No abstract

Background and Aims Inactivating mutations in MYO5B cause severe neonatal diarrhea in Microvillus Inclusion Disease. Loss of active MYO5B causes the formation of pathognomonic inclusions and aberrations in brush border enzymes. Methods We developed three mouse models of germline, constitutively intestinal targeted and inducible intestinal targeted deletion of MYO5B. The mice were evaluated for enterocyte cellular morphology. Results Germline MYO5B KO mice showed early diarrhea and failure to thrive with evident microvillus inclusions and loss of apical transporters in the duodenum. IgG was present within inclusions. Apical transporters were lost and inclusions were present in the duodenum, but were nearly absent in the ileum. VillinCre;MYO5BF/F mice showed similar pathology and morphological changes in duodenal enterocytes. In contrast, when MYO5B KO was induced with tamoxifen treatment at 8 weeks of age, VillinCreERT2;MYO5BF/F mice developed severe diarrhea with loss of duodenal brush border enzymes, but few inclusions were observed in enterocytes. However, if tamoxifen is administered to 2-day-old VillinCreERT2;MYO5BF/F mice, prominent microvillus inclusions were observed. Conclusions The microvillus inclusions that develop after MYO5B loss reveal the presence of an unrecognized apical membrane trafficking pathway in neonatal duodenal enterocytes. However, the diarrheal pathology after MYO5B loss is due to deficits in transporter presentation at the apical membrane in duodenal enterocytes.

Compartmentalization of Toll-like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohn's disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell-intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild-type controls, germ-free Rab11a-deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host-microbial homeostasis at least partially via sorting TLRs.

Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a-knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.

Ser-227 phosphorylation of Rab11-FIP2 by Par1b/MARK2 regulates the establishment of polarized epithelial monolayers in three-dimensional MDCK cell cultures and has an ongoing influence on the composition of both adherens and tight junctions in polarized epithelial cells.