Faculty Bibliography

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.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

OBJECTIVES/OBJECTIVE:Few studies have examined the role of non-Clostridium difficile enteric infections in flares of inflammatory bowel disease (IBD). Our objective was to investigate enteric infection detected by multiplex PCR stool testing in patients with IBD. METHODS:We performed a cross-sectional analysis of 9403 patients who underwent 13,231 stool tests with a gastrointestinal pathogen PCR panel during a diarrheal illness from March 2015 to May 2017. Our primary outcome was the presence of an infection. Secondary outcomes included endoscopic and histologic predictors of infection, and IBD outcomes following testing. RESULTS:A total of 277 patients with Crohn's disease (CD), 300 patients with ulcerative colitis (UC), and 8826 patients without IBD underwent 454, 503, and 12,275 tests, respectively. Compared to patients without IBD, patients with IBD were less likely to test positive (CD 18.1%, UC 16.1%, no IBD 26.6%, p < 0.001). Compared to patients without IBD, CD had a higher prevalence of norovirus (p = 0.05) and Campylobacter (p = 0.043), whereas UC had a lower prevalence of norovirus (p = 0.001) and a higher prevalence of Campylobacter (p = 0.013), Plesiomonas (p = 0.049), and Escherichia coli species (p < 0.001). Of 77 patients who underwent endoscopy, there were no major endoscopic or histologic predictors of a positive test. Patients who tested negative were more likely to have IBD therapy escalated (p = 0.004). Enteric infection did not impact IBD outcomes following testing (log-rank 0.224). CONCLUSIONS:Non-Clostridium difficile enteric infections were identified in 17% of symptomatic patients with IBD. Endoscopic and histologic findings may not differentiate flare from infection. Norovirus and E.coli may play an important role in flare of IBD.

As a conserved pathway that lies at the intersection between host defence and cellular homeostasis, autophagy serves as a rheostat for immune reactions. In particular, autophagy suppresses excess type I interferon (IFN-I) production in response to viral nucleic acids. It is unknown how this function of autophagy relates to the intestinal barrier where host-microbe interactions are pervasive and perpetual. Here, we demonstrate that mice deficient in autophagy proteins are protected from the intestinal bacterial pathogen Citrobacter rodentium in a manner dependent on IFN-I signalling and nucleic acid sensing pathways. Enhanced IFN-stimulated gene expression in intestinal tissue of autophagy-deficient mice in the absence of infection was mediated by the gut microbiota. Additionally, monocytes infiltrating into the autophagy-deficient intestinal microenvironment displayed an enhanced inflammatory profile and were necessary for protection against C. rodentium. Finally, we demonstrate that the microbiota-dependent IFN-I production that occurs in the autophagy-deficient host also protects against chemical injury of the intestine. Thus, autophagy proteins prevent a spontaneous IFN-I response to microbiota that is beneficial in the presence of infectious and non-infectious intestinal hazards. These results identify a role for autophagy proteins in controlling the magnitude of IFN-I signalling at the intestinal barrier.