Impact of Developmental Age, Necrotizing Enterocolitis Associated Stress, and Oral Therapeutic Intervention on Mucus Barrier Properties

Abstract

necrotizing enterocolitis (nec) is a devastating gastrointestinal disease of incompletely understood pathophysiology predominantly affecting premature infants. While NEC is associated with microbial invasion of intestinal tissues, and mucus modulates interactions between microbes and underlying tissues, variations in mucus barrier properties with NEC-associated risk factors have not been investigated. This study explored differences in mucus composition (total protein, DNA, mucin content, sialic acid, and immunoregulatory proteins), as well as structural and transport properties, assessed by tracking of particles and bacteria (E. coli and E. cloacae) with developmental age and exposure to NEC stressors in Sprague Dawley rats. Early developmental age (5 day old) was characterized by a more permeable mucus layer relative to 21 day old pups, suggesting immaturity may contribute to exposure of the epithelium to microbes. Exposure to NEC stressors was associated with reduced mucus permeability, which may aid in survival. Feeding with breastmilk as opposed to formula reduces incidence of NEC. Thus, NEC-stressed (N-S) rat pups were orally dosed with breastmilk components lysozyme (N-S-LYS) or docosahexaenoic acid (N-S-DHA). N-S-LYS and N-S-DHA pups had a less permeable mucus barrier relative to N-S pups, which suggests the potential of these factors to strengthen the mucus barrier and thus protect against disease. Necrotizing enterocolitis (NEC) occurs primarily in premature and low birth weight infants (<1500 g), and is associated with a 15-30% mortality rate 1,2. Infants with NEC present with signs of mucosal inflammation in the distal ileum and proximal colon, bloody stool, abdominal distention, and respiratory distress. Depending on disease severity, treatment options include bowel rest, antibiotics, and/or emergency surgical procedures. Children who survive surgical intervention often face severe complications including neurodevelopmental delays 3,4 and long-term neurological and intestinal rehabilitative needs 5,6. The annual cost of caring for infants with NEC is estimated at $500 million to $1 billion. Despite decades of research, the pathophysiology of NEC is still not completely understood 7-11. NEC has been linked to inappropriate immune response, increased epithelial permeability, and altered microbiome 8,12-16. Given the role of microbes in the pathogenesis of NEC, and the central role of intestinal mucus in controlling interactions between commensal bacteria and underlying tissues, we hypothesized that differences in the intestinal mucus barrier associated with immaturity may contribute to development of NEC. Mucus is a dynamic barrier that is continuously secreted, degraded by the microbiome (e.g., mucin sugar cleavage) 17 , and shed from the mucosal surface 18. The mucus barrier is composed of mucin glycoproteins, proteins, and lipids that covalently and non-covalently interact, creating a mesh-like structure with pores ranging

Comparing the permeability of human and porcine small intestinal mucus for particle transport studies

“…2B-E). Significantly lower amounts of total mucin and DNA have recently been found in the small intestinal mucus of 5-day old rat pups relative to 21-day old rats 36 . The difference, caused by gut immaturity, was suggested to contribute to reduced barrier properties of the neonatal mucus.…”

“…The extent of particle interaction with the mucus network, and possible anomalous diffusion in mucus, was determined by fitting particle MSD vs. Δt to MSD = 4DΔt α , where D is the diffusion coefficient independent of time, and α is the anomalous exponent that has been used to classify the type of particle motion by providing insight into the magnitude of particle transport obstruction 32,34,35 . Individual particle motion type was characterised according to the α-value, where 0.8 < α < 1.0 is considered diffusive 36 , and α < 0.8 represents various degrees of immobilised transport. For each condition used in this study, a proportion of diffusive particles (% of total particles) was calculated.…”

Colloidal transport of lipid digesta in human and porcine small intestinal mucus

“…Because of this, the mean microviscosity experienced by the latex beads diffusing in the piglet mucus was roughly half the value recorded for the pig counterpart (Macierzanka et al, 2014). The impact of developmental age on differences in chemical composition, structural properties and permeability to particles of small intestinal mucus was studied recently in rats (Lock et al, 2020). The gut immaturity was suggested to be responsible for significantly lower concentrations of mucin and DNA in the ileal mucus of 5 day old rat pups relative to 21 day old rats.…”

“…The small intestinal mucus is a viscoelastic hydrogel made from a number of components, amongst which the MUC2 mucin glycoprotein is the major gel-forming macromolecule responsible for the porous structure and rheological properties of the mucus (Corfield, Carroll, Myerscough, & Probert, 2001;Round et al, 2012). Several studies have also pointed out at the importance of extracellular DNA in maintaining the microstructural organisation and high viscosity of the mucus as well as its barrier properties with regard to penetrability to particles and bacteria (Lock et al, 2020;Macierzanka et al, 2014).…”

“…In the latter case, one of regularly used methods is multiple-particle tracking (MPT), which relies on simultaneous real-time monitoring of the displacement of hundreds or thousands of individual particles (Lai, Wang, Wirtz, & Hanes, 2009). Evaluation of the diffusivity of identical-in-size nano-or micro-particles allows for probing the permeability, microrheology, and heterogeneity in microstructural organisation of mucus (Lai, Wang, Hida, Cone, & Hanes, 2010;Lock et al, 2020;Macierzanka, Mackie, & Krupa, 2019). This method has been used to determine the role of the size and surface chemistry of particles on their transport in mucus (Maisel, Ensign, Reddy, Cone, & Hanes, 2015;Yildiz, McKelvey, Marsac, & Carrier, 2015).…”