HIV-1 transmission remains a significant public health concern, with sexual contact among MSM being the primary mode of transmission in the US. Anal intercourse, particularly receptive anal intercourse, carries the highest risk. Recent research has highlighted the crucial roles of gut microbiome dysbiosis and systemic inflammation in HIV-1 pathogenesis. Gut dysbiosis can increase local inflammation, activate CD4⁺ T cells, and increase CCR5 expression, making MSM vulnerable to infection. Translocation of gut bacteria into the bloodstream leads to systemic immune activation, further promoting HIV-1 dissemination. Elevated pro-inflammatory cytokines also contribute to increased susceptibility to infection. This study aimed to comprehensively analyze the associations and interactions among self-reported sexual behaviors, systemic inflammatory cytokine levels, and gut microbiome composition before HIV-1 infection in MSM, using a mediation analysis framework to determine whether changes in biomarkers mediate the effect of sexual behavior on HIV-1 infection.

Previous research has established links between altered gut microbiome profiles (increased Prevotella, decreased Bacteroides) and increased plasma inflammatory cytokines preceding HIV-1 infection in MSM. The odds of HIV-1 infection were highly correlated with the Prevotella/Bacteroidaceae ratio. While these studies confirmed gut microbiome dysbiosis before infection, the interactions between sexual behavior, biomarkers, and microbiome composition in relation to HIV-1 infection remained largely unexplored. Studies on the mediating role of the gut microbiome in various health outcomes, such as the effect of diet on immune function, have demonstrated the importance of this approach.

This study utilized longitudinal data from the Multicenter AIDS Cohort Study (MACS), analyzing data collected before and after HIV-1 infection. Participants were categorized into sexual exposure groups based on the number of partners with whom they engaged in receptive anal intercourse. Plasma levels of inflammatory cytokines (sCD14, sCD163, IL-6, LBP, CRP) were measured. Gut microbiome composition was profiled using 16S rRNA gene sequencing, analyzing alpha and beta diversity and the abundance of specific bacterial taxa. Mediation analyses, using natural effect models, were employed to assess whether cytokine and microbiome changes mediate the relationship between sexual behavior (receptive anal intercourse partners) and HIV-1 infection. ANCOM-BC was used to identify differentially abundant bacterial species between HIV-1 infected and uninfected individuals, and between different sexual exposure groups. Substance use, antibiotic usage, and STI status were considered as potential confounders or covariates.

The study found a significant increase in plasma sCD14 levels with increasing sexual exposure groups. While alpha diversity did not show significant association with exposure groups, several bacterial species showed significant changes in abundance. Specifically, *A. muciniphila*, *B. caccae*, *B. fragilis*, *B. uniformis*, *Bacteroides* spp., and *Butyricimonas* spp. showed decreased abundance in higher sexual exposure groups and in HIV-1 infected individuals. Mediation analysis revealed a significant natural direct effect (NDE) of sexual exposure on HIV-1 infection, even when controlling for biomarker levels. Cytokines (sCD14 and sCD163) and several bacterial taxa significantly mediated the effect of sexual exposure on HIV-1 infection. The combined effect of cytokines and microbiome as mediators was greater than their individual effects. Substance use was positively associated with the number of receptive anal intercourse partners and HIV-1 infection risk, but did not significantly mediate the relationship between sexual behavior and infection.

This study provides strong evidence supporting the hypothesis that sexual behaviors, particularly high-risk receptive anal intercourse, drive gut microbiome dysbiosis and systemic inflammation, increasing susceptibility to HIV-1 infection in MSM. The observed reduction in beneficial bacterial species, such as those within the Bacteroides genus and *A. muciniphila*, suggests a disruption of the gut’s protective mechanisms. The significant mediation effects of both cytokines and microbiome highlight the complex interplay of these factors in HIV-1 transmission. The findings underscore the importance of considering these biological factors in understanding HIV-1 risk among MSM and developing effective prevention strategies. The study’s findings also highlight the need for interventions targeting gut microbiome health and inflammation to reduce HIV-1 risk.

This study demonstrates a significant association between high-risk sexual behavior, gut microbiome dysbiosis, systemic inflammation, and HIV-1 infection in MSM. The findings highlight the importance of these biological factors in HIV-1 acquisition and suggest potential targets for prevention strategies. Future research should investigate the causal mechanisms underlying these relationships and explore potential therapeutic interventions to modulate gut microbiome composition and inflammation to reduce HIV-1 risk.

The study's reliance on a single stool sample per participant limits the ability to assess the full temporal dynamics of microbiome changes. The study's data are from the early years of the AIDS epidemic before the availability of effective anti-HIV-1 therapy, which might affect the generalizability of the findings to the current context. Furthermore, the study's focus on a specific subset of biomarkers might not fully capture the complexity of the interactions between sexual behavior and HIV-1 infection.