The Regulatory Mechanism Of TIM-3 In ANCA Associated Vasculitis

Objective:Neutrophil cytoplasmic antibody associated vasculitis(AAV)is a systemic autoimmune disease.The pathogenesis of AAV is the disorder of immune system,neutrophil cytoplasmic antibodies increase abnormally,and Antineutrophil cytoplasmic antibody(ANCA)stimulation primes neutrophils to undergo a respiratory burst,degranulation of toxic proteins and adhesion to endothelium,also can prime neutrophils to form more neutrophil extracellular traps,led to inflammation of vessel wall and cellulose-like necrosis.Abnormal feature of the neutrophils in ANCA associated vasculitis,which promoted aberrannt NETs formation,had pay attention by more and more reserchers.NETs can release the chromatin fibers and antibacterial cytoplasmic protein.Among these proteins were histones,myeloperoxidase,antibacterial peptides LL37,the serine protease neutrophil elastase,calprotectin,defensins and actin.NETs can also transport these protein components to dendritic cells for further processing and presentation.We know that immune responses in our bodies are regulated by positive and negative signals triggered by the interaction of activation and inhibitory receptors.Inhibitory receptors,also known as immune checkpoints,are important regulator of immune responses and maintain immune balance.When the function of inhibitory receptor is disturbed,the tolerance of the immune system is broken down,which can induce the generation of autoantibodies and the emergence of autoimmune diseases.T cell Ig and mucin domain-containing protein 3(TIM-3)is an important inhibitory receptor molecule which expressed on the surface of T cells,and plays an important role in autoimmune diseases.The main function of TIM-3 on T cells is to inhibit T cell function.TIM-3 is not only express on T cells,but also sustainability express on innate immune cells.TIM-3 is actively involved in regulating macrophage/monocyte activity.TIM-3 also acts as a NK cell maturation marker and is necessary for their cytotoxicity.However,there were few studies on the expression of TIM-3 on Dendritic cells(DCs),especially in autoimmune diseases.Scientists have carried out detailed studies on the function of TIM-3 on T cells in many autoimmune diseases.In recent years,the incidence of ANCA associated vasculitis is increasing,and anti-myeloperoxidase positive AAV(MPO-AAV)was dominant in Chinese people Therefore,this study aimed to explore the regulation of immune cells surface TIM-3 to NETs mediated immune response in MPO-AAV patients,and also to explore possible mechanisms.Methods:Twenty untreated patients with MPO-AAV and 20 health controls were included in this study.Peripheral blood mononuclear cells(PBMCs)in these subjects was isolated,flow cytometry was used to analyze TIM-3 expression on surface of T cell subsets.Clinical laboratory data were collected.Correlation between the levels of CRP or anti-myeloperoxidase antibodies with TIM-3 expression was calculated.Flow cytometry was also used to analysis peripheral DCs surface TIM-3 and TLR4expression in PBMCs.Besides PBMCs,circulation neutrophils and serum were also separation from blood of these research objects.Spontaneous NETosis percentages were observed by immunofluorescence with free serum stimulation.The serum levels of IL-6,IL-10 and TNF-αwere detected by electrochemical luminescence instrument,and the correlation with the percentage of NETosis was analysis.In animal experiments,bone marrow induced DCs(BMDCs)and neutrophils were isolated.Then isolated neutrophils were stimulated with 50n M PMA to form NETosis.After co-culture of mouse BMDCs with PMA induced NETs,DCs activation markers(MHCⅡand CD86),cytokine levels(IL-1βand IL-6)and TLR4 expression were measured.And the effect of TIM-3 blockade to this co-culture system was also detected.Changes of the activation markers were determined by flow cytometry,and the changes of cytokine secretion levels in the cultured supernatant were determined by ELISA.Results:The percentages of CD4~+T cells and CD8~+T cells in lymphocytes of MPO-AAV patients were increased,but there was no statistical difference.The percentage of Treg cells in lymphocytes decreased significantly in MPO-AAV patients.More important,serum CRP level and anti-MPO concentration in MPO-AAV patients were correlated with percentage of Treg cells.Then we detected the expression of TIM-3 on CD4~+T cell,CD8~+T cells and Treg cells,but we did not found the difference between MPO-AAV patients and health controls.The expression of TIM-3 on T cells was low,so we gated DCs in PBMCs,evaluated the expression of TIM-3 and TLR4 on DCs surface in MPO-AAV patients and health controls.Compared with health controls,we detected a significantly lower expression of TIM-3 on DCs.The expression of TIM-3 on DCs negatively correlated with BVAS,which also negatively correlated with MPO-ANCA concentration.Meanwhile,the expression of TLR4on DCs was significantly increased.Compared with health controls,the expression of IL-6and TNF-αwere significantly increased in patients with MPO-AAV,but there was no difference in IL-10 expression.Accompany with the higher expression of cytokines,we also detected the higher produce of NETs spontaneously in MPO-AAV.More interesting,the expression of IL-6 and TNF-αin MPO-AAV patient correlated with the percentage of NETosis.Since NETosis was a prominent feature in MPO-AAV.We sought to examine whether induced NETs could promote DCs maturation.We first successfully induced bone marrow derived dendritic cells formation.We also successfully isolated mouse bone marrow granulocytes,which were successfully induced to form NETs under the stimulation of PMA.In animal experience,NETs fragments stimulation increased the expression of CD86 and MHCⅡactivation molecules on DCs surface.Moreover,to assessment of DCs secreted proinflammatory cytokines expression in culture supernatants after NETs fragments stimulation,the results revealed that NETs could significantly increase cytokines level of IL-1βand IL-6.We also measured TLR4 expression after NETs fragments stimulation.We found a significantly increased expression of TLR4 after NETs stimulation.More interesting,we used anti-TIM-3 antibody as a blocking antibody to block the role of TIM-3,and we observed higher cytokines secretion by BMDCs.Conclusion:In our study,we found that the percentage of Treg cells decreased in MPO-AAV patients,and positively correlated with the level of anti-MPO autoantibodies in serum,suggesting that Treg cells play an important role in controlling the secretion of autoantibodies.We also found that the percentage of Treg cells correlated with CRP level,suggesting that Treg cells play a certain role in regulating inflammatory responses,and the specific mechanism also remains to be study.We detected the expression of TIM-3 on T cell subsets,we found that TIM-3expression was very lower on T cells subsets in PBMCs,which may be due to the lower expression of TIM-3 on na?ve T cells.The positive correlations between spontaneous NETosis forming and the expression of IL-6 or TNF-αin MPO-AAV patient,which supported that NETosis seems to be involved in AAV pathogenesis.TIM-3 was decreased on DCs surface of MPO-AAV patients and correlated with BVAS,which supported the negative regulation of TIM-3 on DCs surface in MPO-AAV.In animal model,NETs stimulation could increase DCs maturation and TLR4expression,and anti-TIM-3 antibody treatment could enhance BMDCs cytokine expression.We concluded NETs stimulation to induced BMDCs maturation,and DCs surface TIM-3played an important role in NETs mediated immune balance.TIM-3 on DC surface might play an important regulatory role in TLR4-mediated innate immune response.The decrease of TIM-3 might be one of the important pathogenesis of MPO-AAV.On the one hand,the down-regulated expression of TIM-3 in MPO-AAV may damage the negative regulatory pathway after TLR4 activation,and which increased the secretion of cytokines furtherly,leading to the expansion of inflammatory response.On the other hand,the down-regulation of TIM-3 expression may lead to presentation of autoantigens by dendritic cells and induce the production of more autoantibodies furtherly.The cross-talk between the TIM-3 and TLR4pathways may make TIM-3 one of the important mechanisms for negative regulation of TLR4-mediated immune response.However,what about the specific mechanism of TIM-3signaling on NETs-TLR4-mediated signaling pathway and what is the downstream signaling molecular?These are the direction of our next researches.