Neutrophil Extracellular Traps (NETs) Can Active Monocytes In ANCA-associated Vasculitis

ObjectiveAnti-neutrophil cytoplasmic antibody associated vasculitis(ANCA-associated vasculitis,AAV)are defined by an autoimmune small vessel inflammation.Renal involvement is characterized by pauci-immune focal segmental necrotizing glomerulonephritis(Pi FSGN),coupled with extracapillary proliferation and crescent formation,which leads to rapid renal failure.Recent datas suggest therapy which tagart deplete B-cell has high recurrence rates,relapsing disease in up to 50% in patients with proteinase 3-ANCA.Hence,it is urgent to do further study on the pathogenesis of AAV and propose more effective therapy.Monocytes derived from bone marrow hematopoietic stem cells,are an important component of innate and acquired immunity.Monocytes circulate and patrol blood vessels and enter tissue where differentiate into dendritic cells(DCs)or macrophages.It plays an important role in the clearance of pathogenic microorganisms and endogenous substances,presenting antigen,and the activity and regulation of T cells function,and secreting various cytokines,etc.Studies have shown that abnormal activation of monocytes participate in pathophysiological process of some diseases.Anti-neutrophil cytoplasmic antibodies(ANCA)is serological marker for diagnosis of ANCA associated vasculitis,ANCA recognize antigens that are in the primary granules of neutrophils and the peroxidase-positive lysosomes of monocytes,including both proteinase 3(PR3)and myeloperoxidase(MPO).ANCA-mediated activation of neutrophils leads to degranulation and production of reactive oxygen species,consequently release of neutrophil extracellular traps(NETs).NETs release self-DNA decorated with antimicrobial proteins,such as MPO,PR3,Cathelicidin LL37 and etc,which induce autoantibodies and autoimmunity.Deregulated NETs generation have been associated with autoimmune diseases,including AAV.Numerous studies have demonstrated abnormal secretion of proinflammatory cytokines play a role in pathogenesis of AAV,such as interleukin-1?(IL-1?),interleukin-6(IL-6),interferon-?(IFN-?),monocyte chemoattractant protein-1(MCP-1)but it is still unclear that the effect of NETs on the human monocytes proinflammationtory cytokines release.This project intends to observe the effect of NETs on the human monocytes proinflammationtory cytokines release,including IL-1?,IL-12,IFN-?,TNF-?,IL-6,MCP-1,IL-10 and study the influence of NETs on monocytes activation.MethodsNeutrophils from peripheral blood of healthy donor were isolated by Polymorphprep gradient separation.NETs was initiated by phorbol myristate acetate(PMA)and validated by fluorescence staining.Peripheral blood mononuclear cells were obtained by Ficoll density gradient separation.Monocytes were isolated from PBMC using magnetic activated cell sorting(MACS)anti-CD14 microbead.Monocytes were divided into two groups: control group and NETs group which monocytes were stimulated by NETs.After twelve hours' culture,IL-1?,IL-6,TNF-a,IL-12,MCP-1,IFN-?,IL-10 were analyzed using LEGENDplex reagents.Results1.The percentage of neutrophils obtained by Polymorphprep gradient separation was 98%.Fluorescence staining analyzed with a confocal microscope show NETs release self-DNA.2.The percentage of monocytes obtained by MACS anti-CD14 microbead was95.3%.3.NETs significantly increased the secretion of IL-1?(P<0.05).4.NETs significantly increased the secretion of IFN-?(P<0.01).5.NETs significantly increased the secretion of TNF-?(P<0.01).6.NETs significantly increased the secretion of MCP-1(P<0.05).7.NETs significantly increased the secretion of IL-6(P<0.01).8.IL-10 and IL-12 levels in both groups had no significant difference.ConclusionThese data demonstrate NETs can significantly promote IL-1?,IFN-?,TNF-?,MCP-1 and IL-6 secretion in human monocytes.