Autoantibody-mediated Erythrophagocytosis Increases Tuberculosis Susceptibility In HIV/AIDS Patients

Background: Tuberculosis caused by Mycobacterium tuberculosis(MTB)infection is one of the 10 most common causes of mortality worldwide.Human immunodeficiency virus(HIV)infection is a strong risk factor for disease progression in TB and is thus associated with poor treatment outcomes.The HIV-mediated depletion of CD4 T-cells that typically confers a protective immune response to MTB infection is likely a main driver of the increased prevalence of active TB in countries with a high HIV burden.Interestingly,increased TB risk has also been reported among patients with HIV and normal CD4 T-cell counts.Indeed,besides CD4 T-cell loss,macrophage function is also altered during HIV infection.Macrophage-driven innate immunity has been increasingly recognized to have a critical role in the host defense against TB;fine-tuning macrophage fate and function is essential to MTB infection outcomes.However,the mechanisms underlying how HIV infection impairs macrophage-mediated defenses against MTB remain to be elucidated.HIV infection can induce the production of various autoantibodies.It is reported that 20-40% of patients with HIV are positive for anti-red blood cell(RBC)autoantibodies,which can be detected using a direct antiglobulin test(DAT).The presence of anti-RBC autoantibodies can sensitize RBCs in vivo and trigger accelerated RBC phagocytosis and destruction by macrophages,which termed erythrophagocytosis.We therefore hypothesized that anti-RBC autoantibodies might impair macrophage functions to fight against TB by enhancing erythrophagocytosis.Objective: 1.To define the presence of anti-RBC autoantibodies(positive DAT)in patients with HIV and its association with TB susceptibility.2.To define erythrophagocytosis influences macrophage-mediated immunity against MTB infection.3.To clarify the mechanisms underlying how erythrophagocytosis inhibits the bactericidal activity against MTB in macrophagesMethods: 1.Univariate and multivariate analyses were used to investigate the presence of anti-RBC autoantibodies(positive DAT)in patients with HIV and its association with tuberculosis susceptibility.2.By flow cytometry and PKH26-labeling,the uptake of RBCs isolated from HIV/AIDS patients with negative and positive DAT and healthy donors by macrophages was determined.3.Commercial anti-human RBC Ig G was used to mimic RBC sensitization in vivo.By using Fc ? receptor and C1 q receptor blocking antibodies,the macrophagic engulfment of sensitized RBCs were determined by flow cytometry.4.By CFU counting,the erythrophagocytosis influences macrophage-mediated immunity against MTB infection was investigated.5.Through confocal and WB methods to examine the role of autophagy in erythrophagocytosis-mediated impairment in bacterial control.6.By employing specific inhibitor Zn PPIX and si RNA to inhibit the expression of HO-1,the autophagy and bactericidal activity against MTB in macrophages was further evaluated.Results: 1.10.5 %(28/267)cases of patients with HIV/AIDS were DAT positive in the study.2.DAT-positivity was significantly associated with TB incidence in both univariate and multivariate analyses(OR = 11.96,95 % CI,4.68 – 30.93;OR = 12.65,95 % CI,3.33 – 52.75,respectively).3.Autoantibodies against RBCs enhanced erythrophagocytosis,which significantly correlated with the agglutination degree of DAT(R = 0.99,p < 0.05).4.The enhanced erythrophagocytosis can be completely inhibited by Fc?receptor blocker treatment.5.Commercial anti-human RBC Ig G can be used to mimic RBC sensitization in vivo.6.Erythrophagocytosis significantly impaired the bactericidal activity of macrophages against MTB.7.Phagosome maturation and p62-dependent xenophagy of H37Ra-infected macrophages was unaffected by erythrophagocytosis.8.Sensitized RBC treatment inhibited the expression of LC3 B and autophagic flux in MTB-infected macrophages.9.Silencing ATG5,a key component for autophagy,completely abrogated the effect of erythrophagocytosis on macrophage bactericidal activity against MTB.10.Sensitized RBC treatment upregulated the transcript and protein levels of HO-1 in H37Ra-infected macrophages.11.HO-1 inhibited by Zn PPIX or si RNA in H37Ra-infected macrophages significantly abrogated the inhibitory effect of erythrophagocytosis on autophagy and bactericidal activity against MTB.Conclusions: 1.Enhanced anti-RBC autoantibody production in patients with HIV is associated with increased risk of development of active tuberculosis.2.Enhanced erythrophagocytosis in patients with HIV is mediated by anti-RBC autoantibodies.3.Enhanced erythrophagocytosis impairs macrophage bactericidal activity against M.tuberculosis by inhibiting autophagy.4.Erythrophagocytosis inhibits macrophage-mediated autophagy via heme oxygenase-1(HO-1)expression.