Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression.

Objective: Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive. Materials and Methods: Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation. Results: Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro. Conclusion: These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.

The novel mechanism of human norovirus induced diarrhea: Activation of PKD2 caused by HuNoVs destroyed AQP3 expression through AP2γ in intestinal epithelial cells.

Our previous work has demonstrated protein kinase D2 (PKD2) played a critical influence in experimental colitis in animal. However, the role of PKD2 in human norovirus (HuNoVs)-induced diarrhea remained unknown. Aquaporin 3 (AQP3) expression, a critical protein mediating diarrhea, was assessed by western blot, qRT-PCR in intestinal epithelial cells (IECs). Luciferase, IF, IP and ChIP assay were used to explore the mechanism through which HuNoVs regulated AQP3. Herein, we found that AQP3 expression was drastically decreased in IECs in response to VP1 transfection, the major capsid protein of HuNoVs, or HuNoVs infection. Mechanistically, HuNoVs triggered phosphorylation of PKD2 through TLR2/MyD88/IRAK4, which further inhibited AP2γ activation and nuclear translocation, leading to suppress AQP3 transactivation in IECs. Most importantly, PKD2 interacted with MyD88/IRAK4, and VP1 overexpression enhanced this complex form, which, in turn, to increase PKD2 phosphorylation. In addition, endogenous PKD2 interacted with AP2γ, and this interaction was enhanced in response to HuNoVs treatment, and subsequently resulting in AP2γ phosphorylation inhibition. Moreover, inhibition of PKD2 activation could reverse the inhibitory effect of HuNoVs on AQP3 expression. In summary, we established a novel mechanism that HuNoV inhibited AQP3 expression through TLR2/MyD88/IRAK4/PKD2 signaling pathway, targeting PKD2 activity could be a promising strategy for prevention of HuNoVs-induced gastroenteritis.