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Summary: The intricate interplay between gut microbes and the onset of experimental autoimmune encephalomyelitis (EAE) remains poorly understood. Here, we uncover remarkable similarities between CD4+ T cells in the spinal cord and their counterparts in the small intestine. Furthermore, we unveil a synergistic relationship between the microbiota, particularly enriched with the tryptophan metabolism gene EC:1.13.11.11, and intestinal cells. This symbiotic collaboration results in the biosynthesis of kynurenic acid (KYNA), which modulates the recruitment and aggregation of GPR35-positive macrophages. Subsequently, a robust T helper 17 (Th17) immune response is activated, ultimately triggering the onset of EAE. Conversely, modulating the KYNA-mediated GPR35 signaling in Cx3cr1+ macrophages leads to a remarkable amelioration of EAE. These findings shed light on the crucial role of microbial-derived tryptophan metabolites in regulating immune responses within extraintestinal tissues.

Diet and gut microbial metabolites mediate host immune responses and are central to the maintenance of intestinal health. The metabolite-sensing G-protein coupled receptors (GPCRs) bind metabolites and trigger signals that are important for the host cell function, survival, proliferation and expansion. On the contrary, inadequate signaling of these metabolite-sensing GPCRs most likely participate to the development of diseases including inflammatory bowel diseases (IBD). In the intestine, metabolite-sensing GPCRs are highly expressed by epithelial cells and by specific subsets of immune cells. Such receptors provide an important link between immune system, gut microbiota and metabolic system. Member of these receptors, GPR35, a class A rhodopsin-like GPCR, has been shown to be activated by the metabolites tryptophan-derived kynurenic acid (KYNA), the chemokine CXCL17 and phospholipid derivate lysophosphatidic acid (LPA) species. There have been studies on GPR35 in the context of intestinal diseases since its identification as a risk gene for IBD. In this review, we discuss the pharmacology of GPR35 including its proposed endogenous and synthetic ligands as well as its antagonists. We elaborate on the risk variants of GPR35 implicated in gut-related diseases and the mechanisms by which GPR35 contribute to intestinal homeostasis.

Summary: Single-nucleotide polymorphisms in the gene encoding G protein-coupled receptor 35 (GPR35) are associated with increased risk of inflammatory bowel disease. However, the mechanisms by which GPR35 modulates intestinal immune homeostasis remain undefined. Here, integrating zebrafish and mouse experimental models, we demonstrate that intestinal Gpr35 expression is microbiota dependent and enhanced upon inflammation. Moreover, murine GPR35+ colonic macrophages are characterized by enhanced production of pro-inflammatory cytokines. We identify lysophosphatidic acid (LPA) as a potential endogenous ligand produced during intestinal inflammation, acting through GPR35 to induce tumor necrosis factor (Tnf) expression in macrophages. Mice lacking Gpr35 in CX3CR1+ macrophages aggravate colitis when exposed to dextran sodium sulfate, which is associated with decreased transcript levels of the corticosterone-generating gene Cyp11b1 and macrophage-derived Tnf. Administration of TNF in these mice restores Cyp11b1 expression and intestinal corticosterone production and ameliorates DSS-induced colitis. Our findings indicate that LPA signals through GPR35 in CX3CR1+ macrophages to maintain TNF-mediated intestinal homeostasis.

Background & Aims: Hypoxia-associated pathways influence the development of inflammatory bowel disease. Adaptive responses to hypoxia are mediated through hypoxia-inducible factors, which are regulated by iron-dependent hydroxylases. Signals reflecting oxygen tension and iron levels in enterocytes regulate iron metabolism. Conversely, iron availability modulates responses to hypoxia. In the present study we sought to elucidate how iron influences the responses to hypoxia in the intestinal epithelium. Methods: Human subjects were exposed to hypoxia, and colonic biopsy specimens and serum samples were collected. HT-29, Caco-2, and T84 cells were subjected to normoxia or hypoxia in the presence of iron or the iron chelator deferoxamine. Changes in inflammatory gene expression and signaling were assessed by quantitative polymerase chain reaction and Western blot. Chromatin immunoprecipitation was performed using antibodies against nuclear factor (NF)-κB and primers for the promoter of tumor necrosis factor (TNF) and interleukin (IL)1β. Results: Human subjects presented reduced levels of ferritin in the intestinal epithelium after hypoxia. Hypoxia reduced iron deprivation–associated TNF and IL1β expression in HT-29 cells through the induction of autophagy. Contrarily, hypoxia triggered TNF and IL1β expression, and NF-κB activation in Caco-2 and T84 cells. Iron blocked autophagy in Caco-2 cells, while reducing hypoxia-associated TNF and IL1β expression through the inhibition of NF-κB binding to the promoter of TNF and IL1β. Conclusions: Hypoxia promotes iron mobilization from the intestinal epithelium. Hypoxia-associated autophagy reduces inflammatory processes in HT-29 cells. In Caco-2 cells, iron uptake is essential to counteract hypoxia-induced inflammation. Iron mobilization into enterocytes may be a vital protective mechanism in the hypoxic inflamed mucosa. Keywords: Inflammatory Bowel Disease, Autophagy, Deferoxamine, Caco-2

Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn’s patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy.

Background & Aims: A novel family of proton-sensing G-proteinâcoupled receptors, including ovarian cancer G-proteinâcoupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1α. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells. Methods: OGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and intestinal mucosa of inflammatory bowel disease (IBD) patients. Changes in OGR1 expression in MonoMac6 (MM6) cells under hypoxia were determined upon stimulation with tumor necrosis factor (TNF), in the presence or absence of nuclear factor-κB (NF-κB) inhibitors. To study the molecular mechanisms involved, chromatin immunoprecipitation analysis of the OGR1 promoter was performed. Results: OGR1 expression was significantly higher in tumor tissue compared with normal murine colon tissue. Hypoxia positively regulated the expression of OGR1 in MM6 cells, mouse peritoneal macrophages, primary human intestinal macrophages, and colonic tissue from IBD patients. In MM6 cells, hypoxia-enhanced TNF-induced OGR1 expression was reversed by inhibition of NF-κB. In addition to the effect of TNF and hypoxia, OGR1 expression was increased further at low pH. Chromatin immunoprecipitation analysis showed that HIF-1α, but not NF-κB, binds to the promoter of OGR1 under hypoxia. Conclusions: The enhancement of TNF- and hypoxia-induced OGR1 expression under low pH points to a positive feed-forward regulation of OGR1 activity in acidic conditions, and supports a role for OGR1 in the pathogenesis of IBD. Keywords: Ovarian Cancer G-ProteinâCoupled Receptor, Inflammation, Inflammatory Bowel Disease, TDAG8, GRP65

Gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. Its lack in intestinal macrophages of Crohn's disease (CD) patients is correlated with loss of tolerance against the host gut flora. Gp96 has been stablished to be an essential chaperone for Toll-like receptors (TLRs). We studied the impact of gp96-knockdown on TLR-function in macrophages. TLR2 and TLR4 expression was only decreased but not abolished when gp96 was knocked-down in cell lines, whereas in a monocyte/macrophage specific knock-out mouse model (LysMCre) TLR4 was abolished, while TLR2 was still present. Lipopolysaccharide (LPS)-induced NF-κB activation was still observed in the absence of gp96, and gp96-deficient macrophages were able to up-regulate surface TLR4 upon LPS treatment, suggesting that there is another chaperone involved in the folding of TLR4 upon stress responses. Moreover, LPS-dependent pro-inflammatory cytokines were still expressed, although to a lesser extent in the absence of gp96, which reinforces the fact that gp96 is involved in regulating signaling cascades downstream of TLR4 are impaired upon loss of gp96. In addition, we have also found a reduced phosphorylation of ERK and p38 kinases and an impaired response upon CSF1R activation in gp96 deficient macrophages. Our findings indicate that the loss of gp96 not only impairs TLR4 signaling, but is also associated with a diminished phosphorylation of ERK and mitogen-activated stress kinases resulting in an impaired signalling through several receptors, including CSF1R.

Increasing evidence has indicated that diet and metabolites, including bacteria- and host-derived metabolites, orchestrate host pathophysiology by regulating metabolism, immune system and inflammation. Indeed, autoimmune diseases such as inflammatory bowel disease (IBD) are associated with the modulation of host response to diets. One crucial mechanism by which the microbiota affects the host is signaling through G protein-coupled receptors (GPCRs) termed metabolite-sensing GPCRs. In the gut, both immune and nonimmune cells express GPCRs and their activation generally provide anti-inflammatory signals through regulation of both the immune system functions and the epithelial integrity. Members of GPCR family serve as a link between microbiota, immune system and intestinal epithelium by which all these components crucially participate to maintain the gut homeostasis. Conversely, impaired GPCR signaling is associated with IBD and other diseases, including hepatic steatosis, diabetes, cardiovascular disease, and asthma. In this review, we first outline the signaling, function, expression and the physiological role of several groups of metabolite-sensing GPCRs. We then discuss recent findings on their role in the regulation of the inflammation, their existing endogenous and synthetic ligands and innovative approaches to therapeutically target inflammatory bowel disease.