Autophagy-ER stress crosstalk controls mucus secretion and susceptibility to gut inflammation

ABSTRACT

Mucus secretion from colonic goblet cells is an important host defense mechanism against the harsh lumenal environment. Yet how mucus secretion is regulated is not well understood. We discovered that constitutive activation of macroautophagy/autophagy via BECN1 (beclin 1) relieves endoplasmic reticulum (ER) stress in goblet cells, which in turn produce a thicker and less penetrable mucus barrier. Pharmacological reduction of the ER stress or activation of the unfolded protein response (UPR) in mice, regardless of autophagy activation, lead to excess mucus secretion. This regulation of mucus secretion by ER stress is microbiota-dependent and requires the activity of the intracellular sensor NOD2 (nucleotide-binding oligomerization domain containing 2). Excess mucus production in the colon alters the gut microbiota and protects from chemical- and infection-driven inflammation. Our findings provide new insights into the mechanisms by which autophagy regulates mucus secretion and susceptibility to intestinal inflammation.

Abbreviations:BECN1– Beclin 1; ER– endoplasmic reticulum; UPR – unfolded protein response; NOD2 – nucleotide-binding oligomerization domain containing 2; IBD– inflammatory bowel disease; BCL2- B cell leukemia/lymphoma 2; TUDCA- tauroursodeoxycholic acid; ATG16L1– autophagy related 16 like 1; LRRK2- leucine-rich repeat kinase 2.

The intestinal epithelium is constantly exposed to the microbiota. Sensing these microbes by the immune system can trigger a proinflammatory response. To preserve homeostasis and prevent chronic inflammation, colonic goblet cells produce and secrete mucus to create a protective barrier that covers the intestinal epithelium. This mucus barrier restricts microbial contact with the host and limits microbial invasion, thus limiting proinflammatory responses. Impairment in the mucus barrier, or goblet cell dysfunction, are associated with infections and inflammatory conditions such as inflammatory bowel disease (IBD). Despite their importance, it was not known how goblet cells regulate the amount of mucus that they secrete.

We have previously shown that autophagy plays a crucial role in secretion from intestinal Paneth cells. Thus, we attempted to unveil how mucus secretion is regulated in goblet cells by examining the role of autophagy in this process [1]. First, we used an animal model in which autophagy is constitutively active. We hypothesized that, as autophagy is crucial for secretion, constant activation of autophagy will enhance secretion. We chose to use a mouse model in which the autophagy-driving protein BECN1 is not inhibited. The engagement of BECN1 requires phosphorylation of BCL2 (B cell leukemia/lymphoma 2), as this protein sequesters BECN1 to the cytoplasm during steady state conditions. In the Becn1^F121A mice, BECN1 cannot bind BCL2 and it is thus always active. We found that preventing BCL2 from binding to BECN1 increases mucus secretion and improves intestinal barrier function by limiting penetration of bacterial antigens. Conversely, genetic inhibition of autophagy in mice by increasing BECN1-BCL2 interaction impairs mucus secretion from goblet cells.

Next, we wanted to understand how autophagy facilitates mucus secretion. We found that expression levels of transcripts encoding key proteins in the unfolded protein response (UPR) pathway are dramatically downregulated in Becn1^F121A mice. Indeed, previous work has shown that the UPR and autophagy are interlinked, as activation of autophagy can reduce ER stress by degrading part of this compartment. Thus, we hypothesized that constitutively active autophagy drives mucus secretion by relieving ER stress. To test this, we pharmacologically reduced ER stress in wild-type mice by treating them with the chemical chaperone TUDCA. We found that this treatment elevates mucus secretion rates from goblet cells, leading to a thicker mucus layer, as seen in Becn1^F121A animals. Accordingly, treating Becn1^F121A mice with an ER stress inducer reverses the enhanced mucus secretion seen in these mice. This finding underscores the crucial involvement of ER stress in modulating mucus secretion, further emphasizing the significance of the UPR-autophagy crosstalk in maintaining proper intestinal function.

We then tested whether immune-microbe interaction regulates this mucus secretion process. We found that the presence of gut microbiota and activation of the intracellular bacterial sensor NOD2 regulate ER stress-mediated mucus secretion. The absence of microbiota hinders the expansion of both the mucus layer and mucus secretion rate in response to reduced ER stress. Similarly, reducing ER stress in mice lacking the Nod2 gene does not facilitate excess mucus secretion. Therefore, the presence of the microbiota is likely sensed by NOD2, which primes excess mucus secretion in response to the suppression of ER stress in colonic goblet cells.

Our findings highlight that ER stress acts as a cell-intrinsic switch that enables goblet cells to regulate mucus secretion. The production and secretion of mucus present a formidable challenge for the secretory machinery within goblet cells. This challenge arises due to the composition of mucus, which consists of large proteins. Production and proper folding of these large proteins exerts significant pressure on the ER, along with the need of retaining a high secretory capacity to maintain constant renewal of the mucus barrier. Thus, goblet cells are particularly vulnerable to ER stress. To restore cellular homeostasis, cells activate autophagy to reduce ER stress and allow goblet cells to intactly perform their secretory functions.

Our observation that autophagy is required to maintain mucus production by relieving ER stress, and that NOD2 plays a critical role in facilitating this mucus secretion (Figure 1), provides a possible explanation for the high prevalence of risk mutations in autophagy genes (ATG16L1 and LRRK2) and NOD2 in IBD patients. Moreover, we found that overproduction of mucus confers protection from chemical- and infection-driven colitis by boosting intestinal barrier function in mice. Indeed, penetration of the mucus barrier by microbes is a hallmark of IBD.

Figure 1.

Autophagy facilitates mucus secretion from goblet cells. (A) Constitutive activation of autophagy in intestinal goblet cells leads to (B) reduction of ER stress levels, which facilitates (C) mucus secretion. This process depends on (i) the presence of the microbiota, which is likely (ii) sensed by the intracellular microbial sensor NOD2. Excess mucus secretion then results in (D) improved barrier function and reduces susceptibility to colitis. Created with BioRender.com.

Our work raises a few interesting questions. First, it remains unclear exactly how autophagy reduces ER stress in goblet cells. While the crosstalk between the UPR and activation of autophagy has been demonstrated, the mechanism by which autophagy reduces ER stress is not fully defined. One possibility is that misfolded mucus-forming proteins are removed and degraded by canonical autophagy. Another possibility is that recycling of whole ER fragments by reticulophagy reduces the UPR by clearing misfolded mucus-forming proteins. How this reduction in ER stress is then translated into higher rates of mucus secretion is still mysterious.

Another question is how NOD2 orchestrates excess mucus secretion when ER stress levels are reduced. Several cell types in the intestine, including goblet cells, express the cytosolic innate immune sensor NOD2, which senses conserved motifs in bacterial peptidoglycans. NOD2 has been extensively recognized as a key susceptible gene for the development of Crohn disease, a form of IBD. Previous research has demonstrated that loss of NOD2 leads to abnormalities in goblet cells in the small intestine, resulting in a decrease in the number of goblet cells and reduced mucus production. However, the mechanism by which NOD2 preserves goblet cell viability has not been elucidated. Our study now links NOD2 to autophagy, ER stress and control of mucus production. Indeed, NOD2 has been previously shown to recruit ATG16L1 to expanding phagophores. Of course, the possibility that NOD2 is needed in a non-epithelial cell and orchestrates mucus secretion from goblet cells via an intercellular signaling pathway is also a possible scenario.

Overall, understanding the mechanisms that regulate mucus secretion may offer a new potential strategy to restore barrier integrity and drive remission in IBD.

Funding Statement

The Azrieli Foundation Early Career Faculty Fellowship (SB)Israeli Science Foundation (ISF) grants 925/19 and 1851/19 (SB)European Crohn’s and Colitis Organization (ECCO) Grant (SB)Mizutani Foundation for Glycoscience (SB)European Research Council (ERC) Starting Grant GCMech 101039927 (SB)

Disclosure statement

No potential conflict of interest was reported by the author(s).