Dextran Sulfate Sodium Colitis Research Articles

A Novel IBD‐Associated Mouse Pathobiont E. coli Causes Disease and Prevents Recovery from Colitis

IntroductionExpansion of intestinal pathobionts, such as adherent‐invasive E. coli (AIEC), are strongly implicated in the pathogenesis of Inflammatory Bowel Disease (IBD) as AIEC increases pro‐inflammatory cytokine production and alters tight junction protein regulation, suggesting a potential mechanism of pathogen‐induced barrier dysfunction and inflammation. In mice lacking the IBD risk gene, protein tyrosine phosphatase non‐receptor Type 2 (PTPN2), we previously showed increased abundance of a novel mouse AIEC (mAIEC) which had sequence match identity of a 250bp segment to the human IBD‐associated AIEC, LF82. We functionally validated the ability of mAIEC to adhere to and invade intestinal epithelial cells in vitro. The aim of this study was to determine if mAIEC can cause disease in vivo and whether it requires other bacteria to support its pathogenicity in mice.Methods(1) Germ‐free ~12‐week old C57Bl/6 male and female mice; or (2) wild‐type 10‐week old confirmed segmented filamentous bacteria (SFB)‐free C57Bl/6 female mice (JAX labs, Stock# 000664) ± 3% dextran‐sodium sulfate (DSS) for 7 days housed in a specific pathogen‐free (SPF) vivarium; were orally gavaged with PBS or gavaged with 109 CFU of either pure mAIEC or K12 at exponential‐phase growth. Germ‐free mice received fecal microbiome transplant (FMT) by oral gavage of fresh caecal content from a Ptpn2‐KO (KO‐FMT) mouse at day 22 post‐mAIEC colonization. Disease activity index (DAI) was assessed by measuring weight loss, physical activity, general appearance, and stool consistency.ResultsmAIEC infected SFB‐free mice had a significant decrease in body weight development (P = 0.002, n = 5) but no apparent increase in DAI compared with the control E. coli K12 or PBS conditions. Infection of germ‐free mice with mAIEC did not alter body weight development compared with K12 infected mice. However, recolonization with KO‐FMT post‐mAIEC infection caused sustained weight loss in male but not female mice compared with K12 mice (P = 0.02, n = 3). To identify the effects of mAIEC infection on inflammation, SFB‐free mice were orally gavaged with mAIEC on days 0–3 of DSS administration. mAIEC+DSS treated mice showed a more severe drop in body weight development, increased disease severity (P < 0.001, n = 4–5), and a worse barrier defect associated with translocation of bacteria to the spleen and liver (P ≤ 0.002, n = 3–5) compared with DSS+PBS, DSS+K12 or non‐DSS treated PBS, mAIEC or K12 alone control groups. When administered to mice with established DSS colitis, mAIEC prevented recovery from DSS‐induced colitis compared with K12 and PBS conditions (P ≤ 0.009, n = 5).ConclusionsWe describe for the first time a novel IBD‐associated mouse AIEC that can cause or worsen disease in a host, but requires other bacteria to support its pathogenicity. This study generates new insights into environment‐microbiome interactions in IBD.Support or Funding InformationCrohn’s and Colitis Foundation Senior Research Award (D.F.M.); NIH‐2R01‐DK091281 (D.F.M.); American Gastroenterological Association IBD Research Award (D.F.M.). UCR Office of Research & Economic Development Pilot Award (D.F.M. & J.B.).

Nix-Mediated Mitophagy Modulates Mitochondrial Damage During Intestinal Inflammation.

Aims: Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study was to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies. Results: In the intestinal epithelium of patients with ulcerative colitis, we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type mice undergoing experimental colitis. An increase in NIX expression was found to depend on stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the Nix promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global Nix-/- mouse to dextran sodium sulfate colitis and found that these mice developed worse disease. In addition, Nix-/- mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria. Innovation: These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis. Conclusion: NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.

Obacunone Protects Against Ulcerative Colitis in Mice by Modulating Gut Microbiota, Attenuating TLR4/NF-κB Signaling Cascades, and Improving Disrupted Epithelial Barriers.

Obacunone, a natural limonoid compound abundantly distributed in citrus fruits, possesses various biological properties, such as antitumor, antioxidant, and antiviral activities. Recent studies suggested an anti-inflammatory activity of obacunone in vitro, but its efficacy on intestinal inflammation remains unknown. This study was designed to evaluate the effects and mechanisms of obacunone in ameliorating intestinal inflammation in a mouse model of ulcerative colitis (UC). We found that obacunone efficiently alleviated the severity of dextran sulfate sodium (DSS)-induced mouse UC by modulating the abnormal composition of the gut microbiota and attenuating the excessive activation of toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling. The intestinal epithelial barrier was disrupted in DSS colitis mice, which was associated with activation of inflammatory signaling cascades. However, obacunone promoted the expression of tight junction proteins (TJP1 and occludin) and repressed the activation of inflammatory signaling cascades. In summary, our findings demonstrated that obacunone attenuated the symptoms of experimental UC in mice through modulation of the gut microbiota, attenuation of TLR4/NF-κB signaling cascades, and restoration of intestinal epithelial barrier integrity.

Near infrared readouts offer sensitive and rapid assessments of intestinal permeability and disease severity in inflammatory bowel disease models

Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts—namely high background—IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

Expanding the utility of the dextran sulfate sodium (DSS) mouse model to induce a clinically relevant loss of intestinal barrier function.

BackgroundInflammatory bowel disease (IBD) is a family of debilitating disorders that affects more than 1 million people in the United States. Many animal studies of IBD use a dextran sulfate sodium (DSS) mouse model of colitis that induces rapid and severe colitis symptoms. Although the typical seven-day DSS model is appropriate for many studies, it destroys intestinal barrier function and results in intestinal permeability that is substantially higher than what is typically observed in patients. As such, therapies that enhance or restore barrier integrity are difficult or impossible to evaluate.MethodsWe identify administration conditions that result in more physiologically relevant intestinal damage by systematically varying the duration of DSS administration. We administered 3.0% DSS for four to seven days and assessed disease metrics including weight, fecal consistency, intestinal permeability, spleen weight, and colon length. Histology was performed to assess the structural integrity of the intestinal epithelium.ResultsExtended exposure (seven days) to DSS resulted in substantial, unrecoverable loss of intestinal structure and intestinal permeability increases of greater than 600-fold. Attenuated DSS administration durations (four days) produced less severe symptoms by all metrics. Intestinal permeability increased only 8-fold compared to healthy mice, better recapitulating the 2–18 fold increases in permeability observed in patients. The attenuated model retains the hallmark properties of colitis against which to compare therapeutic candidates. Our results demonstrate that an attenuated DSS colitis model obtains clinically relevant increases in intestinal permeability, enabling the effective evaluation of therapeutic candidates that promote barrier function.

Caveolin-1 Scaffolding Domain Peptide Regulates Colon Endothelial Cell Survival through JNK Pathway.

It has been reported that pathological angiogenesis contributes to both experimental colitis and inflammatory bowel disease. Recently, we demonstrated that endothelial caveolin-1 plays a key role in the pathological angiogenesis of dextran sodium sulfate (DSS) colitis. However, the molecular mechanism of caveolin-1 regulation of endothelial function is unknown. In this study, we examined how the antennapedia- (AP-) conjugated caveolin-1 scaffolding domain (AP-Cav) modulates vascular endothelial growth factor- (VEGF-) dependent colon endothelial cell angiogenic responses, as seen during colitis. We used mouse colon endothelial cells and found that AP-Cav significantly inhibited VEGF-mediated bromodeoxyuridine (BrdU) incorporation into colon microvascular endothelial cells. AP-Cav significantly blunted VEGF-dependent extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation at 10 minutes and 2 hours after stimulation, compared with the AP control peptide. AP-Cav + VEGF-A treatment also significantly increased c-Jun N-terminal kinase (JNK) phosphorylation at 2 hours. AP-Cav + VEGF-A treatment significantly downregulated retinoblastoma (Rb) protein levels, upregulated cleaved caspase-3 protein levels at 4 hours, and induced apoptosis. Thus, our study suggests that disruption of endothelial caveolin-1 function via the AP-Cav diverts VEGF signaling responses away from endothelial cell proliferation and toward apoptosis through the inhibition of mitogen-activated protein (MAP) kinase signaling and the induction of JNK-associated apoptosis.

TRAF5 Deficiency Ameliorates the Severity of Dextran Sulfate Sodium Colitis by Decreasing TRAF2 Expression in Nonhematopoietic Cells.

TNFR-associated factor 5 (TRAF5) is a cytosolic adaptor protein and functions as an inflammatory regulator. However, the in vivo function of TRAF5 remains unclear, and how TRAF5 controls inflammatory responses in the intestine is not well understood. In this study, we found that intestinal epithelial cells from Traf5-/- mice expressed a significantly lower level of NF-κB-regulated proinflammatory genes, such as Tnf, Il6, and Cxcl1, as early as day 3 after dextran sulfate sodium (DSS) exposure when compared with wild-type mice. The intestinal barrier integrity of DSS-treated Traf5-/- mice remained intact at this early time point, and Traf5-/- mice showed decreased body weight loss and longer colon length at later time points. Surprisingly, the protein level of TRAF2, but not TRAF3, was reduced in colon tissues of Traf5-/- mice after DSS, indicating the requirement of TRAF5 for TRAF2 protein stability in the inflamed colon. Experiments with bone marrow chimeras confirmed that TRAF5 deficiency in nonhematopoietic cells caused the attenuated colitis. Our in vitro experiments demonstrated that proinflammatory cytokines significantly promoted the degradation of TRAF2 protein in Traf5-/- nonhematopoietic cells in a proteasome-dependent manner. Collectively, our data suggest a novel regulatory function of TRAF5 in supporting the proinflammatory function of TRAF2 in nonhematopoietic cells, which may be important for acute inflammatory responses in the intestine.

A44 NOVEL AND HIGHLY SENSITIVE FLOW CYTOMETRIC BASED METHOD FOR CONTINUOUS TRACKING OF INTESTINAL PERMEABILTIY

Abstract Background Inflammatory bowel diseases (IBD) are chronic, inflammatory conditions of the intestinal tract. In addition to a complex mixture of genetic, and environmental factors, increased intestinal permeability is also thought to be involved. Despite its diagnostic importance, there is no reliable yet minimally invasive way to measure intestinal permeability in patients and animal models. Currently, most assays involve the detection of orally given sugar molecules in either the urine (MLR method) or the plasma (FITC-dextran). These methods offer only limited accuracy, and do not allow continual tracking of intestinal permeability within the same animal due to the requirement of euthanization. Herein, we describe a novel cytometric-based method using an ingested dietary protein (ovalbumin (OVA)) and flow cytometry. We show our method is reliable, highly sensitive, minimally invasive and allows for the continuous tracking of intestinal permeability within the same individual using small blood volumes. Aims To compare the utility of our bead-based method of intestinal permeability measurement to the FITC-Dextran method. Methods Wildtype (WT) C57BL/6 mice, and mucin 2-deficient (Muc2-/-) mice (known to suffer a leaky gut) at baseline, or mice given chemical or infection-induced colitis were gavaged with a solution containing OVA protein and FITC-Dextran. After 6 hours, mice were euthanized to collect plasma for spectrophotometry (FITC-Dextran), or for flow cytometry (bead-based ELISA) analyses. Results We found that both OVA and FITC-dextran were detectable in plasma samples of the Muc2-/- mice, but not the WT mice, 6 hours after oral co-administration, however the bead-based method produced more consistent readings. Immunostaining of tissue sections with the same antibodies also showed that OVA can readily diffuse through the gut epithelium of Muc2-/- mice but not the WT mice, validating the specificity of the antibodies. We also tested our assay on WT mice undergoing dextran sodium sulfate (DSS) colitis, or infected with Citrobacter rodentium. We found that our method could detect statically significant changes in intestinal permeability 2 to 3 days earlier than the FITC-dextran method, while providing greatly reduced variability between technical repeats. More importantly, we were able to use a small volume (5 μL) of whole blood collected via tail poke, to measure intestinal permeability without requiring euthanization of the mice. This allowed the continuous tracking of permeability changes within the same animal. Conclusions We conclude that the bead-based method is more sensitive and reliable than the FITC-Dextran method as tested in several murine colitis models. More importantly, we showed that the bead-based method allows continual tracking of intestinal permeability within the same animal, enabling time course measurements. Funding Agencies CAG, CCC, CIHR, NRCBC Children’s Research Institute

Inhibitory effect of Bifidobacterium bifidum ATCC 29521 on colitis and its mechanism

Probiotics are known to be beneficial in preventing different diseases in model animals, including inflammatory bowel disease. However, there are few studies on probiotics related to miRNA regulation and disease status. In this article, the beneficial role and mechanisms of the probiotic strain Bifidobacterium bifidum ATCC 29521 have been studied in ulcerative colitis using dextran sodium sulphate (DSS) model. Male C57JBL/6 mice were randomly divided into three groups (n=7): Normal group, dextran sulphate sodium (DSS) group, and Bifido group gavage with Bifidobacterium bifidum ATCC 29521 (2×108 CFU/day). Our strain restored the DSS-caused damage by regulating the expression of immune markers and tight junction proteins (TJP) in the colon; briefly by up-regulating ROS-scavenging enzymes (SOD1, SOD2, CAT, and GPX2), anti-inflammatory cytokines (IL-10, PPARγ, IL-6), TJP's (ZO-1, MUC-2, Claudin-3, and E Cadherin-1) and downregulating inflammatory genes (TNF-α, IL-1β) in Bifido group mice. Inflammatory markers appeared to be regulated by NF-κB nuclear P65 subunit, and its translocation was inhibited in Bifido group mice colon. In addition, the expression of inflammatory genes and colonic TJP were also associated with the restoration of miRNAs (miR-150, miR-155, miR-223) in B. bifidum ATCC 29521 treated Bifido group. The dysbiosis executed by DSS was restored in the Bifido group, demonstrating that B. bifidum ATCC 29521 possessed a probiotic role in our DSS colitis mouse model. B. bifidum ATCC 29521 exhibited its probiotic role through its anti-inflammatory role by modulating miRNA-associated TJP and NF-κB regulation and by partially restoring dysbiosis.

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.

Role of the δ-Opioid Receptor in 2 Murine Models of Colitis.

Crohn disease and ulcerative colitis, collectively referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the gastrointestinal tract. Currently, the etiology of IBD is unknown, and immunosuppressive therapies have become the standard of care to reduce the inflammation; however, these agents only induce remission 50% of the time in patients and can have serious side effects. Recently, endogenous opioids and opioid receptors have been shown to play a role in the mediation of inflammation. In addition, opioid receptor blockade with a nonselective antagonist, naltrexone, has been shown to reduce colitis in both murine models and human subjects. The goal of the current study was to determine if the antiinflammatory effects of naltrexone are mediated through the delta (δ) opioid receptor. Male C57BL/6NCrl (6 to 8 wk.; n = 110) and female BALB/cAnNCrl (6-8 wk.; n = 91) mice were studied using 2 animal models of chemically induced colitis: dextran sodium sulfate (DSS) and 2, 4, 6-trinitrobenzenesulfonic acid (TNBS). The selective δ-receptor antagonists naltrindole and 7-benzylidenenaltrexone were administered to examine the role of the δ-opioid receptor in colonic inflammation. The quantitative measurement of colitis activity, colon weight and length, Hct, WBC count, and gross and microscopic aberrations were analyzed. Administration of naltrexone in the DSS colitis model significantly improved overall disease activity indices on day 5 of therapy. The use of δ-antagonists and naltrexone had limited to no effect on TNBS colitis. Similar findings were obtained by using the DSS colitis model. Based on the current findings, the authors conclude that naltrexone therapy has limited effect on the improvement of colitis in 2 murine models; however, the δ-opioid receptor was not responsible for mediating the effects.

P015 Tricellulin overexpression protects against elevated macromolecule uptake in DSS colitis mice

Abstract Background In Inflammatory bowel disease, intestinal homeostasis cannot be sustained due to an overreaction of the immune system and a disturbed epithelial barrier due to an impaired tight junction (TJ). At tricellular TJs (tTJs), which are formed where three cells meet, the TJ network is basolaterally expanded and forms a central tube which would be potentially large enough to allow for a paracellular passage of macromolecules. Under normal conditions, the tTJ protein tricellulin (Tric) seals the central tube against luminal uptake. However, in ulcerative colitis (UC), Tric is downregulated, resulting in increased uptake of macromolecules including antigens which may further support the inflammatory processes. Thus, we aimed to identify whether or not overexpression of Tric has a protective effect against inflammation. Methods We generated intestinal epithelial cell-specific overexpression of Tric in mice (B6.C-Tg(Vil-cre) Rosa26tgGFP-Tric). In Tric-overexpressing (Tric-OE) and in wild-type (wt) mice experimental colitis was induced by 3% dextran sodium sulphate (DSS) for 6 days. The effect was assessed by determining the disease activity index (DAI: weight loss, stool consistency, and stool bleeding) and the colon dimensions. Using chamber experiments were performed to determine permeabilities for ions and macromolecules of different sizes (4 and 10 kDa). Results No difference in weight loss was observed between wt and Tric-OE mice upon DSS treatment. However, Tric-OE mice suffered less from diarrhoea and had less bloody stools compared with wt mice resulting in a lower DAI. In addition, DSS caused a reduced colon length which was less prominent in Tric-OE mice than in wt mice. Functional analysis revealed higher permeabilities for Na+ and Cl− after DSS treatment with no significant differences between Tric-OE and wt mice. In contrast, permeabilities for macromolecules of both sizes were unchanged after DSS treatment in Tric-OE mice but elevated in wt-mice. Conclusion Tric-overexpression prevents the epithelial barrier from elevated paracellular uptake of macromolecules during DSS colitis without affecting ion permeability. This protects against DSS colitis as indicated by less affected clinical and structural outcome parameters, most likely via a reduction in a load of immunologically active macromolecules in the subepithelium.

P001 The novel DYRK1a inhibitor VRN024219 alleviates disease severity on the IBD mouse models by modulating T-cell differentiation

Abstract Background DYRK1A belongs to dual-specificity tyrosine (Y) phosphorylation regulated kinase (DYRK) family which is known to be activated through autophosphorylation of tyrosine residues in the activation loop and phosphorylates their substrates on serine and threonine residues. Other members of this family include DYRK1B, DYRK2, DYRK3, and DYRK4. Studies have revealed that DYRK kinase family plays an important role in regulating cell proliferation and apoptosis. DYRK1A has been reported to be strongly expressed in the brain and known to regulate various functions. However, the role and underlying mechanisms of DYRK1a in inflammation in general, specifically in IBD, remain poorly understood. Accordingly, we present the underlying mechanisms of DYRK1a on the course of IBD by using the novel DYRK1a inhibitor VRN024219. Methods First, we tested the effects of our compound VRN024219 on T-cell differentiation using naïve CD4 T cells extracted from the mouse spleen. Then we assessed the efficacy and mechanism of VRN024219 on the dextran sodium sulphate (DSS) and T-cell transfer-induced experimental colitis that mimics human ulcerative colitis (UC), comparing to that of Tofacitinib. Finally, we evaluated the effect of VRN024219 on pro-inflammatory cytokines such as interleukin (IL) −17A, IL-6, and tumour necrosis factor (TNF) α expressed by peripheral blood mononuclear cells (PBMCs) from 20 UC patient samples (Severance Hospital, Seoul, Korea). Results When VRN024219 was treated to splenocyte, the compound significantly downregulated Th17 and enhanced T reg cell differentiation. Protein levels of IL-17a, IL-6, and TNF α were increased in the DSS-induced colitis mice, whereas administration of DYRK1a inhibitor VRN024219 substantially improved clinical score than that of Tofacitinib-treated group. Additionally from T-cell transfer-induced colitis model, VRN024219 treated group demonstrated a larger population of ROR γ T-cell than that of tofacitinib Finally, the protein levels of proinflammatory cytokines were significantly down-regulated in VRN024219 treated 20 patient samples. Conclusion Our data provide clear evidence that the novel DYRK1a inhibitor plays a protective role in DSS- and T-cell transfer-induced colitis which was closely related to a Th17/Treg modulation. Thus, VRN024219 might a promising candidate for a new drug for IBD.

P075 Muc5ac expression protects the colonic barrier in experimental colitis

Abstract Background The mucus gel layer (MGL) lining the colon is integral to exclusion of bacteria and maintaining intestinal homeostasis in health and disease. MGL defects allowing bacteria to directly contact the colonic surface are commonly observed in ulcerative colitis (UC). The major macromolecular component of the colonic MGL is the secreted gel-forming mucin, MUC2, whose expression is essential for homeostasis in health. In UC, another gel-forming mucin, MUC5AC is induced. In mice, Muc5ac is protective during intestinal helminth infection. Here, we tested the expression and functional role of MUC5AC/Muc5ac in colitis patient biopsies and murine colitis. Methods We measured MUC5AC/Muc5ac expression in UC patient biopsies and during acute dextran sulphate sodium (DSS) colitis. We performed DSS-colitis in mice deficient in Muc5ac (Muc5ac−/−) to model the potential functional role of Muc5ac in colitis. To assess MGL integrity, we quantified bacterial–epithelial interaction and translocation to mesenteric lymph nodes (MLNs). Antibiotic treatment was performed to directly investigate the role of colonic bacteria in our murine colitis studies. Results Colonic MUC5AC/Muc5ac mRNA expression increased significantly in active UC and murine colitis. Muc5ac−/− mice experienced worsened injury and inflammation in DSS-colitis compared with controls. This was associated with increased bacterial–epithelial contact and translocation to the MLN. Antibiotic treatment normalised colitis severity in Muc5ac−/− mice to that of antibiotic treated controls. Conclusion We demonstrate for the first time that MUC5AC/Muc5ac induction in acute colitis controls injury by reducing bacterial breach of the MGL. Therefore, developing strategies to induce MUC5AC expression may protect the intestinal barrier in UC.

OP37 Fibrogenesis in chronic DSS colitis is driven by an innate lymphoid cell-independent innate immune response

Abstract Background Obtaining insights in the pathogenesis of intestinal fibrosis is a priority for improving outcomes in inflammatory bowel diseases (IBD). Studies in murine models and human organ fibrosis indicate a role for innate immunity pathways in fibrosis. The aim of this study was to dissect the role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis. Methods A chronic 3-cycles dextran sulphate sodium (DSS) model was induced in wild-type (WT), recombinant activating gene (RAG)-deficient (lacking adaptive immunity), RAG−/− common γ-chain (ɣc)−/− (lacking ILC) and anti-CD90.2 treated (ILC depleted) C57Bl/6 RAG−/− mice. One cycle of DSS comprised 1 week of DSS administration followed by 2 weeks of recovery with normal drinking water. Colonic lamina propria cells were isolated and CD45+Lineage−CD127+CD90.2+ ILC, Ly6C+ monocytes and Ly6G+ neutrophils were identified after staining by flow cytometry. Inflammation and fibrosis were scored by macroscopic and HE and Martius Scarlet Blue staining and fibrosis was evaluated by hydroxyproline quantification. For analysis Kruskal–Wallis testing with multiple Dunn’s comparisons was performed. Results In RAG-1−/− mice chronic inflammation and fibrosis developed similarly as in WT mice, with elevated KLRG-1+ ILC2 (68.90 vs. 48.00% of ILC, p = 0.012) after repeated DSS exposure as compared with control mice (Figure 1). Chronic colitis could also be induced in RAG−/−ɣc−/− or ILC depleted RAG−/− mice (ILC: 0.99 vs. 25.70% of CD45+ cells, p = 0.029), with no attenuation of fibrosis (p > 0.99) as compared with chronic DSS exposed RAG-1−/− mice despite the absence of ILC (Figure 2). Colon length decrease was more pronounced in RAG−/−yc−/− as compared with RAG-1−/− mice after chronic DSS colitis (7.15 vs. 8.30 cm, p = 0.046), while hydroxyproline levels and thickness of mucosa and muscularis propria were not different in RAG−/−yc−/− as compared with RAG-1−/− mice after chronic DSS. Moreover, after the second cycle of DSS a slower recovery of weight was seen in RAG−/−yc−/− mice as compared with RAG-1−/− mice (d31: 88.64 vs. 111.0% of initial weight, p < 0.001; d35 p = 0.001; d39 p = 0.006 and d42 p = 0.002) (Figure 3). In absence of ILC, RAG−/−yc−/− mice increased lamina propria neutrophils (19.10 vs. 5.91% of CD45+ cells, p = 0.004) and monocytes (11.80 vs. 3.25% of CD45+ cells, p = 0.004) may represent an alternative source of inflammation. Conclusion These data argue against a pro-fibrotic role of ILC in the induction of fibrosis in chronic DSS colitis, and suggest a protective and recovery-enhancing role of ILC after repeated intestinal injury.

Fecal IgA Levels and Gut Microbiota Composition Are Regulated by Invariant Natural Killer T Cells.

The gut microbiota is a key element to support host homeostasis and the development of the immune system. The relationship between the microbiota and immunity is a 2-way road, in which the microbiota contributes to the development/function of immune cells and immunity can affect the composition of microbes. In this context, natural killer T cells (NKT cells) are distinct T lymphocytes that play a role in gut immunity and are influenced by gut microbes. In our work, we investigated the involvement of invariant NKT cells (iNKT) in intestinal homeostasis. We found that iNKT-deficient mice (iNKT-KO) had reduced levels of fecal IgA and an altered composition of the gut microbiota, with increased Bacteroidetes. The absence of iNKT cells also affected TGF-β1 levels and plasma cells, which were significantly reduced in knockout (KO) mice. In addition, when submitted to dextran sodium sulfate colitis, iNKT-KO mice had worsening of colitis when compared with wild-type (WT) mice. To further address iNKT cell contribution to intestinal homeostasis, we adoptively transferred iNKT cells to KO mice, and they were submitted to colitis. Transfer of iNKT cells improved colitis and restored fecal IgA levels and gut microbiota. Our results indicate that intestinal NKT cells are important modulators of intestinal homeostasis and that gut microbiota composition may be a potential target in the management of inflammatory bowel diseases.

292 Differential gene expression analysis for piglets supplied dietary prebiotics and arachidonic acid for gastrointestinal disturbances

Abstract Gastrointestinal (GI) disturbances cause significant economic losses in the swine industry. Dietary-based approaches have been well applied to control the inflammation and optimize microbial colonization of the GI tract. This study aimed to screen differentially expressed (DE) RNAs in piglets supplied with arachidonic acid (ARA) or prebiotics in an acute dextran sodium sulfate (DSS) colitis model. Suckling pigs (n = 48) from 6 litters were randomly assigned into 4 diet groups: 0.5% ARA, 0.5% ARA + PRE (4g/L galactooligosaccharide +4 g/L polydextrose), 2.5% ARA, and 2.5% ARA + PRE. On day 17–21, half of the pigs (n = 24) were treated with 0.625 g DSS/kg BW to induce colitis. RNA samples (n = 48) were isolated from the mucosal layer of GI tract on day 22 for the gene expression analysis via RNAseq. The GLIMMIX procedure of SAS (v.9.4) was used to fit the statistical models to gene counts, were the main effects and interactions of PRE, ARA, and DSS were fit in the model as fixed effects and litter as random. Genes with FDR < 0.05 were mapped to the KEGG pathway. A total of 133 DE genes (88 up-regulated, 45 down-regulated) were identified in pigs with colitis compared to healthy ones. PRE and ARA affected gene expression differently but with no interaction effect. PRE supplement decreased the total DE genes from 83 (59 up-regulated, 24 down-regulated) to 33 (16 up-regulated and 17 down-regulated) in pigs with colitis compared with healthy ones. A total of 37 DE genes (21 up-regulated, 16 down-regulated), and 49 DE genes (32 up-regulated, 17 down-regulated) were identified in pigs with colitis compared to healthy ones supplied with 0.5% and 2.5% ARA, respectively. DE genes identified in this study are involved in various signaling pathways with supplied prebiotics significantly changing the gene expression in pigs afflicted with colitis.

Repeated Oral Administration of a KDEL-tagged Recombinant Cholera Toxin B Subunit Effectively Mitigates DSS Colitis Despite a Robust Immunogenic Response.

Cholera toxin B subunit (CTB), a non-toxic homopentameric component of Vibrio cholerae holotoxin, is an oral cholera vaccine antigen that induces an anti-toxin antibody response. Recently, we demonstrated that a recombinant CTB variant with a Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention motif (CTB-KDEL) exhibits colon mucosal healing effects that have therapeutic implications for inflammatory bowel disease (IBD). Herein, we investigated the feasibility of CTB-KDEL for the treatment of chronic colitis. We found that weekly oral administration of CTB-KDEL, dosed before or after the onset of chronic colitis, induced by repeated dextran sodium sulfate (DSS) exposure, could significantly reduce disease activity index scores, intestinal permeability, inflammation, and histological signs of chronicity. To address the consequences of immunogenicity, mice (C57BL/6 or C3H/HeJ strains) were pre-exposed to CTB-KDEL then subjected to DSS colitis and CTB-KDEL treatment. While the pre-dosing of CTB-KDEL elicited high-titer anti-drug antibodies (ADAs) of the immunoglobin A (IgA) isotype in the intestine of C57BL/6 mice, the therapeutic effects of CTB-KDEL were similar to those observed in C3H/HeJ mice, which showed minimal ADAs under the same experimental conditions. Thus, the immunogenicity of CTB-KDEL does not seem to impede the protein’s mucosal healing efficacy. These results support the development of CTB-KDEL for IBD therapy.

Wheel running behaviour in group-housed female mice indicates disturbed wellbeing due to DSS colitis.

Voluntary wheel running (VWR) behaviour is a sensitive indicator of disturbed wellbeing and used for the assessment of individual experimental severity levels in laboratory mice. However, monitoring individual VWR performance usually requires single housing, which itself might have a negative effect on wellbeing. In consideration of the 3Rs principle, VWR behaviour was evaluated under group-housing conditions. To test the applicability for severity assessment, this readout was evaluated in a dextran sodium sulphate (DSS) induced colitis model. For continuous monitoring, an automated system with integrated radio-frequency identification technology was used, enabling detection of individual VWR. After a 14-day adaptation period mice demonstrated a stable running performance. Analysis during DSS treatment in combination with repeated facial vein phlebotomy and faecal sampling procedure resulted in significantly reduced VWR behaviour during the course of colitis and increased VWR during disease recovery. Mice submitted to phlebotomy and faecal sampling but no DSS treatment showed less reduced VWR but a longer-lasting recovery. Application of a cluster model discriminating individual severity levels based on VWR and body weight data revealed the highest severity level in most of the DSS-treated mice on day 7, but a considerable number of control mice also showed elevated severity levels due to sampling procedures alone. In summary, VWR sensitively indicated the course of DSS colitis severity and the impact of sample collection. Therefore, monitoring of VWR is a suitable method for the detection of disturbed wellbeing due to DSS colitis and sampling procedure in group-housed female laboratory mice.