The Role Of Staphylococcus Aureus In HIV-associated Anti-CD4 Autoantibody Production

Background: The advent of antiretroviral therapy(ART)successfully suppressed human immunodeficiency virus(HIV)replication,restored CD4+ T cell count,decreased acquired immunodeficiency syndrome(AIDS)-associated morbidity and mortality,and turned AIDS into a treatable chronic disease.Nevertheless,approximately 20% of people with HIV(PWH)fail to restore their CD4+T cell counts to the level similar to healthy individuals with ART despite viral suppression in the periphery,who were defined as immunological non-responders(INRs).INRs present severe immune perturbation and significantly increased morbidity and mortality of AIDS-related and non-AIDS-related diseases including liver disease,nephropathy,cardiovascular disease,HIV-related neurological disorders,and malignancies.As reported,the mechanisms for poor CD4+ T cells reconstitution are not fully understood,with explanations and risk factors ranging from low nadir CD4+ T cells,persistent immune activation,thymic hypoplasia,and fibrosis.In our published work,we were the first group to detect increased plasma autoreactive anti-CD4 IgGs in the blood of HIV+ INRs(on ART with CD4 cell counts < 350 cells/u L)and demonstrate the pathogenic nature of these IgGs in mediating CD4+ T cell death via antibody-dependent cell-mediated cytotoxicity(ADCC).However,the specific and direct mechanism of anti-CD4 autoantibody production in HIV infection remains obscure.Staphylococcus aureus colonization is associated with autoantibody production and autoimmune disease pathogenesis.However,whether S.aureus affects anti-CD4 autoantibody production in HIV remains unknown.Objective: In this study,we propose to use the peripheral blood mononuclear cells(PBMCs)and plasma from PWH to prove the correlation between S.aureus and the production of anti-CD4 autoantibodies.Furtherly,we will investigate the causal effect of Staphylococcus aureus in promoting the production of anti-CD4 autoantibodies using C57BL/6J mice and their spleen B cells as well as its possible mechanism.Methods:1.To investigate HIV-associated IgG autoantibody profile and the unique characteristics of anti-CD4 IgGs1)We used the autoantigen microarray to assess 87 autoantibody IgGs(auto IgGs)from uninfected controls,untreated PWH(HIV+/ART-na?ve),and virologically suppressed PWH on ART.2)The plasma levels of anti-CD4 IgG,IgA,IgM,and associated IgG subclasses autoantibodies in PWH were evaluated using ELISA.3)Plasma IgG anti-CD4 autoantibodies were purified from HIV-infected patients and co-cultured with isolated CD4+ T cells as well as NK cells to detect the cytotoxicity of CD4 autoantibodies.2.Associations between plasma anti-CD4 IgG levels and markers of circulating S.aureus antigen translocation from clinical samples in treated HIV1)The antibody levels against S.aureus antigens in human plasma were measured by ELISA and were correlated with plasma anti-CD4 IgG autoantibody levels.2)Gene expression profiles of human B cells from PWH and healthy controls were investigated using RNA-seq.Pathway enrichment analysis were performed using the Cytoscape software platform to discover the activation of signaling pathways associated with S.aureus and the corresponding altered cellular functions.3.The causal effect analysis of S.aureus translocation and the production of anti-CD4 autoantibodies as well as its specific mechanism1)C57BL/6J mice were administrated with peptidoglycan(PGN)from different species of bacteria(S.aureus and Bacillus Subtilis)via intraperitoneal injection(i.p.)and the serum levels of anti-CD4 autoantibodies were evaluated with ELISA.2)The percentages of CD4+ T cells in peripheral blood,spleen,and gut lymphoid nodes were assessed using flow cytometry.3)The class switch recombination(CSR)and proliferation of mouse spleen B cells and peripheral human B cells were evaluated with magnetic-activated cell sorting and flow cytometry.4)Using a murine TLR2 reporter cell line to investigate the different degrees of TLR2 activation by different bacterial species PGNs in vitro.Results:1.The plasma levels of anti-CD4 IgGs were specifically elevated in some PWH with ART and the main increased subclasses were IgG1 and IgG3.2.The plasma anti-CD4 IgG levels were positively correlated with the markers of circulating S.aureus antigen translocation in treated HIV.3.Administration of S.aureus PGN in C57BL/6J mice resulted in anti-CD4 IgG antibody production and gut lymph node CD4+ T cell decrease in vivo.4.S.aureus PGN drove IgG2 b and IgG3 class switch recombination(CSR)of spleen B cells via TLR2 signaling pathway in vitro.Conclusion: This study firstly reported the role of S.aureus PGN in the pathogenic anti-CD4 IgG production via CSR and provided a possible option for treating CD4+ T cells depletion in HIV.