Role of TNF signaling in CX3CR1hi macrophages in the regulation of TLS function - Title: Investigating the role of TNF signaling in CX3CR1hi macrophages in the regulation of Tertiary Lymphoid Structure Formation & their Immunological Consequences in Inflammatory Bowel Disease Tumor Necrosis Factor (TNF) is a cytokine crucial for inflammation, immune regulation, & development of secondary lymphoid organs. TNF signaling is detrimental in inflammatory bowel disease (IBD). High levels of soluble TNF receptor 2 (TNFR2) & the polymorphisms in the TNFR2 gene are associated with Crohn’s Disease (CD). Biologics that neutralize TNF considerably improved IBD treatment. However, nearly a third of patients do not respond initially & some patients become refractory, with the reasons for this variability remaining unclear. Tertiary lymphoid structures (TLS) are disorganized lymphoid aggregates found near intestinal inflammatory lesions in IBD patients. Our prior work in Salmonella colitis showed that TLS are initiated by a distinct subset of antigen-presenting macrophages. TLS reduce infection progression, however, the mechanisms that drive TLS formation in IBD & their role in IBD are largely unknown. Increased TNF production can lead to the development of IBD-like colitis & TLS formation. Our preliminary data in Salmonella colitis show that TNF is required for TLS development & mucosal IgA response to Salmonella through TNFR1 signaling in mucosal macrophages. Similarly, Tnfrsf1(TNFR1)–/–Il10–/– mice develop colitis at an earlier age than Il10–/– mice, suggesting a protective role of TNF in IBD that remains unexplored. On the other hand, recent single-cell RNA sequencing analysis of intestinal mucosa in IBD patients identified cellular modules (enriched in IgG plasma cells, inflammatory mononuclear phagocytes, & activated T & stromal cells) named GIMATS that were associated with resistance to anti-TNF therapy. If the GIMATS represent TLS, this suggests that the progression of TLS to the preferential IgG response is independent of TNF. Thus, I hypothesize that TNFR1 signaling in macrophages supports TLS development with a pathobiont-specific protective IgA response, while excessive inflammation promotes the evolution of TLS to pathogenic IgG production, which is TNF-independent. This proposal aims to link TNF signaling in mucosal macrophage subsets to TLS formation & function in IBD, addressing the gaps in understanding TNF’s role in IBD pathogenesis & resistance to TNF therapy. We will use mice deficient in TNF & a transgenic mouse model with TNF receptors selectively removed from mucosal macrophages to assess TNF’s influence on TLS composition & function in Salmonella & Il10–/– colitis. Aim 1 will establish the contribution of macrophage-intrinsic TNFR signaling to colitis progression. Aim 2 will determine the role of macrophage-intrinsic TNFR signaling in TLS formation & antibody responses. Aim 3 will determine how macrophage-intrinsic TNFR signaling regulates the cellular composition of TLS identified in the single-cell RNAseq dataset of TLS from Salmonella & Il10–/– mice we have generated.
