Meghali P. Nighot

Lipopolysaccharide Regulation of Intestinal Tight Junction Permeability Is Mediated by TLR4 Signal Transduction Pathway Activation of FAK and MyD88

Gut-derived bacterial LPS plays an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor in necrotizing enterocolitis and inflammatory bowel disease. The defective intestinal tight junction barrier was shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, causes an increase in intestinal tight junction permeability (TJP) via a TLR4-dependent process; however, the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal tight junction barrier using in vitro and in vivo model systems. LPS caused a TLR4-dependent activation of membrane-associated adaptor protein focal adhesion kinase (FAK) in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and -independent pathways. Small interfering RNA silencing of MyD88 prevented an LPS-induced increase in TJP. LPS caused MyD88-dependent activation of IL-1R–associated kinase 4. TLR4, FAK, and MyD88 were colocalized. Small interfering silencing of TLR4 inhibited TLR4-associated FAK activation, and FAK knockdown prevented MyD88 activation. In vivo studies also confirmed that the LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented an LPS-induced increase in intestinal permeability. Additionally, high-dose LPS–induced intestinal inflammation was dependent on the TLR4/FAK/MyD88 signal transduction axis. To our knowledge, our data show for the first time that the LPS-induced increases in intestinal TJP and intestinal inflammation were regulated by TLR4-dependent activation of the FAK/MyD88/IL-1R–associated kinase 4 signaling pathway.

Chloride Channel ClC-2 is a Key Factor in the Development of DSS-Induced Murine Colitis

Background:Previously, it was shown that the chloride channel ClC-2 modulates intestinal tight junction (TJ) barrier function. The aim of the present study was to investigate the role of ClC-2 in epithelial barrier function and recovery in the event of epithelial injury. Methods:The role of ClC-2 was investigated in TJ barrier function in dextran sodium sulfate (DSS)-induced colitis in ClC-2 knockout mice and ClC-2 knockdown intestinal Caco-2 cells. Barrier function was measured electrophysiologically and by transepithelial mannitol fluxes. Selected TJ and associated proteins were Western blotted, cytokines were measured using quantitative PCR, and human colonic biopsies were examined with immunohistochemistry. Results:ClC-2−/− mice had a higher disease activity index, higher histological scores, and increased paracellular permeability compared with wild-type mice when treated with DSS. DSS-treated ClC-2−/− mice had increased claudin-2 expression, greater loss of occludin in the membrane, increased association of occludin with caveolin-1, and significantly increased tumor necrosis factor-&agr; and interleukin-1&bgr; messenger RNA. ClC-2 knockdown in human intestinal Caco-2 cells resulted in a greater loss of epithelial resistance in the event of epithelial injury. The restoration of colonic barrier function after DSS colitis was delayed in ClC-2−/− mice. In human colonic biopsies, the protein and messenger RNA expression of ClC-2 was found to be reduced in patients with ulcerative colitis. Conclusions:ClC-2 plays a critical role in experimental colitis in that its absence increases disease activity, reduces barrier function and recovery, and perturbs TJs. Furthermore, ClC-2 expression is markedly reduced in the colon of human patients with ulcerative colitis.

Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion.

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H+] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84–95%, P<0.005) in ClC-2-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.

The Chloride Channel CLC-2 is Involved in Organization of Murine Gastric Glands

BACKGROUND: Primary cilia are necessary for Hedgehog (Hh) signaling and other pathways. Anterograde ciliary transport is mediated by kinesin-II, a heterotrimeric protein composed of two motor subunits, KIF3A, KIF3B, and a non-motor subunit KAP3. Intraflagellar transport protein 88 (IFT88) participates in protein transport along the cilia. Conventional null mice for KIF3A and IFT88 are not viable. Sonic hedgehog (Shh) is highly expressed in gastric epithelial cells of the corpus compared to the antrum. Therefore we examined the stomach of mice conditionally null for KIF3A and IFT88 to define the role of primary cilia in this organ. METHODS: KIF3A and IFT88 mice harboring one null allele and loxP sites on the other allele on a ROSA26 reporter background were crossed to Shh-Cre mice (KIF3A-/ FL and IFT88-/FL respectively.) KIF3A+/FL, IFT88+/FL, and wild type (WT) littermates were analyzed at 6 and 8 months. Cell lineages were identified by immunohistochemistry in paraffin-sections, and by β-galactosidase (β-gal) staining for the Cre-expressing cells. Acetylated-α-tubulin was used as the marker for cilia. RESULTS: Epithelial cells in the antrum and corpus expressed primary cilia. β-gal staining confirmed variegated glandular Cre expression in all epithelial cells. Gastric epithelial cells did not express cilia in the IFT88-/FL mice, while KIF3A-/FL mice showed reduced numbers of cilia in the corpus versus controls (0.59+0.04 cilia/gland, N=8 vs. 0.72+0.06 cilia/gland, N=14), but no changes in the antrum (0.94+0.05 vs. 1.08+0.09 cilia/gland.) KIF3A-/FL and IFT88-/FL mice exhibited different phenotypes. By 6 months, KIF3A-/FL mice showed mucous pit hyperplasia in the corpus. Mucous neck and zymogenic lineages were not expanded, but parietal cells were still present. The KIF3A-/FL mice phenotype resembled the mucous pit hyperplasia observed in the Shh conditionally null mice generated with H+K+ATPase-Cre. In contrast, the phenotype of the IFT88-/FL mice showed parietal cell atrophy, and proximal corpus hyperplasia due to expansion of the mucous neck cell compartment assessed by TFF2 expression (SPEM) by 4 months of age. We have previously observed that gastrinand ghrelin-secreting cells exhibit primary cilia, but none of the mouse models showed differences in the number of these endocrine cells.CONCLUSION:The IFT88-/FLmice exhibited amore severe phenotype than the KIF3A-/FL mice even though both molecules affect primary cilia function. The phenotype of these two mouse models suggests that Hh signaling is necessary for the maintenance of the corpus epithelium. Also, gastric epithelial cilia might be important for the function but not differentiation of gastrinand ghrelin-secreting cells.

Indomethacin Induces Gastric Epithelial Barrier Dysfunction via a p38 MAPK-Dependent Mechanism in MKN-28 Cells

Background and AimsIL-33 is an IL-1 related cytokine and like IL-1a has been suggested to amplify the immune response during tissue injury. IL-33 is expressed in endothelium and mucosal epithelium and is a ligand for the receptor ST2. Recently IL-33 has been shown to be a crucial amplifier of immunity that can modulate the response in a number of models of innate-type mucosal immunity. Significantly IL-33 is involved in the development of DSS induced colitis. Additionally we have demonstrated that IL-11, a member of the IL-6 family of cytokines, is also important in DSS induced colitis and others have demonstrated an antiinflammatory role for IL-11 in intestinal inflammation. Here we examine the effects of IL11 on the normal colon and establish that IL-11 is upstream of Bmp1 and IL-33, and that it regulates mucosal proliferation via Reg3β in a STAT3-dependent manner. MethodsNormal mice were treated with 5 ug of recombinant human IL-11 (rhIL-11) every 6 hours and culled either 6 hours, 24 hours of 5 days after the initial dose. Colons were removed for mRNA and protein analysis. ResultsIL-11 is a ligand for gp130 and should initiate both the Ras/MAPK/ERK and STAT1/STAT3 signalling pathways. Chronic IL-11 treatment of normal mice activates STAT3 (Tyr705) but not ERK1/2 (Thr202/Tyr204), and acute IL-11 caused a drop in ERK1/2 activation. This is a very similar to the signalling outcome downstream of IL-11 in the gastric mucosa, where signalling emanating from gp130 activation by IL-11 is skewed towards STAT3. Expression of SOCS3, a bona fide STAT3 responsive gene, is also increased in the colon following IL-11 treatment. In the gastric antrum IL-11 treatment is pro-proliferative. The same is true of the colonic response to IL-11 with a 1.6 fold increase in the expression of the proliferation marker PCNA by immunoblot. IL-11 also caused an increase in the expression of the pro-proliferative gene Reg3β and the mRNAs involved in BMP signalling Bmp1 and Bmp1rb. However, most significantly IL-11 treatment caused a large and significant increase in the expression of IL-33 and other mediators of the innate immune response in the absence of any significant damage to the mucosa. ConclusionsColonic tissue responds to IL-11 largely by activation of STAT3 signalling. There are three major outcomes resulting from this signalling; 1) increased proliferation and expression of pro-proliferative genes, 2) increased expression of components of BMP signalling and 3) activation of IL-33 and the innate immune system.