Faculty Bibliography

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19. Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchioalveolar lavage fluid from critically ill COVID-19 patients was associated with reduced ICU and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Introduction: Multiplex gastrointestinal pathogen panels (GI PCR) for the detection of enteric infection offer advantages in turnaround time and sensitivity compared to conventional modalities of stool testing. Previous research on GI PCR has focused almost exclusively on hospitalized patients, however, most cases of gastroenteritis are managed in the outpatient setting. Our aim was to compare GI PCR to conventional stool testing in outpatients with gastroenteritis and evaluate the impact on clinical decision-making and management.
Method(s): We performed a retrospective study of outpatients presenting with acute gastrointestinal symptoms at an academicmedical center from September 2015 to April 2019 who received stool testing with GI PCR, and from February 2009 to April 2012 who received stool culture and ova and parasite exam ("conventional testing"). Patients who received GI PCR were matched by age and sex to patients who received conventional testing. We recorded pathogens isolated, demographic data, presenting symptoms, risk factors, antibiotic therapy, and downstream associated healthcare utilization including abdominal imaging, endoscopy, emergency room visits, hospitalizations, and abdominal surgeries. Univariate and multivariate linear regression was used to estimate the effect of testing on empiric antibiotic therapy.
Result(s): We identified 1,018 outpatients who received GI PCR or conventional stool testing. (n = 509 each). A pathogen was isolated in 208 (41%) patients with GI PCR and 49 (10%, P< 0.01, Table 1) with conventional culture. There were no significant differences in overall rates of antibiotic therapy or average duration of therapy, but GI PCR was associated with less empiric therapy (47% vs 70%, P < 0.01), and higher rates of narrowing (13 vs 4%) and discontinuation (3 vs 0%, P < 0.01) of empiric antibiotics after the initial visit, with a 1.6-fold lower likelihood (95% CI 1.04-2.57, P= 0.04) of initiating empiric antibiotics on multivariable analysis. There were no significant differences in other outcomes.
Conclusion(s): GI PCR testing detected more pathogens compared to conventional stool testing, without a significant increase in antibiotics prescribed. PCR testing was associated with changes in prescribing patterns for antibiotics, with a shift towards less empiric therapy

Introduction: Traveler's diarrhea is the most common travel related illness and is often encountered in the outpatient setting. Most pathogens that cause traveler's diarrhea are poorly characterized by conventional stool testing such as stool culture and ova and parasite exam. Multiplex polymerase chainreaction based gastrointestinal pathogen panels (GI PCR) offer advantages in sensitivity and turnaround time, however their impact on clinical management is unclear. Our aim was to evaluate the clinical impact of GI PCR testing in outpatients presenting with traveler's diarrhea.
Method(s): We performed a retrospective study comparing outpatients presenting with acute gastrointestinal symptoms at an academic medical center who received stool testing with GI PCR from September 2015 to April 2019, to patients who presented with similar symptoms from February 2009 to April 2012, when GI PCR was unavailable, and received stool culture and ova and parasite exam ("conventional testing"). Patients who received GI PCR were matched by age and sex to patients who received conventional testing. We recorded pathogens isolated, demographic data, history of travel within 30 days of presentation, presenting symptoms and rates of antibiotic therapy.
Result(s): We identified 1,018 outpatients who received GI PCR or conventional stool testing. (n5509 each). There were 70 patients in the conventional cohort and 78 in the GI PCR cohort, respectively, with traveler's diarrhea. In patients who received GI PCR, we observed higher diagnostic yield in patients with traveler's diarrhea who received GI PCR, compared to patients without a history of recent travel (54 vs. 39%, p50.01, Table 1). There was no difference in diagnostic yield in patients who received conventional stool testing (9 vs 10%, p50.74). Patients with travelers' diarrhea were more likely to have pathogenic species of Escherichia coli (E. coli) identified on testing (45 vs. 22%, p, 0.01, Table 2), particularly Enteroaggregative E. coli (23 vs. 6%, p, 0.01). Patients with traveler's diarrhea were more likely to receive antibiotics whether they received GI PCR (38 vs. 27%, p50.04) or conventional testing (35 vs. 25%, p50.06).
Conclusion(s): GI PCR was associated with a higher diagnostic yield in patients presenting with travelers' diarrhea. Patients with travelers' diarrhea were significantly more likely to have pathogenic E. coli species identified on PCR testing, with up to 45% of patients testing positive

The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate that the gut microbiome is directly affected by SARS-CoV-2 infection in a dose-dependent manner in a mouse model, causally linking viral infection and gut microbiome dysbiosis. Comparison with stool samples collected from 97 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients suggest that bacteria translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID 19.

The contributions of the viral component of the microbiome-the virome-to the development of innate and adaptive immunity are largely unknown. Here, we systematically defined the host response in mice to a panel of eukaryotic enteric viruses representing six different families. Infections with most of these viruses were asymptomatic in the mice, the magnitude and duration of which was dependent on the microbiota. Flow cytometric and transcriptional profiling of mice mono-associated with these viruses unveiled general adaptations by the host, such as lymphocyte differentiation and IL-22 signatures in the intestine, as well as numerous viral-strain-specific responses that persisted. Comparison with a dataset derived from analogous bacterial mono-association in mice identified bacterial species that evoke an immune response comparable with the viruses we examined. These results expand an understanding of the immune space occupied by the enteric virome and underscore the importance of viral exposure events.

BACKGROUND & AIMS/OBJECTIVE:Restorative proctocolectomy with ileal pouch-anal anastomosis is a surgical procedure in patients with ulcerative colitis refractory to medical therapies. Pouchitis, the most common complication, is inflammation of the pouch of unknown etiology. To define how the intestinal immune system is distinctly organized during pouchitis, we analyzed tissues from patients with and without pouchitis and from patients with ulcerative colitis using single-cell RNA sequencing (scRNA-seq). METHODS:We examined pouch lamina propria CD45+ hematopoietic cells from intestinal tissues of ulcerative colitis patients with (n=15) and without an ileal pouch-anal anastomosis (n=11). Further in silico meta-analysis was performed to generate transcriptional interaction networks and identify biomarkers for patients with inflamed pouches. RESULTS:In addition to tissue-specific signatures, we identified a population of IL1B/LYZ+ myeloid cells and FOXP3/BATF+ T cells that distinguish inflamed tissues which we further validated in other single cell RNA-seq datasets from IBD patients. Cell type specific transcriptional markers obtained from single-cell RNA-sequencing was used to infer representation from bulk RNA sequencing datasets, which further implicated myeloid cells expressing IL1B and S100A8/A9 calprotectin as interacting with stromal cells, and Bacteroidiales and Clostridiales bacterial taxa. We found that non-responsiveness to anti-integrin biologic therapies in ulcerative colitis patients was associated with the signature of IL1B+/LYZ+ myeloid cells in a subset of patients. CONCLUSIONS:Features of intestinal inflammation during pouchitis and ulcerative colitis are similar, which may have clinical implications for the management of pouchitis. scRNA-seq enables meta-analysis of multiple studies, which may facilitate the identification of biomarkers to personalize therapy for IBD patients.