Investigating the intestinal mucus barriers of wild mice

Abstract

The intestinal mucosal barrier is critical to health. It must continue to function in the variable conditions of the intestinal lumen, and is influenced by many intrinsic and extrinsic factors, such as age, sex, diet and infection. Mucus is a fundamental component of the intestinal mucosal barrier and is primarily composed of mucins. Mucins are highly glycosylated proteins (up to 80% glycan, by weight) and are arranged in a large polymeric network to form the macromolecular structure of the mucus gel. Although laboraotry studies have attempted to identify associations between mucin glycosylation and ecologically relevant parameters individually, no study has yet been conducted that aims to examine the interplay between multiple intrinsic and extrinsic factors, and their combined influence on mucin glycosylation at the intestinal barrier. To determine the role of ecological parameters in shaping the glycosylation of the intestinal mucus barrier, a population of wild Mus musculus domesticus was studied. Goblet cells containing mucins with predominantly acidic glycans were observed in the proximal colons of wild mice. By analysing the presence of acidic glycan containing goblet cells in the context of ecological variables recorded for each mouse associations between acidic goblet cells and potential ecological drivers were explored. Acidic glycan containing goblet cells were found to significantly associate with the sex of mice, and the scaled mass index of individuals. Molecular analysis of gene expression in the proximal colons of mice indicated that expression of sialyltransferase enzymes that produce such acidic glycan structures was associated with the expression of the Esr1 gene. Esr1 encodes the nuclear estrogen receptor α. Finally, the extent of acidic mucin glycosylation in proximal colon goblet cells was predicted using a random forest regression model. Following observations that sialic acid in the mucin glycans is related to estrogen receptor expression, laboratory studies were devised to examine the hypothesis that the acidic glycan content of mucus is influenced by systemic estrogen signalling. In vitro and in vivo experiments indicated that the occurrence of sialic acid and acidic sulphated glycan structures in the mucus increased in response to estrogen treatment in mucin-producing cell lines. Additionally, the extent of mucin sulphation changed significantly over the course of the estrus cycle in mice, and male mice fed and estrogenic diet displayed increased acidic mucin glycans. Finally, a wild isolate of the nematode gastrointestinal parasite Trichuris muris was isolated from the Isle of May (M-isolate). The E-isolate of Trichuris muris is an extensively studied laboratory mouse model of human parasite infection. Infections of the wild isolate in C57BL/6 mice were examined, in comparison to the well-understood laboratory-maintained E-isolate. Development of the immune response was delayed in C57BL/6 mice infected with the wild isolate, with antigen-specific mesenteric lymph node cytokine production reduced when larvae were at the L2 stage in M-infected mice compared to those infected with the E-isolate. This was accompanied by the M-isolate establishing chronic infections at doses where the E-isolate was expelled. Combined, these studies have highlighted the important role of ecologically relevant parameters such as scaled mass index and sex on the glycosylation of intestinal mucins in a wild mouse population. This data suggests, for the first time, the role of systemic estrogen signalling on a non-reproductive mucus barrier in wild animals. This work underscores the importance of studying mucus in ecologically relevant contexts. It also provides a starting point for further work aiming to develop more holistic models of the regulation of mucin-glycan structures. Understanding the processes regulating mucin glycosylation could help treatment and prevention of inflammatory diseases characterised by aberrant

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Thornton (Supervisor) & Kathryn Else (Supervisor)