Impact of Exogenous Estrogen on Innate Immune Responses in Individuals with HIV - Project Summary Sex-based differences in HIV pathogenesis are shaped by both chromosomal and steroidal factors, yet the isolated effects of estradiol on innate immunity remain poorly defined. Estradiol modulates type I interferon (IFN- I) responses through estrogen receptor (ER) signaling, while X-linked genes such as toll-like receptor 7 (TLR7) amplify IFN-I production in individuals with two X chromosomes. Studying people with HIV (PWH) who have an XY karyotype (XY-PWH) and receive estradiol provides a unique biological model to disentangle effects of chromosomes from those of estradiol exposure on antiviral immunity. This K23 proposal will define how estradiol affects innate immune regulation, focusing on plasmacytoid dendritic cells (pDCs), natural killer (NK) cells, and the IFN-I pathway in blood and rectal mucosa, a major site of HIV persistence. Two complementary cohorts will be leveraged: (1) a longitudinal cohort (ACTG A5403) of XY-PWH sampled before and after estradiol initiation (N=30), and (2) a prospective cross-sectional cohort of XY-PWH with and without estradiol exposure (N=20 per group). Aim 1 will evaluate estradiol-associated differences in pDC and NK cell function across systemic and mucosal compartments. Advanced immunologic assays, including high-parameter flow cytometry, cytokine profiling, pDC functional assays (TLR7 stimulation), NK cytotoxicity assays (K562 co-culture), and single-cell RNA sequencing (scRNAseq), will define cellular activation, functional responsiveness, and transcriptional signatures. Aim 2 will investigate direct ER-mediated regulation of NK cell antiviral function. Cleavage under targets and release using nuclease (CUT&RUN) will map ER binding to IFN-related gene promoters (e.g., STAT1, STAT2, IRF9), providing mechanistic insight into estradiol-driven transcriptional programs. Together, these studies will establish how estradiol exposure modulates innate immune responses in XY-PWH, with implications for viral control, reservoir persistence, and immune activation. Integration with existing HIV reservoir data will enhance mechanistic interpretation. The training plan will provide the candidate, Dr. Hastie, with advanced expertise in flow cytometry, functional assays, scRNAseq, and epigenomic profiling, complemented by formal coursework, biostatistical support, and structured mentorship. Dr. Hastie has already acquired proficiency in high-resolution anoscopy and will leverage this expertise to perform mucosal sampling for paired blood–tissue analyses. This distinctive capability enables translational research that few investigators are equipped to carry out, enhancing the study’s innovation. This work addresses a critical NIH priority by defining biological mechanisms underlying sex differences in antiviral immunity. Findings will inform precision immunology strategies and guide care for populations receiving estradiol treatment, ultimately improving HIV care and outcomes for all.
