Murine Dextran Sulfate Sodium Colitis Research Articles

Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

Bacteroides thetaiotaomicron relieves colon inflammation by activating aryl hydrocarbon receptor and modulating CD4+T cell homeostasis

Inflammatory bowel disease (IBD) is a form of nonspecific chronic intestinal inflammation associated with gut microbiome dysbiosis. Modulating the composition of the intestinal flora may be a viable means of alleviating such inflammatory pathology. Bacteroides thetaiotaomicron (B. thetaiotaomicron) is a symbiotic intestinal microbe that has been associated with IBD, although the mechanistic basis for this association remains to be clarified. In this present study, we determined that B. thetaiotaomicron can alleviate colonic inflammation through mechanisms associated with the modulation of tryptophan metabolism and T cell subsets within inflamed intestinal tissues. Specifically, we found that B. thetaiotaomicron promotes the preferential differentiation of anti-inflammatory Treg/Th2 cells while suppressing the relative differentiation of pro-inflammatory Th1/Th17 cells, thereby decreasing inflammation within the colon. At a molecular level, B. thetaiotaomicron treatment was linked to altered CpG methylation within the Foxp3 promoter that was associated with enhanced Treg cell functionality. In a murine dextran sulfate sodium (DSS) colitis model system, B. thetaiotaomicron increased the levels of the aryl hydrocarbon receptor (AHR) ligands indole metabolites-indole acetic acid (IAA) and indole propionic acid (IPA), thereby increasing AHR activation that is related to changes of transcription factor expression profiles within T cells. In summary, our data suggest that B. thetaiotaomicron can activate AHR and modulate CD4+ T cell differentiation profiles in a murine DSS colitis model system, suggesting that this bacterium may be of therapeutic relevance for the treatment of IBD.

Weight loss is a sufficient and economical single outcome measure of murine dextran sulfate sodium colitis.

Inflammatory bowel diseases (IBD: Crohn's disease and ulcerative colitis) are becoming common around the world without a cure. Animal models of colitis have become instrumental in IBD research. The dextran sulfate sodium (DSS) induced murine colitis model is likely the most utilized due to its simplicity and reproducibility with over 4000 publications on PubMed, where weight loss is the most commonly used and reliable positive correlate. We predicted at current state of art, that the DSS colitis model can be optimized by using weight loss as a single cost‐saving outcome measure. Twenty recent and consecutive publications using the DSS model in PubMed were selected for review. Guarded cost estimations for additional outcome measures of colitis beyond weight loss were performed. In all manuscripts (100%), weight loss corroborated the conclusions. Average excess cost for examining additional measures of colitis was approximately $6700 per publication. Two studies (10.5%) were estimated to have spent over $20,000 in excess. Additional measures of colitis either supported the final conclusions found with weight loss, or lead to indeterminate results. Potential annual savings from following our guidance were calculated to be over $60,000 for and IBD lab. We conclude that weight loss is a sufficient, objective, and economical outcome measure of DSS‐induced colitis in mice.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.

Tu1702 Vitamin K Deficiency Deteriorates Murine DSS Colitis Through IL-6 Production From B Cells

Objective: Bone mineral density (BMD) is decreased in patients with inflammatory bowel disease (IBD). Reduced BMD in these patients may be multifactorial. Our recent research demonstrated that serum undercarboxylated osteocalcin (ucOC) levels, which are widely used as a surrogate marker of vitamin K deficiency, were significantly higher in patients with Crohn's disease (CD) than healthy subjects. Vitamin K has been shown to ameliorate liver injury through inhibition of inflammatory cytokine production. We aimed to evaluate the effect of vitamin K on inflammatory responses in the intestine in mice. Methods: CD4, CD11band CD19-positive subpopulations were isolated from splenocytes using the Magnetic Cell Sorting (MACS) separator and these cells were incubated in vitro in the presence or absence of vitamin K2 for 24 h. The mRNA and the culture supernatant of the purified cells were collected. The levels of IL-6 and TNF-α were measured by quantitative RT-PCR and ELISA. Male 8-week-old C57/BL6J mice were fed with the vitamin K deficient diets (K-def) or supplemented diets (K-sup) for 10 days, and then the mice were administered orally with dextran sulfate sodium (DSS) in drinking water for 7 days. Histologic scores of the colon were analyzed 3 days after the cessation of DSS administration. The level of IL6 production from the colonic lamina propria (LP) mononuclear cells was measured by ELISA in the K-def and the K-sup groups. Proportion of the cells costained with CD4 and CD25 was analyzied by flow cytometry. Results: Vitamin K2 reduced the production of IL6 by the splenocytes cultured in vitro, while the expression of TNF-α showed no significant difference. Expression of IL-6 in CD19-positive B cells, but not in CD4and CD11b-positive cells, was significantly decreased in the presence of vitamin K2. The histologic score of DSS colitis was significantly lower in K-sup group than in K-def group. In addition, the expression of IL-6 from LP cells was significantly lower in K-sup group than in the K-def group. The proportion of CD4 and CD25-positive regulatory T cells did not significantly differ between the K-sup and K-def groups. Conclusion: Vitamin K has a suppressive effect on IL-6 production especially in B cells. Vitamin K deficiency deteriorates murine DSS colitis through IL6 production from B cells. Vitamin K may have a potential for the treatment target of IBD.

Tu1703 Analysis of the Protease MT1-MMP and Its Substrates As Biomarkers and Therapeutic Targets in Inflammatory Bowel Disease

Objective: Bone mineral density (BMD) is decreased in patients with inflammatory bowel disease (IBD). Reduced BMD in these patients may be multifactorial. Our recent research demonstrated that serum undercarboxylated osteocalcin (ucOC) levels, which are widely used as a surrogate marker of vitamin K deficiency, were significantly higher in patients with Crohn's disease (CD) than healthy subjects. Vitamin K has been shown to ameliorate liver injury through inhibition of inflammatory cytokine production. We aimed to evaluate the effect of vitamin K on inflammatory responses in the intestine in mice. Methods: CD4, CD11band CD19-positive subpopulations were isolated from splenocytes using the Magnetic Cell Sorting (MACS) separator and these cells were incubated in vitro in the presence or absence of vitamin K2 for 24 h. The mRNA and the culture supernatant of the purified cells were collected. The levels of IL-6 and TNF-α were measured by quantitative RT-PCR and ELISA. Male 8-week-old C57/BL6J mice were fed with the vitamin K deficient diets (K-def) or supplemented diets (K-sup) for 10 days, and then the mice were administered orally with dextran sulfate sodium (DSS) in drinking water for 7 days. Histologic scores of the colon were analyzed 3 days after the cessation of DSS administration. The level of IL6 production from the colonic lamina propria (LP) mononuclear cells was measured by ELISA in the K-def and the K-sup groups. Proportion of the cells costained with CD4 and CD25 was analyzied by flow cytometry. Results: Vitamin K2 reduced the production of IL6 by the splenocytes cultured in vitro, while the expression of TNF-α showed no significant difference. Expression of IL-6 in CD19-positive B cells, but not in CD4and CD11b-positive cells, was significantly decreased in the presence of vitamin K2. The histologic score of DSS colitis was significantly lower in K-sup group than in K-def group. In addition, the expression of IL-6 from LP cells was significantly lower in K-sup group than in the K-def group. The proportion of CD4 and CD25-positive regulatory T cells did not significantly differ between the K-sup and K-def groups. Conclusion: Vitamin K has a suppressive effect on IL-6 production especially in B cells. Vitamin K deficiency deteriorates murine DSS colitis through IL6 production from B cells. Vitamin K may have a potential for the treatment target of IBD.

Longitudinal analysis of inflammation and microbiota dynamics in a model of mild chronic dextran sulfate sodium-induced colitis in mice

To characterize longitudinally the inflammation and the gut microbiota dynamics in a mouse model of dextran sulfate sodium (DSS)-induced colitis. In animal models, the most common method used to trigger colitis is based on the oral administration of the sulfated polysaccharides DSS. The murine DSS colitis model has been widely adopted to induce severe acute, chronic or semi-chronic colitis, and has been validated as an important model for the translation of mice data to human inflammatory bowel disease (IBD). However, it is now clear that models characterized by mild intestinal damage are more accurate for studying the effects of therapeutic agents. For this reason, we have developed a murine model of mild colitis to study longitudinally the inflammation and microbiota dynamics during the intestinal repair processes, and to obtain data suitable to support the recovery of gut microbiota-host homeostasis. All plasma cytokines evaluated, except IL-17, began to increase (P < 0.05), after 7 d of DSS administration. IL-17 only began to increase 4 d after DSS withdrawal. IL-1β and IL-17 continue to increase during the recovery phase, even when clinical signs of colitis had disappeared. IL-6, IL-10 and IFN-γ reached their maxima 4 d after DSS withdrawal and decreased during the late recovery phase. TNFα reached a peak (a three- fold increase, P < 0.05), after which it slightly decreased, only to increase again close to the end of the recovery phase. DSS administration induced profound and rapid changes in the mice gut microbiota. After 3 d of DSS administration, we observed a major reduction in Bacteroidetes/Prevotella and a corresponding increase in Bacillaceae, with respect to control mice. In particular, Bacteroidetes/Prevotella decreased from a relative abundance of 59.42%-33.05%, while Bacillaceae showed a concomitant increase from 2.77% to 10.52%. Gut microbiota rapidly shifted toward a healthy profile during the recovery phase and returned normal 4 d after DSS withdrawal. Cyclooxygenase 2 expression started to increase 4 d after DSS withdrawal (P < 0.05), when dysbiosis had recovered, and continued to increase during the recovery phase. Taken together, these data indicated that a chronic phase of intestinal inflammation, characterized by the absence of dysbiosis, could be obtained in mice using a single DSS cycle. Dysbiosis contributes to the local and systemic inflammation that occurs in the DSS model of colitis; however, chronic bowel inflammation is maintained even after recovery from dysbiosis.

Multifunctional role of dextran sulfate sodium for in vivo modeling of intestinal diseases

BackgroundInflammatory bowel diseases (IBDs) are chronic, relapsing disorders that affect the gastrointestinal tract of millions of people and continue to increase in incidence each year. While several factors have been associated with development of IBDs, the exact etiology is unknown. Research using animal models of IBDs is beginning to provide insights into how the different factors contribute to disease development. Oral administration of dextran sulfate sodium (DSS) to mice induces a reproducible experimental colitis that models several intestinal lesions associated with IBDs. The murine DSS colitis model can also be adapted to quantify intestinal repair following injury. Understanding the mechanistic basis behind intestinal repair is critical to development of new therapeutics for IBDs because of their chronic relapsing nature.ResultsThe murine DSS colitis model was adapted to provide a system enabling the quantification of severe intestinal injury with impaired wound healing or mild intestinal injury with rapid restoration of mucosal integrity, by altering DSS concentrations and including a recovery phase. We showed that through a novel format for presentation of the clinical disease data, the temporal progression of intestinal lesions can be quantified on an individual mouse basis. Additionally, parameters for quantification of DSS-induced alterations in epithelial cell populations are included to provide insights into mechanisms underlying the development of these lesions. For example, the use of the two different model systems showed that toll-like receptor 9, a nucleic acid-sensing pattern recognition receptor, is important for protection only following mild intestinal damage and suggests that this model is superior for identifying proteins necessary for intestinal repair.ConclusionsWe showed that using a murine DSS-induced experimental colitis model system, and presenting data in a longitudinal manner on a per mouse basis, enhanced the usefulness of this model, and provided novel insights into the role of an innate immune receptor in intestinal repair. By elucidating the mechanistic basis of intestinal injury and repair, we can begin to understand the etiology of IBDs, enabling development of novel therapeutics or prophylactics.