Autophagy Controls Epithelial Proteolytic Homeostasis of the Intestinal Mucosa

Background Crohn's Disease (CD) is a chronic relapsing inflammatory bowel disease (IBD) with mucosal ulcerations affecting all of the digestive tract. Intestinal barrier breakdown, dys‐regulated autophagy and excessive proteolytic activity in the intestinal mucosa are common features of IBD. However, possible links between these features have not been addressed. Aims We aimed at investigating the impact of autophagy on the proteolytic activity released by enterocytes and goblets cells and its consequences on the intestinal barrier function. Methods Human intestinal epithelial cells including Caco‐2 (enterocyte) and HT29MTX (goblet cells) were stimulated with autophagy inducers such as nutrient starvation (NS, using Earle's balanced salt solution) and rapamycin (0.5μM). At 2, 4, 6 and 24 hours after autophagy induction, the activity and the expression of both serine and cysteine proteases in cell culture supernatants was investigated by enzyme assay and qPCR. In addition, using transwell systems, paracellular permeability, IL1β and IL8 secretion and the expression of the resistin‐like molecule β (RLMβ) were monitored at 6 and 24 hours after autophagy induction. Cysteine protease involvement in intestinal barrier function was assessed by adding to experiments, a cysteine protease inhibitor (E64, 50μM). Results Activation of autophagy by NS or rapamycin reduced the secretion of serine proteases by Caco‐2 cells, while it increased the secretion of cysteine proteases at 2, 4, 6 and 24 hours. Moreover, at 6 and 24 hours, after autophagy induction, mRNA expression of three major serine proteases (PRSS1, 2 and 3) were reduced. Activation of autophagy in HT29MTX cells also increased the secretion of cysteine proteases. Using transwells, we observed that the modulation of serine and cysteine proteases triggered by autophagy only affected the apical secretion of these proteases. We have evidenced that autophagy increased paracellular permeability of human intestinal epithelial monolayers, and their secretion of IL‐1β, while it decreased the secretion of IL‐8 and the expression of RLMβ. In the presence of cysteine protease inhibitor (E64), autophagy failed to increase paracellular permeability and to decrease IL‐8 secretion. In contrast, E64 treatment aggravated IL‐1β secretion. Among the epithelial cysteine proteases (Cathepsins B, D, L and K), we observed that autophagy drastically increased (40‐fold) Cathepsin B mRNA expression. Conclusions Taken together these data evidence that autophagy modified the proteolytic balance of human enterocytes and goblet cells towards an increased cysteine protease activity released on the apical compartment. Through the release of cysteine protease activity, autophagy modifies key elements of epithelial homeostasis such as epithelial permeability and the production of inflammatory mediators. Which proteases are involved in these alterations and what are the consequences of autophagy‐induced cysteine protease activity on mucosa and microbiota homeostasis needs further investigation. Support or Funding Information Agence Nationale de la Recherche (ANR) and European Research Council (ERC).