Jose Duarte

Mast cells trigger epithelial barrier dysfunction, bacterial translocation and postoperative ileus in a mouse model

Background  Abdominal surgery involving bowel manipulation commonly results in inflammation of the bowel wall, which leads to impaired intestinal motility and postoperative ileus (POI). Mast cells have shown to play a key role in the pathogenesis of POI in mouse models and human studies. We studied whether mast cells contribute to the pathogenesis of POI by eliciting intestinal barrier dysfunction.

Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages

Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn’s disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.

Mo1738 Efficacy of a Novel JAK1-Specific Inhibitor in a Mouse Model of Acute and Chronic Colitis

Introduction and aim: Indoleamine 2,3-dioxygenase (IDO) is expressed in innate immune cells and acts as the first rate-limiting step in the tryptophan (TRP) catabolism along the kynurenine pathway. Decreased serum TRP levels have been associated with active inflammation in Crohns disease (CD) due to induction of IDO (Gupta, Inflamm Bowel Dis 2012). We studied the effect of infliximab-induced downregulation of inflammation on serum levels of TRP and kynurenine, as well as mucosal IDO expression in patients with inflammatory bowel disease (IBD). Methods: Serum samples and endoscopically-derived ileal or colonic mucosal biopsies were obtained from controls and patients with active IBD, and this before and after their first treatment with infliximab. Short-term clinical response and mucosal healing were assessed by experienced clinicians at 4 or 10 weeks after a single infusion or induction schedule, respectively (Ferrante, Inflamm Bowel Dis 2007; Schnitzler, GUT 2009). Serum TRP and kynurenine were measured by high performance liquid chromatography, and IDO mucosal gene expression was measured by Affymetrix Human Genome U133 Plus 2.0 Arrays. Data were analyzed with SPSS software using non-parametric tests and p-values of 9-fold increased in active IBD patients before infliximab therapy vs. controls (pileum=0.001 and pcolon<0.001). Although the IDOmucosal expression levels significantly decreased after infliximab therapy in IBD responders when compared to their baseline samples (pshort-term response and pmucosal healing<0.001), the colonic expression still remained significantly higher than controls (pshort-term response<0.001 and pmucosal healing=0.002). Serum TRP levels were significantly decreased (p<0.001) and the kynurenine/ tryptophan (K/T) ratio was significantly increased (p=0.001) in active IBD patients when compared to controls. However, we found no significant effect of infliximab therapy on both the TRP levels and K/T ratio. Conclusion: This study demonstrates that mucosal IDO expression levels are increased and serum TRP levels decreased in IBD patients with active disease. Of note, after controlling the inflammation with infliximab therapy, both levels remained impaired in IBD responders showing a complete mucosal healing. This persistent upregulation of IDO in patients with complete colonic healing may explain why mucosal ulcers recur very early if patients do not receive maintenance therapy with infliximab.

320 Neuro-Immune Regulation of TH17 Lymphocytes in Mouse Intestine

Background: Upon allergen encounter, food allergy typically leads to mast cell degranulation, an influx of Th2 immune cells together with increases in antigen specific antibodies. We showed that vagal nerve stimulation (VNS) increased Tregs and reduced mast cell activation in a mouse model of food allergy (DDW 2012). Here we aimed to test the hypothesis that, in contrast to VNS, vagotomy (VGX) would worsen food allergy. Methods: We sensitised balb/c mice to ovalbumin (OVA) adsorbed to aluminium-hydroxide (day 0&14) followed by intra-gastric gavages with OVA, from day 28 onwards. Sub-diaphragmatic VGX was performed at least one week prior to OVA gavage, together with pyloroplasty, where pyloroplasty alone served as control. Diarrhoea was scored for 1hr by analysing pellets, where 0 was normal faecal pellets, and 3 represents over diarrhoea. We determined serum mast cell proteases (mouse mast cell protease 1, MMCP-1), antibody titres (OVA specific IgG1 and IgG2a) and identified immune T-cell subsets in the mesenteric lymph nodes, lamina propria (LP) and Peyers patches. Cytokines released upon OVA challenge from mesenteric lymph nodes were measured by cytometric bead array. Further, qPCR was used to determine cytokine mRNA. OVA-specific Treg proliferation was assessed by adoptive transfer of Naive DO11.10 T-cells. Results: VGX increased serum MMCP-1 levels from 1120 ± 136 vs 2841 ± 964 ng/mL (n=7, p,0.05) 1hr after the fourth OVA gavage, coinciding with increased diarrhoea scores (from 1.1 to 2.8 arbitrary score, P , 0.05) but no change in serum IgG1 or IgG2a. VGX did not change the population of Tregs (CD25+/foxp3+) in the MLN (14.2 ± 1.9 controls vs VGX 15.0 ± 2.0% of CD4+) or LP (45.4 ± 2.9 to 42.0 ± 8.0% of CD4+). Similarly, no change in Th1 (IFN γ+), Th17 (IL-17+) or Th2 (IL-4+/IL-5+) cells was found after VGX in the mesenteric lymph nodes as compared to control. OVA specific Tregs (CD25+/foxp3+% of CD4+/DO11.10+) in the mesenteric lymph nodes (4.6 ± 0.4 to 6.2 ± 0.7%), LP (81.1 ± 7.0 vs. 80.5 ± 4.2%) or Peyers patches (24.5 ± 8.1 vs. 26.7 ± 6.4%) remained unchanged after VGX compared to controls (n=8-12). Jejunal mRNA levels (over RPL32 expression) for IL-13, IL-5, TNFα and IFNγ were not affected by VGX. Conclusion: As opposed to VNS, VGX increases the severity of food allergy, as shown by increased mast cell activation and diarrhoea scores compared to sham operated mice. CD4+ T cell skewing and cytokine production was however not affected by VGX. Our data indicate that reduced vagal tone renders mice more susceptible to develop food allergy further underscoring the role of the vagal innervation in modulating the mucosal immune system.