Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies

Abstract

Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an in situ hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due to crypt destruction. In summary, we have applied a strategy for identifying target-specific antibodies in formalin fixed biopsy samples and highlighted that (published) antibodies may not correctly identify the intended antigen in tissues fixed using this method. Furthermore, we have developed and, for the first time, validated an in situ hybridization assay for detection of claudin-2 mRNA, suitable for use as a supportative method in clinical trials. Using our validated assay, we have demonstrated that increased claudin-2 expression correlates with the severity of ulcerative colitis, where crypt destruction is not seen.

IL-22 Increases Permeability of Intestinal Epithelial Tight Junctions by Enhancing Claudin-2 Expression

“…Multiple studies have demonstrated that expression of IL-22 and Claudin-2 are increased in inflamed intestinal tissues (14,16,17,34,58). Our results identify a signaling mechanism that links IL-22 stimulation to the increased expression of Claudin-2.…”

“…Our results identify a signaling mechanism that links IL-22 stimulation to the increased expression of Claudin-2. Claudin-2 is normally expressed on epithelial cells within the small intestine and its expression is upregulated on colon epithelial cells in chronic inflammatory bowel diseases (13)(14)(15)(16)(17)59). We found that IL-22 induced upregulation of Claudin-2 in Caco-2 cells, human small intestinal epithelium, and mouse colonic epithelial cells, suggesting that IL-22 mediates upregulation of Claudin-2 in a broad range of intestinal epithelial tissues.…”

“…In healthy adults, Claudin-2 is abundantly expressed in both crypt and villus cells of both the duodenum and jejunum; whereas its expression in the colon is notably lower than small intestine and is restricted to undifferentiated crypt cells (13)(14)(15)(16)(17). However, expression of Claudin-2 is markedly upregulated in both small and large intestines in immune-or infection-mediated diseases, such as Crohn's disease (14), ulcerative colitis (18,19), HIV infection (20), and gluten-sensitive enteropathy (21).…”

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma

“…CLDN2, usually located in gut epithelia, helps in pore formation, thus regulates paracellular transport through epithelial cells (Rosenthal et al, 2010 ). Its over expression has also been linked to H. pylori -induced inflammatory bowel disease, ulcer and carcinoma (Randall et al, 2016 ). Similarly, a gap junction protein CX32, found in the epithelium of the gastrointestinal tract (GIT), when mislocalized or having altered function could lead to gastric carcinoma (Jee et al, 2011 ).…”

Inflammatory cytokines down-regulate the barrier-protective prostasin-matriptase proteolytic cascade early in experimental colitis

“…E-cadherin, occludin, zonula occludens-1 (ZO-1)) (11,12), leading to increased paracellular permeability to macromolecules (11,12), and also by increasing the expression and junctional localization of permeability-associated tight junction proteins such as claudin-2, which forms paracellular pores mediating cation and water flux (13,14). Elevated claudin-2 expression is found in inflamed villus epithelium of patients with active IBD, correlating with disease severity (15,16). Studies using human colonic epithelial monolayers show that claudin-2 mRNA and protein are specifically induced by Th2 cytokines IL-13 and IL-4, but not Th1 cytokines TNF␣ or IFN␥ (8,13,17).…”