SUMMARY The intestinal microbiota is important for induction of inflammatory bowel disease (IBD). IBD is associated with complex shifts in microbiota composition, but it is unclear whether specific bacterial subsets induce IBD and, if so, whether their proportions in the microbiota are altered during disease. Here we fulfilled Koch’s postulates in host-genotype-specific fashion using a mouse model of IBD with human-relevant disease-susceptibility mutations. From screening experiments we isolated common commensal Bacteroides species, introduced them into antibiotic-pretreated mice, and quantitatively re-isolated them in culture. The bacteria colonized IBD-susceptible and non-susceptible mice equivalently, but induced disease exclusively in susceptible animals. Conversely, commensal Enterobacteriaceae were >100-fold enriched during spontaneous disease but an Enterobacteriaceae isolate failed to induce disease in antibiotic-pretreated mice despite robust colonization. We thus demonstrate that IBD-associated microbiota alterations do not necessarily reflect underlying disease etiology. These findings establish important experimental criteria and a conceptual framework for understanding microbial contributions to IBD.

Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi-vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3-positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3-positive vacuole-associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion.

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound-associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE 2 ) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear b-catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE 2 -Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE 2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury.

Summary Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically-susceptible mice (dnKO) that develops spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron (B. theta), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta, which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase-dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagyrelated 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.