic acids research
Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.
Contrast agents and renal cell apoptosis.
Romano Giulia,Briguori Carlo,Quintavalle Cristina,Zanca Ciro,Rivera Natalia V,Colombo Antonio,Condorelli Gerolama
European heart journal
AIMS:Contrast media (CM) induce a direct toxic effect on renal tubular cells. This toxic effect may have a role in the pathophysiology of contrast nephropathy. METHODS AND RESULTS:We evaluated (i) the cytotoxicity of CM [both low-osmolality (LOCM) and iso-osmolality (IOCM)], of iodine alone, and of an hyperosmolar solution (mannitol 8%) on human embryonic kidney (HEK 293), porcine proximal renal tubular (LLC-PK1), and canine Madin-Darby distal tubular renal (MDCK) cells; and (ii) the effectiveness of various antioxidant compounds [n-acetylcysteine (NAC), ascorbic acid and sodium bicarbonate] in preventing CM cytotoxicity. The cytotoxicity of CM was assessed at different time points, with different methods: cell viability, DNA laddering, flow cytometry, and caspase activation. Both LOCM and IOCM produced a concentration- and time-dependent increase in cell death as assessed by the different methods. On the contrary, iodine alone and hyperosmolar solution did not induce any significant cytotoxic effect. There was not any significant difference in the cytotoxic effect between LOCM and IOCM. Furthermore, both LOCM and IOCM caused a marked increase in caspase-3 and -9 activities and poly(ADP-ribose) fragmentation, while no effect on caspase-8/-10 was observed, thus indicating that the CM activated apoptosis mainly through the intrinsic pathway. Both CM induced an increase in protein expression levels of pro-apoptotic members of the Bcl2 family (Bim and Bad). NAC and ascorbic acid but not sodium bicarbonate had a dose-dependent protective effect on renal cells after 3 h incubation with high dose (200 mg iodine/mL) of both LOCM and IOCM. CONCLUSION:Both LOCM and IOCM induce a dose-dependent renal cell apoptosis. NAC and ascorbic acid but not sodium bicarbonate prevent this contrast-induced apoptosis.
The SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE.
Hagberg Niklas,Joelsson Martin,Leonard Dag,Reid Sarah,Eloranta Maija-Leena,Mo John,Nilsson Magnus K,Syvänen Ann-Christine,Bryceson Yenan T,Rönnblom Lars
Annals of the rheumatic diseases
OBJECTIVES:Genetic variants in the transcription factor are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic risk allele rs7574865[T] affects the function of immune cells in SLE. METHODS:Peripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ cells on IL-12 stimulation were determined by flow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. RESULTS:In resting PBMCs, the risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8 T cells from risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8 and CD4 T cells. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from risk patients, respectively. CONCLUSIONS:T cells from patients with SLE carrying the risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment.
Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses.
Gaucher Denis,Therrien René,Kettaf Nadia,Angermann Bastian R,Boucher Geneviève,Filali-Mouhim Abdelali,Moser Janice M,Mehta Riyaz S,Drake Donald R,Castro Erika,Akondy Rama,Rinfret Aline,Yassine-Diab Bader,Said Elias A,Chouikh Younes,Cameron Mark J,Clum Robert,Kelvin David,Somogyi Roland,Greller Larry D,Balderas Robert S,Wilkinson Peter,Pantaleo Giuseppe,Tartaglia Jim,Haddad Elias K,Sékaly Rafick-Pierre
The Journal of experimental medicine
Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.
Activation of Hedgehog Signaling Promotes Development of Mouse and Human Enteric Neural Crest Cells, Based on Single-Cell Transcriptome Analyses.
BACKGROUND & AIMS:It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. METHODS:We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufu, Ptch1, and Gli3 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1p75) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. RESULTS:Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. CONCLUSIONS:In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons.
Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.
He Zhengxiang,Chen Lili,Chen Grace,Smaldini Paola,Bongers Gerold,Catalan-Dibene Jovani,Furtado Glaucia C,Lira Sergio A
BACKGROUND & AIMS:Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS:We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS:Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha (PDGRFA) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS:In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.
Suppressive and Gut-Reparative Functions of Human Type 1 T Regulatory Cells.
Cook Laura,Stahl Martin,Han Xiao,Nazli Aisha,MacDonald Katherine N,Wong May Q,Tsai Kevin,Dizzell Sara,Jacobson Kevan,Bressler Brian,Kaushic Charu,Vallance Bruce A,Steiner Theodore S,Levings Megan K
BACKGROUND & AIMS:T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3 (forkhead box protein 3)-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin (IL) 10. IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in the treatment of inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. METHODS:We obtained blood and colon biopsy samples from patients with Crohn's disease or ulcerative colitis or healthy individuals (controls). CD4 T cells were isolated from blood samples and stimulated with anti-CD3 and anti-CD28 beads, and Tr1 cells were purified by using an IL10 cytokine-capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4CD25CD127 cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3 and anti-CD28 beads, and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells, and transepithelial electrical resistance was measured to determine epithelial cell barrier function. RESULTS:Phenotypes of Tr1 cells isolated from control individuals vs patients with Crohn's disease or ulcerative colitis did not differ significantly after expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1B and tumor necrosis factor from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy individuals and patients with Crohn's disease or ulcerative colitis secreted IL22, which promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. CONCLUSIONS:Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate immune response). They also secrete IL22 to promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.
Monocytes inhibit hepatitis C virus-induced TRAIL expression on CD56 NK cells.
Mele Dalila,Mantovani Stefania,Oliviero Barbara,Grossi Giulia,Lombardi Andrea,Mondelli Mario U,Varchetta Stefania
Journal of hepatology
BACKGROUND & AIMS:Natural killer (NK) cells play an important role in the pathogenesis of hepatitis C virus (HCV) infection. We have previously shown that culture-derived HCV (HCVcc) enhance tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) expression on healthy NK cells, but not on those from patients infected with HCV, which was likely dependent on accessory cells. Here we sought to elucidate the mechanisms involved in altered TRAIL upregulation in this setting. METHODS:Peripheral blood mononuclear cells (PBMC) from controls and patients infected with HCV were exposed to HCVcc. Cell depletions were performed to identify cells responsible for NK cell activation. Flow cytometry and ELISA were used to identify the cytokines involved in the NK activation process. RESULTS:In patients infected with HCV, soluble factors secreted by control PBMC restored the ability of NK cells to express TRAIL. Of note, CD14+ cell depletion had identical effects upon virus exposure and promoted increased degranulation. Moreover, increased concentrations of interleukin (IL)-18 binding protein a (IL-18BPa) and IL-36 receptor antagonist (IL-36RA) were observed after PBMC exposure to HCVcc in patients with HCV. HCVcc-induced NK cell TRAIL expression was inhibited by IL-18BPa and IL-36RA in control subjects. There were statistically significant correlations between IL-18BPa and indices of liver inflammation and fibrosis, supporting a role for this protein in the pathogenesis of chronic HCV infection. CONCLUSIONS:During chronic HCV infection, monocytes play a key role in negative regulation of NK cell activation, predominantly via secretion of inhibitors of IL-18 and IL-36. LAY SUMMARY:Coordination and collaboration between immune cells are essential to fight pathogens. Herein we show that during HCV infection monocytes secrete IL-18 and IL-36 inhibitory proteins, reducing NK cell activation, and consequently inhibiting their ability to express TRAIL and kill target cells.
Cycling CD4+ T cells in HIV-infected immune nonresponders have mitochondrial dysfunction.
The Journal of clinical investigation
Immune nonresponder (INR) HIV-1-infected subjects are characterized by their inability to reconstitute the CD4+ T cell pool after antiretroviral therapy. This is linked to poor clinical outcome. Mechanisms underlying immune reconstitution failure are poorly understood, although, counterintuitively, INRs often have increased frequencies of circulating CD4+ T cells in the cell cycle. While cycling CD4+ T cells from healthy controls and HIV+ patients with restored CD4+ T cell numbers complete cell division in vitro, cycling CD4+ T cells from INRs do not. Here, we show that cells with the phenotype and transcriptional profile of Tregs were enriched among cycling cells in health and in HIV infection. Yet there were diminished frequencies and numbers of Tregs among cycling CD4+ T cells in INRs, and cycling CD4+ T cells from INR subjects displayed transcriptional profiles associated with the impaired development and maintenance of functional Tregs. Flow cytometric assessment of TGF-β activity confirmed the dysfunction of Tregs in INR subjects. Transcriptional profiling and flow cytometry revealed diminished mitochondrial fitness in Tregs among INRs, and cycling Tregs from INRs had low expression of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α) and transcription factor A for mitochondria (TFAM). In vitro exposure to IL-15 allowed cells to complete division, restored the expression of PGC1α and TFAM, and regenerated mitochondrial fitness in the cycling Tregs of INRs. Our data suggest that rescuing mitochondrial function could correct the immune dysfunction characteristic of Tregs in HIV-1-infected subjects who fail to restore CD4+ T cells during antiretroviral therapy.
Longitudinal profiling of human blood transcriptome in healthy and lupus pregnancy.
Hong Seunghee,Banchereau Romain,Maslow Bat-Sheva L,Guerra Marta M,Cardenas Jacob,Baisch Jeanine,Branch D Ware,Porter T Flint,Sawitzke Allen,Laskin Carl A,Buyon Jill P,Merrill Joan,Sammaritano Lisa R,Petri Michelle,Gatewood Elizabeth,Cepika Alma-Martina,Ohouo Marina,Obermoser Gerlinde,Anguiano Esperanza,Kim Tae Whan,Nulsen John,Nehar-Belaid Djamel,Blankenship Derek,Turner Jacob,Banchereau Jacques,Salmon Jane E,Pascual Virginia
The Journal of experimental medicine
Systemic lupus erythematosus carries an increased risk of pregnancy complications, including preeclampsia and fetal adverse outcomes. To identify the underlying molecular mechanisms, we longitudinally profiled the blood transcriptome of 92 lupus patients and 43 healthy women during pregnancy and postpartum and performed multicolor flow cytometry in a subset of them. We also profiled 25 healthy women undergoing assisted reproductive technology to monitor transcriptional changes around embryo implantation. Sustained down-regulation of multiple immune signatures, including interferon and plasma cells, was observed during healthy pregnancy. These changes appeared early after embryo implantation and were mirrored in uncomplicated lupus pregnancies. Patients with preeclampsia displayed early up-regulation of neutrophil signatures that correlated with expansion of immature neutrophils. Lupus pregnancies with fetal complications carried the highest interferon and plasma cell signatures as well as activated CD4 T cell counts. Thus, blood immunomonitoring reveals that both healthy and uncomplicated lupus pregnancies exhibit early and sustained transcriptional modulation of lupus-related signatures, and a lack thereof associates with adverse outcomes.
Human lymphoma mutations reveal CARD11 as the switch between self-antigen-induced B cell death or proliferation and autoantibody production.
Jeelall Yogesh S,Wang James Q,Law Hsei-Di,Domaschenz Heather,Fung Herman K H,Kallies Axel,Nutt Stephen L,Goodnow Christopher C,Horikawa Keisuke
The Journal of experimental medicine
Self-tolerance and immunity are actively acquired in parallel through a poorly understood ability of antigen receptors to switch between signaling death or proliferation of antigen-binding lymphocytes in different contexts. It is not known whether this tolerance-immunity switch requires global rewiring of the signaling apparatus or if it can arise from a single molecular change. By introducing individual CARD11 mutations found in human lymphomas into antigen-activated mature B lymphocytes in mice, we find here that lymphoma-derived CARD11 mutations switch the effect of self-antigen from inducing B cell death into T cell-independent proliferation, Blimp1-mediated plasmablast differentiation, and autoantibody secretion. Our findings demonstrate that regulation of CARD11 signaling is a critical switch governing the decision between death and proliferation in antigen-stimulated mature B cells and that mutations in this switch represent a powerful initiator for aberrant B cell responses in vivo.
B and T cells collaborate in antiviral responses via IL-6, IL-21, and transcriptional activator and coactivator, Oct2 and OBF-1.
Karnowski Alex,Chevrier Stephane,Belz Gabrielle T,Mount Adele,Emslie Dianne,D'Costa Kathy,Tarlinton David M,Kallies Axel,Corcoran Lynn M
The Journal of experimental medicine
A strong humoral response to infection requires the collaboration of several hematopoietic cell types that communicate via antigen presentation, surface coreceptors and their ligands, and secreted factors. The proinflammatory cytokine IL-6 has been shown to promote the differentiation of activated CD4(+) T cells into T follicular helper cells (T(FH) cells) during an immune response. T(FH) cells collaborate with B cells in the formation of germinal centers (GCs) during T cell-dependent antibody responses, in part through secretion of critical cytokines such as IL-21. In this study, we demonstrate that loss of either IL-6 or IL-21 has marginal effects on the generation of T(FH) cells and on the formation of GCs during the response to acute viral infection. However, mice lacking both IL-6 and IL-21 were unable to generate a robust T(FH) cell-dependent immune response. We found that IL-6 production in follicular B cells in the draining lymph node was an important early event during the antiviral response and that B cell-derived IL-6 was necessary and sufficient to induce IL-21 from CD4(+) T cells in vitro and to support T(FH) cell development in vivo. Finally, the transcriptional activator Oct2 and its cofactor OBF-1 were identified as regulators of Il6 expression in B cells.
Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.
Huang Z,Petty J T,O'Quinn B,Longmire J L,Brown N C,Jett J H,Keller R A
Nucleic acids research
A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.
Tryptophan hydroxylase-1 regulates immune tolerance and inflammation.
Nowak Elizabeth C,de Vries Victor C,Wasiuk Anna,Ahonen Cory,Bennett Kathryn A,Le Mercier Isabelle,Ha Dae-Gon,Noelle Randolph J
The Journal of experimental medicine
Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity.
Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory.
Kaji Tomohiro,Ishige Akiko,Hikida Masaki,Taka Junko,Hijikata Atsushi,Kubo Masato,Nagashima Takeshi,Takahashi Yoshimasa,Kurosaki Tomohiro,Okada Mariko,Ohara Osamu,Rajewsky Klaus,Takemori Toshitada
The Journal of experimental medicine
One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.
Neuropilin 1 deficiency on CD4+Foxp3+ regulatory T cells impairs mouse melanoma growth.
Hansen Wiebke,Hutzler Marina,Abel Simone,Alter Christina,Stockmann Christian,Kliche Stefanie,Albert Juliane,Sparwasser Tim,Sakaguchi Shimon,Westendorf Astrid M,Schadendorf Dirk,Buer Jan,Helfrich Iris
The Journal of experimental medicine
Infiltration of Foxp3(+) regulatory T (T reg) cells is considered to be a critical step during tumor development and progression. T reg cells supposedly suppress locally an effective anti-tumor immune response within tumor tissues, although the precise mechanism by which T reg cells infiltrate the tumor is still unclear. We provide evidence that Neuropilin 1 (Nrp-1), highly expressed by Foxp3(+) T reg cells, regulates the immunological anti-tumor control by guiding T reg cells into the tumor in response to tumor-derived vascular endothelial growth factor (VEGF). We demonstrate for the first time that T cell-specific ablation of Nrp-1 expression results in a significant breakdown in tumor immune escape in various transplantation models and in a spontaneous, endogenously driven melanoma model associated with strongly reduced tumor growth and prolonged tumor-free survival. Strikingly, numbers of tumor-infiltrating Foxp3(+) T reg cells were significantly reduced accompanied by enhanced activation of CD8(+) T cells within tumors of T cell-specific Nrp-1-deficient mice. This phenotype can be reversed by adoptive transfer of Nrp-1(+) T reg cells from wild-type mice. Thus, our data strongly suggest that Nrp-1 acts as a key mediator of Foxp3(+) T reg cell infiltration into the tumor site resulting in a dampened anti-tumor immune response and enhanced tumor progression.
TNF-related apoptosis-inducing ligand (TRAIL) exerts therapeutic efficacy for the treatment of pneumococcal pneumonia in mice.
Steinwede Kathrin,Henken Stefanie,Bohling Jennifer,Maus Regina,Ueberberg Bianca,Brumshagen Christina,Brincks Erik L,Griffith Thomas S,Welte Tobias,Maus Ulrich A
The Journal of experimental medicine
Apoptotic death of alveolar macrophages observed during lung infection with Streptococcus pneumoniae is thought to limit overwhelming lung inflammation in response to bacterial challenge. However, the underlying apoptotic death mechanism has not been defined. Here, we examined the role of the TNF superfamily member TNF-related apoptosis-inducing ligand (TRAIL) in S. pneumoniae-induced macrophage apoptosis, and investigated the potential benefit of TRAIL-based therapy during pneumococcal pneumonia in mice. Compared with WT mice, Trail(-/-) mice demonstrated significantly decreased lung bacterial clearance and survival in response to S. pneumoniae, which was accompanied by significantly reduced apoptosis and caspase 3 cleavage but rather increased necrosis in alveolar macrophages. In WT mice, neutrophils were identified as a major source of intraalveolar released TRAIL, and their depletion led to a shift from apoptosis toward necrosis as the dominant mechanism of alveolar macrophage cell death in pneumococcal pneumonia. Therapeutic application of TRAIL or agonistic anti-DR5 mAb (MD5-1) dramatically improved survival of S. pneumoniae-infected WT mice. Most importantly, neutropenic mice lacking neutrophil-derived TRAIL were protected from lethal pneumonia by MD5-1 therapy. We have identified a previously unrecognized mechanism by which neutrophil-derived TRAIL induces apoptosis of DR5-expressing macrophages, thus promoting early bacterial killing in pneumococcal pneumonia. TRAIL-based therapy in neutropenic hosts may represent a novel antibacterial treatment option.
Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound self-antigen.
Taylor Justin J,Martinez Ryan J,Titcombe Philip J,Barsness Laura O,Thomas Stephanie R,Zhang Na,Katzman Shoshana D,Jenkins Marc K,Mueller Daniel L
The Journal of experimental medicine
B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen-specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen-specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens.
Chronic lymphocytic leukemia after chronic myeloid leukemia in the same patient: two different genomic events and a common treatment?
D'Arena Giovanni,Gemei Marica,Luciano Luigiana,D'Auria Fiorella,Deaglio Silvia,Statuto Teodora,Bianchino Gabriella,Grieco Vitina,Mansueto Giovanna,Guariglia Roberto,Pietrantuono Giuseppe,Martorelli Maria Carmen,Villani Oreste,Del Vecchio Luigi,Musto Pellegrino
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
Continuous expression of the transcription factor e2-2 maintains the cell fate of mature plasmacytoid dendritic cells.
Ghosh Hiyaa S,Cisse Babacar,Bunin Anna,Lewis Kanako L,Reizis Boris
The interferon-producing plasmacytoid dendritic cells (pDCs) share common progenitors with antigen-presenting classical dendritic cells (cDCs), yet they possess distinct morphology and molecular features resembling those of lymphocytes. It is unclear whether the unique cell fate of pDCs is actively maintained in the steady state. We report that the deletion of transcription factor E2-2 from mature peripheral pDCs caused their spontaneous differentiation into cells with cDC properties. This included the loss of pDC markers, increase in MHC class II expression and T cell priming capacity, acquisition of dendritic morphology, and induction of cDC signature genes. Genome-wide chromatin immunoprecipitation revealed direct binding of E2-2 to key pDC-specific and lymphoid genes, as well as to certain genes enriched in cDCs. Thus, E2-2 actively maintains the cell fate of mature pDCs and opposes the "default" cDC fate, in part through direct regulation of lineage-specific gene expression programs.
Epigenomic plasticity enables human pancreatic α to β cell reprogramming.
Bramswig Nuria C,Everett Logan J,Schug Jonathan,Dorrell Craig,Liu Chengyang,Luo Yanping,Streeter Philip R,Naji Ali,Grompe Markus,Kaestner Klaus H
The Journal of clinical investigation
Insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development. Using ChIP sequencing and RNA sequencing analysis, we determined the epigenetic and transcriptional landscape of human pancreatic α, β, and exocrine cells. We found that, compared with exocrine and β cells, differentiated α cells exhibited many more genes bivalently marked by the activating H3K4me3 and repressing H3K27me3 histone modifications. This was particularly true for β cell signature genes involved in transcriptional regulation. Remarkably, thousands of these genes were in a monovalent state in β cells, carrying only the activating or repressing mark. Our epigenomic findings suggested that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets. Indeed, we show that treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets. Thus, mammalian pancreatic islet cells display cell-type-specific epigenomic plasticity, suggesting that epigenomic manipulation could provide a path to cell reprogramming and novel cell replacement-based therapies for diabetes.
Epitope-specific airway-resident CD4+ T cell dynamics during experimental human RSV infection.
Guvenel Aleks,Jozwik Agnieszka,Ascough Stephanie,Ung Seng Kuong,Paterson Suzanna,Kalyan Mohini,Gardener Zoe,Bergstrom Emma,Kar Satwik,Habibi Maximillian S,Paras Allan,Zhu Jie,Park Mirae,Dhariwal Jaideep,Almond Mark,Wong Ernie Hc,Sykes Annemarie,Del Rosario Jerico,Trujillo-Torralbo Maria-Belen,Mallia Patrick,Sidney John,Peters Bjoern,Kon Onn Min,Sette Alessandro,Johnston Sebastian L,Openshaw Peter J,Chiu Christopher
The Journal of clinical investigation
BACKGROUNDRespiratory syncytial virus (RSV) is an important cause of acute pulmonary disease and one of the last remaining major infections of childhood for which there is no vaccine. CD4+ T cells play a key role in antiviral immunity, but they have been little studied in the human lung.METHODSHealthy adult volunteers were inoculated i.n. with RSV A Memphis 37. CD4+ T cells in blood and the lower airway were analyzed by flow cytometry and immunohistochemistry. Bronchial soluble mediators were measured using quantitative PCR and MesoScale Discovery. Epitope mapping was performed by IFN-γ ELISpot screening, confirmed by in vitro MHC binding.RESULTSActivated CD4+ T cell frequencies in bronchoalveolar lavage correlated strongly with local C-X-C motif chemokine 10 levels. Thirty-nine epitopes were identified, predominantly toward the 3' end of the viral genome. Five novel MHC II tetramers were made using an immunodominant EFYQSTCSAVSKGYL (F-EFY) epitope restricted to HLA-DR4, -DR9, and -DR11 (combined allelic frequency: 15% in Europeans) and G-DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labeling revealed enrichment of resident memory CD4+ T (Trm) cells in the lower airway; these Trm cells displayed progressive differentiation, downregulation of costimulatory molecules, and elevated CXCR3 expression as infection evolved.CONCLUSIONSHuman infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of Trm recognizing novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific CD4+ cells, potentially accelerating the development of effective vaccines.TRIAL REGISTRATIONClinicalTrials.gov NCT02755948.FUNDINGMedical Research Council, Wellcome Trust, National Institute for Health Research.
visualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption.
Matsuura Yoshinobu,Kikuta Junichi,Kishi Yuika,Hasegawa Tetsuo,Okuzaki Daisuke,Hirano Toru,Minoshima Masafumi,Kikuchi Kazuya,Kumanogoh Atsushi,Ishii Masaru
Annals of the rheumatic diseases
OBJECTIVES:Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destruction has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted . METHODS:Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry. RESULTS:Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation. CONCLUSIONS:Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens.
Increased circulating fibrocytes in asthma with chronic airflow obstruction.
Wang Chun-Hua,Huang Chien-Da,Lin Horng-Chyuan,Lee Kang-Yun,Lin Shu-Min,Liu Chien-Ying,Huang Kuo-Hsiung,Ko Yu-Shien,Chung Kian Fan,Kuo Han-Pin
American journal of respiratory and critical care medicine
RATIONALE:A proportion of patients with asthma present with chronic airflow obstruction (CAO). We hypothesized that this effect may result from increased activity of circulating fibroblast-like progenitor cells (fibrocytes) that could home to the airway mucosal wall. OBJECTIVES:To compare the proportion, proliferation, and differentiation of circulating fibrocytes from patients with asthma with CAO or no airflow obstruction (NOA) and control subjects. METHODS:We investigated circulating fibrocytes in 11 patients with asthma with CAO and a rapid decline in FEV(1), 9 patients with asthma with NOA, and 10 nonasthmatic control subjects. Blood nonadherent non-T (NANT) cells were incubated with fetal calf serum or each patient's own serum and fibrocytes expressing CD34, CD45, and collagen I with alpha-smooth muscle actin were identified by flow cytometry. MEASUREMENTS AND MAIN RESULTS:A higher percentage of circulating fibrocytes in NANT cells was found in patients with CAO when compared with patients with NOA and control subjects. In CAO, the slope of the yearly decline in FEV(1) correlated with circulating fibrocytes (r = -0.756, n = 11, P < 0.01). When NANT cells from patients with CAO were cultured in the patients' own sera, more fibrocytes were detected than when cultured in sera from patients with NOA or from normal subjects. An anti-transforming growth factor (TGF)-beta(1)-neutralizing antibody inhibited alpha-smooth muscle actin-positive fibrocyte transformation from NANT cells of patients with CAO. Serum TGF-beta(1) levels were higher in patients with CAO than in patients with NOA or in normal subjects. CONCLUSIONS:Circulating fibrocytes are increased in patients with asthma with CAO and can be transformed by TGF-beta(1) to myofibroblasts. Fibrocytes may contribute to airway obstruction in asthma.
Effects of Low FODMAP Diet on Symptoms, Fecal Microbiome, and Markers of Inflammation in Patients With Quiescent Inflammatory Bowel Disease in a Randomized Trial.
Cox Selina R,Lindsay James O,Fromentin Sébastien,Stagg Andrew J,McCarthy Neil E,Galleron Nathalie,Ibraim Samar B,Roume Hugo,Levenez Florence,Pons Nicolas,Maziers Nicolas,Lomer Miranda C,Ehrlich S Dusko,Irving Peter M,Whelan Kevin
BACKGROUND & AIMS:There is limited evidence that a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) reduces gut symptoms in quiescent inflammatory bowel disease (IBD). We performed a randomized, controlled trial to investigate the effects of a low FODMAP diet on persistent gut symptoms, the intestinal microbiome, and circulating markers of inflammation in patients with quiescent IBD. METHODS:We performed a single-blind trial of 52 patients with quiescent Crohn's disease or ulcerative colitis and persistent gut symptoms at 2 large gastroenterology clinics in the United Kingdom. Patients were randomly assigned to groups that followed a diet low in FODMAPs (n = 27) or a control diet (n = 25), with dietary advice, for 4 weeks. Gut symptoms and health-related quality of life were measured using validated questionnaires. Stool and blood samples were collected at baseline and end of trial. We assessed fecal microbiome composition and function using shotgun metagenomic sequencing and phenotypes of T cells in blood using flow cytometry. RESULTS:A higher proportion of patients reported adequate relief of gut symptoms following the low FODMAP diet (14/27, 52%) than the control diet (4/25, 16%, P=.007). Patients had a greater reduction in irritable bowel syndrome severity scores following the low FODMAP diet (mean reduction of 67; standard error, 78) than the control diet (mean reduction of 34; standard error, 50), although this difference was not statistically significant (P = .075). Following the low FODMAP diet, patients had higher health-related quality of life scores (81.9 ± 1.2) than patients on the control diet (78.3 ± 1.2, P = .042). A targeted analysis revealed that in stool samples collected at the end of the study period, patients on the low FODMAP diet had significantly lower abundance of Bifidobacterium adolescentis, Bifidobacterium longum, and Faecalibacterium prausnitzii than patients on control diet. However, microbiome diversity and markers of inflammation did not differ significantly between groups. CONCLUSIONS:In a trial of the low FODMAP diet vs a control diet in patients with quiescent IBD, we found no significant difference after 4 weeks in change in irritable bowel syndrome severity scores, but significant improvements in specific symptom scores and numbers reporting adequate symptom relief. The low FODMAP diet reduced fecal abundance of microbes believed to regulate the immune response, compared with the control diet, but had no significant effect on markers of inflammation. We conclude that a 4-week diet low in FODMAPs is safe and effective for managing persistent gut symptoms in patients with quiescent IBD. www.isrctn.com no.: ISRCTN17061468.
NLRP3 Inflammasome Regulates Development of Systemic Inflammatory Response and Compensatory Anti-Inflammatory Response Syndromes in Mice With Acute Pancreatitis.
Sendler Matthias,van den Brandt Cindy,Glaubitz Juliane,Wilden Anika,Golchert Janine,Weiss Frank Ulrich,Homuth Georg,De Freitas Chama Laura L,Mishra Neha,Mahajan Ujjwal Mukund,Bossaller Lukas,Völker Uwe,Bröker Barbara M,Mayerle Julia,Lerch Markus M
BACKGROUND & AIMS:Pancreatitis starts with primarily sterile local inflammation that induces systemic inflammatory response syndrome, followed by compensatory anti-inflammatory response syndrome (CARS). We investigated the mechanisms of these processes in mice and human serum. METHODS:We induced severe acute pancreatitis by partial duct ligation with caerulein stimulation or intraperitoneal injection of l-arginine in mice with deletion of interleukin (IL)12B, NLRP3, or IL18 and in mice given MCC950, a small molecule inhibitor of the NLRP3-inflammasome. Pancreata were collected from mice and analyzed by histology, and cytokine levels were measured in serum samples. We measured activation of adaptive immune responses in mice with pancreatitis by flow cytometry analysis of T cells (CD25 and CD69) isolated from the spleen. Differentiation of T-helper (Th1) cells, Th2 cells, and T-regulatory cells was determined by nuclear staining for TBET, GATA3, and FOXP3. We performed transcriptome analysis of mouse lymph nodes and bone marrow-derived macrophages after incubation with acini. We measured levels of cytokines in serum samples from patients with mild and severe acute pancreatitis. RESULTS:Activation of the adaptive immune response in mice was initiated by macrophage-derived, caspase 1-processed cytokines and required activation of NLRP3 (confirmed in serum samples from patients with pancreatitis). Spleen cells from mice with pancreatitis had increases in Th2 cells but not in Th1 cells. Bone marrow-derived macrophages secreted IL1B and IL18, but not IL12, after co-incubation with pancreatic acini. T-cell activation and severity of acute pancreatitis did not differ significantly between IL12B-deficient and control mice. In contrast, NLRP3- or IL18-deficient mice had reduced activation of T cells and no increase in Th2 cell-mediated responses compared with control mice. The systemic type 2 immune response was mediated by macrophage-derived cytokines of the IL1 family. Specifically, IL18 induced a Th2 cell-mediated response in the absence of IL12. MCC950 significantly reduced neutrophil infiltration, T-cell activation, and disease severity in mice. CONCLUSIONS:In mice with severe pancreatitis, we found systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome developed in parallel. Infiltrating macrophages promote inflammation and simultaneously induce a Th2 cell-mediated response via IL18. Inhibition of NLRP3 reduces systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome and might be used to treat patients with severe pancreatitis.
Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma.
Planagumà Anna,Kazani Shamsah,Marigowda Gautham,Haworth Oliver,Mariani Thomas J,Israel Elliot,Bleecker Eugene R,Curran-Everett Douglas,Erzurum Serpil C,Calhoun William J,Castro Mario,Chung Kian Fan,Gaston Benjamin,Jarjour Nizar N,Busse William W,Wenzel Sally E,Levy Bruce D
American journal of respiratory and critical care medicine
RATIONALE:Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A(4) (LXA(4)) is an arachidonic acid-derived mediator that serves as an agonist for resolution of inflammation. OBJECTIVES:Airway levels of LXA(4), as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma. METHODS:Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA(4) and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA(4) receptors were monitored by flow cytometry. MEASUREMENTS AND MAIN RESULTS:Individuals with severe asthma had significantly less LXA(4) in bronchoalveolar lavage fluids (11.2 +/- 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 +/- 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA(4) receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes. CONCLUSIONS:Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.
Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis.
Jensen Kim B,Driskell Ryan R,Watt Fiona M
In this protocol, we describe how to isolate keratinocytes from adult mouse epidermis, fractionate them into different sub-populations on the basis of cell surface markers and examine their function in an in vivo skin reconstitution assay with disaggregated neonatal dermal cells. We also describe how the isolated keratinocytes can be subjected to clonal analysis in vitro and in vivo and how to enrich for hair follicle-inducing dermal papilla cells in the dermal preparation. Using these approaches, it is possible to compare the capacity of different populations of adult epidermal stem cells to proliferate and to generate progeny that differentiate along the different epidermal lineages. Isolating, fractionating and grafting cells for the skin reconstitution assay is normally spread over 2 d. Clonal growth in culture is assessed after 14 d, while evaluation of the grafts is carried out after 4-5 weeks.
Multi-parameter phenotypic profiling: using cellular effects to characterize small-molecule compounds.
Feng Yan,Mitchison Timothy J,Bender Andreas,Young Daniel W,Tallarico John A
Nature reviews. Drug discovery
Multi-parameter phenotypic profiling of small molecules provides important insights into their mechanisms of action, as well as a systems level understanding of biological pathways and their responses to small molecule treatments. It therefore deserves more attention at an early step in the drug discovery pipeline. Here, we summarize the technologies that are currently in use for phenotypic profiling--including mRNA-, protein- and imaging-based multi-parameter profiling--in the drug discovery context. We think that an earlier integration of phenotypic profiling technologies, combined with effective experimental and in silico target identification approaches, can improve success rates of lead selection and optimization in the drug discovery process.
p63 regulates olfactory stem cell self-renewal and differentiation.
Fletcher Russell B,Prasol Melanie S,Estrada Jose,Baudhuin Ariane,Vranizan Karen,Choi Yoon Gi,Ngai John
The olfactory epithelium is a sensory neuroepithelium that supports adult neurogenesis and tissue regeneration following injury, making it an excellent model for investigating neural stem cell regulation in vivo. Previous studies have identified the horizontal basal cell (HBC) as the neural stem cell of the postnatal olfactory epithelium. However, the molecules and pathways regulating HBC self-renewal and differentiation are unknown. In the present study, we demonstrate that the transcription factor p63, a member of the p53 tumor suppressor gene family known to regulate stem cell dynamics in other epithelia, is highly enriched in HBCs. We show that p63 is required cell autonomously for olfactory stem cell renewal and further demonstrate that p63 functions to repress HBC differentiation. These results provide critical insight into the genetic regulation of the olfactory stem cell in vivo and more generally provide an entrée toward understanding the coordination of stem cell self-renewal and differentiation.
Adaptive NK cells in people exposed to correlate with protection from malaria.
Hart Geoffrey T,Tran Tuan M,Theorell Jakob,Schlums Heinrich,Arora Gunjan,Rajagopalan Sumati,Sangala A D Jules,Welsh Kerry J,Traore Boubacar,Pierce Susan K,Crompton Peter D,Bryceson Yenan T,Long Eric O
The Journal of experimental medicine
How antibodies naturally acquired during infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. -infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of -infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to antigens is therefore warranted in the design of malaria vaccines.
Impaired Tumor-Infiltrating T Cells in Patients with Chronic Obstructive Pulmonary Disease Impact Lung Cancer Response to PD-1 Blockade.
Biton Jérôme,Ouakrim Hanane,Dechartres Agnès,Alifano Marco,Mansuet-Lupo Audrey,Si Han,Halpin Rebecca,Creasy Todd,Bantsimba-Malanda Claudie,Arrondeau Jennifer,Goldwasser François,Boudou-Rouquette Pascaline,Fournel Ludovic,Roche Nicolas,Burgel Pierre-Régis,Goc Jeremy,Devi-Marulkar Priyanka,Germain Claire,Dieu-Nosjean Marie-Caroline,Cremer Isabelle,Herbst Ronald,Damotte Diane
American journal of respiratory and critical care medicine
RATIONALE:Patients with chronic obstructive pulmonary disease (COPD) have a higher prevalence of lung cancer. The chronic inflammation associated with COPD probably promotes the earliest stages of carcinogenesis. However, once tumors have progressed to malignancy, the impact of COPD on the tumor immune microenvironment remains poorly defined, and its effects on immune-checkpoint blockers' efficacy are still unknown. OBJECTIVES:To study the impact of COPD on the immune contexture of non-small cell lung cancer. METHODS:We performed in-depth immune profiling of lung tumors by immunohistochemistry and we determined its impact on patient survival (n = 435). Tumor-infiltrating T lymphocyte (TIL) exhaustion by flow cytometry (n = 50) was also investigated. The effectiveness of an anti-PD-1 (programmed cell death-1) treatment (nivolumab) was evaluated in 39 patients with advanced-stage non-small cell lung cancer. All data were analyzed according to patient COPD status. MEASUREMENTS AND MAIN RESULTS:Remarkably, COPD severity is positively correlated with the coexpression of PD-1/TIM-3 (T-cell immunoglobulin and mucin domain-containing molecule-3) by CD8 T cells. In agreement, we observed a loss of CD8 T cell-associated favorable clinical outcome in COPD patients. Interestingly, a negative prognostic value of PD-L1 (programmed cell death ligand 1) expression by tumor cells was observed only in highly CD8 T cell-infiltrated tumors of COPD patients. Finally, data obtained on 39 patients with advanced-stage non-small cell lung cancer treated by an anti-PD-1 antibody showed longer progression-free survival in COPD patients, and also that the association between the severity of smoking and the response to nivolumab was preferentially observed in COPD patients. CONCLUSIONS:COPD is associated with an increased sensitivity of CD8 tumor-infiltrating T lymphocytes to immune escape mechanisms developed by tumors, thus suggesting a higher sensitivity to PD-1 blockade in patients with COPD.
Genetic and Pharmacologic Inhibition of the Chemokine Receptor CXCR2 Prevents Experimental Hypertension and Vascular Dysfunction.
Wang Lei,Zhao Xue-Chen,Cui Wei,Ma Yong-Qiang,Ren Hua-Liang,Zhou Xin,Fassett John,Yang Yan-Zong,Chen Yingjie,Xia Yun-Long,Du Jie,Li Hui-Hua
BACKGROUND:The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. METHODS:Angiotensin II (490 ng·kg·min) or deoxycorticosterone acetate (DOCA) salt-induced mouse hypertensive models in genetic ablation, pharmacologic inhibition of CXCR2, and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time polymerase chain reaction [PCR] analysis), vascular remodeling (studied by haematoxylin and eosin and Masson's trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by nicotinamide adenine dinucleotide phosphate [NADPH] oxidase activity, dihydroethidium staining, and quantitative real-time PCR analysis). Moreover, the blood CXCR2 cells in normotensive controls and hypertension patients were analyzed by flow cytometry. RESULTS:Angiotensin II significantly upregulated the expression of CXCR2 mRNA and protein and increased the number of CD45 CXCR2 cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of proinflammatory cells into the vascular wall, and expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity, and expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type mice with CXCR2 bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). It is important to note that CXCR2 blockade reversed established hypertension induced by angiotensin II or DOCA-salt challenge (n=10 per group). Furthermore, we demonstrated that CXCR2 proinflammatory cells were higher in hypertensive patients (n=30) compared with normotensive individuals (n=20). CONCLUSIONS:Infiltration of CXCR2 cells plays a pathogenic role in arterial hypertension and vascular dysfunction. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat hypertension.
In Situ Programmable DNA Circuit-Promoted Electrochemical Characterization of Stemlike Phenotype in Breast Cancer.
Cao Ya,Yu Xiaomeng,Han Bing,Dong Langjian,Xu Jingjing,Dai Yuhao,Li Genxi,Zhao Jing
Journal of the American Chemical Society
Breast cancer is one of the most common malignant diseases among women worldwide, and the existence of breast cancer stem cells is closely associated with poor outcomes. Herein, we report an electrochemical phenotyping method to characterize the stemlike phenotype in breast cancer, offering a low-cost but robust choice other than the highly expensive and experience-dependent flow cytometry. Specially, after immune-magnetic beads-assisted enrichment, an in situ programmable DNA circuit is designed using capture probes to bring in the toeholds for DNA assembly and effector probes to accelerate the removal of background signals. The electrochemical phenotyping method could sensitively determine breast cancer stem cells in a wide linear range and exhibit desirable accuracy and reliability. The method can not only monitor the phenotypic transition of breast cancer cells and the drug-reversed effect but also determinate stemlike phenotype in the mice bearing breast cancer xenograft tumor. Overall, the electrochemical phenotyping method may provide promising technical support for precise management of breast tumors.
Preexisting and de novo humoral immunity to SARS-CoV-2 in humans.
Ng Kevin W,Faulkner Nikhil,Cornish Georgina H,Rosa Annachiara,Harvey Ruth,Hussain Saira,Ulferts Rachel,Earl Christopher,Wrobel Antoni G,Benton Donald J,Roustan Chloe,Bolland William,Thompson Rachael,Agua-Doce Ana,Hobson Philip,Heaney Judith,Rickman Hannah,Paraskevopoulou Stavroula,Houlihan Catherine F,Thomson Kirsty,Sanchez Emilie,Shin Gee Yen,Spyer Moira J,Joshi Dhira,O'Reilly Nicola,Walker Philip A,Kjaer Svend,Riddell Andrew,Moore Catherine,Jebson Bethany R,Wilkinson Meredyth,Marshall Lucy R,Rosser Elizabeth C,Radziszewska Anna,Peckham Hannah,Ciurtin Coziana,Wedderburn Lucy R,Beale Rupert,Swanton Charles,Gandhi Sonia,Stockinger Brigitta,McCauley John,Gamblin Steve J,McCoy Laura E,Cherepanov Peter,Nastouli Eleni,Kassiotis George
Science (New York, N.Y.)
Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.
Dotted Core-Shell Nanoparticles for T -Weighted MRI of Tumors.
Shen Zheyu,Song Jibin,Zhou Zijian,Yung Bryant C,Aronova Maria A,Li Yan,Dai Yunlu,Fan Wenpei,Liu Yijing,Li Zihou,Ruan Huimin,Leapman Richard D,Lin Lisen,Niu Gang,Chen Xiaoyuan,Wu Aiguo
Advanced materials (Deerfield Beach, Fla.)
Gd-based T -weighted contrast agents have dominated the magnetic resonance imaging (MRI) contrast agent market for decades. Nevertheless, they are reported to be nephrotoxic and the U.S. Food and Drug Administration has issued a general warning concerning their use. In order to reduce the risk of nephrotoxicity, the MRI performance of the Gd-based T -weighted contrast agents needs to be improved to allow a much lower dosage. In this study, novel dotted core-shell nanoparticles (FeGd-HN3-RGD2) with superhigh r value (70.0 mM s ) and very low r /r ratio (1.98) are developed for high-contrast T -weighted MRI of tumors. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and histological analyses show good biocompatibility of FeGd-HN3-RGD2. Laser scanning confocal microscopy images and flow cytometry demonstrate active targeting to integrin α β positive tumors. MRI of tumors shows high tumor ΔSNR for FeGd-HN3-RGD2 (477 ± 44%), which is about 6-7-fold higher than that of Magnevist (75 ± 11%). MRI and inductively coupled plasma results further confirm that the accumulation of FeGd-HN3-RGD2 in tumors is higher than liver and spleen due to the RGD2 targeting and small hydrodynamic particle size (8.5 nm), and FeGd-HN3-RGD2 is readily cleared from the body by renal excretion.
Cellular origin and pathophysiology of chronic lymphocytic leukemia.
Seifert Marc,Sellmann Ludger,Bloehdorn Johannes,Wein Frederik,Stilgenbauer Stephan,Dürig Jan,Küppers Ralf
The Journal of experimental medicine
The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5(+) B cells and mutated CLL derives from a distinct, previously unrecognized CD5(+)CD27(+) post-germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5(+) B cells, providing independent evidence for a CD5(+) B cell derivation of CLL. Notably, these CD5(+) B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.
Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus.
McCaughtry Tom M,Etzensperger Ruth,Alag Amala,Tai Xuguang,Kurtulus Sema,Park Jung-Hyun,Grinberg Alex,Love Paul,Feigenbaum Lionel,Erman Batu,Singer Alfred
The Journal of experimental medicine
The thymus generates T cells with diverse specificities and functions. To assess the contribution of cytokine receptors to the differentiation of T cell subsets in the thymus, we constructed conditional knockout mice in which IL-7Rα or common cytokine receptor γ chain (γ(c)) genes were deleted in thymocytes just before positive selection. We found that γ(c) expression was required to signal the differentiation of MHC class I (MHC-I)-specific thymocytes into CD8(+) cytotoxic lineage T cells and into invariant natural killer T cells but did not signal the differentiation of MHC class II (MHC-II)-specific thymocytes into CD4(+) T cells, even into regulatory Foxp3(+)CD4(+) T cells which require γ(c) signals for survival. Importantly, IL-7 and IL-15 were identified as the cytokines responsible for CD8(+) cytotoxic T cell lineage specification in vivo. Additionally, we found that small numbers of aberrant CD8(+) T cells expressing Runx3d could arise without γ(c) signaling, but these cells were developmentally arrested before expressing cytotoxic lineage genes. Thus, γ(c)-transduced cytokine signals are required for cytotoxic lineage specification in the thymus and for inducing the differentiation of MHC-I-selected thymocytes into functionally mature T cells.
Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress.
Austin Wayne R,Armijo Amanda L,Campbell Dean O,Singh Arun S,Hsieh Terry,Nathanson David,Herschman Harvey R,Phelps Michael E,Witte Owen N,Czernin Johannes,Radu Caius G
The Journal of experimental medicine
Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1(-/-) erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK(-/-) counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK "tune" dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.
β-Amyloid Precursor Protein Intracellular Domain Controls Mitochondrial Function by Modulating Phosphatase and Tensin Homolog-Induced Kinase 1 Transcription in Cells and in Alzheimer Mice Models.
Goiran Thomas,Duplan Eric,Chami Mounia,Bourgeois Alexandre,El Manaa Wejdane,Rouland Lila,Dunys Julie,Lauritzen Inger,You Han,Stambolic Vuk,Biféri Maria-Grazia,Barkats Martine,Pimplikar Sanjay W,Sergeant Nicolas,Colin Morvane,Morais Vanessa A,Pardossi-Piquard Raphaelle,Checler Frédéric,Alves da Costa Cristine
BACKGROUND:Mitophagy and mitochondrial dynamics alterations are two major hallmarks of neurodegenerative diseases. Dysfunctional mitochondria accumulate in Alzheimer's disease-affected brains by yet unexplained mechanisms. METHODS:We combined cell biology, molecular biology, and pharmacological approaches to unravel a novel molecular pathway by which presenilins control phosphatase and tensin homolog-induced kinase 1 (Pink-1) expression and transcription. In vivo approaches were carried out on various transgenic and knockout animals as well as in adeno-associated virus-infected mice. Functional readout and mitochondrial physiology (mitochondrial potential) were assessed by combined procedures including flow cytometry, live imaging analysis, and immunohistochemistry. RESULTS:We show that presenilins 1 and 2 trigger opposite effects on promoter transactivation, messenger RNA, and protein expression of Pink-1. This control is linked to γ-secretase activity and β-amyloid precursor protein but is independent of phosphatase and tensin homolog. We show that amyloid precursor protein intracellular domain (AICD) accounts for presenilin-dependent phenotype and upregulates Pink-1 transactivation in cells as well as in vivo in a Forkhead box O3a-dependent manner. Interestingly, the modulation of γ-secretase activity or AICD expression affects Pink-1-related control of mitophagy and mitochondrial dynamics. Finally, we show that parkin acts upstream of presenilins to control Pink-1 promoter transactivation and protein expression. CONCLUSIONS:Overall, we delineate a molecular cascade presenilins-AICD-Forkhead box O3a linking parkin to Pink-1. Our study demonstrates AICD-mediated Pink-1-dependent control of mitochondrial physiology by presenilins. Furthermore, it unravels a parkin-Pink-1 feedback loop controlling mitochondrial physiology that could be disrupted in neurodegenerative conditions.
Analysis of a wild mouse promoter variant reveals a novel role for FcγRIIb in the control of the germinal center and autoimmunity.
Espéli Marion,Clatworthy Menna R,Bökers Susanne,Lawlor Kate E,Cutler Antony J,Köntgen Frank,Lyons Paul A,Smith Kenneth G C
The Journal of experimental medicine
Genetic variants of the inhibitory Fc receptor FcγRIIb have been associated with systemic lupus erythematosus in humans and mice. The mechanism by which Fcgr2b variants contribute to the development of autoimmunity is unknown and was investigated by knocking in the most commonly conserved wild mouse Fcgr2b promoter haplotype, also associated with autoimmune-prone mouse strains, into the C57BL/6 background. We found that in the absence of an AP-1-binding site in its promoter, FcγRIIb failed to be up-regulated on activated and germinal center (GC) B cells. This resulted in enhanced GC responses, increased affinity maturation, and autoantibody production. Accordingly, in the absence of FcγRIIb activation-induced up-regulation, mice developed more severe collagen-induced arthritis and spontaneous glomerular immune complex deposition. Our data highlight how natural variation in Fcgr2b drives the development of autoimmune disease. They also show how the study of such variants using a knockin approach can provide insight into immune mechanisms not possible using conventional genetic manipulation, in this case demonstrating an unexpected critical role for the activation-induced up-regulation of FcγRIIb in controlling affinity maturation, autoantibody production, and autoimmunity.
Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression.
Powers Sarah E,Mandal Malay,Matsuda Satoshi,Miletic Ana V,Cato Matthew H,Tanaka Azusa,Rickert Robert C,Koyasu Shigeo,Clark Marcus R
The Journal of experimental medicine
Ubiquitously expressed D-type cyclins are required for hematopoiesis but are dispensable in other cell lineages. Furthermore, within different hematopoietic progenitor populations the D-type cyclins play nonredundant roles. The basis of this lineage and developmental specificity is unknown. In pro-B cells we demonstrate four distinct nuclear D-type cyclin compartments, including one cyclin D3 fraction associated with CDK4 and another phosphoinositide 3-kinase-regulated fraction not required for proliferation. A third fraction of cyclin D3 was associated with the nuclear matrix and repression of >200 genes including the variable (V) gene segments Igkv1-117, Iglv1, and Igh-VJ558. Consistent with different subnuclear compartments and functions, distinct domains of cyclin D3 mediated proliferation and Igk V gene segment repression. None of the cyclin D3 nuclear compartments overlapped with cyclin D2, which was distributed, unbound to CDK4, throughout the nucleus. Furthermore, compartmentalization of the cyclins appeared to be lineage restricted because in fibroblasts, cyclin D2 and cyclin D3 occupied a single nuclear compartment and neither bound CDK4 efficiently. These data suggest that subnuclear compartmentalization enables cyclin D3 to drive cell cycle progression and repress V gene accessibility, thereby ensuring coordination of proliferation with immunoglobulin recombination.
Transcriptional regulator early growth response gene 2 (Egr2) is required for T cell anergy in vitro and in vivo.
Zheng Yan,Zha Yuanyuan,Driessens Gregory,Locke Frederick,Gajewski Thomas F
The Journal of experimental medicine
T cell receptor engagement in the absence of costimulation results in a hyporesponsive state termed anergy. Understanding the transcriptional regulation of other T cell differentiation states has provided critical information regarding the biology of T cell regulation in vivo. However, the transcriptional regulation of T cell anergy has been poorly understood. Using the key anergy target gene diacylglycerol kinase (DGK) α as a focal point, we identified early growth response gene 2 (Egr2) as a central transcription factor that regulates the anergic state. Conditional Egr2 deletion in peripheral T cells abolishes induced expression of DGK-α and other anergy genes and restores Ras/MAPK signaling, IL-2 production, and proliferation upon attempted anergy induction. Using superantigen- and tumor-induced anergy models, we found that Egr2 is necessary for anergy induction in vivo. Collectively, our results implicate Egr2 as an essential transcriptional regulator of the T cell anergy program.
Congenital B cell lymphocytosis explained by novel germline CARD11 mutations.
Snow Andrew L,Xiao Wenming,Stinson Jeffrey R,Lu Wei,Chaigne-Delalande Benjamin,Zheng Lixin,Pittaluga Stefania,Matthews Helen F,Schmitz Roland,Jhavar Sameer,Kuchen Stefan,Kardava Lela,Wang Wei,Lamborn Ian T,Jing Huie,Raffeld Mark,Moir Susan,Fleisher Thomas A,Staudt Louis M,Su Helen C,Lenardo Michael J
The Journal of experimental medicine
Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.
A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis.
Zhang Lin,Orban Martin,Lorenz Michael,Barocke Verena,Braun Daniel,Urtz Nicole,Schulz Christian,von Brühl Marie-Luise,Tirniceriu Anca,Gaertner Florian,Proia Richard L,Graf Thomas,Bolz Steffen-Sebastian,Montanez Eloi,Prinz Marco,Müller Alexandra,von Baumgarten Louisa,Billich Andreas,Sixt Michael,Fässler Reinhard,von Andrian Ulrich H,Junt Tobias,Massberg Steffen
The Journal of experimental medicine
Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.
IL-18 induces emphysema and airway and vascular remodeling via IFN-γ, IL-17A, and IL-13.
Kang Min-Jong,Choi Je-Min,Kim Bo Hye,Lee Chang-Min,Cho Won-Kyung,Choe Gina,Kim Do-Hyun,Lee Chun Geun,Elias Jack A
American journal of respiratory and critical care medicine
RATIONALE:Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation, alveolar destruction, and airway and vascular remodeling. However, the mechanisms that lead to these diverse alterations have not been defined. OBJECTIVES:We hypothesized that IL-18 plays a central role in the pathogenesis of these lesions. METHODS:We generated and characterized lung-specific, inducible IL-18 transgenic mice. MEASUREMENTS AND MAIN RESULTS:Here we demonstrate that the expression of IL-18 in the mature murine lung induces inflammation that is associated with the accumulation of CD4(+), CD8(+), CD19(+), and NK1.1(+) cells; emphysema; mucus metaplasia; airway fibrosis; vascular remodeling; and right ventricle cardiac hypertrophy. We also demonstrate that IL-18 induces type 1, type 2, and type 17 cytokines with IFN-γ-inhibiting macrophage, lymphocyte, and eosinophil accumulation while stimulating alveolar destruction and genes associated with cell cytotoxicity and IL-13 and IL-17A inducing mucus metaplasia, airway fibrosis, and vascular remodeling. We also highlight interactions between these responses with IL-18 inducing IL-13 via an IL-17A-dependent mechanism and the type 1 and type17/type 2 responses counterregulating each another. CONCLUSIONS:These studies define the spectrum of inflammatory, parenchymal, airway, and vascular alterations that are induced by pulmonary IL-18; highlight the similarities between these responses and the lesions in COPD; and define the selective roles that type 1, type 2, and type 17 responses play in the generation of IL-18-induced pathologies.
Nuclear factor-κB1 controls the functional maturation of dendritic cells and prevents the activation of autoreactive T cells.
Dissanayake Dilan,Hall Håkan,Berg-Brown Nancy,Elford Alisha R,Hamilton Sara R,Murakami Kiichi,Deluca Leslie Summers,Gommerman Jennifer L,Ohashi Pamela S
Mature dendritic cells (DCs) are crucial for the induction of adaptive immune responses and perturbed DC homeostasis can result in autoimmune disease. Either uncontrolled expansion or enhanced survival of DCs can result in a variety of autoimmune diseases in mouse models. In addition, increased maturation signals, through overexpression of surface Toll-like receptors (TLRs) or stimulation by type I interferon (IFN), has been associated with systemic autoimmunity. Whereas recent studies have focused on identifying factors required for initiating the maturation process, the possibility that resting DCs also express molecules that 'hold' them in an immature state has generally not been considered. Here we show that nuclear factor-κB1 (NF-κB1) is crucial for maintaining the resting state of DCs. Self-antigen-pulsed unstimulated DCs that do not express NF-κB1 were able to activate CD8(+) T lymphocytes and induce autoimmunity. We further show that NF-κB1 negatively regulates the spontaneous production of tumor necrosis factor-α (TNF-α), which is associated with increased granzyme B expression in cytotoxic T lymphocytes (CTLs). These findings provide a new perspective on functional DC maturation and a potential mechanism that may account for pathologic T cell activation.
Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging.
Temme Sebastian,Baran Paul,Bouvain Pascal,Grapentin Christoph,Krämer Wolfgang,Knebel Birgit,Al-Hasani Hadi,Moll Jens Mark,Floss Doreen,Schrader Jürgen,Schubert Rolf,Flögel Ulrich,Scheller Jürgen
Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic "cargo internalization receptors" (CIRs) for tracking of individual cell populations by H/F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For F MR visibility, the GFP carrier was equipped with "contrast cargo", in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1-3). Exposure of CIR cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence microscopy as well as flow cytometry and enabled visualization by H/F MRI. In particular, expression of CIR2/3 resulted in substantial incorporation of F cargo and readily enabled in vivo visualization of GFP-PFC recruitment to transplanted CIR cells by H/F MRI in mice. Competition experiments with blood immune cells revealed that CIR cells are predominantly loaded with GFP-PFCs even in the presence of cells with strong phagocytotic capacity. Importantly, binding and internalization of GFP-PFCs did not result in the activation of signaling cascades and therefore does not alter cell physiology. Overall, this approach represents a versatile in vivo imaging platform for tracking of individual cell populations by making use of cell-type-specific CIR mice.
Revised response criteria for malignant lymphoma.
Cheson Bruce D,Pfistner Beate,Juweid Malik E,Gascoyne Randy D,Specht Lena,Horning Sandra J,Coiffier Bertrand,Fisher Richard I,Hagenbeek Anton,Zucca Emanuele,Rosen Steven T,Stroobants Sigrid,Lister T Andrew,Hoppe Richard T,Dreyling Martin,Tobinai Kensei,Vose Julie M,Connors Joseph M,Federico Massimo,Diehl Volker,
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:Standardized response criteria are needed to interpret and compare clinical trials and for approval of new therapeutic agents by regulatory agencies. METHODS:The International Working Group response criteria (Cheson et al, J Clin Oncol 17:1244, 1999) were widely adopted, but required reassessment because of identified limitations and the increased use of [18F]fluorodeoxyglucose-positron emission tomography (PET), immunohistochemistry (IHC), and flow cytometry. The International Harmonization Project was convened to provide updated recommendations. RESULTS:New guidelines are presented incorporating PET, IHC, and flow cytometry for definitions of response in non-Hodgkin's and Hodgkin's lymphoma. Standardized definitions of end points are provided. CONCLUSION:We hope that these guidelines will be adopted widely by study groups, pharmaceutical and biotechnology companies, and regulatory agencies to facilitate the development of new and more effective therapies to improve the outcome of patients with lymphoma.
Identification of a γc Receptor Antagonist That Prevents Reprogramming of Human Tissue-resident Cytotoxic T Cells by IL15 and IL21.
BACKGROUND & AIMS:Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. METHODS:We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8 T-cell lines, or CD8 T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. RESULTS:Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. CONCLUSIONS:We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.
Helicobacter pylori-induced peptic ulcer disease is associated with inadequate regulatory T cell responses.
Robinson K,Kenefeck R,Pidgeon E L,Shakib S,Patel S,Polson R J,Zaitoun A M,Atherton J C
BACKGROUND AND AIMS:Helicobacter pylori infection is the major cause of peptic ulceration and gastric adenocarcinoma. To address the hypothesis that the human acquired immune response to H. pylori influences pathogenesis, we characterised the gastric T helper (Th) and regulatory T cell (Treg) response of infected patients. METHODS:The human gastric CD4(+) T cell response of 28 donors who were infected with H. pylori and 44 who were not infected was analysed using flow cytometry. The T cell associated mucosal cytokine response was analysed by real-time polymerase chain reaction assay of samples from 38 infected and 22 uninfected donors. Recombinant interleukin 10 (IL10) was added to co-cultures of H. pylori and AGS cells and its suppressive effects upon inflammatory responses were measured. RESULTS:We found that the H. pylori-specific response consists of both T helper 1 and 2 subsets with high levels of IL10-secreting Tregs. People with peptic ulcer disease had a 2.4-fold reduced CD4(+)CD25(hi)IL10(+) Treg response (p = 0.05) but increased Th1 and Th2 responses (Th1: 3.2-fold, p = 0.038; Th2: 6.1-fold, p = 0.029) compared to those without ulcers. In vitro studies showed that IL10 inhibited IL8 expression and activation of nuclear factor kappa B induced by H. pylori in gastric epithelial cells, and enhanced H. pylori growth in a bacterial-cell co-culture model. CONCLUSIONS:Together our data suggest that H. pylori induces a regulatory T cell response, possibly contributing to its peaceful coexistence with the human host, and that ulcers occur when this regulatory response is inadequate.
Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression.
Stoeckius Marlon,Maaskola Jonas,Colombo Teresa,Rahn Hans-Peter,Friedländer Marc R,Li Na,Chen Wei,Piano Fabio,Rajewsky Nikolaus
Caenorhabditis elegans is one of the most prominent model systems for embryogenesis, but collecting many precisely staged embryos has been impractical. Thus, early C. elegans embryogenesis has not been amenable to most high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect staged C. elegans embryos by fluorescence-activated cell sorting (eFACS). In a proof-of-principle experiment, we found that a single eFACS run routinely yielded tens of thousands of almost perfectly staged 1-cell stage embryos. As the earliest embryonic events are driven by posttranscriptional regulation, we combined eFACS with second-generation sequencing to profile the embryonic expression of small, noncoding RNAs. We discovered complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including microRNAs and 26G-RNAs, during embryogenesis.
Extracellular polymeric substances are transient media for microbial extracellular electron transfer.
Xiao Yong,Zhang Enhua,Zhang Jingdong,Dai Youfen,Yang Zhaohui,Christensen Hans E M,Ulstrup Jens,Zhao Feng
Microorganisms exploit extracellular electron transfer (EET) in growth and information exchange with external environments or with other cells. Every microbial cell is surrounded by extracellular polymeric substances (EPS). Understanding the roles of three-dimensional (3D) EPS in EET is essential in microbiology and microbial exploitation for mineral bio-respiration, pollutant conversion, and bioenergy production. We have addressed these challenges by comparing pure and EPS-depleted samples of three representative electrochemically active strains viz Gram-negative MR-1, Gram-positive sp. WS-XY1, and yeast using technology from electrochemistry, spectroscopy, atomic force microscopy, and microbiology. Voltammetry discloses redox signals from cytochromes and flavins in intact MR-1 cells, whereas stronger signals from cytochromes and additional signals from both flavins and cytochromes are found after EPS depletion. Flow cytometry and fluorescence microscopy substantiated by -acetylglucosamine and electron transport system activity data showed less than 1.5% cell damage after EPS extraction. The electrochemical differences between normal and EPS-depleted cells therefore originate from electrochemical species in cell walls and EPS. The 35 ± 15-nm MR-1 EPS layer is also electrochemically active itself, with cytochrome electron transfer rate constants of 0.026 and 0.056 s for intact MR-1 and EPS-depleted cells, respectively. This surprisingly small rate difference suggests that molecular redox species at the core of EPS assist EET. The combination of all the data with electron transfer analysis suggests that electron "hopping" is the most likely molecular mechanism for electrochemical electron transfer through EPS.
Phase 2 Trial of Gemcitabine, Cisplatin, plus Ipilimumab in Patients with Metastatic Urothelial Cancer and Impact of DNA Damage Response Gene Mutations on Outcomes.
Galsky Matthew D,Wang Huan,Hahn Noah M,Twardowski Przemyslaw,Pal Sumanta K,Albany Costantine,Fleming Mark T,Starodub Alexander,Hauke Ralph J,Yu Menggang,Zhao Qianqian,Sonpavde Guru,Donovan Michael J,Patel Vaibhav G,Sfakianos John P,Domingo-Domenech Josep,Oh William K,Akers Nicholas,Losic Bojan,Gnjatic Sacha,Schadt Eric E,Chen Rong,Kim-Schulze Seunghee,Bhardwaj Nina,Uzilov Andrew V
BACKGROUND:Chemotherapy may exert immunomodulatory effects, thereby combining favorably with the immune checkpoint blockade. The pharmacodynamic effects of such combinations, and potential predictive biomarkers, remain unexplored. OBJECTIVE:To determine the safety, efficacy, and immunomodulatory effects of gemcitabine and cisplatin (GC) plus ipilimumab and explore the impact of somatic DNA damage response gene alterations on antitumor activity. DESIGN, SETTING, AND PARTICIPANTS:Multicenter single arm phase 2 study enrolling 36 chemotherapy-naïve patients with metastatic urothelial cancer. Peripheral blood flow cytometry was performed serially on all patients and whole exome sequencing of archival tumor tissue was performed on 28/36 patients. INTERVENTION:Two cycles of GC followed by four cycles of GC plus ipilimumab. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:The primary endpoint was 1-yr overall survival (OS). Secondary endpoints included safety, objective response rate, and progression-free survival. RESULTS AND LIMITATIONS:Grade ≥3 adverse events occurred in 81% of patients, the majority of which were hematologic. The objective response rate was 69% and 1-yr OS was 61% (lower bound 90% confidence interval: 51%). On exploratory analysis, there were no significant changes in the composition and frequency of circulating immune cells after GC alone. However, there was a significant expansion of circulating CD4 cells with the addition of ipilimumab which correlated with improved survival. The response rate was significantly higher in patients with deleterious somatic DNA damage response mutations (sensitivity=47.6%, specificity=100%, positive predictive value=100%, and negative predictive value=38.9%). Limitations are related to the sample size and single-arm design. CONCLUSIONS:GC+ipilimumab did not achieve the primary endpoint of a lower bound of the 90% confidence interval for 1-yr OS of >60%. However, within the context of a small single-arm trial, the results may inform current approaches combining chemotherapy plus immunotherapy from the standpoint of feasibility, appropriate cytotoxic backbones, and potential predictive biomarkers. TRIAL REGISTRATION:ClinicalTrials.gov NCT01524991. PATIENT SUMMARY:Combining chemotherapy and immune checkpoint blockade in patients with metastatic urothelial cancer is feasible. Further studies are needed to refine optimal combinations and evaluate tests that might identify patients most likely to benefit.
Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells.
Levenberg Shulamit,Ferreira Lino S,Chen-Konak Limor,Kraehenbuehl Thomas P,Langer Robert
Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain >80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.
IL-35-mediated induction of a potent regulatory T cell population.
Collison Lauren W,Chaturvedi Vandana,Henderson Abigail L,Giacomin Paul R,Guy Cliff,Bankoti Jaishree,Finkelstein David,Forbes Karen,Workman Creg J,Brown Scott A,Rehg Jerold E,Jones Michael L,Ni Hsiao-Tzu,Artis David,Turk Mary Jo,Vignali Dario A A
Regulatory T cells (T(reg) cells) have a critical role in the maintenance of immunological self-tolerance. Here we show that treatment of naive human or mouse T cells with IL-35 induced a regulatory population, which we call 'iT(R)35 cells', that mediated suppression via IL-35 but not via the inhibitory cytokines IL-10 or transforming growth factor-β (TGF-β). We found that iT(R)35 cells did not express or require the transcription factor Foxp3, and were strongly suppressive and stable in vivo. T(reg) cells induced the generation of iT(R)35 cells in an IL-35- and IL-10-dependent manner in vitro and induced their generation in vivo under inflammatory conditions in intestines infected with Trichuris muris and within the tumor microenvironment (B16 melanoma and MC38 colorectal adenocarcinoma), where they contributed to the regulatory milieu. Thus, iT(R)35 cells constitute a key mediator of infectious tolerance and contribute to T(reg) cell-mediated tumor progression. Furthermore, iT(R)35 cells generated ex vivo might have therapeutic utility.
Plasma cells negatively regulate the follicular helper T cell program.
Pelletier Nadége,McHeyzer-Williams Louise J,Wong Kurt A,Urich Eduard,Fazilleau Nicolas,McHeyzer-Williams Michael G
B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (T(FH) cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated T(FH) cells. Mice lacking plasma cells showed altered T(FH) cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific T(FH) cell program that controls ongoing B cell immunity.
Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.
Miao Edward A,Leaf Irina A,Treuting Piper M,Mao Dat P,Dors Monica,Sarkar Anasuya,Warren Sarah E,Wewers Mark D,Aderem Alan
Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.
In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells.
Machado Léo,Esteves de Lima Joana,Fabre Odile,Proux Caroline,Legendre Rachel,Szegedi Anikó,Varet Hugo,Ingerslev Lars Roed,Barrès Romain,Relaix Frédéric,Mourikis Philippos
State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest.
Cell-type-based analysis of microRNA profiles in the mouse brain.
He Miao,Liu Yu,Wang Xiaowo,Zhang Michael Q,Hannon Gregory J,Huang Z Josh
MicroRNAs (miRNA) are implicated in brain development and function but the underlying mechanisms have been difficult to study in part due to the cellular heterogeneity in neural circuits. To systematically analyze miRNA expression in neurons, we have established a miRNA tagging and affinity-purification (miRAP) method that is targeted to cell types through the Cre-loxP binary system in mice. Our studies of the neocortex and cerebellum reveal the expression of a large fraction of known miRNAs with distinct profiles in glutamatergic and GABAergic neurons and subtypes of GABAergic neurons. We further detected putative novel miRNAs, tissue or cell type-specific strand selection of miRNAs, and miRNA editing. Our method thus will facilitate a systematic analysis of miRNA expression and regulation in specific neuron types in the context of neuronal development, physiology, plasticity, pathology, and disease models, and is generally applicable to other cell types and tissues.
Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells.
The Journal of clinical investigation
Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.
TNFα inhibitors restrict T cell activation and cycling via Notch-1 signalling in inflammatory bowel disease.
Werner Lael,Berndt Uta,Paclik Daniela,Danese Silvio,Schirbel Anja,Sturm Andreas
BACKGROUND:Tumour necrosis factor α (TNFα) inhibitors such as adalimumab and infliximab are frequently prescribed for inflammatory bowel disease (IBD). Despite the clinical success of TNFα inhibitors, their physiological mode of action is not fully understood. The aim of this study was to investigate the mode of action of anti-TNFα agents in IBD. METHODS:It was hypothesised that Notch mediates anti-TNFα action in T cells. A study was carried out to identify Notch-1 as a link by which anti-TNFα antibodies mediate their inhibitory functions. RESULTS:TNFα inhibitors induced T cell apoptosis, inhibited activation, reduced cytokine secretion and restricted cell cycling. TNFα blockade at several levels showed that TNFα is responsible for inducing apoptosis by anti-TNFα but not for cell cycle restriction. By linking Notch and TNFα it was shown that (1) Notch-1 mucosal expression differs in inflamed and non-inflamed mucosa and increases in response to anti-TNFα treatment; (2) Notch-1 function is regulated by TNFα inhibitors; (3) Notch-1 binds to TNFα; and (4) Notch-1 inhibition prevents anti-TNFα-induced T cell cycle arrest but not apoptosis. CONCLUSIONS:TNFα inhibitors potently inhibit T cell function. By demonstrating for the first time that Notch-1 mediates the inhibitory effects of adalimumab and infliximab on T cell cycling, this study reveals a new mode of action and also an underlying signalling pathway by which biological agents act in IBD.
Regulation of human lung alveolar multipotent cells by a novel p38α MAPK/miR-17-92 axis.
Oeztuerk-Winder Feride,Guinot Anna,Ochalek Anna,Ventura Juan-Jose
The EMBO journal
The cellular and molecular mechanisms that control lung homeostasis and regeneration are still poorly understood. It has been proposed that a population of cells exists in the mouse lung with the potential to differentiate into all major lung bronchioalveolar epithelium cell types in homeostasis or in response to virus infection. A new population of E-Cad/Lgr6(+) putative stem cells has been isolated, and indefinitely expanded from human lungs, harbouring both, self-renewal capacity and the potency to differentiate in vitro and in vivo. Recently, a putative population of human lung stem cells has been proposed as being c-Kit(+). Unlike Integrin-α6(+) or c-Kit(+) cells, E-Cad/Lgr6(+) single-cell injections in the kidney capsule produce differentiated bronchioalveolar tissue, while retaining self-renewal, as they can undergo serial transplantations under the kidney capsule or in the lung. In addition, a signalling network involving the p38α pathway, the activation of p53 and the regulation of the miR-17-92 cluster has been identified. Disruption of the proper cross-regulation of this signalling axis might be involved in the promotion of human lung diseases.
Diesel exhaust inhalation increases thrombus formation in man.
Lucking Andrew J,Lundback Magnus,Mills Nicholas L,Faratian Dana,Barath Stefan L,Pourazar Jamshid,Cassee Flemming R,Donaldson Kenneth,Boon Nicholas A,Badimon Juan J,Sandstrom Thomas,Blomberg Anders,Newby David E
European heart journal
AIMS:Although the mechanism is unclear, exposure to traffic-derived air pollution is a trigger for acute myocardial infarction (MI). The aim of this study is to investigate the effect of diesel exhaust inhalation on platelet activation and thrombus formation in men. METHODS AND RESULTS:In a double-blind randomized crossover study, 20 healthy volunteers were exposed to dilute diesel exhaust (350 microg/m(3)) and filtered air. Thrombus formation, coagulation, platelet activation, and inflammatory markers were measured at 2 and 6 h following exposure. Thrombus formation was measured using the Badimon ex vivo perfusion chamber. Platelet activation was assessed by flow cytometry. Compared with filtered air, diesel exhaust inhalation increased thrombus formation under low- and high-shear conditions by 24% [change in thrombus area 2229 microm(2), 95% confidence interval (CI) 1143-3315 microm(2), P = 0.0002] and 19% (change in thrombus area 2451 microm(2), 95% CI 1190-3712 microm(2), P = 0.0005), respectively. This increased thrombogenicity was seen at 2 and 6 h, using two different diesel engines and fuels. Diesel exhaust also increased platelet-neutrophil and platelet-monocyte aggregates by 52% (absolute change 6%, 95% CI 2-10%, P = 0.01) and 30% (absolute change 3%, 95% CI 0.2-7%, P = 0.03), respectively, at 2 h following exposure compared with filtered air. CONCLUSION:Inhalation of diesel exhaust increases ex vivo thrombus formation and causes in vivo platelet activation in man. These findings provide a potential mechanism linking exposure to combustion-derived air pollution with the triggering of acute MI.
Single-cell dissection of transcriptional heterogeneity in human colon tumors.
Dalerba Piero,Kalisky Tomer,Sahoo Debashis,Rajendran Pradeep S,Rothenberg Michael E,Leyrat Anne A,Sim Sopheak,Okamoto Jennifer,Johnston Darius M,Qian Dalong,Zabala Maider,Bueno Janet,Neff Norma F,Wang Jianbin,Shelton Andrew A,Visser Brendan,Hisamori Shigeo,Shimono Yohei,van de Wetering Marc,Clevers Hans,Clarke Michael F,Quake Stephen R
Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.
Study of cellular DNA content by flow cytometry in primary bladder carcinomas. Significance of monoclonal and multiclonal varieties of DNA aneuploidy.
Campanella R,Russo A,Plaja S,Bazan V,Pavone C,Corselli G,Pavone-Macaluso M
A prospective study of cellular DNA content by flow cytometry was performed on a nonconsecutive series of 67 patients undergoing diagnostic and/or therapeutic transurethral resection for primary urothelial bladder carcinoma. DNA-aneuploidy was present in 82% of the cases (55/67), while multiclonality was found in 45% of the DNA-aneuploid cases (25/55). DNA-ploidy was much more strictly correlated with histological grading (p less than 0.005) than with papillary or non-papillary growth pattern (p less than 0.05) or T staging (p less than 0.05). Of 26 patients with a minimum follow-up of 24 months, 100% (6/6) of cases with DNA-diploid neoplasias showed no signs of disease relapse, versus 10% (2/20) of those with DNA-aneuploid neoplasias (p less than 0.001). Furthermore, tumoral progression occurred in 10 of 20 cases (50%) with aneuploid DNA content. In this latter group of 10 cases a multiclonal DNA-aneuploid pattern was found, with a significant difference (p less than 0.001).
DNA flow cytometry and neo-adjuvant chemotherapy/radiotherapy in operable muscle-invasive bladder carcinoma. A preliminary report.
Jacobsen A B,Berner A,Juul M,Ous S,Pettersen E O,Fosså S D
Fifty-five patients with muscle-invasive transitional cell carcinoma of the bladder were treated with preoperative cisplatin-based chemotherapy followed by radiotherapy (20 Gy within 1 week) and cystectomy. DNA flow cytometry (FCM) was performed in paraffin-embedded tissue obtained by transurethral resection immediately before therapy. Together with the T-category and histological grade, DNA ploidy and S-phase fraction (SPF) were evaluated for the ability to predict the response to chemotherapy/radiotherapy and survival: a low T-category, but neither DNA ploidy nor SPF, was predictive for the response to neo-adjuvant treatment. The T-category was not related to the patients' survival. In the Cox regression analysis, SPF was an independent prognostic parameter together with response to the precystectomy therapy. We concluded that, in spite of remaining technical problems, paraffin-embedded tissue from bladder carcinoma is suitable for DNA FCM. Contrary to the situation in superficial bladder cancer, DNA ploidy is not related to the clinical outcome in muscle-invasive bladder carcinoma treated by neo-adjuvant chemo-/radiotherapy and cystectomy. SPF seems to be a clinically worthwhile parameter with significance that has to be further studied in larger series.
Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.
Paust Silke,Gill Harvinder S,Wang Bao-Zhong,Flynn Michael P,Moseman E Ashley,Senman Balimkiz,Szczepanik Marian,Telenti Amalio,Askenase Philip W,Compans Richard W,von Andrian Ulrich H
Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.
A genetically selective inhibitor demonstrates a function for the kinase Zap70 in regulatory T cells independent of its catalytic activity.
Au-Yeung Byron B,Levin Susan E,Zhang Chao,Hsu Lih-Yun,Cheng Debra A,Killeen Nigel,Shokat Kevan M,Weiss Arthur
To investigate the role of the kinase Zap70 in T cells, we generated mice expressing a Zap70 mutant whose catalytic activity can be selectively blocked by a small-molecule inhibitor. We found that conventional naive, effector and memory T cells were dependent on the kinase activity of Zap70 for their activation, which demonstrated a nonredundant role for Zap70 in signals induced by the T cell antigen receptor (TCR). In contrast, the catalytic activity of Zap70 was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This Zap70 kinase-independent pathway was sufficient for the suppressive activity of regulatory T cells (T(reg) cells), which was unperturbed by inhibition of the catalytic activity of Zap70. Our results indicate Zap70 is a likely therapeutic target.
CD1a-autoreactive T cells are a normal component of the human αβ T cell repertoire.
de Jong Annemieke,Peña-Cruz Victor,Cheng Tan-Yun,Clark Rachael A,Van Rhijn Ildiko,Moody D Branch
CD1 activates T cells, but the function and size of the possible human T cell repertoires that recognize each of the CD1 antigen-presenting molecules remain unknown. Using an experimental system that bypasses major histocompatibility complex (MHC) restriction and the requirement for defined antigens, we show that polyclonal T cells responded at higher rates to cells expressing CD1a than to those expressing CD1b, CD1c or CD1d. Unlike the repertoire of invariant natural killer T (NKT) cells, the CD1a-autoreactive repertoire contained diverse T cell antigen receptors (TCRs). Functionally, many CD1a-autoreactive T cells homed to skin, where they produced interleukin 22 (IL-22) in response to CD1a on Langerhans cells. The strong and frequent responses among genetically diverse donors define CD1a-autoreactive cells as a normal part of the human T cell repertoire and CD1a as a target of the T(H)22 subset of helper T cells.
A role for IL-27p28 as an antagonist of gp130-mediated signaling.
Stumhofer Jason S,Tait Elia D,Quinn William J,Hosken Nancy,Spudy Björn,Goenka Radhika,Fielding Ceri A,O'Hara Aisling C,Chen Yi,Jones Michael L,Saris Christiaan J M,Rose-John Stefan,Cua Daniel J,Jones Simon A,Elloso Merle M,Grötzinger Joachim,Cancro Michael P,Levin Steven D,Hunter Christopher A
The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.
Bacillus Calmette-Guérin strain differences have an impact on clinical outcome in bladder cancer immunotherapy.
Rentsch Cyrill A,Birkhäuser Frédéric D,Biot Claire,Gsponer Joël R,Bisiaux Aurélie,Wetterauer Christian,Lagranderie Micheline,Marchal Gilles,Orgeur Mickael,Bouchier Christiane,Bachmann Alexander,Ingersoll Molly A,Brosch Roland,Albert Matthew L,Thalmann George N
BACKGROUND:Whether the commonly used bacillus Calmette-Guérin (BCG) strains Connaught and Tice confer different treatment responses in non-muscle-invasive bladder cancer (NMIBC) is unknown. OBJECTIVES:To compare clinical efficacy, immunogenicity, and genetics of BCG Connaught and Tice. DESIGN, SETTING, AND PARTICIPANTS:A prospective randomized single-institution trial with treatment of 142 high-risk NMIBC patients with BCG Connaught or Tice. INTERVENTION:Patients were randomized to receive six instillations of BCG Connaught or Tice. For experimental studies, BCG strains were compared in C57Bl/6 mice. Bladders and lymphoid tissues were analyzed by cytometry and the latter cultivated to detect live BCG. BCG genomic DNA was sequenced and compared with reference genomes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:Recurrence-free survival was the primary end point of the clinical study. The Kaplan-Meier estimator was used for estimating survival and time-to-event end points. Nonparametric tests served for the analysis of the in vivo results. RESULTS AND LIMITATIONS:Treatment with BCG Connaught conferred significantly greater 5-yr recurrence-free survival compared with treatment with BCG Tice (p=0.0108). Comparable numbers of patients experienced BCG therapy-related side effects in each treatment group (p=0.09). In mice, BCG Connaught induced stronger T-helper cell 1-biased responses, greater priming of BCG-specific CD8(+) T cells, and more robust T-cell recruitment to the bladder than BCG Tice. Genome sequencing of the BCG strains revealed candidate genes potentially involved in the differential clinical responses. CONCLUSIONS:BCG strain may have an impact on treatment outcome in NMIBC immunotherapy. PATIENT SUMMARY:We compared the efficacy of two commonly used bacillus Calmette-Guérin (BCG) strains for the treatment of NMIBC and found that treatment with BCG Connaught prevented recurrences more efficiently than BCG Tice. Comparison of the immunogenicity of the two strains in mice indicated superior immunogenicity of BCG Connaught. We also identified genetic differences that may explain the differential efficacy of the Connaught and Tice BCG strains. TRIAL REGISTRATION:NCT00003779.
TLR4CXCR4 plasma cells drive nephritis development in systemic lupus erythematosus.
Ma Kongyang,Li Jingyi,Wang Xiaohui,Lin Xiang,Du Wenhan,Yang Xi,Mou Fangxiang,Fang Yongfei,Zhao Yanbin,Hong Xiaoping,Chan Kwok Wah,Zhang Xiaoming,Liu Dongzhou,Sun Lingyun,Lu Liwei
Annals of the rheumatic diseases
OBJECTIVES:In patients with systemic lupus erythematosus (SLE), immune tolerance breakdown leads to autoantibody production and immune-complex glomerulonephritis. This study aimed to identify pathogenic plasma cells (PC) in the development of lupus nephritis. METHODS:PC subsets in peripheral blood and renal tissue of patients with SLE and lupus mice were examined by flow cytometry and confocal microscopy, respectively. Sorting-purified PCs from lupus mice were adoptively transferred into -deficient recipients, in which immune-complex deposition and renal pathology were investigated. In culture, PCs from lupus mice and patients with SLE were treated with a TLR4 inhibitor and examined for autoantibody secretion by enzyme-linked immunospot assay (ELISPOT). Moreover, lupus mice were treated with a TLR4 inhibitor, followed by the assessment of serum autoantibody levels and glomerulonephritis activity. RESULTS:The frequencies of TLR4CXCR4 PCs in peripheral blood and renal tissue were found significantly increased with the potent production of anti-dsDNA IgG, which were associated with severe renal damages in patients with SLE and mice with experimental lupus. Adoptive transfer of TLR4CXCR4 PCs from lupus mice led to autoantibody production and glomerulonephritis development in -deficient recipients. In culture, TLR4CXCR4 PCs from both lupus mice and patients with SLE showed markedly reduced anti-dsDNA IgG secretion on TLR4 blockade. Moreover, in vivo treatment with TLR4 inhibitor significantly attenuated autoantibody production and renal damages in lupus mice. CONCLUSIONS:These findings demonstrate a pathogenic role of TLR4CXCR4 PCs in the development of lupus nephritis and may provide new therapeutic strategies for the treatment of SLE.
Circulating tumor cells as prognostic markers in neuroendocrine tumors.
Khan Mohid S,Kirkwood Amy,Tsigani Theodora,Garcia-Hernandez Jorge,Hartley John A,Caplin Martyn E,Meyer Tim
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:To determine the prognostic significance of circulating tumor cells (CTCs) in patients with neuroendocrine cancer. PATIENTS AND METHODS:In this single-center prospective study, 176 patients with measurable metastatic neuroendocrine tumors (NETs) were recruited. CTCs were measured using a semiautomated technique based on immunomagnetic separation of epithelial cell adhesion molecule-expressing cells. RESULTS:Overall, 49% patients had ≥ one CTC, 42% had ≥ two CTCs, and 30% had ≥ five CTCs in 7.5 mL blood. Presence of CTCs was associated with increased burden, increased tumor grade, and elevated serum chromogranin A (CgA). Using a 90-patient training set and 85-patient validation set, we defined a cutoff of < one or ≥ one as the optimal prognostic threshold with respect to progression-free survival (PFS). Applying this threshold, the presence of ≥ one CTC was associated with worse PFS and overall survival (OS; hazard ratios [HRs], 6.6 and 8.0, respectively; both P < .001). In multivariate analysis, CTCs remained significant when other prognostic markers, grade, tumor burden, and CgA were included. Within grades, presence of CTCs was able to define a poor prognostic subgroup. For grade 1, HRs were 5.0 for PFS (P = .017) and 7.2 for OS (P = .023); for grade 2, HRs were 3.5 for PFS (P = .018) and 5.2 for OS (P = .036). CONCLUSION:CTCs are a promising prognostic marker for patients with NETs and should be assessed in the context of clinical trials with defined tumor subtypes and therapy.
Opposing chemokine gradients control human thymocyte migration in situ.
Halkias Joanna,Melichar Heather J,Taylor Kayleigh T,Ross Jenny O,Yen Bonnie,Cooper Samantha B,Winoto Astar,Robey Ellen A
The Journal of clinical investigation
The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.
IL-33-dependent induction of allergic lung inflammation by FcγRIII signaling.
Tjota Melissa Y,Williams Jesse W,Lu Tiffany,Clay Bryan S,Byrd Tiara,Hrusch Cara L,Decker Donna C,de Araujo Claudia Alves,Bryce Paul J,Sperling Anne I
The Journal of clinical investigation
Atopic asthma is a chronic inflammatory disease of the lungs generally marked by excessive Th2 inflammation. The role of allergen-specific IgG in asthma is still controversial; however, a receptor of IgG-immune complexes (IgG-ICs), FcγRIII, has been shown to promote Th2 responses through an unknown mechanism. Herein, we demonstrate that allergen-specific IgG-ICs, formed upon reexposure to allergen, promoted Th2 responses in two different models of IC-mediated inflammation that were independent of a preformed T cell memory response. Development of Th2-type airway inflammation was shown to be both FcγRIII and TLR4 dependent, and T cells were necessary and sufficient for this process to occur, even in the absence of type 2 innate lymphoid cells. We sought to identify downstream targets of FcγRIII signaling that could contribute to this process and demonstrated that bone marrow-derived DCs, alveolar macrophages, and respiratory DCs significantly upregulated IL-33 when activated through FcγRIII and TLR4. Importantly, IC-induced Th2 inflammation was dependent on the ST2/IL-33 pathway. Our results suggest that allergen-specific IgG can enhance secondary responses by ligating FcγRIII on antigen-presenting cells to augment development of Th2-mediated responses in the lungs via an IL-33-dependent mechanism.
CD40 ligation reverses T cell tolerance in acute myeloid leukemia.
Zhang Long,Chen Xiufen,Liu Xiao,Kline Douglas E,Teague Ryan M,Gajewski Thomas F,Kline Justin
The Journal of clinical investigation
Spontaneous antigen-specific T cell responses can be generated in hosts harboring a variety of solid malignancies, but are subverted by immune evasion mechanisms active within the tumor microenvironment. In contrast to solid tumors, the mechanisms that regulate T cell activation versus tolerance to hematological malignancies have been underexplored. A murine acute myeloid leukemia (AML) model was used to investigate antigen-specific T cell responses against AML cells inoculated i.v. versus s.c. Robust antigen-specific T cell responses were generated against AML cells after s.c., but not i.v., inoculation. In fact, i.v. AML cell inoculation prevented functional T cell activation in response to subsequent s.c. AML cell challenge. T cell dysfunction was antigen specific and did not depend on Tregs or myeloid-derived suppressor cells (MDSCs). Antigen-specific TCR-Tg CD8+ T cells proliferated, but failed to accumulate, and expressed low levels of effector cytokines in hosts after i.v. AML induction, consistent with abortive T cell activation and peripheral tolerance. Administration of agonistic anti-CD40 Ab to activate host APCs enhanced accumulation of functional T cells and prolonged survival. Our results suggest that antigen-specific T cell tolerance is a potent immune evasion mechanism in hosts with AML that can be reversed in vivo after CD40 engagement.
RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3.
Han Wei,Xin Zhongshuai,Zhao Zhiqiang,Bao Wen,Lin Xihua,Yin Bin,Zhao Jizong,Yuan Jiangang,Qiang Boqin,Peng Xiaozhong
The Journal of clinical investigation
PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3-mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.
Bacterial killing by complement requires membrane attack complex formation via surface-bound C5 convertases.
Heesterbeek Dani Ac,Bardoel Bart W,Parsons Edward S,Bennett Isabel,Ruyken Maartje,Doorduijn Dennis J,Gorham Ronald D,Berends Evelien Tm,Pyne Alice Lb,Hoogenboom Bart W,Rooijakkers Suzan Hm
The EMBO journal
The immune system kills bacteria by the formation of lytic membrane attack complexes (MACs), triggered when complement enzymes cleave C5. At present, it is not understood how the MAC perturbs the composite cell envelope of Gram-negative bacteria. Here, we show that the role of C5 convertase enzymes in MAC assembly extends beyond the cleavage of C5 into the MAC precursor C5b. Although purified MAC complexes generated from preassembled C5b6 perforate artificial lipid membranes and mammalian cells, these components lack bactericidal activity. In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium, MACs need to be assembled locally by the C5 convertase enzymes. Our data indicate that C5b6 rapidly loses the capacity to form bactericidal pores; therefore, bacterial killing requires both conversion of C5 and immediate insertion of C5b67 into the membrane. Using flow cytometry and atomic force microscopy, we show that local assembly of C5b6 at the bacterial surface is required for the efficient insertion of MAC pores into bacterial membranes. These studies provide basic molecular insights into MAC assembly and bacterial killing by the immune system.
Accelerated telomere shortening in leukocyte subpopulations of patients with coronary heart disease: role of cytomegalovirus seropositivity.
Spyridopoulos Ioakim,Hoffmann Jedrzej,Aicher Alexandra,Brümmendorf Tim H,Doerr Hans W,Zeiher Andreas M,Dimmeler Stefanie
BACKGROUND:Shortening of mean telomere length (TL) in white blood cells is correlated with the development of coronary heart disease (CHD) and with increased mortality due to infectious disease. The goal of the present study was to investigate whether telomere shortening in CHD is restricted to specific peripheral blood lymphocyte and/or myeloid cell subpopulations. Results were correlated to TL in CD34+ hematopoietic peripheral blood stem cells and progenitor cells obtained from the same individual patients. METHODS AND RESULTS:TL was measured by multicolor flow cytometry-fluorescent in situ hybridization in 12 leukocyte subpopulations after immunomagnetic bead sorting. We investigated TL in 14 young (mean age 25 years) and 13 older (mean age 65 years) healthy male volunteers and in 25 age-matched patients with CHD (mean age 65 years). We show that TL in granulocytes and monocytes mirrors TL of CD34+ peripheral blood stem cells and progenitor cells extremely well (r=0.95, P<0.0001) in patients and in healthy adults. TL was approximately 0.5 kilobases (kb) shorter in leukocytes from patients with CHD than in their age-matched control subjects. This difference was identical for CD34+ peripheral blood stem cells and progenitor cells, monocytes, granulocytes, B lymphocytes, and CD4+ T cells, including their memory and naïve subpopulations. Surprisingly, only in cytotoxic CD8+ T lymphocytes, we found a substantially increased TL deficit of 1.0 kb in CHD patients as opposed to control subjects. Further analysis revealed that TL shortening was particularly pronounced in CD8+CD28(-) T cells obtained from cytomegalovirus-seropositive CHD patients, whereas such a difference was not observed in healthy cytomegalovirus-positive as opposed to cytomegalovirus-negative control subjects. Finally, TL shortening of CD8+CD45(RA+) T cells was correlated with the decrease in left ventricular function in CHD patients (r=0.629, P=0.001). CONCLUSIONS:Telomere shortening in patients with CHD could potentially be attributed to either inherited TL shortening or acquired accelerated telomere shortening restricted to the hematopoietic system, which affects the baseline TL of all peripheral blood cell populations, including peripheral blood stem cells and progenitor cells. In addition, cytomegalovirus-seropositive patients but not healthy control subjects exhibited further shortening of their cytotoxic T lymphocytes. Surprisingly, TL shortening of CD8+ T lymphocytes in CHD patients demonstrated a very strong correlation with cardiac dysfunction, which suggests a mechanistic link between CHD and immunosenescence.
VEGF-B Promotes Endocardium-Derived Coronary Vessel Development and Cardiac Regeneration.
Räsänen Markus,Sultan Ibrahim,Paech Jennifer,Hemanthakumar Karthik Amudhala,Yu Wei,He Liqun,Tang Juan,Sun Ying,Hlushchuk Ruslan,Huan Xiuzheng,Armstrong Emma,Khoma Oleksiy-Zakhar,Mervaala Eero,Djonov Valentin,Betsholtz Christer,Zhou Bin,Kivelä Riikka,Alitalo Kari
BACKGROUND:Recent discoveries have indicated that, in the developing heart, sinus venosus and endocardium provide major sources of endothelium for coronary vessel growth that supports the expanding myocardium. Here we set out to study the origin of the coronary vessels that develop in response to vascular endothelial growth factor B (VEGF-B) in the heart and the effect of VEGF-B on recovery from myocardial infarction. METHODS:We used mice and rats expressing a VEGF-B transgene, VEGF-B-gene-deleted mice and rats, apelin-CreERT, and natriuretic peptide receptor 3-CreERT recombinase-mediated genetic cell lineage tracing and viral vector-mediated VEGF-B gene transfer in adult mice. Left anterior descending coronary vessel ligation was performed, and 5-ethynyl-2'-deoxyuridine-mediated proliferating cell cycle labeling; flow cytometry; histological, immunohistochemical, and biochemical methods; single-cell RNA sequencing and subsequent bioinformatic analysis; microcomputed tomography; and fluorescent- and tracer-mediated vascular perfusion imaging analyses were used to study the development and function of the VEGF-B-induced vessels in the heart. RESULTS:We show that cardiomyocyte overexpression of VEGF-B in mice and rats during development promotes the growth of novel vessels that originate directly from the cardiac ventricles and maintain connection with the coronary vessels in subendocardial myocardium. In adult mice, endothelial proliferation induced by VEGF-B gene transfer was located predominantly in the subendocardial coronary vessels. Furthermore, VEGF-B gene transduction before or concomitantly with ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function. CONCLUSIONS:The myocardial VEGF-B transgene promotes the formation of endocardium-derived coronary vessels during development, endothelial proliferation in subendocardial myocardium in adult mice, and structural and functional rescue of cardiac tissue after myocardial infarction. VEGF-B could provide a new therapeutic strategy for cardiac neovascularization after coronary occlusion to rescue the most vulnerable myocardial tissue.
High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.
Jarjour Jordan,West-Foyle Hoku,Certo Michael T,Hubert Christopher G,Doyle Lindsey,Getz Melissa M,Stoddard Barry L,Scharenberg Andrew M
Nucleic acids research
Experimental analysis and manipulation of protein-DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein-DNA interactions.
Circulating endothelial progenitor cells in systemic sclerosis: association with disease severity.
Avouac J,Juin F,Wipff J,Couraud P O,Chiocchia G,Kahan A,Boileau C,Uzan G,Allanore Y
Annals of the rheumatic diseases
BACKGROUND:Heterogeneous data have been reported regarding the detection and number of circulating endothelial progenitor cells (EPCs) in systemic sclerosis (SSc). OBJECTIVE:We investigated the number of circulating EPCs using recent recommendations and we quantified their late outgrowth in patients with SSc and healthy controls. PATIENTS AND METHODS:EPCs, defined as Lin-/7AAD-/CD34+/CD133+/VEGFR-2+ cells, were quantified in 50 patients with SSc (mean age: 55 (16) years, disease duration: 9 (9) years) and 26 controls (mean age: 53 (19) years) by cell sorting/flow cytometry and by counting late outgrowth colony-forming units (CFU). RESULTS:Patients with SSc displayed higher circulating EPC counts than controls (median 86 (5-282) vs 49 (5-275)) EPCs for 1 million Lin- mononuclear cells; p = 0.01). Lower EPC counts were associated with the higher Medsger's severity score (p = 0.01) and with the presence of past and/or current digital ulcers (p = 0.026). There was no difference for the number of late outgrowth EPC-CFUs between patients with SSc and controls in cell culture evaluation. The formation of colonies was associated with higher levels of circulating EPCs (p = 0.02) and the number of colonies correlated with levels of EPCs (R = 0.73, p = 0.0004), validating our combination of fluorescence-activated cell sorter surface markers. CONCLUSIONS:We quantified circulating EPCs with an accurate combination of markers herein validated. Our data demonstrate increased circulating EPC levels in SSc, supporting their mobilisation from bone marrow. Furthermore, the subset of patients with digital vascular lesions and high severity score displayed low EPC counts, suggesting increased homing at this stage. The predictive value of this biomarker now warrants further evaluation.