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Multi-system pathophysiology and molecular signalling in a Ccr6-deficient murine model of spontaneous colitis

Ranasinghe, HR ORCID: 0000-0003-3634-2689 2020 , 'Multi-system pathophysiology and molecular signalling in a Ccr6-deficient murine model of spontaneous colitis', Research Master thesis, University of Tasmania.

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Abstract

Background: Inflammatory Bowel Disease (IBD) is an immune-deficient gut diseases complex [Crohn’s Disease (CD) and Ulcerative Colitis (UC)] which cause serious digestive problems from prolonged inflammation. A multitude of immunological, microbial and environmental reasons have been named as its causative agents although its true aetiology is still unknown. There is no known definite treatment regime, nor a permanent cure has yet been established for these serious chronic illnesses in the gut. This study has attempted to investigate the influence of the C-C Chemokine Receptor 6 (CCR6) and its sole ligand C-C Chemokine Ligand (CCL20) as a potential therapeutic target for IBD given the important immunological role played by this chemokine pair. The immunomodulatory behaviour of the CCR6/CCL20 axis on multi-system pathophysiology was investigated at two clinically significant time points, using a Ccr6-deficient mouse model of spontaneous colitis. Methods: Four groups of mice, [(i) C57BL/6J which was the Wild Type abbreviated as WT and having (Winnie +/+, Ccr6 +/+) genotype (ii) homozygous Ccr6-/- mutant of the WT having (Winnie +/+, Ccr6-/-) genotype (iii) Winnie x Ccr6 -/-, the double knockout experimental model derived from the WT background (denoted by Winnie m/m, Ccr6-/-) and (iv) Winnie, also derived from the WT background, having the (m/m, Ccr6+/+) genotype] were utilized and (1) colonic inflammatory parameters, (2) colonic histology, (3) histology of spleen, kidney and liver (4) T and B lymphocyte quantification in the spleen and mesenteric lymph nodes (MLN) by flow cytometry, (5) colonic CCL20, phosphorylated phosphoinositide – 3 – kinase (PI3KP) and phosphorylated protein kinase B also known as (AktP) expression by immunohistochemistry, and (6) cytokine expression in the colon by real time – polymerase chain reaction (RT-PCR) were evaluated. Results: CCR6 influenced upregulation of inflammation during murine colitis in the gut, spleen and liver while renal histology remained unaffected. Marked focal lobular inflammation with reactive nuclear features were observed in hepatocytes and a significant neutrophil infiltration in red pulp with extra medullary hemopoiesis existed in the spleen. These changes were considerably reduced in the gut, spleen and liver of Winnie x Ccr6-/- [also known as the Ccr6-deficient-Winnie] with elevated goblet cell numbers and a conspicuously high mucus production in the colonic epithelium. In its spleen, the splenic lymphocyte percentage during early inflammation remained static but declined considerably towards the acute inflammatory stage compared to the WT, indicating decreased lymphocyte egression. The reverse of splenic hemopoiesis was seen in the MLN of the same model which had suppressed lymphocyte presence at 8 weeks which was restored with increased presence during acute disease. Cytokine expression in the Winnie x Ccr6-/- was below that of Winnie in all the cytokines tested. As an effect of CCR6 deficiency during chronic colitis, Winnie x Ccr6-/- produced upregulation of AKTP and downregulation of PI3KP in its molecular signalling apparatus, compared to those of Winnie. Conclusion: CCR6/CCL20 are known to produce detrimental effects in the gut and this is further substantiated by our study. We report inflammation and tissue injury in the spleen and liver during chronic colitis in the Winnie model for the first time. Results indicated that Ccr6 deficiency in the experimental colitis model contributed towards ameliorating colitis. Consequently, they displayed reduced inflammation in the gut, spleen and liver. These findings demonstrate an intricate networking role for CCR6 in pre-clinical immune activation, which was substantially diminished in Winnie x Ccr6-/- and need clinical investigation to determine whether it could provide newer clinical therapies in colitis.

Item Type: Thesis - Research Master
Authors/Creators:Ranasinghe, HR
Keywords: Inflammatory Bowel Disease, Spontaneous Colitis, Winnie, CCR6, CCL20, Immunology
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Copyright 2020 the author

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