Background
Females have lower viral setpoints and are more likely to control HIV infection without ART than males, though the mechanisms remain unclear. The X-chromosome-encoded ssRNA receptor TLR7 may contribute to this difference as evasion of X-inactivation in females can increase TLR7 expression. TLR7 activation induces type-I Interferon (IFN) release and production of pro-inflammatory cytokines, but its role in HIV infection across sexes is not fully understood. We investigated cytokine production and gene expression in ART-treated females and males with HIV upon ex-vivo PBMC stimulation with the TLR7 agonist Imiquimod (IMQ).
Methods
The 2000HIV study (NCT03994835) was divided into a Discovery cohort (201 females, 1161 males) and a Validation cohort (46 females, 252 males). PBMCs were stimulated with 5 µg/mL IMQ for 24 h, and IL-1β, IL-1RA, IL-6, IL-8, MCP1, and MIP1α levels were measured by ELISA in supernatants. Cytokine production between sexes was compared using a rank-based regression model, adjusting for age, ethnicity, lymphocyte/monocyte ratio, COVID-19 vaccination, and time of inclusion. Bulk RNA sequencing was conducted on PBMCs from the 2000HIV-TRAINED cohort (27 females, 43 males) (NCT04968717), assessed differential gene expression (DEG), adjusting for age, ethnicity, and COVID-19 vaccination. Gene set enrichment analysis (GSEA) was performed using the HALLMARK gene sets.
Results
IMQ stimulation led to lower IL-1β production in PBMCs from females compared to males (p=0.002) and a trend towards lower IL-8 levels (p=0.054). DEGs included 43 up- and 40 downregulated genes in females compared to males. Downregulated genes in females included CRISP3, WNT5A, ARG1, and MMP8 (p=1.864×10-5, p=0.041, p=0.002, p=0.002, respectively), while upregulated genes included interferon-stimulated genes like IRF7, IFI6, IFIT3, ISG15 (p=0.004, p=0.004, p=0.018, p=0.015, respectively), and the anti-viral factor APOBEC3A (p= 0.042). GSEA revealed enriched “Interferon alpha response” and “Interferon-gamma response” pathways in females (p=1.195×10-19, p=1.194×10-19) compared to males.
Conclusions
Females with HIV show enhanced IFN pathway gene expression but lower pro-inflammatory IL-1β production upon TLR7 activation compared to males with HIV. This suggests TLR7 activation drives a stronger antiviral response in females, favoring IFN responses over pro-inflammatory pathways. Sex-biased immune response differences in people with HIV should be considered in the development of TLR7-agonist therapies.