Mucus Barriers to Microparticles and Microbes are Altered in Hirschsprung's Disease

Mucus forms a protective hydrogel layer over the intestinal epithelium, presenting a selective and robust barrier to the uptake of particulates and microbe invasion. Disease can alter mucus production and composition, thus potentially modifying mucosal barrier properties. Hirschsprung’s disease (HD) is a developmental abnormality of the nervous system often complicated by intestinal infection. An investigation of colonic mucus barrier properties in an HD animal model, endothelin receptor B mutant mice, revealed significantly reduced microsphere (passive) and microbe (active) transport rates (7-fold and 3.6-fold, respectively, in proximal colonic mucus) relative to wild-type. Transport differences were evident in both the ganglionic and aganglionic colon segments, in agreement with the risk of Hirschsprung’s disease-associated enterocolitis after surgery to remove aganglionic colon segments. The development of therapies aimed at altering colonic mucus barrier properties could be explored towards preventing the onset of enterocolitis in Hirschsprung’s disease. Keywords: mucus, Hirschsprung’s disease, nanoparticle diffusion, microbe transport, intestine 1. Introduction Mucus, a highly complex hydrogel composed of mucin glycoproteins with a pore size reported to be approximately 10–500 nm,[1] provides a barrier against particulate materials and biological pathogens. The barrier properties of mucus are a combined result of physical effects, such as size exclusion and hydrodynamic drag, and intermolecular interactions between diffusing entities and mucus components.[1–5] The significance of the mucus barrier in controlling access to the underlying epithelium raises the question of whether mucosal structural and/or chemical changes occur in disease states, potentially contributing to pathogen invasion. Hirschsprung’s disease (HD), also known as intestinal aganglionosis, is a developmental abnormality of the enteric nervous system that affects 1 in 5,000 live births.[6, 7] HD is characterized by the lack of ganglion cells along a variable length of the distal intestine, resulting in the absence of peristalsis in the aganglionic segment and dilation of the colon proximally.[6, 7] HD is associated with the development of Hirschsprung’s-associated enterocolitis (HAEC), a severe inflammation of the intestinal mucosa that presents with abdominal distension, diarrhea, and fever and can progress to sepsis and death.[6, 7] HAEC, the leading cause of death in HD, occurs in up to 50% of untreated HD cases, and the risk persists even after surgical removal of the aganglionic intestine, suggesting that the abnormality associated with HAEC extends beyond the region of aganglionosis.[6] It is thought that HD leads to changes in colonic microbiota, innate immunity, and epithelial barrier properties,[8] any of which may contribute to the risk of developing enterocolitis. Although these theories are being investigated, little attention has been focused on the potential contributory role of mucus barrier properties in this disease. Recent insight into changes in HD mucosal properties has been gained by investigating mucus turnover rate, mucin gene expression, and histological structure.[8–13] These studies have shown that HD is associated with lowered mucus turnover rate, lowered mucin concentrations, abnormal mucin ratios (neutral mucins:acidic-sulphomucins),[13] and differences in mucin producing goblet cell size and proliferation compared to the healthy state.[14] These results therefore support differences in mucus quantity and composition in HD, but do not specifically test potential changes in mucus barrier properties that may play a role in microbe invasion and in the development of enterocolitis. Given the important role of mucus in protecting underlying epithelia from microbial invasion, we hypothesize that the inherent barrier properties of colonic mucus are altered in HD. To test this hypothesis, transport of passively diffusing entities (polystyrene microspheres) and actively moving microbes (Escherichia coli) through colonic mucus in an HD animal model were investigated. In this work, live imaging and multiple particle tracking (MPT),[15–21] a powerful technique enabling non-disruptive investigation of transport across the mucosal layer, was employed. Particle and microbe trajectories were used to calculate time-averaged mean squared displacements (MSDs) and average velocities to quantitatively characterize transport across mucus layers. Results supported differences in colonic mucus barriers between HD and wild-type (WT) animals. A deeper understanding of the mechanisms underlying mucus barrier differences could lead to therapeutic treatments to modulate mucus barrier properties and potentially prevent the onset of HAEC.