Intestinal Epithelial Barrier Maturation by Enteric Glial Cells Is GDNF-Dependent.

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAPcre x Ai14floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.

Desmoglein2 Regulates Claudin2 Expression by Sequestering PI-3-Kinase in Intestinal Epithelial Cells.

Inflammation-induced reduction of intestinal desmosomal cadherin Desmoglein 2 (Dsg2) is linked to changes of tight junctions (TJ) leading to impaired intestinal epithelial barrier (IEB) function by undefined mechanisms. We characterized the interplay between loss of Dsg2 and upregulation of pore-forming TJ protein Claudin2. Intraperitoneal application of Dsg2-stablising Tandem peptide (TP) attenuated impaired IEB function, reduction of Dsg2 and increased Claudin2 in DSS-induced colitis in C57Bl/6 mice. TP blocked loss of Dsg2-mediated adhesion and upregulation of Claudin2 in Caco2 cells challenged with TNFα. In Dsg2-deficient Caco2 cells basal expression of Claudin2 was increased which was paralleled by reduced transepithelial electrical resistance and by augmented phosphorylation of AKTSer473 under basal conditions. Inhibition of phosphoinositid-3-kinase proved that PI-3-kinase/AKT-signaling is critical to upregulate Claudin2. In immunostaining PI-3-kinase dissociated from Dsg2 under inflammatory conditions. Immunoprecipitations and proximity ligation assays confirmed a direct interaction of Dsg2 and PI-3-kinase which was abrogated following TNFα application. In summary, Dsg2 regulates Claudin2 expression by sequestering PI-3-kinase to the cell borders in intestinal epithelium.

Enteroids Generated from Patients with Severe Inflammation in Crohn's Disease Maintain Alterations of Junctional Proteins.

BACKGROUND: The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn's disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients' specimens. METHODS: Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. RESULTS: Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. CONCLUSIONS: These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease.

Impaired intestinal epithelial barrier (IEB) function with loss of desmosomal junctional protein desmoglein 2 (DSG2) is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). While previous studies have reported that glial cell line-derived neurotrophic factor (GDNF) promotes IEB function, the mechanisms are poorly understood. We hypothesized that GDNF is involved in the loss of DSG2, resulting in impaired IEB function as seen in IBD. In the inflamed intestine of patients with IBD, there was a decrease in GDNF concentrations accompanied by a loss of DSG2, changes of the intermediate filament system, and increased phosphorylation of p38 MAPK and cytokeratins. DSG2-deficient and RET-deficient Caco2 cells revealed that GDNF specifically recruits DSG2 to the cell borders, resulting in increased DSG2-mediated intercellular adhesion via the RET receptor. Challenge of Caco2 cells and enteroids with proinflammatory cytokines as well as dextran sulfate sodium-induced (DSS-induced) colitis in C57Bl/6 mice led to impaired IEB function with reduced DSG2 mediated by p38 MAPK-dependent phosphorylation of cytokeratins. GDNF blocked all inflammation-induced changes in the IEB. GDNF attenuates inflammation-induced impairment of IEB function caused by the loss of DSG2 through p38 MAPK-dependent phosphorylation of cytokeratin. The reduced GDNF in patients with IBD indicates a disease-relevant contribution to the development of IEB dysfunction.