ORF17-01 - Knockout of TLR4 Preserves Gut Microbiome Diversity and Reduces Ovarian Immune Cell Infiltration in a PCOS-Like Mouse Model

Polycystic Ovary Syndrome (PCOS) is characterized by reproductive and metabolic dysfunction, with emerging evidence implicating gut dysbiosis in disease pathogenesis. Our previous work demonstrated that Toll-like receptor 4 (TLR4) knockout mice were protected against Letrozole (LET)-induced PCOS-like symptoms. Here, we investigate the mechanistic role of TLR4 in mediating gut microbiome alterations, immune cell populations, and vagus nerve-dependent gut-reproductive axis communication in PCOS pathophysiology. Peripubertal female C57BL6J (WT) and TLR4-/- mice (n=40 per group) were treated with LET or placebo for five weeks. Using 16S rRNA sequencing of fecal samples, we confirmed microbial dysbiosis in LET-treated WT mice. Strikingly, LET-treated TLR4-/- mice maintained a 1-1.5 fold increase in Shannon and Chao1 alpha diversity indices and exhibited greater beta diversity by Bray-Curtis dissimilarity analysis compared to LET-treated WT mice. Furthermore, the Firmicutes-to-Bacteroidetes ratio in LET-treated TLR4-/- mice closely resembled placebo-treated animals. Spectral flow cytometric analysis of mesenteric lymph nodes, critical immune surveillance sites integrating gut signals, revealed significant alterations in T cell, myeloid, B cell, and dendritic cell populations in LET-treated WT mice. In contrast, immune populations in LET-treated TLR4-/- mice remained comparable to placebo controls, suggesting preserved gut-immune homeostasis. This alteration extended to the ovaries, where CD45+ immune cell infiltration increased over 2% only in LET-treated WT mice. The parallel dysregulation across gut microbiome, gut-associated lymph nodes, and ovaries suggested coordinated signaling. We hypothesized that the vagus nerve, which connects the gastrointestinal tract and hypothalamic-pituitary-gonadal (HPG) axis, serves as this critical link. To test this, we performed subdiaphragmatic vagotomy (SDV) (n=11 per group). SDV mice treated with LET demonstrated reduced body weight, enhanced glucose tolerance, and decreased luteinizing hormone (LH) levels, with all parameters returning to baseline comparable to placebo and sham controls, establishing vagal signaling as a critical mediator of PCOS-like pathology. Collectively, our data reveal a novel gut-reproductive axis in PCOS pathogenesis. TLR4 deletion preserves microbiome diversity and gut-immune homeostasis, thereby reducing aberrant vagal signaling that would otherwise propagate reproductive and metabolic disturbances. This work identifies the TLR4-microbiome-vagus nerve pathway as a novel therapeutic target.