No abstract

Increased small intestinal permeability has been proposed to be an integral element, along with genetic makeup and environmental triggers, in the pathogenies of chronic inflammatory diseases (CIDs). We identified zonulin as a master regular of intercellular tight junctions linked to the development of several CIDs. We aim to study the role of zonulin-mediated intestinal permeability in the pathogenesis of CIDs. Zonulin transgenic Hp2 mice (Ztm) were subjected to dextran sodium sulfate (DSS) treatment for 7 days, followed by 4–7 days recovery and compared to C57Bl/6 (wild-type) mice. Intestinal permeability was measured in vivo and ex vivo, and weight, histology, and survival were monitored. To mechanistically link zonulin-dependent impairment of small intestinal barrier function with clinical outcome, Ztm were treated with the zonulin inhibitor AT1001 added to drinking water in addition to DSS. We observed increased morbidity (more pronounced weight loss and colitis) and mortality (40–70% compared with 0% in wild type) at 11 days post-DSS treatment in Ztm compared with wild-type mice. Both in vivo and ex vivo measurements showed an increased intestinal permeability at baseline in Ztm compared to wild-type mice, which was exacerbated by DSS treatment and was associated with upregulation of zonulin gene expression (fourfold in the duodenum, sixfold in the jejunum). Treatment with AT1001 prevented the DSS-induced increased intestinal permeability both in vivo and ex vivo without changing zonulin gene expression and completely reverted morbidity and mortality in Ztm. Our data show that zonulin-dependent small intestinal barrier impairment is an early step leading to the break of tolerance with subsequent development of CIDs.

The interleukin-2-deficient (IL-2(-/-)) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2(-/-) mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2(-/-) mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-kappaB (p65) activation with the inhibitory p50 form of NF-kappaB predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

The balanced interplay between epithelial barrier, immune system, and microbiota maintains gut homeostasis, while disruption of this interplay may lead to inflammation. Paracellular permeability is governed by intercellular tight-junctions (TJs). Zonulin is, to date, the only known physiological regulator of intestinal TJs. We used a zonulin transgenic mouse (Ztm) model characterized by increased small intestinal permeability to elucidate the role of a primary impaired gut barrier on microbiome composition and/or immune profile. Ztm exhibit an altered gene expression profile of TJs in the gut compared to wild-type mice (WT): Claudin-15, Claudin-5, Jam-3, and Myosin-1C are decreased in the male duodenum whereas Claudin-15, Claudin-7, and ZO-2 are reduced in the female colon. These results are compatible with loss of gut barrier function and are paralleled by an altered microbiota composition with reduced abundance of the genus Akkermansia, known to have positive effects on gut barrier integrity and strengthening, and an increased abundance of the Rikenella genus, associated to low-grade inflammatory conditions. Immune profile analysis shows a subtly skewed distribution of immune cell subsets toward a pro-inflammatory phenotype with more IL-17 producing adaptive and innate-like T cells in Ztm. Interestingly, microbiota “normalization” involving the transfer of WT microbiota into Ztm, did not rescue the altered immune profile. Our data suggest that a primary impaired gut barrier causing an uncontrolled trafficking of microbial products leads to a latent pro-inflammatory status, with a skewed microbiota composition and immune profile that, in the presence of an environmental trigger, as we have previously described (1), might promote the onset of overt inflammation and an increased risk of chronic disease.

LTbetaR expression is critical for GALT control mechanisms and intact mucosal immunity. PN reduces LTbetaR expression, PP lymphocytes, and intestinal IgA production. Exogenous LTbetaR stimulation reverses PN-induced depression of gut mucosal immunity.