RSV G Activates The Alternative Pathway Of Complement And Promotes HGECs To Recruit CD16~+ Monocytes To Participate In IgAN Pathogenesis

Background:Clinical development and exacerbation of IgA nephropathy(IgAN)are frequently preceded by episodes of upper respiratory tract infection.Our previous study found that Respiratory Syncytial Virus(RSV)infection can aggravate renal damage in IgAN mice.The RSV G glycoprotein(RSV G)is an important RSV attachment protein that stimulates host neutralization antibody responses.It was found that RSV G could simulate CX3CL1 promoting CX3CR1~+leukocytes recruitment in infected tissues.Monocytes express the chemokine receptor CX3CR1,which binds the chemokine CX3CL1.Gross hematuria in patients with IgAN correlates significantly with renal expression and plasma levels of CX3CL1 and elevation of the number of CD16~+monocytes in urine.RSV G not only induce the expression of CX3CL1 and other chemokines in airway epithelial cells,but also promote the production of inflammatory factors by monocytes.Then,whether RSV G can promote glomerular endothelial cells chemotaxis of CD16~+monocytes through CX3CL1-CX3CR1 axis to participate in the pathogenesis of IgAN remains to be confirmed.Alternative pathway of complement(APC)activation plays an important role in the pathogenesis of IgAN.Our previous study showed that C3a and C5a level in serum and kidney,C3aR and C5aR expression was increased in RSV infected IgAN mice,while administration of C3a R and C5a R1 antagonist could alleviate the kidney damage.RSV could activate APC(alternative pathway of complement)after infection of airway epithelial cells.The SARS-Co V-2 spike protein binds to heparin sulfate on the cell surface and interferes with factor H function on the cell surface resulting APC activation.Heparan sulfate is a glycosaminoglycan widely present on the cell surface,extracellular matrix and within the basement membrane.RSV G directly binds to heparin sulfate expressed at the surface of the host cell.However,it is not clear whether RSV G can activate the APC on the surface of glomerular endothelial cells and participate in the pathogenesis of IgAN.Methods:Clinical data of IgAN patients were collected,the proportion of monocyte subtypes and the expression of chemokine receptor in peripheral blood of IgAN patients were analyzed by flow cytometry.The correlations between monocytes subtype and indicators of renal injury were analyzed.The expression of chemokines in plasma and renal tissue of IgAN patients was detected by ELISA and immunofluorescence respectively.Immunofluorescence detected the deposition of RSV G and F in IgAN patients’renal tissue.Monocytes associated expression in vitro induced by TNF-αand RSV G in the activated primary human renal glomerular endothelial cells(HGECs)was measured by RT-PCR.The co-localization of RSV G with membrane attack complex(C5b-9)was observed in the kidney of IgAN patients by immunofluorescence.Peripheral blood mononuclear cells were isolated from healthy controls and IgAN patients.and the complement reaction of RSV G on glomerular endothelial cells was detected by in vitro complement activation assay.The chemoattractant activity of HGECs on CD16~+monocytes was observed after APC activation.Results:(1)Compared to healthy controls,Increased CD16~+monocytes in IgAN patients were observed.Further analysis showed that decreased CD16~+monocytes in IgAN patients with crescent formation compared those without crescent formation.Besides,IgAN patients had a significant increased CX3CR1 expression in CD16~+monocytes and the elevated plasma level of CX3CL1.The proportion of CD16~+monocytes was positively correlated with the s IgA and urinary protein-creatinine ratio in IgAN patients.The infiltration degree of CD16~+macrophages in renal tissue were significantly increased in IgAN patients.CX3CL1 was expressed in glomerular endothelial cells.CX3CR1 inhibitors can significantly suppressed monocytes chemotaxis.(2)RSV G was deposited in 11.7%IgAN patients’renal tissues.RSV G not only induces CX3CL1 expression in HGECs,but also mimics CX3CL1 enhancing chemotaxis of HGECs on CD16~+monocytes.(3)C5b-9 deposition induced by RSV G in glomerular endothelial cells was inhibited by anti-C5Ab and D factor inhibitor.After complement activation,complement chemotactic fragments,are deposited on glomerular endothelial cells and bind to corresponding receptors,further promotes chemotaxis of CD16~+monocytes.Conclusion:RSV G could promote glomerular endothelial cells to recruit CD16~+monocytes via the CX3CL1-CX3CR1 axis and activation of the complement alternative pathway to participate in the pathogenesis of IgAN.