Integrated transcriptomic profiling of rectal and adipose HIV-1 reservoirs relative to matched PBMCs in ART-treated individuals.

Background: HIV-1 persistence in anatomical reservoirs remains the primary obstacle to a functional cure, even under suppressive antiretroviral therapy (ART). While gut-associated lymphoid tissue (GALT) is a well-recognized site of viral sequestration, the role of adipose tissue as an immunometabolic sanctuary, and its distinct host immune environment compared with peripheral blood remain under-characterized. Methods: In this study, we quantified the HIV-1 DNA reservoir using droplet digital PCR (ddPCR) in rectal tissues and subcutaneous adipose tissues (SAT), each paired with matched peripheral blood mononuclear cells (PBMCs) from ART-treated individuals. To characterize the host transcriptomic landscape associated with viral persistence, RNA-seq was performed across these compartments. An external healthy human tissue dataset (GSE120795) was integrated to perform background-identity filtering. Key differentially expressed signatures and enriched pathways were further validated using RT-qPCR and Western blot. Results: The ddPCR analysis confirmed significantly higher viral DNA loads in both rectal and adipose tissues relative to matched PBMCs, identifying them as major anatomical reservoirs. Transcriptomic profiling after background filtering revealed divergent host responses: rectal reservoirs were primarily characterized by extracellular matrix (ECM) reorganization and epithelial barrier dysfunction, whereas SAT exhibited prominent dysregulation in cell cycle progression and immunometabolic signaling. Notably, cross-tissue enrichment analysis identified the PI3K-Akt signaling pathway as a common transcriptomeic feature across these anatomical sites. Validation experiments confirmed that fibrosis-associated hub gene Conclusion: Our study provides a comprehensive landscape of tissue-specific transcriptomic remodeling in rectal and adipose HIV-1 reservoirs. These findings suggest that persistent viral sequestration is associated with distinct microenvironmental alterations, ranging from fibrotic shifts to metabolic dysregulation, which share common molecular features such as potentially linked to the PI3K-Akt axis. This work underscores the importance of targeting anatomical sanctuaries beyond the peripheral blood in future HIV-1 cure strategies.