Faculty Bibliography

Although Western diet and dysbiosis are the most prominent environmental factors associated with inflammatory bowel diseases (IBDs), the corresponding host factors and cellular mechanisms remain poorly defined. Here we report that the TSC1/mTOR pathway in the gut epithelium represents a metabolic and innate immune checkpoint for intestinal dysfunction and inflammation. mTOR hyperactivation triggered by Western diet or Tsc1 ablation led to epithelium necroptosis, barrier disruption, and predisposition to dextran sulfate sodium-induced colitis and inflammation-associated colon cancer. Mechanistically, our results uncovered a critical role for TSC1/mTOR in restraining the expression and activation of RIPK3 in the gut epithelium through TRIM11-mediated ubiquitination and autophagy-dependent degradation. Notably, microbiota depletion by antibiotics or gnotobiotics attenuated RIPK3 expression and activation, thereby alleviating epithelial necroptosis and colitis driven by mTOR hyperactivation. mTOR primarily impinged on RIPK3 to potentiate necroptosis induced by TNF and by microbial pathogen-associated molecular patterns (PAMPs), and hyperactive mTOR and aberrant necroptosis were intertwined in human IBDs. Together, our data reveal a previously unsuspected link between the Western diet, microbiota, and necroptosis and identify the mTOR/RIPK3/necroptosis axis as a driving force for intestinal inflammation and cancer.

Autophagy is a cell biological process that promotes resilience in the face of environmental perturbations. Given that infectious agents represent a major type of environmental threat, it follows that the autophagy pathway is central to the outcome of host-microbe interactions. Detailed molecular studies have revealed intricate ways in which autophagy suppresses or enhances the fitness of infectious agents, particularly intracellular pathogens such as viruses that require the host cell machinery for replication. Findings in animal models have reinforced the importance of these events that occur within individual cells and have extended the role of autophagy to extracellular microbes and immunity at the whole organism level. These functions impact adaptation to bacteria that are part of the gut microbiota, which has implications for the etiology of chronic disorders such as inflammatory bowel disease. Despite major advances in how autophagy regulates inflammatory reactions toward microbes, many challenges remain, including distinguishing autophagy from closely related pathways such as LC3-associated phagocytosis. Here, we review the role of autophagy in microbial pathogenesis at the level of organismal biology. In addition to providing an overview of the prominent function of autophagy proteins in host-microbe interactions, we highlight how observations at the cellular level are informing pathogenesis studies and offer our perspective on the future directions of the field.

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)^1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin⁴-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues^2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.

Despite being a staple of our science, the process of pre-publication peer review has few agreed-upon standards defining its goals or ideal execution. As a community of reviewers and authors, we assembled an evaluation format and associated specific standards for the process as we think it should be practiced. We propose that we apply, debate, and ultimately extend these to improve the transparency of our criticism and the speed with which quality data and ideas become public.

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria, indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here, we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells, which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition, we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges.

INTRODUCTION: Rapid, highly sensitive and specific multiplex polymerase chain reaction-based stool assays for gastrointestinal pathogens (GI PCR) are increasingly being used alternatively to conventional stool culture. We investigated the concordance between simultaneous GI PCR and stool culture with an ova and parasite (O&P) exam in outpatients presenting with symptoms of infectious gastroenteritis.
METHOD(S): We performed a cross-sectional study of outpatients who received a FilmArray GI PCR test for acute diarrhea at an academic medical center from September 2015 to February 2019 to identify patients who had a concomitant stool culture with an ova and parasite exam (conventional testing) at the same time, on the same stool sample. The primary outcome was detection of an infection on GI PCR or conventional stool testing. Correlation was evaluated using McNemar's test for pathogens detected on both tests. Other categorical variables were compared with Chi-square analysis.
RESULT(S): We identified 150 outpatients who received GI PCR and stool culture with an ova and parasite exam for an episode of acute gastroenteritis. 106 (71%) patients had a pathogen isolated on GI PCR for 144 total pathogens including 128 (88%) bacteria, 13 (9%) viruses, and 3 (2%) parasites; 21 (14%) patients had a pathogen isolated on conventional testing for 18 total pathogens including 9 (50%) bacteria and 9 (50%) parasites (Table 1). Multiple pathogens were found in 38 (26%) GI PCR tests. PCR testing most commonly identified Enteropathogenic Escherichia coli (EPEC), representing 42 (33%) positive PCR tests. Conventional testing most commonly identified Campylobacter jejuni with 13 (54%) positive tests. Of 28 total C. jejuni infections, 15 (54%) were positive only on PCR, 3 (10%) only on conventional testing, and 10 (36%) on both modalities, showing that conventional testing missed 54% of all infections (P=0.008). Conventional testing missed 4/6 (67%, P=0.125) Salmonella infections and 9/14 (64%, P=0.0215) Yersinia infections, nor did it detect any viral or diarrheagenic E. coli infections. Overall, PCR detected 144 of 191 (75%) of possible pathogens whereas conventional testing detected 47 of 179 possible pathogens (26%).
CONCLUSION(S): GI PCR testing identified multiple pathogens unidentified by conventional testing, such as enteric viruses and pathogenic strains of E. coli. Conventional testing missed 88% of enteric bacteria showing poor concordance between simultaneous GI PCR testing and stool culture with an ova and parasite exam. (Table Presented)

The cellular degradative pathway of autophagy prevents unrestrained inflammatory signaling by removing intracellular microbes, damaged organelles, and other factors that trigger immune reactions. Consistent with this function, a common variant of the autophagy gene ATG16L1 is associated with susceptibility to inflammatory bowel disease (IBD), a disorder characterized by a chronic immune reaction directed against the gut microbiota. We recently contributed to our understanding of the link between autophagy and inflammatory signaling in the intestine by demonstrating that autophagy proteins including ATG16L1 are necessary in the epithelium to prevent a spontaneous type I interferon response to the gut microbiota. Enhanced innate immunity that occurs upon autophagy inhibition is protective in mouse models of infection by an enteric bacterial pathogen and acute epithelial injury. Although avoiding excess immune reactions towards the microbiota is necessary to prevent IBD, these observations indicate that autophagy hampers productive immunity at the intestinal epithelial barrier in certain contexts. Here, we discuss how this counterintuitive consequence of autophagy inhibition can be reconciled with the established beneficial role of the pathway.

Tissue-resident macrophages are the most abundant immune cell population in healthy adipose tissue. Adipose tissue macrophages (ATMs) change during metabolic stress and are thought to contribute to metabolic syndrome. Here, we studied ATM subpopulations in steady state and in response to nutritional and infectious challenges. We found that tissue-resident macrophages from healthy epididymal white adipose tissue (eWAT) tightly associate with blood vessels, displaying very high endocytic capacity. We refer to these cells as vasculature-associated ATMs (VAMs). Chronic high-fat diet (HFD) results in the accumulation of a monocyte-derived CD11c⁺CD64⁺ double-positive (DP) macrophage eWAT population with a predominant anti-inflammatory/detoxifying gene profile, but reduced endocytic function. In contrast, fasting rapidly and reversibly leads to VAM depletion, while acute inflammatory stress induced by pathogens transiently depletes VAMs and simultaneously boosts DP macrophage accumulation. Our results indicate that ATM populations dynamically adapt to metabolic stress and inflammation, suggesting an important role for these cells in maintaining tissue homeostasis.