Toll-like receptor 4-induced interleukin-1 defines the intestinal microbiome and mucosal immune response in arthritis-prone IL-1 receptor antagonist deficient mice

Background/Purpose: Interleukin-1 (IL-1) plays a pivotal role in inflammation and autoimmunity. Mice deficient in the IL-1 receptor antagonist (IL-1Ra-/-) spontaneously develop a T cell-driven autoimmune arthritis, which we previously showed to depend on the presence of commensal microbiota. Recent findings suggest alteration of intestinal microbiome in new-onset rheumatoid arthritis (RA). The aim of this study was to investigate the role of IL-1 receptor signaling and the involvement of Toll-like receptor (TLR) 2 and TLR4 in defining the intestinal microbiota and the associated mucosal and systemic immune response. Methods: Multiplex 454 pyrosequencing of V5 and V6 hyper-variable regions of fecal bacterial 16S rRNA was used to define intestinal microbial communities in BALB/c wild type (WT), IL-1Ra-/- and IL-1Ra/TLR double knock-out (DKO) mice. For gene sequencing analysis, a customized workflow based on Quantitative Insights Into Microbial Ecology (QIIME version 1.2) was adopted. Intestinal T cell differentiation was studied in lamina propria lymphocytes using flow cytometry and gene expression was assessed by qPCR. Results: Excessive IL-1R signaling strongly affected the composition of intestinal microbiota and resulted in a significant reduction in species diversity compared to WT mice. Both alpha diversity (number of unique taxonomic entities) and phylogenetic diversity (PD) whole tree (based on taxonomic distance) were significantly diminished in IL-1Ra-/-mice compared to WT. Interestingly, the loss of species diversity was absent in IL-1Ra/TLR4 DKO, but not IL-1Ra/TLR2 DKO mice, suggesting that IL-1R-driven skewing of bacterial diversity depends on TLR4. IL-1Ra-/- mice exhibited significantly increased abundance of the genus Helicobacter and reduced Prevotella (p=0.008 and p=0.004, respectively). Importantly, significant alterations in the genera Xylanibacter, Prevotella, Streptococcus, and Ruminococcus were markedly normalized in TLR4, but not TLR2, deficient mice, identifying a role for TLR4 in IL-1 mediated shifts in microbial community. In line with the relevance of intestinal microbiota in mucosal helper T cell polarization, IL-1Ra-/- mice had greatly increased Th1 and Th17 in small intestine lamina propria, while Treg proportions were unaffected. Also, small intestine lamina propria lymphocytes produced increased levels of IL-17 when stimulated with PMA and ionomycin ex vivo. Although expression of IL-1 itself remained unaltered, intestinal IL-23p19 mRNA expression was increased in IL-1Ra-/- mice. Interestingly, mucosal expression of both IL-1β and IL-23 was significantly diminished in IL-1Ra/TLR4 DKO mice. Moreover, splenic expression of IL-6 and RORγt was increased in IL-1Ra-/-, and suppressed in IL-1Ra/TLR4 DKO mice. Conclusion: These data indicate a clear role for the IL-1 pathway in defining the intestinal microbiota and mucosal immune response in autoimmune prone mice, potentially driven by TLR4.Understanding the molecular and cellular mechanisms linking the intestinal T cell response with extra-intestinal disease may help identify novel therapeutic targets in autoimmune diseases including RA.