Amelioration Of Colitis Research Articles

Dietary fiber guar gum-induced shift in gut microbiota metabolism and intestinal immune activity enhances susceptibility to colonic inflammation

ABSTRACT With an increasing interest in dietary fibers (DFs) to promote intestinal health and the growth of beneficial gut bacteria, there is a continued rise in the incorporation of refined DFs in processed foods. It is still unclear how refined fibers, such as guar gum, affect the gut microbiota activity and pathogenesis of inflammatory bowel disease (IBD). Our study elucidated the effect and underlying mechanisms of guar gum, a fermentable DF (FDF) commonly present in a wide range of processed foods, on colitis development. We report that guar gum containing diet (GuD) increased the susceptibility to colonic inflammation. Specifically, GuD-fed group exhibited severe colitis upon dextran sulfate sodium (DSS) administration, as evidenced by reduced body weight, diarrhea, rectal bleeding, and shortening of colon length compared to cellulose-fed control mice. Elevated levels of pro-inflammatory markers in both serum [serum amyloid A (SAA), lipocalin 2 (Lcn2)] and colon (Lcn2) and extensive disruption of colonic architecture further affirmed that GuD-fed group exhibited more severe colitis than control group upon DSS intervention. Amelioration of colitis in GuD-fed group pre-treated with antibiotics suggest a vital role of intestinal microbiota in GuD-mediated exacerbation of intestinal inflammation. Gut microbiota composition and metabolite analysis in fecal and cecal contents, respectively, revealed that guar gum primarily enriches Actinobacteriota, specifically Bifidobacterium. Guar gum also altered multiple genera belonging to phyla Bacteroidota and Firmicutes. Such shift in gut microbiota composition favored luminal accumulation of intermediary metabolites succinate and lactate in the GuD-fed mice. Colonic IL-18 and tight junction markers were also decreased in the GuD-fed group. Importantly, GuD-fed mice pre-treated with recombinant IL-18 displayed attenuated colitis. Collectively, unfavorable changes in gut microbiota activity leading to luminal accumulation of lactate and succinate, reduced colonic IL-18, and compromised gut barrier function following guar gum feeding contributed to increased colitis susceptibility.

BACTERIAL SPHINGOLIPIDS EXACERBATE COLITIS BY INHIBITING ILC3-DERIVED IL-22 PRODUCTION

Abstract Commensal bacteria of the Bacteroidetes phylum are the primary producers of sphingolipids in the gut lumen. These lipids serve dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells (iNKT). Sphingolipid composition is significantly altered in fecal samples of patients with inflammatory bowel disease (IBD). However, the specific mechanisms by which bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remain unclear. In this study, we investigated the impact of bacterial sphingolipids on intestinal inflammation by mono-colonizing mice with Bacteroides fragilis strains that either express or lack sphingolipids during DSS-induced colitis. We discovered that B. fragilis sphingolipids exacerbate intestinal inflammation. Mice mono-colonized with B. fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin-22 (IL-22) by innate lymphoid cell type 3 (ILC3). Consistent with the inhibitory effect of sphingolipids on IL-22 production, mice colonized with B. fragilis lacking sphingolipids showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production following DSS treatment compared to those colonized with B. fragilis producing sphingolipids. Additionally, colitis severity in mice colonized with B. fragilis lacking sphingolipids was exacerbated upon IL-22 blockade. Furthermore, our study reveals that bacterial sphingolipids restrict epithelial IL-18 production following DSS treatment and interfere with IL-22 production by a subset of ILC3 cells expressing both the interleukin-18 receptor (IL-18R) and major histocompatibility complex class II (MHC II). These findings indicate that B. fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL-18 expression, resulting in compromised production of IL-22 by ILC3 cells. Work Model

Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production

Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear. We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function invivo was investigated by monoclonal antibody injection. B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II. B fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.

Amelioration of Colitis by a Gut Bacterial Consortium Producing Anti-Inflammatory Secondary Bile Acids

The Integrative Human Microbiome Project and other cohort studies have indicated that inflammatory bowel disease is accompanied by dysbiosis of gut microbiota, decreased production of secondary bile acids, and increased levels of primary bile acids. Secondary bile acids, such as ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), have been reported to be anti-inflammatory, yet it remains to be studied whether introducing selected bacteria strains to restore bile acid metabolism of the gut microbiome can alleviate intestinal inflammation. In this study, we screened human gut bacterial strains for bile acid metabolism and designed a consortium of three species, including Clostridium AP sp000509125, Bacteroides ovatus, and Eubacterium limosum, and named it BAC (bile acid consortium). We showed that the three-strain gut bacterial consortium BAC is capable of converting conjugated primary bile acids taurochenodeoxycholic acid and glycochenodeoxycholic acid to secondary bile acids UDCA and LCA in vitro. Oral gavage treatment with BAC in mice resulted in protective effects against dextran sulfate sodium (DSS)-induced colitis, including reduced weight loss and increased colon length. Furthermore, BAC treatment increased the fecal level of bile acids, including UDCA and LCA. BAC treatment enhanced intestinal barrier function, which may be attributed to the increased activation of the bile acid receptor TGR5 by secondary bile acids. Finally, we examined the remodeling of gut microbiota by BAC treatment. Taken together, the three-strain gut bacterial consortium BAC restored the dysregulated bile acid metabolism and alleviated DSS-induced colitis. Our study provides a proof-of-concept demonstration that a rationally designed bacterial consortium can reshape the metabolism of the gut microbiome to treat diseases. IMPORTANCE Secondary bile acids have been reported to be anti-inflammatory, yet it remains to be studied whether introducing selected bacteria strains to restore bile acid metabolism of the gut microbiome can alleviate intestinal inflammation. To address this gap, we designed a consortium of human gut bacterial strains based on their metabolic capacity to produce secondary bile acids UDCA and LCA, and we evaluated the efficacy of single bacterial strains and the bacterial consortium in treating the murine colitis model. We found that oral gavage of the bacterial consortium to mice restored secondary bile acid metabolism to increase levels of UDCA and LCA, which induced the activation of TGR5 to improve gut-barrier integrity and reduced the inflammation in murine colitis. Overall, our study demonstrates that rationally designed bacterial consortia can reshape the metabolism of the gut microbiome and provides novel insights into the application of live biotherapeutics for treating IBD.

Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

Background and AimsCurrent management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium. Wnt signaling may also be modulated with the recently developed Wnt mimetics, recombinant antibody-based molecules mimicking endogenous Wnts.MethodsWe first compared the epithelial healing effects of RSPO2 and a Wnt mimetic with broad Fzd specificity in an acute dextran sulfate sodium mouse colitis model. Guided by Fzd expression patterns in the colon epithelium, we also examined the effects of Wnt mimetics with subfamily Fzd specificities.ResultsIn the DSS model, Wnt mimetics repaired damaged colon epithelium and reduced disease activity and inflammation and had no apparent effect on uninjured tissue. We further identified that the FZD5/8 and LRP6 receptor–specific Wnt mimetic, SZN-1326-p, was associated with the robust repair effect. Through a range of approaches including single-cell transcriptome analyses, we demonstrated that SZN-1326-p directly impacted epithelial cells, driving transient expansion of stem and progenitor cells, promoting differentiation of epithelial cells, histologically restoring the damaged epithelium, and secondarily to epithelial repair, reducing inflammation.ConclusionsIt is feasible to design Wnt mimetics such as SZN-1326-p that impact damaged intestine epithelium specifically and restore its physiological functions, an approach that holds promise for treating epithelial damage in inflammatory bowel disease.

Probiotics in Intestinal Mucosal Healing: A New Therapy or an Old Friend?

Inflammatory bowel disease (IBD), Crohn’s disease, and ulcerative colitis are characterized by chronic and relapsing inflammation, while their pathogenesis remains mostly unelucidated. Gut commensal microbiota seem to be one of the various implicated factors, as several studies have shown a significant decrease in the microbiome diversity of patients with IBD. Although the question of whether microbiota dysbiosis is a causal factor or the result of chronic inflammation remains unanswered, one fact is clear; active inflammation in IBD results in the disruption of the mucus layer structure, barrier function, and also, colonization sites. Recently, many studies on IBD have been focusing on the interplay between mucosal and luminal microbiota, underlining their possible beneficial effect on mucosal healing. Regarding this notion, it has now been shown that specific probiotic strains, when administrated, lead to significantly decreased inflammation, amelioration of colitis, and improved mucosal healing. Probiotics are live microorganisms exerting beneficial effects on the host’s health when administered in adequate quantity. The aim of this review was to present and discuss the current findings on the role of gut microbiota and their metabolites in intestinal wound healing and the effects of probiotics on intestinal mucosal wound closure.

Protective Effect of Apium graveolens L. (Celery) Seeds Extracts and Luteolin on Acetic Acid-Induced Colitis in Rats.

Introduction:Anti-oxidant, antispasmodic, anti-inflammatory, and analgesic effects have been reported for Apium graveolens Linn. ) Celery( seeds and its active component luteolin. So, this study was carried out to investigate the protective effects of hexane (AGHE) and methanol (AGME) extracts of A. graveolens seeds and luteolin on acetic acid-induced colitis in rats.Methods:Three doses of AGHE (100, 200, and 400 mg/kg), AGME (200, 400, and 800 mg/kg), and luteolin (5, 10, and 20 mg/kg) were administered orally (p.o.) to separate groups of male Wistar rats, 2 h before ulcer induction (acetic acid 4%) and continued once daily for 4 days. Prednisolone (4 mg/kg) and mesalazine (100 mg/kg) were used as reference and vehicle (2 mL/kg) as control groups. Colon biopsies were taken for weighting, macroscopic and histopathologic evaluation, and measuring myeloperoxidase (MPO) activity.Results:Our findings showed that AGHE (200 and 400 mg/kg), AGME (400 and 800 mg/kg), and luteolin (10 and 20 mg/kg) were effective to reduce colonic ulcer score, area, and index as well as total colitis index, and MPO activity significantly in comparison with controls. Since the lowest doses of extracts and luteolin were not significantly effective to diminish evaluated parameters of colitis, it is concluded that the ameliorative effect was dose related.Conclusion:It is also concluded that both extracts and luteolin, as an important ingredient of celery extract, were effective in the amelioration of colitis in rats, but further clinical and detailed mechanistic experiments are required to introduce these natural agents for colitis treatment or prevention in human.

Lactiplantibacillus plantarum 22A-3-induced TGF-β1 secretion from intestinal epithelial cells stimulated CD103+ DC and Foxp3+ Treg differentiation and amelioration of colitis in mice.

In the present study, we evaluated the anti-inflammatory properties of Lactiplantibacillus plantarum 22A-3 (LP22A3) and attempted to elucidate the underlying molecular mechanism. The oral administration of LP22A3 significantly inhibited body weight reduction and decreased colon shortening and colitis score in mice with dextran sulfate sodium (DSS)-induced colitis. It was demonstrated that the production of the active-form of TGF-β tended to increase in both the intestinal epithelial cells (IECs) of the ileum and serum but not in the colon of non-DSS-treated mice by LP22A3. IL-10 level in serum was also elevated by LP22A3-treatment. The mRNA expression of TGF-β, IL-10 and Foxp3 increased only in the small intestines of LP22A3-treated mice. Both the aldehyde dehydrogenase 1 family member A2 (Aldh1a2) mRNA expression and population of CD103+ dendritic cells (DCs) in the small intestine significantly increased in the LP22A3-treated group. LP22A3 induced TGF-β secretion from the IECs of the small intestine with retinoic acid production probably through TLR2, resulting in an increase in CD103+ DCs and the Foxp3+ Treg population. Both cells secrete a high level of anti-inflammatory cytokines, TGF-β and IL-10 contributing to the protective condition in the intestine and thus making it less susceptible to inflammation. This suggested that oral administration of LP22A-3 may be an alternative therapeutic strategy for IBD.

In vitro and in vivo amelioration of colitis using targeted delivery system of cyclosporine a in New Zealand rabbits

Necessity to develop the efficient targeted delivery of highly potent immunosuppressant for IBD in order to avoid surgical procedure, led to fabrication and evaluation of its anti-inflammatory potential. Previously formulated cyclosporine A (Cyp A) into enteric coated capsules was further evaluated for its site-specificity in the treatment of TNBS induced colitis. Contact angle measurement studies showed excellent spreadability of the developed formulation over the hydrophilic biological tissue substrate. HET-CAM study demonstrates that the formulation prepared is nonirritant to the highly vascular tissues and hence can be used for the immunological sensitive tissues like inflamed intestine in IBD. Further the developed formulation has been characterized for site specificity to distal parts of intestine by pharmacokinetic studies. The appearance of drug in systemic circulation at approximately 5 hours in New Zealand strain of rabbits confirms drug delivery at distal parts of intestine. Significant reduced levels of TNF-α, IL-6 and IL-10 in drug treated animals signifies inhibition of inflammatory reactions at the TNBS treated site. Simultaneously, the change in body weight of same group of animals was observed for 15 days. Results showed a marginal recovery of body weight in Cyp A treated TNBS induced colitis animals. In conclusion, all in vitro and in vivo results confirm the successful site specific delivery and anti-inflammatory efficacy of developed formulation of Cyp A in TNBS induced colitis in New Zealand rabbits.

A264 HELMINTH-INFECTION MOBILIZES HOST AND MICROBIAL FACTORS THAT CO-OPERATE TO SUPPRESS CHEMICAL-INDUCED COLITIS IN MICE

Abstract Background Infection with helminth parasites suppresses inflammation in murine model systems; for example, IL-10 is important in Hymenolepis diminuta-inhibition of DNBS-induced colitis. Bacteria-derived products can have anti-inflammatory effects. Given that infection with H. diminuta, or other parasitic worms, results in perturbation of the gut microbiota, the present study tested a role for bacteria in helminth-suppression of colitis by assessing reciprocity between IL-10 and butyrate signaling in the amelioration of colitis. Aims To determine if a functional relationship exists between IL-10 and butyrate in the inhibition of colitis observed following infection with the lumen-dwelling tapeworm, Hymenolepis diminuta. Methods Colitis was induced in male BALB/c mice by intra-rectal dinitrobenzene sulphonic acid (DNBS) (3 mg/~22g mouse), with necropsy and assessment 3 days later. Mice received either infection with five H. diminuta cysticercoids by gavage or daily butyrate enemas or acetate in their drinking water. Immunostaining assessed IL-10R protein expression on formalin-fixed sections of colon. The murine IEC4.1 epithelial cell line and epithelial organoids were treated with butyrate and mRNA for the IL10Rα chain assessed, as was colonic tissue from mice. Results Mice infected with H. diminuta or receiving butyrate enemas (n=8–12) were protected from DNBS-induced colitis as gauged by colon length, and macroscopic disease and histopathology scores. Addition of acetate to the drinking water resulted in a more modest anti-colitic effect. Suppression of colitis was accompanied by increased epithelial expression of IL-10 in butyrate- and H. diminuta-treated mice, with the later also showing upregulation of the IL-10R on lamina propria cells; an effect negated by co-treating the mice with broad spectrum antibiotics. In vitro analyses revealed increased IL10Rα mRNA in butyrate-treated epithelia (n=4). Conclusions This study begins to tease apart the host (i.e. IL-10) and bacterial (i.e. butyrate) molecules that mediate H. diminuta-evoked suppression of colitis in a murine model. These proof-of-principle data suggest that knowledge of the individual patient (i.e. immunological basis of their disease and their microbiota) may be a critical determinant of the success or failure of helminth therapy. Funding Agencies CAG, CCCNSERC

Amelioration of colitis in mice by Leuconostoc lactis EJ-1 by M1 to M2 macrophage polarization.

Dysregulation of immune responses to environmental antigens by the intestine leads to the chronic inflammatory disease, inflammatory bowel disease (IBD). Recent studies have thus sought to identify a dietary component that can inhibit lipopolysaccharide (LPS)-induced nuclear factor-kappa beta (NF-κB) signaling to ameliorate IBD. This study assessed if the lactic acid bacteria (LAB) from kimchi, suppresses the expression of tumor necrosis factor-alpha (TNF-α) in peritoneal macrophages induced by LPS. Leuconostoc lactis EJ-1, an isolate from LAB, reduced the expression of interleukin-6 (IL-6) and IL-1β in peritoneal macrophages induced by LPS. The study further tested whether EJ-1 alleviates colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. TNBS significantly increased myeloperoxidase (MPO) expression, macroscopic colitis scores, and colon shortening. Oral administration of L. lactis EJ-1 resulted in an inhibited in TNBS-induced loss in body weight, colon shortening, MPO activity, and NF-κB and inducible nitric oxide synthase expression; it also led to a marked reduction in cyclooxygenase-2 expression. L. lactis EJ-1 also inhibited the TNBS-induced expression of TNF-α, IL-1β, and IL-6; however, it induced the expression of IL-10. The M2 macrophage markers arginase I, IL-10, and CD206 were elevated by EJ-1. Collectively, these results suggest that EJ-1 inhibits the NF-κB signaling and polarizes M1- to M2-macrophage transition, which help in ameliorating colitis.

Alpinetin improves intestinal barrier homeostasis via regulating AhR/suv39h1/TSC2/mTORC1/autophagy pathway

The injury of intestinal epithelial barrier is considered as the key pathophysiological process in response to gastrointestinal infection and inflammation, and plays an important role in the initiation and development of colitis. Alpinetin has been shown to improve intestinal barrier homeostasis under colitis condition, but the mechanism is still unclear. Here, we showed that alpinetin significantly improved transepithelial electrical resistance (TEER) in TNF-α-stimulated Caco-2 cells, which was mainly mediated by inhibiting the apoptosis. Mechanistic studies demonstrated that alpinetin markedly increased the production of autophagosomes, along with obvious regulation of LC3B-II, beclin-1, p62, Atg7 and Atg5 expressions. In addition, it also markedly repressed the activation of mTORC1 signaling pathway, which was ascribed to TSC2 rather than p-AKT, p-ERK, p-AMPKα or PTEN expressions in Caco-2 and NCM460 cells. Furthermore, the enrichment of H3K9me3 at TSC2 promoter region was decreased and ubiquitin proteasome degradation of suv39h1 was increased. Additionally, alpinetin activated aryl hydrocarbon receptor (AhR) and promoted co-localization of AhR with suv39h1 in the cytoplasm. The relationship between alpinetin-regulated AhR/suv39h1/TSC2/mTORC1 signals, autophagy and apoptosis of Caco-2 and NCM460 cells was confirmed by using CH223191, siAhR, siTSC2 and chloroquine. Finally, CH223191 and leucine abolished alpinetin-mediated inhibition of intestinal epithelial cells apoptosis, improvement of intestinal epithelial barrier and amelioration of colitis.

Alteration of Gut Microbiota in Inflammatory Bowel Disease (IBD): Cause or Consequence? IBD Treatment Targeting the Gut Microbiome.

Inflammatory bowel disease (IBD) is a chronic complex inflammatory gut pathological condition, examples of which include Crohn’s disease (CD) and ulcerative colitis (UC), which is associated with significant morbidity. Although the etiology of IBD is unknown, gut microbiota alteration (dysbiosis) is considered a novel factor involved in the pathogenesis of IBD. The gut microbiota acts as a metabolic organ and contributes to human health by performing various physiological functions; deviation in the gut flora composition is involved in various disease pathologies, including IBD. This review aims to summarize the current knowledge of gut microbiota alteration in IBD and how this contributes to intestinal inflammation, as well as explore the potential role of gut microbiota-based treatment approaches for the prevention and treatment of IBD. The current literature has clearly demonstrated a perturbation of the gut microbiota in IBD patients and mice colitis models, but a clear causal link of cause and effect has not yet been presented. In addition, gut microbiota-based therapeutic approaches have also shown good evidence of their effects in the amelioration of colitis in animal models (mice) and IBD patients, which indicates that gut flora might be a new promising therapeutic target for the treatment of IBD. However, insufficient data and confusing results from previous studies have led to a failure to define a core microbiome associated with IBD and the hidden mechanism of pathogenesis, which suggests that well-designed randomized control trials and mouse models are required for further research. In addition, a better understanding of this ecosystem will also determine the role of prebiotics and probiotics as therapeutic agents in the management of IBD.

Novel Selective Estrogen Receptor Modulator Ameliorates Murine Colitis.

Estrogen-receptor-mediated signaling has been suggested to decrease the inflammatory response in monocyte macrophages. Previously, we showed that a novel selective estrogen receptor modulator (SERM2) promotes anti-inflammatory phenotype of monocytes in vitro. In this study, we demonstrate the potential of SERM2 in amelioration of colitis. We utilized a dextran sodium sulfate (DSS)-induced colitis model in FVB/n mice to demonstrate the effects of orally administered SERM2 on the clinical status of the mice and the histopathological changes in the colon, as well as proportion of Mrc-1 positive macrophages. SERM2 nuclear receptor affinities were measured by radioligand binding assays. Orally administered, this compound significantly alleviated DSS-induced colitis in male mice and induced local estrogen receptor activation in the inflamed colon, as well as promoting anti-inflammatory cytokine expression and infiltration of anti-inflammatory monocytes. We show that this novel drug candidate has an affinity to estrogen receptors α and β and progesterone receptors, but not to glucocorticoid receptor, thus expressing unique binding properties compared to other sex steroid receptor ligands. These results indicate that novel drug candidates to alleviate inflammatory conditions of the colon could be found among sex steroid receptor activating compounds.

The Role of Specific Chemokines in the Amelioration of Colitis by Appendicitis and Appendectomy.

The appendix contains abundant lymphoid tissue and is constantly exposed to gut flora. When completed at a young age, appendicitis followed by appendectomy (AA) prevents or significantly ameliorates Inflammatory Bowel Diseases (IBDs) in later life. Inflammatory bowel disease comprises Crohn’s disease and ulcerative colitis. Our murine AA model is the only existing experimental model of AA. In our unique model, AA performed in the most proximal colon limits colitis pathology in the most distal colon by curbing T-helper 17 cell activity, diminishing autophagy, modulating interferon activity-associated molecules, and suppressing endothelin vaso-activity-mediated immunopathology. In the research presented in this paper, we have examined the role of chemokines in colitis pathology with our murine AA model. Chemokines are a family of small cytokines with four conserved cysteine residues. Chemokines induce chemotaxis in adjacent cells with corresponding receptors. All 40 known chemokine genes and 24 chemokine receptor genes were examined for gene expression levels in distal colons three days post-AA and 28 days post-AA. At 28 days post-AA, the chemokine gene CCL5 was significantly upregulated. Furthermore, Gene Set Enrichment Analysis (GSEA) showed upregulation of seven CCL5-associated gene-sets 28 days post-AA in contrast to just one gene-set downregulated at the same time-point. The chemokine gene CXCL11 was significantly upregulated three days post-AA and 28 days post-AA. Evaluation using GSEA showed upregulation of six CXCL11-associated gene sets but no downregulation of any gene set. At 28 days post-AA, CCL17 gene expression was significantly downregulated. There was no expression of any chemokine receptor gene three days post-AA, but CCR10 was the only chemokine receptor gene that displayed differential gene expression (upregulation) 28 days post-AA. No CCR10-associated gene set was upregulated in GSEA in contrast to one downregulated gene set. Our analysis resulted in identifying three new therapeutic targets towards ameliorating colitis: CCL5, CXCL11, and CCL17. While CCL5 and CXCL11 are good therapeutic chemokine candidates to be exogenously administered, CCL17 is a good candidate chemokine to competitively inhibit or limit colitis pathology.

An endogenous aryl hydrocarbon receptor ligand, ITE, induces regulatory T cells and ameliorates experimental colitis.

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that affects millions of people with high morbidity and health care costs. The precise etiology of IBD is unknown, but clear evidence suggests that intestinal inflammation is caused by an excessive immune response to mucosal antigens. Recent studies have shown that activation of the aryl hydrocarbon receptor (AhR) induces regulatory T cells (Tregs) and suppresses autoimmune diseases. In the current study, we investigated if a nontoxic ligand of AhR, 2-(1' H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), can attenuate dextran sodium sulfate-induced colitis. Our studies demonstrated that in mice that received ITE treatment in vivo, colitis pathogenesis, including a decrease in body weight, was significantly reversed along with the systemic and intestinal inflammatory cytokines. ITE increased the expression of Tregs in spleen, mesenteric lymph nodes (MLNs), and colon lamina propria lymphocytes (cLPL) of mice with colitis when compared with controls. This induction of Tregs was reversed by AhR antagonist treatment in vitro. ITE treatment also increased dendritic cells (CD11c+) and decreased macrophages (F4/80+) from the spleen, MLNs, and cLPL in mice with colitis. ITE also reversed the systemic and intestinal frequency of CD4+ T cells during colitis and suppressed inflammatory cytokines including IFN-γ, TNF-α, IL-17, IL-6, and IL-1 as well as induced IL-10 levels. These findings suggest that ITE attenuates colitis through induction of Tregs and reduction in inflammatory CD4+ T cells and cytokines. Therefore, our work demonstrates that the nontoxic endogenous AhR ligand ITE may serve as a therapeutic modality to treat IBD. NEW & NOTEWORTHY We report the novel finding that activation of the aryl hydrocarbon receptor with the nontoxic ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces regulatory T cells (Tregs) and suppresses inflammatory bowel disease (IBD). Our data suggest that ITE diminishes colitis pathology through induction of Tregs; reduces inflammatory cytokines, inflammation score, and macrophage frequency; and induces DCs resulting in amelioration of colitis. Therefore, nontoxic endogenous ITE promotes the induction of Tregs and may be useful for the treatment of IBD.

Amelioration of colitis through blocking lymphocytes entry to Peyer's patches by sphingosine-1-phosphate lyase inhibitor.

Sphingosine-1-phosphate (S1P) receptor 1, a therapeutic target of the S1P1 agonist FTY720, plays a crucial role in lymphocyte migration and is expressed in several cells including naïve T lymphocytes and endothelial cells. 2-Acetyl-4-tetrahydroxybutyl imidazole (THI), an inhibitor of S1P lyase, exhibits immunomodulatory activity through increasing the S1P concentration in the secondary lymphoid organs, but its effects on colitis remain unclear. This study aimed to clarify how THI affects colitis and migration of naïve T lymphocytes in Peyer's patches (PPs). The effect of THI on gut immunity was investigated by analyzing the dextran sulfate sodium (DSS)-induced murine colitis model, lymphocyte components in thoracic duct lymphocytes (TDLs), and microscopic movement of TDLs in PPs. 2-Acetyl-4-tetrahydroxybutyl imidazole ameliorated DSS-induced colitis histologically by causing a significant decrease in colonic lymphocyte infiltration and expression of mucosal pro-inflammatory cytokines. THI suppressed the inflow of naïve T lymphocytes into the thoracic duct. Microscopic observation of PPs in control animals revealed that many TDLs egressed to the stroma and migrated to lymph capillaries after attaching to the high endothelial venules (HEVs). THI or FTY720 treatment in recipient animals blocked lymphocyte egression from the HEVs to the stroma. This is the first study to clarify the ameliorating effects of THI on DSS-induced colitis. Microscopic observations demonstrated the involvement of HEVs in the egression of S1P-dependent gut-tropic T lymphocytes to lymph capillaries. This S1P lyase inhibitor might become a novel immunosuppressant for inflammatory bowel disease therapy by blocking infiltration of lymphocytes through HEVs into the stroma in PPs.

Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation

Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3-RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Effects of dexpanthenol on acetic acid-induced colitis in rats.

While the pathogenesis of acetic acid (AA)-induced colitis is unclear, reactive oxygen species are considered to have a significant effect. The aim of the present study was to elucidate the therapeutic potential of dexpanthenol (Dxp) on the amelioration of colitis in rats. Group I (n=8; control group) was intrarectally administered 1 ml saline solution (0.9%); group II [n=8; AA] was administered 4% AA into the colon via the rectum as a single dose for three consecutive days; group III (n=8; AA + Dxp) was administered AA at the same dosage as group II from day 4, and a single dose of Dxp was administered intraperitoneally; and group IV (n=8; Dxp) was administered Dxp similarly to Group III. Oxidative stress and colonic damage were assessed via biochemical and histologic examination methods. AA treatment led to an increase in oxidative parameters and a decrease in antioxidant systems. Histopathological examination showed that AA treatment caused tissue injury and increased caspase-3 activity in the distal colon and triggered apoptosis. Dxp treatment caused biochemical and histopathological improvements, indicating that Dxp may have an anti-oxidant effect in colitis; therefore, Dxp may be a potential therapeutic agent for the amelioration of IBD.

Mo1828 Reduced Intestinal Leukocyte Exit Heightens the Severity of DSS Colitis in a Murine Model of Lymphovascular Deficiency

process of intestinal inflammation is less understood. While we could demonstrate that HDAC inhibitors induce amelioration of colitis in mice, we are further interested in how far specific HDAC are responsible for inflammatory responses and what are the mechanism behind these effects. Methods: The macrophage cell line RAW 264.7 and the monocyte cell line U937 were analysed for HDAC5-expression using quantitative PCR and Western-blot. The cells were transfected with a HDAC5-encoding plasmid for overexpression or small interfering (si)RNA for knock-down studies. HDAC5 knock out mice were used in dextran sulphate sodium (DSS) induced colitis model. Colon tissue was analysed by histology and lamina propria mononuclear cells (LPMCs) were isolated. All cells were analysed by flow cytometry. Supernatant of colon and cell cultures were analysed by cytometric bead assay. Results: In RAW 264.7 and U937 expression of HDAC5 mRNA down regulated for up to 7 h but presented baseline levels after 24 h. HDAC5-overexpression in RAW 264.7 resulted in an increased LPS-dependent secretion of the pro-inflammatory cytokines tumor necrosis factor alpha, the monocyte chemoattractant protein-1 and interleukin 6. Subsequently, knock-down of HDAC5-mRNA expression by specific siRNA significantly reduced the secretion of these cytokines. These effects were accompanied by increased nuclear factor kappa B activity. Further experimental colitis experiments demonstrate a differential disease pattern for HDAC5 knockout mice compared to wild type mice. This was shown by an increased histological disease score, higher weight loss and severe intestinal bleeding. LPMC isolation followed by extensive FACS analysis of the LPMC confirmed these results by demonstrating changes in macrophage polarization. When the colonic barrier function was analysed in DSS induced colitis experiments, we detected a decreased trans-epithelial resistance and modulated flux of mannitol, HRP and FITC-Dextran. Conclusion: Our data demonstrate a critical function of HDAC5 in the pro-inflammatory immune response of macrophages in the lamina propria as well as a role within the physiological barrier of the gut epithelium, proposing a potential new target for the treatment of inflammatory bowel disease