381 Oral pathogenic bacterial products differently modulate inflammatory responses in HIV+ peripheral blood immune cells and human brain organoids

Objectives/Goals: Our goal is to characterize how peripheral immune cells from people with HIV (HIV+) and control (HIV-) donors respond to stimulation with lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) and lipoteichoic acid from Staphylococcus aureus (Sa-LTA) and differentiate how these affect signaling in brain organoids. Methods/Study Population: To characterize peripheral blood mononuclear cells (PBMCs) response to microbial products, HIV+ and HIV– PBMCs were exposed to Pg-LPS or Sa-LTA for 24 or 48 hours. iNOS expression was assessed by western blot. IL-6 was quantified by ELISA, while TNF-α and IL-10 were measured via Ella multiplex. To differentiate how bacterial products directly affect neurons, human brain organoids (hBOs) were stimulated with Pg-LPS or Sa-LTA for 24 hours. To differentiate how bacterial products indirectly affect neurons, hBOs were co-cultured with PBMCs previously exposed to microbial products (primed). TNF-α, IL-6, and IL-10 levels were quantified by ELISA and multiplex. Neural function and reactivity were analyzed by measuring PSD95, synaptophysin, vGLUT1, pERK/tERK, and GFAP expression with western blot. Results/Anticipated Results: Pg-LPS increased TNF-α in HIV– PBMCs only at 48 h (p=0.033); in HIV+ PBMCs, TNF-α increased at 24 h (p=0.026) but declined by 48 h (p=0.011). HIV+ PBMCs secreted IL-6 at 24 h, which decreased by 48 h (p=0.025). IL-10 secretion remained stable in HIV– PBMCs but was elevated at 24 h versus 48 h in HIV+ PBMCs (p=0.006). No changes in iNOS expression were detected. Sa-LTA did not induce notable effects. In hBOs, direct stimulation with Pg-LPS or Sa-LTA did not alter TNF-α and IL-10 levels, nor neuronal markers. IL-6 was undetectable in Pg-LPS-treated hBOs and decreased in Sa-LTA-treated hBOs. Interestingly, hBOs co-cultured with primed PBMCs had higher astrocyte reactivity (p=0.034) than hBOs directly exposed to Pg-LPS 24h, while hBOs co-cultured with untreated PBMCs showed higher levels of neuronal markers. Discussion/Significance of Impact: HIV+ PBMCs exhibit a pro-inflammatory response to Pg-LPS at 24h, counterbalanced by an anti-inflammatory modulation. Oral pathogen products may be driving distinct immune activation dynamics in PWH and in the brain, potentially contributing to neuroinflammatory mechanisms that trigger neuronal dysfunction and cognitive impairment.