Acute DSS Colitis Research Articles

Suppression of MyD88 disturbs gut microbiota and activates the NLR pathway and hence fails to ameliorate DSS-induced colitis.

Myeloid differentiation factor 88 (MyD88) is the core adaptor for Toll-like receptors defending against microbial invasion and initiating a downstream immune response during microbiota-host interaction. However, the role of MyD88 in the pathogenesis of inflammatory bowel disease is controversial. This study aims to investigate the impact of MyD88 on intestinal inflammation and the underlying mechanism. MyD88 knockout (MyD88-/-) mice and the MyD88 inhibitor (TJ-M2010-5) were used to investigate the impact of MyD88 on acute dextran sodium sulfate (DSS)-induced colitis. Disease activity index, colon length, histological score, and inflammatory cytokines were examined to evaluate the severity of colitis. RNA transcriptome analysis and 16S rDNA sequencing were used to detect the potential mechanism. In an acute DSS-colitis model, the severity of colitis was not alleviated in MyD88-/- mice and TJ-M2010-5-treated mice, despite significantly lower levels of NF-κB activation being exhibited compared to control mice. Meanwhile, 16S rDNA sequencing and RNA transcriptome analysis revealed a higher abundance of intestinal Proteobacteria and an up-regulation of the nucleotide oligomerization domain-like receptors (NLRs) signaling pathway in colitis mice following MyD88 suppression. Further blockade of the NLRs signaling pathway or elimination of gut microbiota with broad-spectrum antibiotics in DSS-induced colitis mice treated with TJ-M2010-5 ameliorated the disease severity, which was not improved solely by MyD88 inhibition. After treatment with broad-spectrum antibiotics, downregulation of the NLR signaling pathway was observed. Our study suggests that the suppression of MyD88 might be associated with unfavorable changes in the composition of gut microbiota, leading to NLR-mediated immune activation and intestinal inflammation.

THE PATHOGENIC ROLE OF MICROBIAL TRIMETHYLAMINE IN IBD ASSOCIATED INTESTINAL FIBROSIS

Abstract INTRODUCTION Intestinal fibrosis significantly contributes to the burden of inflammatory bowel diseases (IBD). No specific anti-fibrotic therapy is available. Two modifiable risk factors that may be exploited to limit fibrosis in IBD are the diet and gut microbiome. In the metaorganismal metabolism model, microbes metabolize dietary components to primary metabolites that may themselves signal through host receptor systems or be further metabolized to secondary metabolites by other microbes or host enzymes to exert a physiological or pathophysiological effect. We hypothesize that gut microbes profoundly influence intestinal fibrosis through small molecule metabolites and may provide novel targets to address the fibrotic burden in IBD. METHODS To understand how the trimethylamine-N-oxide (TMAO) pathway is affected by inflammation in the intestine, LC-MS was used to quantitate a dietary precursor, choline, primary microbial metabolite, trimethylamine (TMA), and secondary metabolite, TMAO, in mice subjected to acute DSS colitis. The cellular effects of choline, TMA, and TMAO on the deposition of fibronectin and collagen I were quantitated in primary human intestinal myofibroblasts (HIMFs). Single cell RNA-sequencing (scRNA-seq) data were interrogated to determine whether expression of the TMA-to-TMAO converting enzyme, FMO3, was downregulated in human intestinal cells of Crohn’s disease patients. RESULTS Acute DSS colitis showed increased TMA and reduced levels of TMAO, in colon tissue, which is consistent with reduced TMA-to-TMAO conversion by FMO3. Additionally, TMA was increased in intestinal contents, which suggests that microbial production of TMA from choline may also be upregulated during intestinal inflammation. In HIMFs, TMA, but not its dietary precursor, choline, nor the secondary metabolite, TMAO, increased the deposition of the extracellular matrix proteins fibronectin and collagen I. scRNA-seq analysis of the mesenchymal compartment of human intestine revealed expression of FMO3 specifically in the fibroblast cluster and downregulation of this expression in Crohn’s disease (CD) strictures compared to controls, an observation that could contribute to TMA accumulation in vivo. CONCLUSION TMA is increased in experimental colitis and activates fibroblasts towards a pro-fibrogenic phenotype. This may be mediated by reduced levels of FMO3. We next aim to measure TMAO pathway metabolites in human tissues, functionally block the TMAO pathway in vivo, and explore TMA signaling through its putative receptor, trace amine-associated receptor 5 (TAAR5). Diet-microbe-host interaction pathways represent an untapped opportunity for therapeutic intervention in multiple human disease states, such as inflammatory bowel diseases (IBD).

P086 Identification of a Novel Immunometabolic Target and Agonist for PLXDC2 for Amelioration of DSS Colitis Model in Mice

Abstract Background PLXDC2 is expressed on the cell surface of macrophage, dendritic and specific mesenchymal and epithelial cells whose activation shifts cellular metabolism and reduces oxidative stress to rebalance the immune response and decrease inflammation. It’s role in IBD pathogenesis is unknown. PLXDC2 activation improves disease severity in rheumatoid arthritis models1, while loss of PLXDC2 exacerbates the severity of disease in DSS colitis2. PX-04 is a novel, first-in-class, orally active PLXDC2 agonist. Here, we report on the consequences of activating PLXDC2 using PX-04 in an acute DSS colitis model. Methods Mice were given DSS in drinking water for seven days to induce disruption of the epithelial layer. At project initiation, mice were 8 weeks of age and began once daily dosing with 20mg/kg of PX-04 24 hours after being placed on DSS. Mice were weighed and scored daily for 15 symptoms of disease (weight loss, diarrhea, rectal bleeding, rectal inflammation, pain, & overall behavior). Colons were collected and digested, and the resultant cellular suspensions were filtered to produce single cell suspensions. Colonic Lamina Propria Immune cells were isolated by Percoll gradient centrifugation. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, Gr1, CX3CR1, CD64) and intracellular (Tbet, RORγT, FOXP3, IFNγ, IL17, IL10, TNFα) antibodies in a sequential live staining in 96-well plates. Data was acquired using a FACS Celesta flow cytometer with FACSDiva software. Results Oral PX-04 treatment decreased the cumulative disease activity of mice challenged with DSS (FIG 1A). Immunologically, PX-04 affected both the adaptive and innate immune responses. First, PX-04 greatly decreased Th17 cells in the colon, while providing a slight increase to regulatory CD4+ T cells (FIG. 1C). A lower proportion of Natural Killer (NK) cells (FIG. 7D) produced IFNγ. Meanwhile, the proportion of TNF producing dendritic cells was decreased by PX-04 treatment. Conclusion Conclusion: PLXDC2 is expressed in cells relevant to IBD pathogenesis. PX-04, which binds to and activates PLXDC2, effectively decreases levels of effector T cells and myeloid cells, as well as overall disease severity in acute DSS colitis, warranting further investigation. 1Tubau-Juni et al. J Immunol 206(Supp) 2Tubau-Juni et al. Sci Rep 10(1)

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

P066 ON104, a novel bioengineered antibody targeting oxidized Macrophage Migration Inhibitory Factor (oxMIF) shows efficacy in two distinct models of experimental colitis

Abstract Background Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic inflammatory cytokine and a primary counter-regulator of glucocorticoids (GCs) that emerged as a pivotal regulator of chronic inflammation including inflammatory bowel diseases (IBD). MIF occurs in two immunologically distinct, conformational isoforms: reduced MIF, ubiquitously present in various tissues and the circulation of healthy subjects, and oxidized MIF (oxMIF), described as the pathogenic and druggable isoform of MIF. In this study we evaluated the anti-inflammatory effects of the anti-oxMIF antibody ON104 in two different mouse models of colitis. Methods The fully human antibody ON104, which specifically targets oxMIF, was generated by advanced antibody engineering technology. The therapeutic potential of the Fc-silenced ON104 antibody was tested in two different models of colitis in mice: the acute model of DSS-induced colitis in C57BL/6 mice and the chronic model of adoptive T-cell transfer in SCID mice. At disease induction, ON104 was given twice a week for 8 weeks (chronic colitis) or three times for 1 week (acute colitis). Body weight, stool consistency, and rectal bleeding were assessed to evaluate colitis severity. At the end of the DSS-induced colitis model, colons were dissected for immuno-histological examinations. Results ON104 antibody, specifically directed against oxMIF, was completely devoid of Fcγ receptors binding and cytokine triggering in the presence of immune cells. Treatment with ON104 in the chronic colitis model substantially attenuated clinical signs of colitis by thwarting body weight loss and restoring stool consistency. In the acute DSS colitis model, oxMIF neutralization was able to reduce mucosal thickness and colonic immune cell infiltration compared to vehicle-treated mice. Conclusion Our findings substantiate the role of oxMIF in the pathogenesis of experimental colitis. Thus, targeting oxMIF may represent a new and promising treatment option for IBD.

COLON TARGETED DELIVERY AND STABILIZATION OF MONOCLONAL ANTIBODIES FOR LOCAL TREATMENT OF INFLAMMATORY BOWEL DISEASES

Abstract In the era of biologics where IBD treatment has been transformed by potent antibody-based therapeutics, a key challenge still remains, to develop orally administered antibody medicines for chronic GI inflammation. This is primarily due to the challenges in accurate oral targeted delivery of drugs to the lower GI tract and the inherent enzymatic instability of antibodies in these harsh luminal environment. As a result, all antibody therapeutics in clinical development and on the market are available only as an injection leading to undesired adverse effects due to high systemic exposure and suboptimal therapeutic response due to insufficient antibody levels at the inflamed tissue. We have developed a suite of delivery technologies that are designed to bypass the harsher stomach and small intestinal conditions of the GI tract and achieve accurate targeted release and enzymatic stabilization of the antibody therapeutics in the colon. The delivery platform comprises of a film coating which is applied on the outside of the pill. The coating is a clinically validated polymer coating comprising of a combination of pH sensitive polymer and polysaccharide that is designed to start releasing drugs only when exposed to the correct pH and/or colonic bacteria, dramatically improving on the inconsistent colonic release profiles of solely pH-based coatings. The second component is in the core of the pill, a cocktail of amino acid-based excipients that are mixed together with the antibody and protects the drug from pancreatic and bacterial origin protease enzymes, allowing high luminal antibody concentration build-up that leads to inflamed tissue/systemic uptake through a combination of active and passive diffusion. We demonstrated in an acute DSS mouse model of colitis the effective colonic delivery and stabilization of an anti-IL6 antibody leading to superior uptake into the tissue compared IP injection. The superior tissue level demonstrated higher downregulation of target IL6. We are working to leverage the delivery technology for oral delivery of a variety of anti-inflammatory and epithelial wound healing protein modalities such as monoclonal antibodies, fusion proteins, cytokines, VHHs and peptides to allow for broad therapeutic interventions in indications such as Crohn’s disease, ulcerative colitis, pouchitis and celiac disease. Mechanism of oral colon targeted delivery of therapeutic antibodies in local treatment of inflammatory bowel disease. In-vivo PK of anti-IL6 antibody in acute DSS colitis mouse model. A. Impact of stabilization on antibody levels in the colon lumen compared to naked antibody and injected antibody. B. Impact of stabilization on colon tissue uptake. C. Effect of tissue conc. on colon tissue IL6 target downregulation.

Enteric glial adenosine 2B receptor signaling mediates persistent epithelial barrier dysfunction following acute DSS colitis

Intestinal epithelial barrier function is compromised in inflammatory bowel disease and barrier dysfunction contributes to disease progression. Extracellular nucleotides/nucleosides generated in gut inflammation may regulate barrier function through actions on diverse cell types. Enteric glia modulate extracellular purinergic signaling and exert pathophysiological effects on mucosal permeability. These glia may regulate inflammation with paracrine responses, theoretically mediated via adenosine 2B receptor (A2BR) signaling. As the cell-specific roles of A2BRs in models of colitis and barrier dysfunction are unclear, we studied glial A2BRs in acute dextran sodium sulfate (DSS) colitis. We performed and validated conditional ablation of glial A2BRs in Sox10CreERT2+/−;Adora2bf/f mice. Overt intestinal disease activity indices in DSS-colitis were comparable between Sox10CreERT2+/–;Adora2bf/f mice and littermate controls. However, ablating glial A2BRs protected against barrier dysfunction following acute DSS-colitis. These benefits were associated with the normalization of tight junction protein expression and localization including claudin-1, claudin-8, and occludin. Glial A2BR signaling increased levels of proinflammatory mediators in the colon and cell-intrinsic regulation of genes including Csf3, Cxcl1, Cxcl10, and Il6. Our studies show that glial A2BR signaling exacerbates immune responses during DSS-colitis and that this adenosinergic cell-specific mechanism contributes to persistent gut epithelial barrier dysfunction.

A53 A NOVEL PH-SENSITIVE OPIOID ANALOGUE PROVIDES SUSTAINED ANALGESIA DURING COLONIC INFLAMMATION AND LACKS COMMON SIDE EFFECTS

BackgroundOpioids provide effective pain relief during flares of inflammatory bowel disease but are limited by serious side effects. We showed that acute administration of a novel pH-sensitive opioid agonist, NFEPP, had potent analgesic effects in inflamed acidified colonic tissues without opioid typical side effects. However, the effects of repeated application of NFEPP during the course of an acute flare of colonic inflammation are unknown.AimsTo assess the analgesic and side effect profile resulting from repeated NFEPP applications during the course of acute colitis.MethodsAcute colitis in C57BL/6 mice was induced via 2.5% dextran sulfate sodium dissolved in drinking water for 5 days. Mice were then randomly group assigned to either vehicle, fentanyl or NFEPP. Drugs and vehicle were administered sc. BID for 5 days (0.4 mg/kg), with a final injection on Day 6 (Day 1–6). Visceral nociception was evaluated by performing visceromotor responses (VMRs) to colorectal distension at different time points during colitis. Side effects were assessed using 1) a combination of oral contrast-enhanced abdominal CT-scans (before/after treatment) and defecation assessments to analyze gastrointestinal transit and isometric tension recordings to evaluate colon motility 2) pulse oximeter measurements to reveal cardiorespiratory effects. Inflammation was assessed by histological scoring, myeloperoxidase activity assay and tissue pH measurement of colon samples.ResultsNFEPP decreased VMRs to colorectal distension over the entire period of acute DSS colitis (Day 6: 40.54%, p<0.001, reduction compared to baseline). However, strongest VMR inhibition was observed at Day 3 (66.7%, p<0.05), concordant with the peak of inflammation (MPO: 3.5 U/mg tissue, p<0.001; tissue Damage-Score: 3.0, p<0.001; tissue pH: ΔpH= -0.28, p=0.001, all compared to healthy control). Treatment with NFEPP did not delay gastrointestinal transit (GIT, p=0.37, compared to baseline) nor fecal output (p=0.45, compared to vehicle) whereas fentanyl decreased transit (GIT: p<0.05; fecal output: p<0.05). Colonic contractile responses evoked by electrical field stimulation were reduced in fentanyl treated mice compared to NFEPP (10 Hz: p<0.05). Fentanyl significantly reduced oxygen saturation (15 min: 85% SpO2, p<0.01) and caused a sustained reduction in heart rate at Day 2 (60 min: Δ BPM -91.8 from baseline, p=0.001) whereas NFEPP did not affect oxygen saturation (p=0.95) and revealed minor, transient effects on heart rate (15 min: Δ BPM -75.6, p<0.01) which recovered after 60 min.ConclusionsProlonged NFEPP administration effectively inhibits visceral nociception during acute colitis in a preclinical mouse model without altering gastrointestinal transit, colon motility and oxygen saturation.Funding AgenciesCCC

Smectite as a Preventive Oral Treatment to Reduce Clinical Symptoms of DSS Induced Colitis in Balb/c Mice.

Natural smectites have demonstrated efficacy in the treatment of diarrhea. The present study evaluated the prophylactic effect of a diosmectite (FI5pp) on the clinical course, colon damage, expression of tight junction (TJ) proteins and the composition of the gut microbiota in dextran sulfate sodium (DSS) colitis. Diosmectite was administered daily to Balb/c mice from day 1 to 7 by oral gavage, followed by induction of acute DSS-colitis from day 8 to 14 (“Control”, n = 6; “DSS”, n = 10; “FI5pp + DSS”, n = 11). Mice were sacrificed on day 21. Clinical symptoms (body weight, stool consistency and occult blood) were checked daily after colitis induction. Colon tissue was collected for histological damage scoring and quantification of tight junction protein expression. Stool samples were collected for microbiome analysis. Our study revealed prophylactic diosmectite treatment attenuated the severity of DSS colitis, which was apparent by significantly reduced weight loss (p = 0.022 vs. DSS), disease activity index (p = 0.0025 vs. DSS) and histological damage score (p = 0.023 vs. DSS). No significant effects were obtained for the expression of TJ proteins (claudin-2 and claudin-3) after diosmectite treatment. Characterization of the microbial composition by 16S amplicon NGS showed that diosmectite treatment modified the DSS-associated dysbiosis. Thus, diosmectites are promising candidates for therapeutic approaches to target intestinal inflammation and to identify possible underlying mechanisms of diosmectites in further studies.

Macrophages Compensate for Loss of Protein Tyrosine Phosphatase N2 in Dendritic Cells to Protect from Elevated Colitis.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.

P025 Identification and validation of predictors for tofacitinib through supervised and unbiased approaches in Inflammatory Bowel Disease

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by an overwhelming gut inflammation, where CD4+ effector T cells are main mediators of the inflammatory response. Tofacitinib, a small molecular drug recently used in IBD patients, blocks the JAK/STAT signaling pathway necessary for CD4+ effector T-cell activation. However, clinical data show that a percentage of patients do not respond to the treatment. Our main goal is to identify biomarkers predicting the response of patients to tofacitinib. Methods Tofacitinib efficacy was studied in vivo in wild type (WT) and T-cell-specific PTPN2 deficient mice (CD4-Cre;Ptpn2 floxed) in which the JAK/STAT signaling pathway is over activated. WT and PTPN2 deficient mice were gavaged with tofacitinib (50mg/kg, twice daily) or vehicle. Acute DSS-colitis was induced. Colitis development was evaluated by weight loss, colonoscopy and histology. CD4+ T cells were isolated from the colon and analyzed by flow cytometry. To study the effect of tofacitinib on T-cell differentiation, we isolated naïve T cells from mouse spleen and polarized them in vitro to different T-cell subsets with or without tofacitinib. CD4+ T cells differentiation and cytokine production were analyzed by flow cytometry. To evaluate the influence of tofacitinib on human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs) from healthy donors and IBD patients were stimulated in presence of tofacitinib, and analyzed by flow cytometry. Results While no protective effect was found after tofacitinib treatment in WT mice, PTPN2 deficient mice were protected from colitis based on less weight loss, lower endoscopic and histological scores. The expression of pro-inflammatory cytokines such as IL-17 and IFN-γ by colonic CD4+ T cells was also decreased by tofacitinib. Consistent with the in vivo observations, in vitro experiments revealed a strong impact of tofacitinib on CD4+ T-cells cytokine production. In PBMCs from IBD patients, IFN-γ and TNF-α expression was strongly impacted. In contrast, in healthy donors, IL-10 was the most impacted cytokine. Finally, tofacitinib decreased the in vitro differentiation of Th1, Th2, Th17, Th22, Treg and Tr1. Conclusion In the T-cell-specific PTPN2 deficient mice, tofacitinib exerts a protective effect after DSS-induced colitis. In line with the in vivo findings, in vitro experiments show that tofacitinib has a strong impact on pro-inflammatory cytokine production, especially in the IBD patients. Taken together, these data suggest that tofacitinib might be suitable primarily for IBD patients where the JAK/STAT signaling pathway is over activated.

5-Aminolevulinic Acid as a Novel Therapeutic for Inflammatory Bowel Disease.

5-Aminolevulinic acid (5-ALA) is a naturally occurring nonprotein amino acid licensed as an optical imaging agent for the treatment of gliomas. In recent years, 5-ALA has been shown to possess anti-inflammatory and immunoregulatory properties through upregulation of heme oxygenase-1 via enhancement of porphyrin, indicating that it may be beneficial for the treatment of inflammatory conditions. This study systematically examines 5-ALA for use in inflammatory bowel disease (IBD). Firstly, the ex vivo colonic stability and permeability of 5-ALA was assessed using human and mouse fluid and tissue. Secondly, the in vivo efficacy of 5-ALA, in the presence of sodium ferrous citrate, was investigated via the oral and intracolonic route in an acute DSS colitis mouse model of IBD. Results showed that 5-ALA was stable in mouse and human colon fluid, as well as in colon tissue. 5-ALA showed more tissue restricted pharmacokinetics when exposed to human colonic tissue. In vivo dosing demonstrated significantly improved colonic inflammation, increased local heme oxygenase-1 levels, and decreased concentrations of proinflammatory cytokines TNF-α, IL-6, and IL-1β in both plasma and colonic tissue. These effects were superior to that measured concurrently with established anti-inflammatory treatments, ciclosporin and 5-aminosalicylic acid (mesalazine). As such, 5-ALA represents a promising addition to the IBD armamentarium, with potential for targeted colonic delivery.

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.

DOP14 TiO2 nanoparticles abrogate the protective effect of the autoimmunity-associated PTPN22R619W variant during acute DSS colitis

DOP14 TiO2 nanoparticles abrogate the protective effect of the autoimmunity-associated PTPN22R619W variant during acute DSS colitis

Administration of the Hyper-immune Bovine Colostrum Extract IMM-124E Ameliorates Experimental Murine Colitis.

Inflammatory bowel disease [IBD] is accompanied by lesions in the epithelial barrier, which allow translocation of bacterial products from the gut lumen to the host's circulation. IMM-124E is a colostrum-based product containing high levels of anti-E.coli-LPS IgG, and might limit exposure to bacterial endotoxins. Here, we investigated whether IMM-124E can ameliorate intestinal inflammation. Acute colitis was induced in WT C57Bl/6J mice by administration of 2.5% dextran sodium sulphate [DSS] for 7 days. T cell transfer colitis was induced via transfer of 0.5 x 106 naïve T cells into RAG2-/- C57Bl/6J mice. IMM-124E was administered daily by oral gavage, either preventively or therapeutically. Treatment with IMM-124E significantly ameliorated colitis in acute DSS colitis and in T cell transfer colitis. Maximum anti-inflammatory effects were detected at an IMM-124E concentration of 100 mg/kg body weight, whereas 25 mg/kg and 500 mg/kg were less effective. Histology revealed reduced levels of infiltrating immune cells and less pronounced mucosal damage. Flow cytometry revealed reduced numbers of effector T helper cells in the intestine, whereas levels of regulatory T cells were enhanced. IMM-124E treatment reduced the DSS-induced increase of serum levels of lipopolysaccharide [LPS]-binding protein, indicating reduced systemic LPS exposure. Our results demonstrate that oral treatment with IMM-124E significantly reduces intestinal inflammation, via decreasing the accumulation of pathogenic T cells and concomitantly increasing the induction of regulatory T cells. Our study confirms the therapeutic efficacy of IMM-124E in acute colitis and suggests that administration of IMM-124E might represent a novel therapeutic strategy to induce or maintain remission in chronic colitis.

Lack of the pH-sensing Receptor TDAG8 [GPR65] in Macrophages Plays a Detrimental Role in Murine Models of Inflammatory Bowel Disease.

Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naïve T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.

OP038 Hobit- and Blimp1-driven tissue resident memory T cells critically control chronic intestinal inflammation

Tissue resident memory T (TRM) cells expressing CD69 and often co-expressing CD103 reside in peripheral tissues without recirculating. In contrast to the “traditional” central and effector memory T-cell subsets, they form a first-line of adaptive immune defense in mucosal tissues. An important role in anti-viral immunity and cancer is well established, but their contribution to inflammatory bowel diseases is unknown. Recently, the transcription factors Hobit and Blimp were identified as crucial regulators of mouse TRM cell development. TRM cell markers in human gut samples from IBD patients and controls were analysed by qPCR, immunohistochemistry and flow cytometry. Hobit × Blimp1 double knockout, Hobit knockout and wildtype mice were used in experimental colitis models. Significantly more TRM cells were identified in active Crohn’s disease and ulcerative colitis compared with gut tissue from control patients (Figure 1A). Similarly, expression of CD69 as main residency marker was elevated in IBD vs. controls and in patients with high vs. low disease activity. The expression of several pro-inflammatory cytokines was significantly correlated with the abundance of CD69 in the inflamed mucosa. Moreover, flare-free survival in both Crohn’s disease and ulcerative colitis patients with high CD69 expression was significantly lower than in patients with low CD69 expression (Figure 1B). Acute DSS colitis in Hobit × Blimp1 double knockout (DKO) mice was markedly less severe compared with wildtype mice as demonstrated by analysis of weight, endoscopy, histology and in vivo imaging of reactive oxygen species (Figure 1C). Chronic DSS colitis and T-cell transfer colitis experiments yielded compatible results. The expression of TRM cell markers and pro-inflammatory cytokines in Hobit × Blimp1 DKO mice were reduced. Corroborating these data, local depletion of colon TRM cells using an alternative method protected from subsequent TNBS-induced colitis. Intestinal numbers of CD69+CD103+ TRM cells in IBD vs. control. (B) Flare-free survival in patients with high vs. low CD69 mRNA expression. (C) Weight curve and endoscopic phenotype of Hobit x Blimp DKO, Hobit KO and WT mice in acute DSS colitis. Our data are consistent with a crucial role of TRM cells in the pathogenesis of IBD. Thus, TRM cells appear to be important for the orchestration of inflammatory networks not only in the context of pathogen defense but also in the case of dysregulated immune cell activation in response to luminal antigens. TRM cells might thus represent an interesting future target for IBD therapy.

An HDAC6 Inhibitor Confers Protection and Selectively Inhibits B-Cell Infiltration in DSS-Induced Colitis in Mice.

Small molecule histone deacetylase (HDAC) inhibitors with anti-inflammatory activity may be candidates for targeting intestinal inflammatory pathways in inflammatory bowel disease (IBD). This study investigated whether treatment with a potent HDAC6 inhibitor, BML-281, could protect against colonic inflammation and prevent inflammatory cell infiltration into the colon to drive disease pathology in a mouse model of acute dextran sodium sulfate (DSS) colitis. Control and acute DSS-colitis mice were treated with BML-281 (1 mg/kg per day s.c. and 10 mg/kg per day s.c.) for 8 days. Changes in disease pathology, colonic structure, function, alterations in inflammatory milieu, together with colonic inflammatory cell flux, were assessed by weight loss and disease activity index in vivo and by flow cytometry, gene expression, and histology ex vivo. Anti-inflammatory responses of BML-281 on human polymorphonuclear leukocytes were assessed in vitro. Administration of BML-281 to DSS-treated mice attenuated colitis, weight loss, and disease pathology, including changes in colon structure and function, by eliciting broad-spectrum anti-inflammatory effects and preventing infiltration and activation of key immune cells in the lamina propria of the intestinal epithelium. Among different immune cells, BML-281 particularly suppressed the infiltration of CD19+ B-cells into the inflamed colonic lamina propria. This study supports the targeting of HDAC6 as an anti-inflammatory strategy for treating colon inflammation progressing to IBD. Some HDAC inhibitors are used in the clinic to treat cancer, and the results here for BML-281 highlight the potential for HDAC6 inhibitors to be used in a clinical setting for preventing and treating colonic inflammation and IBD in humans.

Cell-specific Activation of the Nrf2 Antioxidant Pathway Increases Mucosal Inflammation in Acute but Not in Chronic Colitis.

The transcription factor Nrf2 is a major modulator of the cellular antioxidant response. Oxidative burst of infiltrating macrophages leads to a massive production of reactive oxygen species in inflamed tissue of inflammatory bowel disease patients. This oxidative burst contributes to tissue destruction and epithelial permeability, but it is also an essential part of the antibacterial defence. We therefore investigated the impact of the Nrf2 orchestrated antioxidant response in both acute and chronic intestinal inflammation. To study the role of Nrf2 overexpression in mucosal inflammation, we used transgenic mice conditionally expressing a constitutively active form of Nrf2 [caNrf2] either in epithelial cells or in the myeloid cell lineage. Acute colitis was induced by dextran sulphate sodium [DSS] in transgenic and control animals, and changes in gene expression were evaluated by genome-wide expression studies. Long-term effects of Nrf2 activation were studied in mice with an IL-10-/- background. Expression of caNrf2 either in epithelial cells or myeloid cells resulted in aggravation of DSS-induced acute colitis. Aggravation of inflammation by caNrf2 was not observed in the IL-10-/- model of spontaneous chronic colitis, where even a trend towards reduced prolapse rate was observed. Our findings show that a well-balanced redox homeostasis is as important in epithelial cells as in myeloid cells during induction of colitis. Aggravation of acute DSS colitis in response to constitutive Nrf2 expression emphasises the importance of tight regulation of Nrf2 during the onset of intestinal inflammation.

Changes in the composition of intestinal fungi and their role in mice with dextran sulfate sodium-induced colitis.

Intestinal fungi are increasingly believed to greatly influence gut health. However, the effects of fungi on intestinal inflammation and on gut bacterial constitution are not clear. Here, based on pyrosequencing method, we reveal that fungal compositions vary in different intestinal segments (ileum, cecum, and colon), prefer different colonization locations (mucosa and feces), and are remarkably changed during intestinal inflammation in dextran sulfate sodium (DSS)-colitis mouse models compare to normal controls: Penicillium, Wickerhamomyces, Alternaria, and Candida are increased while Cryptococcus, Phialemonium, Wallemia and an unidentified Saccharomycetales genus are decreased in the guts of DSS-colitis mice. Fungi-depleted mice exhibited aggravated acute DSS-colitis associated with gain of Hallella, Barnesiella, Bacteroides, Alistipes, and Lactobacillus and loss of butyrate-producing Clostridium XIVa, and Anaerostipes compare with normal control. In contrast, bacteria-depleted mice show attenuated acute DSS-colitis. Mice with severely chronic recurrent DSS-colitis show increased plasma (1,3)-β-D-glucan level and fungal translocation into the colonic mucosa, mesenteric lymph nodes and spleen. This work demonstrate the different roles of fungi in acute and chronic recurrent colitis: They are important counterbalance to bacteria in maintaining intestinal micro-ecological homeostasis and health in acutely inflamed intestines, but can harmfully translocate into abnormal sites and could aggravate disease severity in chronic recurrent colitis.