Interactions among the gut microbiome, dysregulated immune responses, and genetic factors contribute to the pathogenesis of inflammatory bowel disease (IBD). Nlrx1−/− mice have exacerbated disease severity, colonic lesions, and increased inflammatory markers. Global transcriptomic analyses demonstrate enhanced mucosal antimicrobial defense response, chemokine and cytokine expression, and epithelial cell metabolism in colitic Nlrx1−/− mice compared to wild-type (WT) mice. Cell-specificity studies using cre-lox mice demonstrate that the loss of NLRX1 in intestinal epithelial cells (IEC) recapitulate the increased sensitivity to DSS colitis observed in whole body Nlrx1−/− mice. Further, organoid cultures of Nlrx1−/− and WT epithelial cells confirm the altered patterns of proliferation, amino acid metabolism, and tight junction expression. These differences in IEC behavior can impact the composition of the microbiome. Microbiome analyses demonstrate that colitogenic bacterial taxa such as Veillonella and Clostridiales are increased in abundance in Nlrx1−/− mice and in WT mice co-housed with Nlrx1−/− mice. The transfer of an Nlrx1−/−-associated gut microbiome through co-housing worsens disease in WT mice confirming the contributions of the microbiome to the Nlrx1−/− phenotype. To validate NLRX1 effects on IEC metabolism mediate gut–microbiome interactions, restoration of WT glutamine metabolic profiles through either exogenous glutamine supplementation or administration of 6-diazo-5-oxo-l-norleucine abrogates differences in inflammation, microbiome, and overall disease severity in Nlrx1−/− mice. The influence NLRX1 deficiency on SIRT1-mediated effects is identified to be an upstream controller of the Nlrx1−/− phenotype in intestinal epithelial cell function and metabolism. The altered IEC function and metabolisms leads to changes in barrier permeability and microbiome interactions, in turn, promoting greater translocation and inflammation and resulting in an increased disease severity. In conclusion, NLRX1 is an immunoregulatory molecule and a candidate modulator of the interplay between mucosal inflammation, metabolism, and the gut microbiome during IBD.

Nucleotide oligomerization domain (NOD) like receptor X1 (NLRX1) has been implicated in viral response, cancer progression and inflammatory disorders; however, its role as a dual modulator of CD4+ T cell function and metabolism has not yet been defined. The loss of NLRX1 results in increased disease severity, populations of T helper 1 and T helper 17, and inflammatory markers (IFNγ, TNFα, and IL-17) in mice with DSS colitis. To further characterize this phenotype, we employed in vitro CD4+ T cell differentiation assays and show that NLRX1 deficient T cells have a greater ability to differentiate into inflammatory phenotype and greater proliferation rates. Further, NLRX1−/− cells have a decreased responsiveness to immune checkpoint pathways and greater rates of lactate dehydrogenase activity. When metabolic effects of the knockout are impaired, NLRX1 deficient cells do not display significant differences in differentiation or proliferation. To confirm the role of NLRX1 specifically in T cells, we used an adoptive transfer model of colitis. Rag2−/− recipient mice of NLRX1−/− naïve or effector T cells experienced increased disease activity and effector T cell populations, while no differences were observed between groups receiving wild-type or NLRX1−/− regulatory T cells. Metabolic effects of NLRX1 deficiency are observed in a CD4-specific knockout of NLRX1 within a C. rodentium model of colitis. The aerobic glycolytic preference in NLRX1−/− effector T cells is combined with a decreased sensitivity to immunosuppressive checkpoint pathways to provide greater proliferative capabilities and an inflammatory phenotype bias leading to increased disease severity.

Helicobacter pylori, the dominant member of the human gastric microbiota, elicits immunoregulatory responses implicated in protective versus pathological outcomes. To evaluate the role of macrophages during infection, we employed a system with a shifted pro-inflammatory macrophage phenotype by deleting PPARγ in myeloid cells, and found a 5 to 10-fold decrease in gastric bacterial loads. Higher levels of colonization in wild-type (WT) mice were associated with increased presence of mononuclear phagocytes (MNPs) and in particular with the accumulation of CD11b+F4/80hiCD64+CX3CR1+ macrophages in the gastric lamina propria. Depletion of phagocytic cells by clodronate liposomes in WT resulted in a reduction of gastric H. pylori colonization compared to non-treated mice. PPARγ-deficient and macrophage-depleted mice presented decreased IL-10-mediated myeloid and T cell regulatory responses early post-infection. IL-10 neutralization during H. pylori infection led to increased IL-17-mediated responses and increased neutrophil accumulation at the gastric mucosa. In conclusion, we report the induction of IL-10-driven regulatory responses mediated by CD11b+F4/80hiCD64+CX3CR1+ MNPs that contribute to maintaining high levels of H pylori loads in the stomach by modulating effector T cell responses at the gastric mucosa.