Genetic ablation of CCR6 confers differential exacerbation in a spontaneous colitis model

Inflammatory bowel disease (IBD) is an immunological imbalance of the gut, characterised by chronic inflammation involving the mucosal lining of the colon. IBD presents in two forms: Crohn's disease (CD) and ulcerative colitis (UC). The increasing global incidence of IBD has necessitated a better understanding of the underlying mechanism of disease progression, and while its aetiology remains obscure, current literature suggests that both genetic susceptibility and environmental aspects have an influence on the course of IBD. Murine models have led to a number of discoveries in IBD pathogenesis. Amongst the many mouse models developed, the Winnie mouse model of colitis closely resembles the condition of human colitis in terms of both symptoms and immunology. A number of studies have reported that Winnie mice are a preclinical resource for better understanding IBD pathogenesis. An emerging mechanism for the pathogenicity of IBD development includes a dysregulated chemokine system. In particular, the CCR6-CCL20 axis has been implicated due to its major role in regulating T cell homeostasis in the intestine. The current literature regarding CCR6-CCL20 is inconclusive as to the exact role of CCR6. The aim of this study was to introduce CCR6 deficiency in the Winnie spontaneous colitis model in order to further elucidate the functional role of CCR6 in colitis and characterise clinical, histopathological and immunological changes. In this present study, the hypothesis was that the genetic ablation of the Ccr6 gene would exacerbate intestinal inflammation. Winnie mice were backcrossed with Ccr6\\(^{-/-}\\) mice to generate Winnie mice with a Ccr6 deficiency. Winnie x Ccr6\\(^{-/-}\\) mice developed normally and did not have any developmental abnormalities. At around 8‚Äö-12 weeks of age, the Winnie x Ccr6\\(^{-/-}\\) mice were clinically assessed, and no significant differences were noted between Winnie and Winnie x Ccr6\\(^{-/-}\\) mice with regard to body weight. There was, however, a significant increase in the colon weight/body weight ratio (¬¨¬±0.026 mg/g), with Winnie x Ccr6\\(^{-/-}\\) mice revealing a significant increase in colon weight by body weight ratio compared to control mice (WT and Ccr6\\(^{-/-}\\) mice) of a similar magnitude to the expected Winnie-only phenotype. Histopathological examination of proximal and distal colonic sections showed no obvious inflammatory changes in the control mice (WT grade-0 and Ccr6\\(^{-/-}\\) grade-0). Winnie mice demonstrated the expected amount of inflammation in the distal colonic segment (grade-6). Surprisingly, there was a differential distribution of inflammation in Winnie x Ccr6\\(^{-/-}\\) mice, where the proximal colon revealed significant inflammation (grade-8) while there was mild inflammation (grade-4) in the distal colon. Immuno-phenotyping of splenocytes and colonic lymphocytes did not reveal any significant changes in the functions of T regulatory cells. To identify the differential regulation in Winnie x Ccr6\\(^{-/-}\\) mice, we cultured ex vivo colonic segments from Winnie, Winnie x Ccr6\\(^{-/-}\\), Ccr6\\(^{-/-}\\) and WT mice to assess the secretion of cytokines. Interestingly, molecular and cytokine assays demonstrated elevated levels of anti-inflammatory cytokine interleukin-10 (IL-10) in the distal colon and increased expression of pro- inflammatory cytokine, interleukin-1˜í‚⧠(IL-1˜í‚â§), in the proximal colon. In Winnie x Ccr6\\(^{-/-}\\) mice, an increased expression of IL-1˜í‚⧠in the proximal coloncorrelated with an exacerbation of inflammation. Increased expression of anti-inflammatory cytokine IL-10 was associated with an amelioration of inflammation in the Winnie x Ccr6\\(^{-/-}\\) distal colon. These results suggest that absence of the Ccr6 gene selectively confers amelioration of colitis in the distal colon but exacerbation in theproximal colon. Overall, this study presents significant and novel findings in the CCR6-deficient spontaneous colitis model. Genetic ablation of Ccr6 conferred regional differential exacerbation between colonic segments of Winnie x Ccr6\\(^{-/-}\\) mice. Findings from this study reveal the hitherto unknown mechanism of Ccr6 differential distribution in the colon, thereby demonstrating an important role for CCR6 in the development of colitis. This will throw more light towards understanding the pathogenesis of colitis in IBD. These findings indicate that CCR6 could potentially serve as a diagnostic marker and anovel therapeutic target.