Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6-7 h.

Enhanced NF-κB activity is involved in the pathology of both forms of inflammatory bowel disease (IBD), Crohn disease (CD) and ulcerative colitis (UC). Here we analyzed the mechanism of proteasome-mediated NF-κB activation in CD and UC. Our studies demonstrate that the subunit composition and the proteolytic function of proteasomes differ between UC and CD. High expression of the immunoproteasome subunits β1i and β2i is characteristic of the inflamed mucosa of CD. In line with this, we found enhanced processing of NF-κB precursor p105 and degradation of inhibitor of NF-κB, IκBα, by immunoproteasomes isolated from the mucosa of CD patients. In comparison with healthy controls and CD patients, UC patients exhibited an intermediate phenotype regarding the proteasome-mediated processing/degradation of NF-κB components. Finally, increased expression of the NF-κB family member c-Rel in the inflamed mucosa of CD patients suggests that p50/c-Rel is important for IFN-γ-mediated induction of immunoproteasomes via IL-12-driven Th1 responses. These findings suggest that distinct proteasome subunits influence the intensity of NF-κB-mediated inflammation in IBD patients.

The pathogenesis of primary sclerosing cholangitis (PSC) remains poorly understood. Since PSC predominantly occurs in patients with inflammatory bowel disease, autoimmunity triggered by activated T cells migrating from the gut to the liver is a possible mechanism. We hypothesized that T cells primed in the gut‐associated lymphoid tissue (GALT) by a specific antigen migrate to the liver and cause cholangitis when they recognize the same antigen on cholangiocytes. We induced ovalbumin‐dependent colitis in mice that express ovalbumin in biliary epithelia (ASBT‐OVA mice) and crossed ASBT‐OVA mice with mice that express ovalbumin in enterocytes (iFABP‐OVA mice). We analyzed T‐cell activation in the GALT and crossreactivity to the same antigen in the liver as well as the effects of colitis per se on antigen‐presentation and T‐cell activation in the liver. Intrarectal application of ovalbumin followed by transfer of CD8 OT‐I T cells led to antigen‐dependent colitis. CD8 T cells primed in the GALT acquired effector function and the capability to migrate to the liver, where they caused cholangitis in a strictly antigen‐dependent manner. Likewise, cholangitis developed in mice expressing ovalbumin simultaneously in biliary epithelia and enterocytes after transfer of OT‐I T cells. Dextran sodium sulfate colitis led to increased levels of inflammatory cytokines in the portal venous blood, induced activation of resident liver dendritic cells, and promoted the induction of T‐cell‐dependent cholangitis. Conclusion: Our data strengthen the notion that immune‐mediated cholangitis is caused by T cells primed in the GALT and provide the first link between colitis and cholangitis in an antigen‐dependent mouse model. (Hepatology 2014;59:601–611)

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Spleen tyrosine kinase (Syk) is expressed widely in hematopoietic and non-hematopoietic cells. The widespread distribution of Syk and its involvement in host defense and allergic reactions, prompted us analyze the influence of microbial exposure on Syk expression. We compared the distribution of Syk in various tissues of germ-free and conventional mice using immunohistochemistry, Western blot analysis and real time RT-PCR. Total Syk expression was similar between germ-free and conventional mice. Since it has been claimed that Syk isoforms are differentially expressed, we studied the distribution and abundance of Syk (L) and Syk (S) isoforms in tissues from these mice. In contrast to previous reports, we found broad tissue expression of Syk (S). Interestingly, in germ-free mice the amount of Syk (S) but not Syk L protein was selectively increased in lung and spleen. In summary, our study reveals new and broad tissue expression of both Syk isoforms and demonstrates that lack of microbial flora results in selectively increased expression of Syk (S) isoform in lung and spleen.