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The association between type 2 diabetes (T2D) pathogenesis and immune-mediated tissue damage and insulin resistance suggests that T2D patients might benefit from the suppression of pathogenic inflammation. Foxp3+ Treg cells are crucial suppressors of inflammation,but the differentiation of Foxp3+ Treg cells is not static and is subject to conversion into IL-17-producing Th17-like cells upon receiving external signals. In this study,we examined the production of IL-17 by Treg cells. Compared to non-T2D controls,T2D patients presented significantly higher levels of IL-17-expressing cells in both Foxp3- CD4 T cells and Foxp3+ Treg cells. The frequencies of IL-17-nonexpressing Foxp3+ Treg cells,on the other hand,were not changed. Interestingly,IL-17-expressing Foxp3+ Treg cells were mutually exclusive from IL-10-expressing and TGF-$\beta$-expressing Foxp3+ Treg cells,suggesting that multiple subpopulations exist within the Foxp3+ Treg cells from T2D patients. In T2D patients,the frequencies of IL-17-expressing Foxp3+ Treg cells were positively correlated with the body mass index (BMI) and the HbA1c levels of T2D patients. The frequencies of IL-10-expressing Treg cells,on the other hand,were inversely associated with the BMI of both non-T2D controls and T2D patients. In addition,the suppressive activity of Treg cells was significantly lower in T2D patients than in non-T2D controls. Together,our study uncovered a dysregulation in Foxp3+ Treg cells from T2D patients,characterized by high IL-17 expression and low suppression activity. View Publication

Understanding of the factors governing immune responses in cancer remains incomplete,limiting patient benefit. In this study,we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time,with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges,including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context,whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection,and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade,revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment. View Publication

To date,the effects of endurance exercise training on lymphocyte physiology at the kinome level are largely unknown. Therefore,the present study used a highly sensitive peptide-based kinase activity profiling approach to investigate if the basal activity of tyrosine (Tyr) and serine/threonine (Ser/Thr) kinases of human lymphocytes is affected by the aerobic endurance training status. Results revealed that the activity of various tyrosine kinases of the FGFR family and ZAP70 was increased,whereas the activity of multiple Ser/Thr kinases such as IKK$\alpha$,CaMK4,PKA$\alpha$,PKC$\alpha$+$\delta$ (among others) was decreased in lymphocytes of endurance trained athletes (ET). Moreover,functional associations between several differentially regulated kinases in ET-derived lymphocytes were demonstrated by phylogenetic mapping and network analysis. Especially,Ser/Thr kinases of the AGC-kinase (protein kinase A,G,and C) family represent exercise-sensitive key components within the lymphocytes kinase network that may mediate the long-term effects of endurance training. Furthermore,KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome pathway analysis indicate that Ras as well as intracellular signaling by second messengers were found to be enriched in the ET individuals. Overall,our data suggest that endurance exercise training improves the adaptive immune competence by modulating the activity of multiple protein kinases in human lymphocytes. View Publication

BACKGROUND Lymphotoxin $\beta$ receptor (LT$\beta$R) plays important roles in the development of the immune system and immune response. At the cellular level,ligand-bound LT$\beta$R activates the pro-inflammatory NF-$\kappa$B pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LT$\beta$R and its ligands are promising therapeutic targets. Here,we studied the consequences of perturbed cellular cholesterol content on LT$\beta$R-induced NF-$\kappa$B signaling. METHODS To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228,and endothelial HUVEC cells different treatment regimens with filipin,methyl-$\beta$-cyclodextrin and simvastatin were applied. LT$\beta$R localization was studied by confocal microscopy. The activity of LT$\beta$R-induced NF-$\kappa$B pathway was assessed by measuring the levels of NF-$\kappa$B pathway inhibitor I$\kappa$B$\alpha$ and phosphorylation of RelA transcription factor by Western blotting. The NF-$\kappa$B transcriptional response,production of chemokines and adhesion molecules were examined by qRT-PCR,ELISA,and Western blotting,respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LT$\beta$R with its protein partners were investigated by immunoprecipitation. RESULTS We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-$\beta$-cyclodextrin impaired LT$\beta$R internalization and potentiated LT$\beta$R-dependent activation of the canonical branch of the NF-$\kappa$B pathway. The latter was manifested by enhanced degradation of I$\kappa$B$\alpha$ inhibitor,elevated RelA phosphorylation,substantial increase in the expression of NF-$\kappa$B target genes encoding,among others,cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1,that favored the adhesion of immune cells (NK and T cells,neutrophils) to A549 cells and HUVECs. Mechanistically,we showed that cholesterol depletion stabilized interactions of ligand-stimulated LT$\beta$R with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LT$\beta$R. Thus,drugs modulating cholesterol levels could potentially improve efficacy of LT$\beta$R-based therapies. Video abstract. View Publication

Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation,vasculopathy,and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc,playing key roles in disease pathogenesis. However,the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes,B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC),showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1 flox/flox LysMCre+/+ mice,and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I,independent of TGF$\beta$ upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation,to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc,the top most upregulated pathways were hallmark IFN-$\gamma$ and IFN-$\alpha$ response. Additionally,several genes previously linked to SSc pathogenesis and fibrosis in general were also induced,including CCL2,CCL7,MMP12,and CXCL10. ANKRD1,a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients,with higher expression of profibrotic markers and activation of interferon responsive genes,thus suggesting that dysregulation of Fli1 in myeloid cells may contribute to SSc pathogenesis. View Publication

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein,we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore,the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFN$\gamma$ production,also Ca2+- and CaM-dependent,was instead not reduced in HNSCC T cells,which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity,but not IFN$\gamma$ production,and reduced their chemotaxis in the presence of adenosine,thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally,1-EBIO enhanced INF$\gamma$ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore,they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers. View Publication

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset,progression,and treatment of a broad spectrum of inflammatory conditions. In this study,we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form,as well as in its partially demannosylated and partially desialylated form,on HEK293 cells expressing TLR-8,and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells,N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent,i.e.,chloroquine (CQN),that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8,it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold,p {\textless} 0.0001),partially demannosylated (0.3-fold,p {\textless} 0.0001) and partially desialylated (0.4-fold,p {\textless} 0.0001) N-glycans. Furthermore,the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN,the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune,infections or other inflammatory disorders. View Publication

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche,but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor,RET,at the cell surface of HSCs,in mediating sustained cellular growth,resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex,GDNF/GFRa1,show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally,we demonstrate that RET drives a multi-faceted intracellular signalling pathway,including key signalling intermediates AKT,ERK1/2,NFkB and p53,responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions. View Publication

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here,we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP,termed the wing region. One mAb targeting the GP2 wing,MR228,showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb,MR235,to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs,resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection. View Publication

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success,yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however,the administration of IL-12 has been associated with immune-related adverse events. Here we show that,after intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive mouse tumours,CBD-IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12,CBD-IL-12 induced sustained intratumoural levels of interferon-$\gamma$,substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy,eliciting complete regression of CPI-unresponsive breast tumours. Furthermore,CBD-IL-12 potently synergized with CPI to eradicate large established melanomas,induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours. View Publication

Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However,it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-$\kappa$B and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+),34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV-) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced,single spliced,and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p {\textless} 0.05). As Tat is synthesized by multiple splicing,the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects,which could indicate a higher Tat activity in these cells despite their resting state. View Publication

Upon immunogenic challenge,lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments,and with resolution,they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation,YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease. View Publication