Chronic Dextran Sulfate Sodium Colitis Research Articles

Microbiome- and Host Inflammasome-Targeting Inhibitor Nanoligomers Are Therapeutic in the Murine Colitis Model.

Autoimmune and autoinflammatory diseases account for more than 80 chronic conditions affecting more than 24 million people in the US. Among these autoinflammatory diseases, noninfectious chronic inflammation of the gastrointestinal (GI) tract causes inflammatory bowel diseases (IBDs), primarily Crohn's and ulcerative colitis (UC). IBD is a complex disease, and one hypothesis is that these are either caused or worsened by compounds produced by bacteria in the gut. While traditional approaches have focused on pan immunosuppressive techniques (e.g., steroids), low remission rates, prolonged illnesses, and an increased frequency of surgical procedures have prompted the search for more targeted and precision therapeutic approaches. IBD is a complex disease resulting from both genetic and environmental factors, but several recent studies have highlighted the potential pivotal contribution of gut microbiota dysbiosis. Gut microbiota are known to modulate the immune status of the gut by producing metabolites that are encoded in biosynthetic gene clusters (BGCs) of the bacterial genome. Here, we show a targeted and high-throughput screening of more than 90 biosynthetic genes in 41 gut anaerobes, through downselection using available bioinformatics tools, targeted gene manipulation in these genetically intractable organisms using the Nanoligomer platform, and identification and synthesis of top microbiome targets as a Nanoligomer BGC cocktail (SB_BGC_CK1, abbreviated as CK1) as a feasible precision therapeutic approach. Further, we used a host-directed immune target screening to identify the NF-κB and NLRP3 cocktail SB_NI_112 (or NI112 for short) as a targeted inflammasome inhibitor. We used these top two microbe- and host-targeted Nanoligomer cocktails in acute and chronic dextran sulfate sodium (DSS) mouse colitis and in TNFΔARE/+ transgenic mice that develop spontaneous Crohn's like ileitis. The mouse microbiome was humanized to replicate that in human IBD through antibiotic treatment, followed by mixed fecal gavage from 10 human donors and spiked with IBD-inducing microbial species. Following colonization, colitis was induced in mice using 1 week of 3% DSS (acute) or 6 weeks of 3 rounds of 2.5% DSS induction for a week followed by 1 week of no DSS (chronic colitis model). Both Nanoligomer cocktails (CK1 and NI112) showed a strong reduction in disease severity, significant improvement in disease histopathology, and profound downregulation of disease biomarkers in colon tissue, as assessed by multiplexed ELISA. Further, we used two different formulations of intraperitoneal injections (IP) and Nanoligomer pills in the chronic DSS colitis model. Although both formulations were highly effective, the oral pill formulation demonstrated a greater reduction in biochemical markers compared to IP. A similar therapeutic effect was observed in the TNFΔARE/+ model. Overall, these results point to the potential for further development and testing of this inflammasome-targeting host-directed therapy (NI112) and more personalized microbiome cocktails (CK1) for patients with recalcitrant IBD.

Eosinophil Depletion as a Potential Therapeutic Strategy in Acute and Chronic Intestinal Inflammation Based on a Dextran Sulfate Sodium Colitis Model.

A role for eosinophils in intestinal inflammation and fibrosis in the context of inflammatory bowel disease has been suggested, yet the precise nature, whether causal or secondary remains debated. Hence, it remains unclear whether targeting eosinophils should be further explored as a treatment option in inflammatory bowel disease. Acute and chronic dextran sulfate sodium colitis was induced in wild-type C57BL/6 mice. Eosinophils were depleted by anti-CCR3 injections before colitis induction in a chronic model and after colitis onset in an acute model in order to investigate the impact of eosinophil depletion on pre-existing colitis. Inflammation was assessed using the disease activity index, macroscopic damage, and histological disease activity score. In the chronic model, fibrosis was assessed by examining colon weight/length ratio, collagen deposition through Martius Scarlet Blue staining, hydroxyproline assay, and COL1A1 expression. Protein and gene expression were assessed using the Meso Scale Discovery platform and real-time quantitative polymerase chain reaction. In the acute and chronic colitis model, eosinophil depletion resulted in reduced disease activity and faster recovery, as observed via the total area under the curve of the disease activity index (P = .004 and P = .02, respectively), macroscopic damage score (P = .009 and P = .08, respectively), and histological disease activity score (P = .09 and P = .002, respectively). In the acute model, the accelerated recovery was accompanied by an increase in interleukin (IL)-10 (P = .03) and a decrease in IL-4 (P = .03) and IL-6 (P = .009). Colon weight/length ratio and collagen deposition were not affected by eosinophil depletion. Eosinophil depletion prevents and decreases intestinal inflammation in a preclinical dextran sulfate sodium model without affecting fibrosis. These results pave the way for exploring eosinophil depletion as a novel treatment modality in addressing intestinal inflammation.

Human umbilical cord/placenta mesenchymal stem cell conditioned medium attenuates intestinal fibrosis in vivo and in vitro

BackgroundA significant unmet need in inflammatory bowel disease is the lack of anti-fibrotic agents targeting intestinal fibrosis. This study aimed to investigate the anti-fibrogenic properties and mechanisms of the conditioned medium (CM) from human umbilical cord/placenta-derived mesenchymal stem cells (UC/PL-MSC-CM) in a murine intestinal fibrosis model and human primary intestinal myofibroblasts (HIMFs).MethodsUC/PL-MSC-CM was concentrated 15-fold using a 3 kDa cut-off filter. C57BL/6 mice aged 7 weeks old were randomly assigned to one of four groups: (1) control, (2) dextran sulfate sodium (DSS), (3) DSS + CM (late-phase treatment), and (4) DSS + CM (early-phase treatment). Chronic DSS colitis and intestinal fibrosis was induced by three cycles of DSS administration. One DSS cycle consisted of 7 days of oral DSS administration (1.75%, 2%, and 2.5% DSS), followed by 14 days of drinking water. UC/PL-MSC-CM was intraperitoneally administered in the late phase (from day 50, 10 times) or early phase (from day 29, 10 times) of DSS cycles. HIMFs were treated with TGF-β1 and co-treated with UC/PL-MSC-CM (10% of culture media) in the cellular model.ResultsIn the animal study, UC/PL-MSC-CM reduced submucosa/muscularis propria thickness and collagen deposition, which improved intestinal fibrosis in chronic DSS colitis. The UC/PL-MSC-CM significantly reduced the expressions of procollagen1A1 and α-smooth muscle actin, which DSS significantly elevated. The anti-fibrogenic effect was more apparent in the UC-MSC-CM or early-phase treatment model. The UC/PL-MSC-CM reduced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs in the cellular model. The UC/PL-MSC-CM downregulated fibrogenesis by suppressing RhoA, MRTF-A, and SRF expression.ConclusionsHuman UC/PL-MSC-CM inhibits TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and chronic DSS colitis-induced intestinal fibrosis. Thus, it may be regarded as a novel candidate for stem cell-based therapy of intestinal fibrosis.

DOP09 Eosinophils exert a pro-inflammatory role in a chronic DSS colitis model without an impact on fibrosis

Abstract Background Eosinophils might play a pro-inflammatory role in inflammatory bowel disease (IBD). However, the role of eosinophils in intestinal fibrosis remains poorly understood, although a pro-fibrotic function has been hypothesized based on data outside the gastrointestinal tract. Therefore, we aimed to unravel the role of eosinophils in chronic intestinal inflammation and fibrosis and to explore eosinophil depletion as a potential therapeutic target. Methods A 3-cycles chronic dextran sodium sulphate (DSS) model was induced in 6-8-week-old C57BL/6 wild type mice. Mice received 3 DSS cycles (1.5% - 2.00% - 2.00%) in which 1 DSS cycle consisted of 1 week of DSS administration followed by 2 weeks of recovery. During the 3 cycles, and starting 3 days prior to colitis induction, anti-CCR3 antibody or isotype injections were given twice weekly to study the effect of the eosinophil depletion (total of 18 injections). The disease activity index (DAI; weight loss, stool consistency and presence of blood) was determined twice weekly. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation) and colonic single cells were isolated and fluorescently stained for flow cytometry. Eosinophils were thereby identified as CD45+ CD11b+ Siglec-F+ CD117- cells. Lastly, a histological active disease score was determined comprising of the sum of neutrophil infiltration, mononuclear cell infiltration, changes in mucosal architecture, goblet cell loss and epithelial defects (Creyns et al., 2019). Intestinal fibrosis was assessed via colon weight/length ratio, COL1A1 gene expression and collagen deposition with a Martius Scarlet Blue (MSB) staining and a colorimetric hydroxyproline assay. Results Blood and colonic eosinophil depletion via anti-CCR3 injections was confirmed via flow cytometry (figure 1). The DAI in the eosinophil depleted group was decreased compared to the isotype injected group (area under the curve 115.4 ± 29 vs 160.6 ± 28; p=0.02). A similar trend was seen in the macroscopic damage score (2.0 ± 1.3 vs 3.9 ± 2.1; p=0.08). Furthermore, a lower histological active disease score was found in the mice in which the eosinophils were depleted compared to the isotype injected mice (8.8 ± 0.8 vs 12.8 ± 1.5; p=0.002) (figure 2). Lastly, no differences in the parameters for fibrosis between the anti-CCR3 injected and isotype injected groups were observed (figure 3). Conclusion Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection from intestinal inflammation in chronic DSS, and could therefore be further explored as a potential therapeutic agent. In contrast, eosinophil depletion does not seem to have any anti-fibrotic effect

DOP33 Single-cell analysis identifies pathological fibroblasts as a new therapeutic target to prevent intestinal fibrosis in Crohn’s disease.

Abstract Background Recurrent episodes of intestinal inflammation and tissue remodeling progressively result in fibro-stenosis and bowel obstruction in Crohn’s disease (CD). This irreversible end-stage complication is the main indication for surgical intervention but is often associated with postoperative recurrence of inflammation and repeated tissue resection. Methods To understand transmural inflammation-induced fibro-stenosis, full-thickness ileum from CD patients (n=10) undergoing ileocecal resection were profiled using single-cell RNA sequencing (scRNA seq) on 10x Genomics platform. Our tissue sampling strategy aimed at recapitulating different disease stages from the same patient including normal ileum, chronic active inflammation and stenotic ileum, allowing a better definition of cell heterogeneity, inter-cellular communication, and tissue organization in CD. Healthy ileum from CRC patients (n=5) undergoing right hemicolectomy were included as external control. Flow cytometry (FACS) was carried out to confirm scRNA seq findings. For functional validation, intestinal fibroblasts were co-cultured with NicheNet-predicted cytokines and FACS-sorted myeloid supernatants. Finally, the chronic dextran sulfate sodium (DSS)-induced colitis model was used to validate the transcription regulation in activated fibroblasts. Results Using scRNA seq, we identified a specific subset of activated fibroblasts during chronic inflammation and fibrosis. FACS showed increasing FAP+ fibroblasts in both inflamed and stenotic ileum, compared to control ileum and proximal ileum (p<0.01). Computational methods predicting ligand-receptor signaling suggest that fibroblast activation is mostly mediated by myeloid cell-derived inflammatory signals resulting in collagen deposition and tissue fibrosis. Intestinal fibroblasts co-cultured with inflammatory monocyte supernatant and predicted ligands expressed higher protein levels of FAP, COL3A1 and showed signs of EMT transformation. Furthermore, stimulated intestinal fibroblasts secreted higher levels of ECM proteins, including COL1 (p<0.001), and COL3A1 (p<0.005). After inhibiting an EMT-related transcription factor identified in the activated fibroblasts, collagen expression and extracellular matrix accumulation were decreased in fibroblasts. Finally, inhibition of EMT transformation in chronic DSS colitis resulted in reduced ECM deposition, compared to vehicle mouse (p<0.05). Conclusion Our findings suggest that inflammatory monocytes mediate local activation of fibroblasts promoting excessive collagen deposition. We furthermore show that targeting activated fibroblasts was able to reduce tissue remodeling and may therefore prevent fibrostenosis in CD.

Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease.

BackgroundImpaired intestinal epithelial barrier is highly affected in inflammatory bowel disease. Transmembrane collagens connecting the epithelial cells to the extracellular matrix have an important role in epithelial cell homeostasis. Thus, we sought to determine whether the transmembrane type 23 collagen could serve as a surrogate marker for disease activity in patients with Crohn's disease and ulcerative colitis.MethodsWe developed an enzyme‐linked immunosorbent assay to detect the ectodomain of type 23 collagen (PRO‐C23) in serum, followed by evaluation of its levels in both acute and chronic dextran sulphate sodium colitis models in rats and human inflammatory bowel disease cohorts. Serum from 44 Crohn's disease and 29 ulcerative colitis patients with active and inactive disease was included.ResultsIn the acute and chronic dextran sulphate sodium‐induced rat colitis model, the PRO‐C23 serum levels were significantly increased after colitis and returned to normal levels after disease remission. Serum levels of PRO‐C23 were elevated in Crohn's disease (p < 0.05) and ulcerative colitis (p < 0.001) patients with active disease compared to healthy donors. PRO‐C23 differentiated healthy donors from ulcerative colitis (area under the curve [AUC]: 0.81, p = 0.0009) and Crohn's disease (AUC: 0.70, p = 0.0124). PRO‐C23 differentiated ulcerative colitis patients with active disease from those in remission (AUC: 0.75, p = 0.0219) and Crohn's disease patients with active disease from those in remission (AUC: 0.68, p = 0.05).ConclusionPRO‐C23 was elevated in rats with active colitis, and inflammatory bowel disease patients with active disease. Therefore, PRO‐C23 may be used as a surrogate marker for monitoring disease activity in ulcerative colitis and Crohn's disease.

Matricellular Protein SPARCL1 Regulates Blood Vessel Integrity and Antagonizes Inflammatory Bowel Disease.

BackgroundThe understanding of vascular plasticity is key to defining the role of blood vessels in physiologic and pathogenic processes. In the present study, the impact of the vascular quiescence marker SPARCL1 on angiogenesis, capillary morphogenesis, and vessel integrity was evaluated.MethodsAngiogenesis was studied using the metatarsal test, an ex vivo model of sprouting angiogenesis. In addition, acute and chronic dextran sodium sulfate colitis models with SPARCL1 knockout mice were applied.ResultsThis approach indicated that SPARCL1 inhibits angiogenesis and supports vessel morphogenesis and integrity. Evidence was provided that SPARCL1-mediated stabilization of vessel integrity counteracts vessel permeability and inflammation in acute and chronic dextran sodium sulfate colitis models. Structure-function analyses of purified SPARCL1 identified the acidic domain of the protein necessary for its anti-angiogenic activity.ConclusionsOur findings inaugurate SPARCL1 as a blood vessel–derived anti-angiogenic molecule required for vessel morphogenesis and integrity. SPARCL1 opens new perspectives as a vascular marker of susceptibility to colitis and as a therapeutic molecule to support blood vessel stability in this disease.

Selective deletion of MyD88 signaling in α-SMA positive cells ameliorates experimental intestinal fibrosis via post-transcriptional regulation.

Background:Intestinal fibrosis leading to strictures remains a significant clinical problem in inflammatory bowel diseases (IBD). The role of bacterial components in activating intestinal mesenchymal cells and driving fibrogenesis is largely unexplored.Methods:Tamoxifen inducible α-SMA promoter Cre mice crossed with floxed MyD88 mice were subjected to chronic dextran sodium sulfate colitis. MyD88 was deleted prior to or after induction of colitis. Human intestinal myofibroblasts (HIMF) were exposed to various bacterial components and assessed for fibronectin (FN) and collagen I (Col1) production. RNA sequencing was performed. Post-transcriptional regulation was assessed by polysome profiling assay.Results:Selective deletion of MyD88 in α-SMA positive cells prior to, but not after induction of experimental colitis decreased the degree of intestinal fibrosis. HIMF selectively responded to flagellin with enhanced FN or Col1 protein production in a MyD88 dependent manner. RNA sequencing suggested minimal transcriptional changes induced by flagellin in HIMF. Polysome profiling revealed higher proportions of FN and Col1 mRNA in the actively translated fractions of flagellin exposed HIMF, which was mediated by eIF2 alpha and 4EBP1.Conclusions:Selectivity of flagellin-induced ECM secretion in HIMF is post-transcriptionally regulated. The results may represent a novel and targetable link between the gut microbiota and intestinal fibrogenesis.

Hippocampal neural stem cells and microglia response to experimental inflammatory bowel disease (IBD).

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a disease associated with dysbiosis, resulting in compromised intestinal epithelial barrier and chronic mucosal inflammation. Patients with IBD present with increased incidence of psychiatric disorders and cognitive impairment. Hippocampus is a brain region where adult neurogenesis occurs with functional implications in mood control and cognition. Using a well-established model of experimental colitis based on the administration of dextran sodium sulfate (DSS) in the drinking water, we sought to characterize the short and long-term effects of colitis on neurogenesis and glia responses in the hippocampus. We show that acute DSS colitis enhanced neurogenesis but with deficits in cell cycle kinetics of proliferating progenitors in the hippocampus. Chronic DSS colitis was characterized by normal levels of neurogenesis but with deficits in the migration and integration of newborn neurons in the functional circuitry of the DG. Notably, we found that acute DSS colitis-induced enhanced infiltration of the hippocampus with macrophages and inflammatory myeloid cells from the periphery, along with elevated frequencies of inflammatory M1-like microglia and increased release of pro-inflammatory cytokines. In contrast, increased percentages of tissue-repairing M2-like microglia, along with elevated levels of the anti-inflammatory cytokine, IL-10 were observed in the hippocampus during chronic DSS colitis. These findings uncover key effects of acute and chronic experimental colitis on adult hippocampal neurogenesis and innate immune cell responses, highlighting the potential mechanisms underlying cognitive and mood dysfunction in patients with IBD.

Fibrogenesis in Chronic DSS Colitis is Not Influenced by Neutralisation of Regulatory T Cells, of Major T Helper Cytokines or Absence of IL-13

Mechanisms underlying fibrogenesis in chronic colitis are largely unknown. There is an urgent need for clinical markers and identification of targets to prevent, treat and limit intestinal fibrosis. This study investigated the contribution of major T cell cytokines and T regulatory cells (Tregs) to inflammation and fibrosis induced in a model of experimental colitis by oral intake of dextran sodium sulphate (DSS) in wild type and IL-13 knock-out C57Bl/6 mice. Inflammation and fibrosis were scored by macroscopic and histological examination and fibrosis was quantified by hydroxyproline. Numbers of Tregs and IFN-γ+, IL-13+ and IL-17A+ CD4+ T helper (Th) cells in mesenteric lymph nodes increased during chronic DSS administration and mRNA for IFN-γ and IL-17 in the inflamed colon tissue was upregulated. However, antibody-mediated neutralisation of IFN-γ or IL-17A/F in a therapeutic setting had no effect on chronic intestinal inflammation and fibrosis. Antibody-mediated depletion of Tregs did not enhance fibrosis, nor did IL-13 deficiency have an effect on the fibrotic disease. These data argue against an important contribution of Tregs and of the cytokines IFN-γ, IL-13, IL-17A, IL-17F in the induction and/or control of fibrosis in this Crohn’s disease like murine model.

Diet Rich in Animal Protein Promotes Pro-inflammatory Macrophage Response and Exacerbates Colitis in Mice.

Diet is a major factor determining gut microbiota composition and perturbances in this complex ecosystem are associated with the inflammatory bowel disease (IBD). Here, we used gnotobiotic approach to analyze, how interaction between diet rich in proteins and gut microbiota influences the sensitivity to intestinal inflammation in murine model of ulcerative colitis. We found that diet rich in animal protein (aHPD) exacerbates acute dextran sulfate sodium (DSS)-induced colitis while diet rich in plant protein (pHPD) does not. The deleterious effect of aHPD was also apparent in chronic DSS colitis and was associated with distinct changes in gut bacteria and fungi. Therefore, we induced acute DSS-colitis in germ-free mice and transferred gut microbiota from aCD or aHPD fed mice to find that this effect requires presence of microbes and aHPD at the same time. The aHPD did not change the number of regulatory T cells or Th17 cells and still worsened the colitis in immuno-deficient RAG2 knock-out mice suggesting that this effect was not dependent on adaptive immunity. The pro-inflammatory effect of aHPD was, however, abrogated when splenic macrophages were depleted with clodronate liposomes. This treatment prevented aHPD induced increase in colonic Ly-6Chigh pro-inflammatory monocytes, but the ratio of resident Ly-6C−/low macrophages was not changed. These data show that the interactions between dietary protein of animal origin and gut microbiota increase sensitivity to intestinal inflammation by promoting pro-inflammatory response of monocytes.

Performance of behavioral assays: the Rat Grimace Scale, burrowing activity and a composite behavior score to identify visceral pain in an acute and chronic colitis model.

The Rat Grimace Scale (RGS), a facial expression scale, quantifies the affective component of pain in rats. The RGS was developed to identify acute and inflammatory pain, and applicability in acute and chronic visceral pain is unknown. The dextran sulfate sodium (DSS) colitis model is commonly used in rats, but pain is rarely assessed, instead, disease progression is monitored with the Disease Activity Index (DAI; assessing fecal blood, stool consistency, and weight loss). The aim of this study was to assess whether the RGS and 2 additional behavioral tools (composite behavior score [CBS] and burrowing) could identify pain in an acute and chronic DSS colitis model. Male and female Sprague-Dawley rats were block randomized to (1) acute colitis (4 days DSS in drinking water); (2) chronic colitis (4 days DSS, 7 days water, and 3 days DSS); or (3) control (14 days water). Disease Activity Index, RGS, CBS, and burrowing assessments were performed daily. Rat Grimace Scale scores increased as DAI scores increased during both acute and chronic phases. Burrowing only decreased during the acute phase. By contrast, CBS scores did not increase significantly during either colitis phase. These data show that the RGS and burrowing did not decrease in a sustained manner during chronic phase visceral pain, and that variables assessed in the DAI are indicative of pain. This suggests that the RGS can be applied to a wider range of pain types and chronicity than originally suggested. These findings increase the application of the RGS as a pain scale and welfare improvement tool.

Disease Development Is Accompanied by Changes in Bacterial Protein Abundance and Functions in a Refined Model of Dextran Sulfate Sodium (DSS)-Induced Colitis

Using the acute dextran sulfate sodium (DSS)-induced colitis model, studies have demonstrated that intestinal inflammation is accompanied by major changes in the composition of the intestinal microbiota. Only little is known about the microbial changes and more importantly their functional impact in the chronic DSS colitis model. We used a refined model of chronic DSS-induced colitis that reflects typical symptoms of the human disease without detrimental weight loss usually observed in DSS models. We sampled cecum and colon content as well as colon mucus from healthy and diseased mouse cohorts ( n = 12) and applied 16S rRNA gene sequencing and metaproteomics. An increase of Prevotella sp. in both colon content and mucus was observed. Functional differences were observed between sample types demonstrating the importance of separately sampling lumen content and mucus. The abundance of Desulfovibrio, a sulfate-reducing bacterium, was positively associated with the carbon metabolism. Lachnoclostridium was positively correlated to both vitamin B6 and tryptophan metabolism. In summary, functional changes in the distal colon caused by DSS treatment were more pronounced in the mucus-associated microbiota than in the microbiota present in the distal colon content.

The EBI2-oxysterol axis promotes the development of intestinal lymphoid structures and colitis

The gene encoding for Epstein-Barr virus-induced G-protein-coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied. We found increased mRNA expression of EBI2 and oxysterol-synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2−/− mice in the IL-10 colitis model. The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2−/− mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures. In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis.

Environmental Factors Modify the Severity of Acute DSS Colitis in Caspase-11-Deficient Mice.

Human and mouse studies implicate the inflammasome in the pathogenesis of inflammatory bowel diseases, though the effects in mice are variable. The noncanonical inflammasome activator caspase-11 (Casp11) reportedly attenuates acute dextran sodium sulfate (DSS) colitis in mice. However, the effects of Casp11 on chronic experimental colitis and factors that influence the impact of Casp11 on acute DSS colitis are unknown. We studied the role of Casp11 in Il10-/- mice and acute and chronic DSS colitis mouse models. We quantified colonic Casp11 mRNA using quantative polymerase chain reaction and colitis using weight loss, blinded histological scoring, IL-12/23p40 secretion by colonic explants, and fecal lipocalin-2. We determined fecal microbial composition using 16S amplicon sequencing. We detected increased colonic Casp11 mRNA in Il10-/- mice with chronic colitis, but not in mice with DSS colitis. The presence of Casp11 did not alter the severity of chronic colitis in DSS-treated or Il10-/- mice. Contrary to prior reports, we initially observed that Casp11 exacerbates acute DSS colitis. Subsequent experiments in the same animal facility revealed no effect of Casp11 on acute DSS colitis. There were pronounced stochastic changes in the fecal microbiome over this time. The majority of bacterial taxa that changed over time in wild-type vs Casp11-/- mice belong to the Clostridiales. Casp11 does not impact chronic experimental colitis, and its effects on acute DSS colitis vary with environmental factors including the microbiota, particularly Clostridiales. Stochastic drifts in intestinal microbiota composition, even in mice in the same housing facility, should be considered when interpreting studies of acute DSS colitis models.

A refined and translationally relevant model of chronic DSS colitis in BALB/c mice.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders of the gastrointestinal tract. Several mouse models for IBD are available, but the acute dextran sulfate sodium (DSS)-induced colitis model is mostly used for preclinical studies. However, this model lacks chronicity and often leads to significant loss of mice. The aim of this study was to establish a refined and translationally relevant model of DSS chronic colitis in BALB/c mice. In the first part, we compared several standard therapeutic (ST) treatments for IBD in the acute DSS colitis model to identify the optimal treatment control for a DSS colitis model as compared to literature data. In the second part, we tested the two most effective ST treatments in a refined model of chronic DSS colitis. Cyclosporine A (CsA) and 6-thioguanine (6-TG) caused considerable reduction of clinical scores in acute DSS colitis. The clinical outcome was confirmed by the results for colon length and by histopathological evaluation. Moreover, CsA and 6-TG considerably reduced mRNA expression of several pro-inflammatory cytokines in spleen and colon. Both compounds also showed a substantial therapeutic effect in the refined model of chronic DSS colitis with regard to clinical scores and histopathology as well as the expression of inflammatory markers. The refined model of chronic DSS colitis reflects important features of IBD and is well suited to test potential IBD therapeutics.

Therapeutic efficacy of a combined sage and bitter apple phytopharmaceutical in chronic DSS-induced colitis

Inflammatory bowel diseases are multifactorial disorders of the gastrointestinal tract with rising incidence worldwide. Current standard therapies are only partially effective and often show severe adverse effects. Thus, novel, more efficient and well-tolerated therapeutic options are urgently needed. We have studied the therapeutic potential of a phytopharmaceutical combining sage and bitter apple (SBA) in the mouse model of chronic dextran sulfate sodium (DSS) colitis. SBA represents a traditional medicine against diarrhea and was shown to exhibit anti-inflammatory effects in vitro. In the chronic DSS colitis model SBA treatment significantly reduced clinical symptoms in a dose-dependent manner. The positive therapeutic effect of SBA was characterized by a decreased histopathological score indicating tissue healing. Moreover, the number of neutrophils as well as the expression of the neutrophil-recruiting chemokine CXCL-1/KC in the colon tissue was significantly reduced, whereas the recruitment of macrophages was induced. Also, the expression of inflammatory markers was significantly suppressed, while the expression of the anti-inflammatory cytokine interleukin-10 was induced in colon tissue following treatment with SBA. Phytopharmaceuticals are increasingly recognized as potential therapeutics in IBD. Thus, based on the results from this study, SBA can be considered as an alternative or supplementary option for IBD therapy.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Combination Therapy of Selective MMP9 Inhibitor and JAK Inhibitor Is Effective to Induce Disease Control in Mouse DSS Model of Colitis

Introduction: MMP9 (matrix metalloproteinase-9) promotes tissue destruction by degrading the basement membrane of epithelia and vasculature, in addition to driving inflammation via activation of cytokines and chemokines. MMP9 knock out animals were protected in DSS (dextran sulfate sodium)-induced colitis. A functional murine analog of the selective anti-MMP9 inhibitory antibody reduced disease activity index (DAI: stool consistency, hemoccults, body weight change) in established chronic DSS colitis. A potent, selective, allosteric antibody that inhibits MMP9 is currently being investigated in clinical trials. Here, a combination therapy treatment with systemic anti-inflammatory agent JAK (janus kinase) inhibitor was investigated. Methods: Colitis was induced in female C57BL/6 (n=15/group) and treatments were administered after the disease induction. Efficacy was assessed via metrics of colitis including DAI and histopathological assessment. Results: All animals were included in the evaluation. Treatment with each therapeutic agent, on its own or in combination, resulted in efficacy with respect to body weight change, stool consistency, hemoccults, colon length and weight, and histopathological assessment. Disease-induced body weight loss was improved in combination as compared to single agents (48% vs. 21-32%, relative to sham; p < 0.05 combination vs. single agents). DAI score was lower in combination vs. single agents (48% vs. 73-63%, relative to vehicle, p < 0.05). Normal stool consistency was better maintained in combination vs. single agents throughout the study period (205% vs. 139-159%, relative to vehicle, p < 0.05). Drug treatment induced early disease control, with no disease at day 12 vs. consistent disease symptoms in control-treated animals. The median ratio of colon weight/ length (mg/ cm) was 29 in combination vs. 32-38 in single agents (40-42 in controls, p < 0.05). The median surface affected by erosion or atrophy inflammation was 19% in combination vs. 38-41% in single agents (relative to vehicle, p < 0.05). Conclusion: The combination of anti-MMP9 inhibitory antibody with JAK inhibitor was significantly more effective than single agents to induce and maintain disease control by slowing disease progression and promoting mucosal healing. Further studies are required to examine combination therapy of selective anti-MMP9 antibody and JAK inhibitor in a clinical setting.

PTH-051 Influence of Iron Supplementation on The Natural History of Colitis

Introduction Iron deficiency anaemia is common in IBD. Iron supplementation may induce or exacerbate colitis in rats (APT 2001;15:1989–99).Dysbiosis of the microbiota is common in IBD and iron contributes to this as it is a growth factor for pathogenic bacteria.We investigated the effect of dietary iron supplementation and/or reduction on the severity of chronic colitis in a murine model using clinical, histological and biochemical parameters. Methods Studies were performed on 6 groups of 8 wild type (WT) C57BL/6 mice.Chronic colitis was induced with 1.25% Dextran Sodium Sulphate (DSS) for 5 days,followed by16 further days on water [for 3 consecutive cycles]. DSS-treated mice were fed one of three diets:low iron [LI] (100 ppm), normal iron [NI] (200 ppm) and high iron[HI] (400 ppm) supplemented chow. Also, 3 non-DSS-treated groups were studied and fed similarly. Half of the mice in each control group were treated with 1 cycle of acute 2% DSS for 5 days at day 53, followed by 5 further days on water. All mice were sacrificed at day 63. Daily weights and clinical features were recorded. Histological colitis was scored using the Bauer score(Gut 2010; 59:1192–99).Faecal calprotectin was measured by ELISA and faecal iron by immunoassay at various time points [day (d) 1, 21, 42 & 63]. Statistical analyses used the Kruskal–Wallis test with post-hoc analysis. Results Oral DSS administration induced colitis in all treated mice. While chronic DSS colitis was not associated with weight loss, there was severe weight loss in acute DSS mice which was greatest in the low iron diet group (p = 0.001 LI vs. HI; p = 0.01 for LI vs. NI). Histologically, the colitis features were more prominent in acute DSS-treated mice ingesting low and high iron diets, with median colitis scores 6 & 5.5 respectively.Cyclic administration of DSS in drinking water resulted in a significant rise in faecal calprotectin, from baseline to d63 in LI (p = 0.05) and for HI (p = 0.01), but this was not significant in the NI group. In acute DSS, the rise was greater in LI and HI (p = 0.001) and less in NI (p = 0.01).Total faecal iron was increased in a dose-dependent manner within 9 weeks in all non-DSS groups. Nevertheless, in chronic DSS groups, p = 0.001 at d1 vs. d63 for all groups [382% change for LI, 331% for NI and 355% for HI].However, in acute DSS p = 0.05 for LI, p = 0.001 NI & HI (d1 vs. d10). Conclusion Changes in nutritional luminal iron exacerbate colitis.Oral administration of DSS causes a reproducible acute colitis, followed by a slow recovery phase with a concomitant chronic inflammation. Chronic colitis was worse in mice fed low or high iron diets, as shown by elevated calprotectin. Faecal iron rose equally in all 3 groups: with iron increases likely arising from diet and bleeding during colitis. Dysbiosis may be a consequence of this change in luminal iron. Disclosure of Interest None Declared