Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection.

Kim Y ，Kessler SP ，Obery DR ，Homer CR ，McDonald C ，de la Motte CA ... -  《-》

Layilin is critical for mediating hyaluronan 35kDa-induced intestinal epithelial tight junction protein ZO-1 in vitro and in vivo.

Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.

Kim Y ，West GA ，Ray G ，Kessler SP ，Petrey AC ，Fiocchi C ，McDonald C ，Longworth MS ，Nagy LE ，de la Motte CA ... -  《-》

Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis.

Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.

Conlin VS ，Wu X ，Nguyen C ，Dai C ，Vallance BA ，Buchan AM ，Boyer L ，Jacobson K ... -  《-》

Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.

Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.

Bellos DA ，Sharma D ，McMullen MR ，Wat J ，Saikia P ，de la Motte CA ，Nagy LE ... -  《-》

Hyaluronan-induced alterations of the gut microbiome protects mice against Citrobacter rodentium infection and intestinal inflammation.

Mao T ，Su CW ，Ji Q ，Chen CY ，Wang R ，Vijaya Kumar D ，Lan J ，Jiao L ，Shi HN ... -  《-》