Immunoregulation Of DC-SIGN On Podocytes In Lupus Nephritis

Lupus nephritis(LN)is a serious complication of systemic lupus erythematosus.The severity of the lesion directly affects the prognosis of systemic lupus erythematosus(SLE).At present,the pathogenesis of LN is still not clear.Currently,the main characterizations of LN include deposition of immune complex(IC)on glomeruli,inflammatory cell infiltration and local immune inflammation,which are regulated by multiple factors.Podocytes,which are localized on the outer basement membrane,are one of the main targets of IC.Podocytes can also play important roles in innate immune and adaptive immune reactions as an immune cell in the glomerular injury caused by various reasons,which actively participate in the process of disease development.Both adaptive immunity and innate immunity participate in the development of LN.In recent years,more attention has been attracted on the immunoregulatory function of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin(DC-SIGN),an important molecule which link innate and adaptive immune responses.DC-SIGN can be expressed not only in immune cells such as dendritic cells and macrophages but also in some epithelial cells.It has been found that DC-SIGN can be expressed in the podocytes of HIV-associated nephritis and mediate the entry of HIV into podocytes and act as a functional receptor involved in virus activity in podocytes.DC-SIGN plays an indispensable role in positive and negative immunoregulation of various infectious diseases and inflammatory diseases by virtue of its intermolecular carbohydrate recognition properties,such as immune recognition,cell adhesion,and positive and negative immunoregulatory functions.Glycobiology is an extension of genomics and proteomics.Abnormally glycosylated immunoglobulin G(IgG)not only changes its immunological function,but also changes its antigenicity,thereby inducing various chronic inflammation and autoimmune diseases.Abnormal glycosylated IgG was found in the serum of patients with autoimmune diseases such as SLE and rheumatoid arthritis.The main manifestation was galactose deficiency at Fc,and expose GlcNAc at the end of the carbohydrate chain.Recently it was found that DC-SIGN,which has a carbohydrate-recognizing property,can bind to GlcNAc in N-glycans of IgG and thereby mediate the function of DC-SIGN to exert adhesion and phagocytose antigens.This gives DC-SIGN more research value in the immune regulation of inflammatory diseases.At present,DC-SIGN is rarely studied in renal inflammatory diseases.Our previous studies have confirmed that DC-SIGN has immunomodulatory effects in the pathogenesis of primary glomerulonephritis and renal tubular interstitial injury,and in the immune complex and inflammation.The study of immunomodulatory effects DC-SIGN in glomeruli has not been reported.To date,the pathogenesis of LN has been more concerned with the role of immune cells.The immunoregulatory effect of renal local podocytes on the LN glomerular injury is relatively rare,which may restrict further interpretation of the pathogenesis and clinically effective treatment of LN.Therefore,this study started from podocyte immune regulation and focused on the expression of DC-SIGN,an immune molecule in the LN microenvironment,to investigate the immune regulation of DC-SIGN in the pathogenesis of LN.This study first observed the expression of DC-SIGN in renal tissue samples from patients with different pathological types of LN and in renal tissues of LN mouse models,and its correlation with proteinuria.It was found that DC-SIGN was inducible in the glomeruli of type III,IV and V LN and MRL/lpr lupus mice,and the glomerular expression of DC-SIGN was related to the degree of proteinuria.Immunofluorescence confirmed that DC-SIGN co-localizes with podocyte marker nephrin,suggesting that DC-SIGN is expressed in podocytes.DC-SIGN is also observed to correlate with renal function progression in MRL/lpr lupus mice.It was found that inhibition of DC-SIGN expression can relieve local renal pathology and renal function by using anti-DC-SIGN intervention.The above results suggest that DC-SIGN in LN podocyte participates in the progress of LN.Then the immunoregulatory effect of DC-SIGN of LN podocytes was observed in vitro using a mouse podocyte cell line and an established mouse podocyte cell line with DC-SIGN shRNA.We found that serum of LN mice could up-regulate the expression of DC-SIGN,MHC II and co-stimulatory molecule CD80 in podocytes,stimulate the proliferation of T cells,and increase the ratio of IFN-?/IL-4 secreted by T cells.Anti-DC-SIGN antibody intervention or DC-SIGN shRNA could down-regulate podocyte DC-SIGN,MHC II and CD80 expression,decrease T cell proliferation,and reduce IFN-?/IL-4 ratio.It was suggested that podocytes in LN could exert DC-like cell function by expressing DC-SIGN and participate in inflammation and immune response in LN.Inhibition of DC-SIGN expression can down-regulate the immune function of the podocyte and produce a preventive effect.At the same time,it was found that DC-SIGN in LN podocytes is involved in the production of IL-6 and IL-23 pro-inflammatory cytokines in podocytes.We further explored the signaling pathways of DC-SIGN in LN podocytes.The activation of Raf-1 signaling molecules is the most critical step of the DCSIGN signaling pathway.We found that phosphorylation of Raf-1(Ser 338)in podocytes stimulated by serum of LN mice reached a peak at 10 min,and phosphorylation was weakened over time.DC-SIGN shRNA significantly attenuated phosphorylation of the Raf-1(Ser 338).Studies have shown that phosphorylation of Raf-1(Ser 338)is associated with the activation of their upstream PAK kinases.Using the PAK inhibitor Toxin B,we found that phosphorylation of Raf-1(Ser338)was significantly inhibited when PAK kinase was inhibited.Crosstalk with the TLR signaling pathway is another important feature of DC-SIGN immune function.Studies have found that DC-SIGN can induce phosphorylation of NF-kB p65 by crosstalk with the TLR signaling pathway and participate in the secretion of some cytokines.We found that after stimulation with LN mouse serum,NF-kB p65 subunit Ser 276 can be activated in podocytes,and DC-SIGN shRNA can weaken the phosphorylation of this site.In addition,activation of MAPK signaling pathway is another downstream pathway of Raf-1 in the DC-SIGN pathway,and is associated with the production of proinflammatory cytokines such as IL-6.We found that serum stimulation of LN mice can activate ERK,while DC-SIGN shRNA can inhibit the phosphorylation of ERK in podocytes,suggesting that there are also MAPK pathway activation in LN podocytes,and may related to the production of pro-inflammatory factors such as IL-6 and IL-23.We hypothesize that GlcNAc in various autoimmune antibodies in LN serum may interact with DC-SIGN in podocytes and then participate in the immune reactions exerted by DC-SIGN.We conducted a preliminary exploration of this.We found that podocyte DC-SIGN in LN could co-immunoprecipitate GlcNAc,suggesting that DC-SIGN may bind to autoantibody through GlcNAc.In addition,anti-GlcNAc antibody interventions inhibited DC-SIGN-mediated T cell proliferation and Th1 cytokine secretion,while podocyte DC-SIGN-mediated proinflammatory cytokine IL-6 and IL-23 production did not change significantly.It suggests that GlcNAc participates in the DC-SIGN-mediated immune response on the podocyte surface,but has no obvious correlation with the production of podocyte derived cytokines.In conclusion,podocytes can be induced the expression of DC-SIGN in LN,which is associated with proteinuria,renal function and local histopathological status.It can stimulate the expression of MHC II and co-stimulatory molecule CD80 in podocytes,stimulate T cell proliferation,induce Th1 cell cytokines secretion,and promote podocyte production of IL-6,IL-23.The key signaling molecule of the DC-SIGN signaling pathway,Raf-1,is activated in the DC-SIGN pathway of LN podocytes.Its activation site is Raf-1(Ser338),and its upstream is PAK kinase.The downstream of Raf-1 including ERK and NF-kB can be phosphorylated,suggesting that MAPK pathway is related to LN podocytes DCSIGN-exerted pro-inflammatory cytokines production,and DC-SIGN signaling pathway may crosstalk with TLR pathway.DC-SIGN in podocytes of LN can interact with GlcNAc.It is speculated that this may be one of the ways that podocytes in LN interact with various autoimmune antibodies,and GlcNAc is involved in regulation of T cell immune function mediated by DC-SIGN in podocyte.Therefore,DC-SIGN in podocytes in LN can be used as a new intervention method or approach for the prevention and treatment of LN.This study provides a new theoretical and experimental basis for the pathogenesis of LN and clinical interventions.