Gut Microbiota enriched in HIV-Infected Subjects Induce Inflammatory Cytokines but not T Regulatory Cells

Background: We co-exist with an abundant microbiota that cooperate with and help regulate our immune system. However, during HIV infection the microbial community in the gut is altered to a distinctive composition which is not consistently restored to that of seronegative subjects by ART. Recently, our group illustrated that in contrast to uninfected subjects, the microbial community in HIV-infected subjects exhibits increased phylogenetic diversity and is Prevotella-rich and Bacteroides-poor. Here we seek to understand the impact of the change in gut microbiota on HIV infection by characterizing innate and adaptive immune responses, including T regulatory cells, to selected gut bacteria that changed with HIV infection. Methodology: Based on our 16S rRNA sequencing from HIV+ and HIV- subjects we chose four characteristic bacteria; Bacteroides dorei, Bacteroides fluxus, which were decreased, and Prevotellaceae copri, Erysipelotrichaceae biforme, which were elevated with HIV infection. PBMCs of 6 HIV-positive and 6 HIV-seronegative subjects were incubated with lysate of these bacteria. Levels of the anti-inflammatory cytokine, IL-10, were evaluated by ELISA, and T regulatory cells were enumerated by flow cytometry after 3 days. Statistical significance was determined using Mann-Whitney T tests. Results: Levels of T regulatory cells increased with all bacteria compared with the unstimulated control, although levels were not significantly different in regard to HIV status. Median levels of T regulatory cells from PBMC cultured with B. fluxus (4.14) and B. dorei (3.94) were greater compared to that of P. copri (2.73) and E. biforme (2.55). Although these values did not reach statistical significant (p = 0.08). In regard to IL-10 production, higher levels were observed in PBMCs stimulated with B. fragilis, species that is known to stimulate IL-10 production by Tregs using a molecular factor called polysaccharide A (PSA). Stimulation with B. fluxus, a species that we predicted to have this same immune phenotype based on the presence of PSA encoding genes in its genome, also resulted in high IL-10 levels.. These levels were significant when comparing B. fluxus to non PSA producers B. dorei (p = 0.008), to E. biforme (p = 0.001) and P. copri (p = 0.0007). Additionally, B. fragilis compared to B. fragilis with a PSA gene knockout was also significant (p = 0.008). Helios, a maker of thymically derived Tregs, was significantly reduced (p = 0.04) in PBMC stimulated with B. fluxus compared to E. biforme. IL-10 production was confirmed to not be derived from monocytes. Conclusions: These results illustrate that HIV infection is associated with a distinct microbial signature that is more proinflammatory. Furthermore, Bacteroides species which are lost during HIV infection are better adapted for inducing T regulatory cells than P. copri and E. biforme that are enriched in the gut during infection. Within the Bacteroides species, genomic PSA operons predict high IL-10 production from Tregs. By evaluating bacterial mediated immune responses these studies will provide important new understanding about the mechanisms and functional aspects of these bacteria in regulating the immune system and their role in HIV disease.