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Acidosis is one of the hallmarks of demyelinating central nervous system (CNS) lesions in multiple sclerosis (MS). The response to acidic pH is primarily mediated by a family of G protein-coupled proton-sensing receptors: OGR1, GPR4 and TDAG8. These receptors are inactive at alkaline pH, reaching maximal activation at acidic pH. Genome-wide association studies have identified a locus within the TDAG8 gene associated with several autoimmune diseases, including MS. Accordingly, we here found that expression of TDAG8, as opposed to GPR4 or OGR1, is upregulated in MS plaques. This led us to investigate the expression of TDAG8 in oligodendrocytes using mouse and human in vitro and in vivo models. We observed significant upregulation of TDAG8 in human MO3.13 oligodendrocytes during maturation and in response to acidic conditions. However, its deficiency did not impact normal myelination in the mouse CNS, and its expression remained unaltered under demyelinating conditions in mouse organotypic cerebellar slices. Notably, our data revealed no expression of TDAG8 in primary mouse oligodendrocyte progenitor cells (OPCs), in contrast to its expression in primary human OPCs. Our investigations have revealed substantial species differences in the expression of proton-sensing receptors in oligodendrocytes, highlighting the limitations of the employed experimental models in fully elucidating the role of TDAG8 in myelination and oligodendrocyte biology. Consequently, the study does not furnish robust evidence for the role of TDAG8 in such processes. Nonetheless, our findings tentatively point towards a potential association between TDAG8 and myelination processes in humans, hinting at a potential link between TDAG8 and the pathophysiology of MS and warrants further research.

The availability of endogenous and dietary carbohydrates in the gastrointestinal tract influences the composition of the gut microbiota. Carbohydrate foraging requires the action of bacterially-encoded glycoside hydrolases, which release mono- and oligosaccharides taken up as carbon sources by multiple microbial taxa. In addition to providing nutrients to the microbiota, the cleavage of host glycans by bacterial glycoside hydrolases may alter the properties of surface glycoproteins involved in cell adhesion and activation processes in the gut lumen. To investigate the impact of bacterial glycoside hydrolase activities on the gut microbial composition and on host glycans during colon inflammation, we increased local glycoside hydrolase activity by supplementing mice with recombinant E. coli expressing specific sialidase, fucosidase and rhamnosidase enzymes during acute colitis induced by dextran sulfate sodium ingestion. Whereas increased fucosidase and rhamnosidase activity did not alter the course of colitis, increased sialidase activity exacerbated disease severity. The effect of increased sialidase activity on inflammation was not caused by changes in the microbial composition given that a similar shift in gut bacteria occurred in all groups of mice supplemented with recombinant E. coli. Increased sialidase activity in the colon of treated mice however significantly altered the distribution of sialic acid on mucosal glycans. Treatment of lamina propria dendritic cells with bacterial sialidase also strongly decreased the density of sialylated ligands to anti-inflammatory siglec lectins, indicating that the remodeling of surface sialylation caused by increased sialidase activity likely accounts for the observed exacerbation of acute colitis in mice.

Abstract Background Genetic variations within the regulatory region of the gene encoding NOD-like receptor pyrin domain containing 3 (NLRP3) have been associated with Crohn’s Disease (CD). NLRP3 is part of the NLRP3-inflammasome that mediates the maturation of IL-1β and IL-18. Carrying the major allele of the single nucleotide polymorphisms (SNPs) rs10733113, rs4353135 and rs55646866 is associated with an increased risk for CD. We here studied the impact of these polymorphisms on clinical characteristics in patients of the Swiss IBD Cohort Study (SIBDCS). Methods We included 981 Crohn’s disease (CD) patients and 690 ulcerative colitis (UC) patients of the SIBDCS. We analyzed whether three CD-associated NLRP3 polymorphisms have an impact on the clinical disease course in these patients. Results In CD patients presence of the major allele (G) of rs10733113 was associated with less surgeries and lower maximal CDAI and a similar trend was observed for rs55646866 and rs4353135. Presence of the major allele of all three SNPs was negatively correlated to maximal CDAI. In UC patients homozygous genotype for the major allele (CC) for rs55646866 was associated with a higher age at diagnosis and a higher MTWAI index. Homozygous genotype for the major allele of all three polymorphisms was associated with a higher number of ambulatory visits and longer hospital stays. Conclusions In CD patients presence of the major allele of all three polymorphisms was associated with markers of a less severe disease course, while in UC the homozygous genotype for all major alleles suggested a more severe disease activity.

Background and Aims: Local extracellular acidification is associated with several conditions, such as ischemia, cancer, metabolic disease, respiratory diseases, and inflammatory bowel disease (IBD). Several recent studies reported a link between IBD and a family of pH-sensing G protein-coupled receptors. Our previous studies point to an essential role for OGR1 (GPR68) in the modulation of intestinal inflammation and fibrosis. In the current study, we evaluated the effects of a novel OGR1 inhibitor in murine models of colitis. Methods: The effects of a novel small-molecule OGR1 inhibitor were assessed in the acute and chronic dextran sulfate sodium (DSS) murine models of colitis. Macroscopic disease indicators of intestinal inflammation were evaluated, and epithelial damage and immune cell infiltration and proliferation were assessed by immunohistochemistry. Results: The OGR1 inhibitor ameliorated clinical parameters in acute and chronic DSS-induced colitis. In mice treated with the OGR1 inhibitor, endoscopy showed no thickening and normal vascularity, while fibrin was not detected. Histopathological findings revealed a decrease in severity of colonic inflammation in the OGR1 inhibitor group when compared to vehicle-DSS controls. In OGR1 inhibitor-treated mice, staining for the macrophage marker F4/80 and cellular proliferation marker Ki-67 revealed a reduction of infiltrating macrophages and slightly enhanced cell proliferation, respectively. This was accompanied by a reduction in pro-inflammatory cytokines, TNF and IL-6, and the fibrosis marker TGF-β1. Conclusion: This is the first report providing evidence that a pharmacological inhibition of OGR1 has a therapeutic effect in murine colitis models. Our data suggest that targeting proton-sensing OGR1 using specific small-molecule inhibitors may be a novel therapeutic approach for the treatment of IBD.

Introduction: Intestinal fibrosis, characterized by excessive deposition of extracellular matrix proteins, is a common and severe clinical complication of inflammatory bowel disease (IBD). However, the mechanisms underlying fibrosis remain elusive, and currently, there are limited effective pharmacologic treatments that target the development of fibrosis. Hypoxia is one of the key microenvironmental factors influencing intestinal inflammation and has been linked to fibrosis. Objective: In the present study, we sought to elucidate the impact of hypoxia on fibrotic gene expression in the intestinal mucosa. Methods: Human volunteers, IBD patients, and dextran sulphate sodium-treated mice were exposed to hypoxia, and colonic biopsies were collected. The human intestinal epithelial cell line Caco-2, human THP-1 macrophages, and primary human gut fibroblasts were subjected to hypoxia, and changes in fibrotic gene expression were assessed. Results: Human volunteers subjected to hypoxia presented reduced transcriptional levels of fibrotic and epithelial-mesenchymal transition markers in the intestinal mucosa. IBD patients showed a trend towards a decrease in tissue inhibitor of metalloproteinase 1 protein expression. In mice, hypoxic conditions reduced the colonic expression of several collagens and matrix metalloproteinases. Hypoxic Caco-2 cells, THP-1 cells, and primary gut fibroblasts showed a significant downregulation in the expression of fibrotic and tissue remodelling factors. Conclusions: Stabilization of hypoxia-inducible factors might represent a novel therapeutic approach for the treatment of IBD-associated fibrosis.

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.

Milk oligosaccharides exert a prebiotic action that contributes to the development of the infant gut microbiota during lactation. Given that milk oligosaccharides remain intact after passage through stomach and small intestine, they can potentially influence the composition of the gut microbiota when ingested as dietary supplements after weaning. To address the regulatory effects of specific oligosaccharides in colitis linked to the microbiota composition, we have supplemented interleukin-10 null (Il10-/-) mice with four fucosylated and sialylated oligosaccharides. We found that oral supplementation with 2-fucosyllactose significantly decreased the severity of colitis as displayed by reduced inflammatory marker expression, histological and diarrhea scores, an increased epithelial integrity and less pronounced colon shortening. Oral supplementation with 2-fucosyllactose led to a marked expansion of the commensal Ruminococcus gnavus, which was accompanied by an enhanced cecal concentration of propionate. Decreased activation of immune cells by R. gnavus was confirmed by reconstitution of antibiotic-treated Il10-/- mice and by stimulation of dendritic cells in vitro. This study demonstrates that post-weaning administration of specific oligosaccharides can shift the composition of the gut microbiota to lessen chronic inflammation as observed in Il10-/- mice. The expansion of R. gnavus sets a positive microbial environment at the cost of pro-inflammatory Gram-negative bacteria, thereby lowering intestinal inflammation.

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.

The concepts on the pathophysiology of intestinal fibrosis in Crohn’s disease (CD) have changed in recent years. Some years ago fibrosis was regarded to be a consequence of long-standing inflammation with subsequent destruction of the gut wall matrix followed by scar formation and collagen deposition. Fibrosis in CD patients appeared to be an irreversible process that could hardly be influenced. Therefore, the main target in CD therapy was to control inflammation to avoid fibrosis development. Many of these assumptions seem to be only partially true. Inflammation may be a necessary prerequisite for the initiation of fibrosis. However, when the pathophysiologic processes that lead to fibrosis in CD patients have been initiated fibrosis development may be independent of inflammation and may continue even when inflammation is under good medical control. Fibrosis in CD also may be reversible. After strictureplasty local collagen deposits decrease or even disappear. With new animal models for intestinal fibrosis on the horizon, we need to spend more efforts on understanding the factors influencing fibrosis in CD patients to finally find specific therapies. In this context, it will be as important to find markers and quantitative imaging tools to have reliable endpoints for clinical trials in fibrosing CD.

Background & Aims: Plasma levels of high-density lipoprotein (HDL) cholesterol are frequently found decreased in patients with inflammatory bowel disease (IBD). Therefore, and because HDL exerts anti-inflammatory activities, we investigated whether HDL and its major protein component apolipoprotein A-I (apoA-I) modulate mucosal inflammatory responses in vitro and in vivo. Methods: The human intestinal epithelial cell line T84 was used as the in vitro model for measuring the effects of HDL on the expression and secretion of tumor necrosis factor (TNF), interleukin-8 (IL-8), and intracellular adhesion molecule (ICAM). Nuclear factor-κB (NF-κB)-responsive promoter activity was studied by dual luciferase reporter assays. Mucosal damage from colitis induced by dextran sodium sulphate (DSS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was scored by colonoscopy and histology in apoA-I transgenic (Tg) and apoA-I knockout (KO) and wild-type (WT) mice. Myeloperoxidase (MPO) activity and TNF and ICAM expression were determined in intestinal tissue samples. Autophagy was studied by Western blot analysis, immunofluorescence, and electron microscopy. Results: HDL and apoA-I down-regulated TNF-induced mRNA expression of TNF, IL-8, and ICAM, as well as TNF-induced NF-κB-responsive promoter activity. DSS/TNBS-treated apoA-I KO mice displayed increased mucosal damage upon both colonoscopy and histology, increased intestinal MPO activity and mRNA expression of TNF and ICAM as compared with WT and apoA-I Tg mice. In contrast, apoA-I Tg mice showed less severe symptoms monitored by colonoscopy and MPO activity in both the DSS and TNBS colitis models. In addition, HDL induced autophagy, leading to recruitment of phosphorylated IκB kinase to the autophagosome compartment, thereby preventing NF-κB activation and induction of cytokine expression. Conclusions: Taken together, the in vitro and in vivo findings suggest that HDL and apoA-I suppress intestinal inflammation via autophagy and are potential therapeutic targets for the treatment of IBD. Keywords: Apolipoprotein A-I, Autophagy, Inflammatory Bowel Disease, NF-κB

ObjectivesHLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders.MethodsThe monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry.ResultsHD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules.ConclusionHD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD).

The life cycle of an intestinal epithelial cell is terminated by apoptosis and/or cell shedding. Apoptotic deletion of epithelial cells from the intact intestinal mucosa is not accompanied by detectable inflammatory response or loss of barrier function. But increased permeability of the epithelial barrier and increased apoptotic rates of epithelial cells have been reported for patients suffering from inflammatory bowel disease. Microbiota can both induce or inhibit apoptosis of intestinal epithelial cells thus contribute to mucosal inflammation or support epithelial integrity respectively. Bacteria-mediated cytokine secretion and altered cell signalling are central to epithelial injury. Tumor necrosis factor (TNF) secreted after exposure to invasive bacteria induces both apoptosis and cell shedding. TNF is the major target gene of the transcription factor nuclear factor-kappa B with both pro- and anti-apoptotic effects. Autophagy promotes both cell survival and “autophagic” cell death. If autophagy is directed against microbes it is termed xenophagy. Inhibition of xenophagy has been shown to decrease cell survival. Endoplasmic reticulum (ER) stress causes misfolded proteins to accumulate in the ER lumen. It was suggested that ER stress and autophagy may interact within intestinal epithelial cells. Apoptosis in response to infection may be well proposed by the host to delete infected epithelial cells or could be a strategy of microbial pathogens to escape from exhausted cells to invade deeper mucosal layers for a prolonged bacterial colonization.