Many studies have suggested a role for gut-resident microbes (the “gut microbiome”) in modulating host health; however, the mechanisms by which they impact systemic physiology remain largely unknown. ...In this study, metabolomic and transcriptional profiling of germ-free and conventionalized mouse liver revealed an upregulation of the Nrf2 antioxidant and xenobiotic response in microbiome-replete animals. Using a Drosophila-based screening assay, we identified members of the genus Lactobacillus capable of stimulating Nrf2. Indeed, the human commensal Lactobacillus rhamnosus GG (LGG) potently activated Nrf2 in the Drosophila liver analog and the murine liver. This activation was sufficient to protect against two models of oxidative liver injury, acetaminophen overdose and acute ethanol toxicity. Characterization of the portal circulation of LGG-treated mice by tandem mass spectrometry identified a small molecule activator of Nrf2, 5-methoxyindoleacetic acid, produced by LGG. Taken together, these data demonstrate a mechanism by which intestinal microbes modulate hepatic susceptibility to oxidative injury.
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•The gut microbiome induces the Nrf2 antioxidant response pathway in the liver•Gut-resident Lactobacilli induce hepatic Nrf2 in both Drosophila and mice•Oral delivery of Lactobacillus rhamnosus GG protects against oxidative liver injury•Lactobacilli-derived 5-methoxyindoleacetic acid activates Nrf2
Saeedi et al. demonstrate that the gut microbiome acts at a distance to activate host antioxidant responses in the liver. These responses can be amplified by exogenous administration of Lactobacilli and protect against oxidative liver injury. Finally, they identify a Lactobacilli-derived small molecule that mediates, in part, these effects.
Adenocarcinomas of the luminal gastrointestinal tract and pancreatobiliary system often show histologic and immunohistochemical overlap, making delineation of the primary site in a metastatic setting ...difficult. Previous studies have shown that site-specific missense mutations in the oncogene KRAS could be used in conjunction with immunohistochemistry to differentiate metastatic pancreatic adenocarcinoma from primary lung adenocarcinoma. In this study, we assessed the patterning of KRAS mutations across sites in the gastrointestinal and pancreatobiliary system. By integrating sequencing data from 44 separate studies, we assessed 2523 KRAS mutations in 7382 distinct cases of adenocarcinoma, including those from the esophagus, stomach, ampulla, biliary system, pancreas, and colon. We found that gastrointestinal adenocarcinomas demonstrate a marked regional variation in the frequency of KRAS mutations, with the most frequent KRAS mutation observed in pancreatic adenocarcinoma (up to 94.9%), whereas the frequency is much lower in adenocarcinomas from the esophagus and stomach (5.4% and 8.7%, respectively). Intriguingly, the pattern of missense mutations showed site specificity as well, with c.35G>T (p.G12V) and c.34G>C (p.G12R) mutations enriched in pancreatic primaries and codon 13 and non–codon 12/13 alterations enriched in gastric primaries (specificity of 98.9% and 93.2%, respectively, with a negative predictive value of 93.6% and 92.93% against pancreatic adenocarcinoma). Furthermore, we found that esophageal and gastric adenocarcinomas show an enrichment in transitional mutations, whereas other sites showed an equal distribution. Importantly, the examination of a validation cohort from our own institution revealed similar trends. These findings indicate that, in addition to providing therapeutic and diagnostic information, KRAS mutational analysis may also prove useful in delineating the site of origin in gastrointestinal adenocarcinomas that share morphologic and immunohistochemical overlap. Moreover, transitional mutations are more frequent in esophageal and gastric adenocarcinomas, reiterating the role of chronic inflammation in the pathogenesis of foregut adenocarcinomas.
Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are ...mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA-mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.
Acetaminophen is the most common cause of acute drug-induced liver injury in the United States. However, research into the mechanisms of acetaminophen toxicity and the development of novel ...therapeutics is hampered by the lack of robust, reproducible, and cost-effective model systems. Herein, we characterize a novel Drosophila-based model of acetaminophen toxicity. We demonstrate that acetaminophen treatment of Drosophila results in similar pathophysiologic alterations as those observed in mammalian systems, including a robust production of reactive oxygen species, depletion of glutathione, and dose-dependent mortality. Moreover, these effects are concentrated in the Drosophila fat body, an organ analogous to the mammalian liver. Utilizing this system, we interrogated the influence of environmental factors on acetaminophen toxicity which has proven difficult in vertebrate models due to cost and inter-individual variability. We find that both increasing age and microbial depletion sensitize Drosophila to acetaminophen toxicity. These environmental influences both alter oxidative stress response pathways in metazoans. Indeed, genetic and pharmacologic manipulations of the antioxidant response modify acetaminophen toxicity in our model. Taken together, these data demonstrate the feasibility of Drosophila for the study of acetaminophen toxicity, bringing with it an ease of genetic and microbiome manipulation, high-throughput screening, and availability of transgenic animals.
Interactions between the microbiota and distal gut are fundamental determinants of human health. Such interactions are concentrated at the colonic mucosa and provide energy for the host epithelium ...through the production of the short-chain fatty acid butyrate. We sought to determine the role of epithelial butyrate metabolism in establishing the austere oxygenation profile of the distal gut. Bacteria-derived butyrate affects epithelial O2 consumption and results in stabilization of hypoxia-inducible factor (HIF), a transcription factor coordinating barrier protection. Antibiotic-mediated depletion of the microbiota reduces colonic butyrate and HIF expression, both of which are restored by butyrate supplementation. Additionally, germ-free mice exhibit diminished retention of O2-sensitive dyes and decreased stabilized HIF. Furthermore, the influences of butyrate are lost in cells lacking HIF, thus linking butyrate metabolism to stabilized HIF and barrier function. This work highlights a mechanism where host-microbe interactions augment barrier function in the distal gut.
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•The mammalian colon exists in a state of relative hypoxia•Hypoxic regions of the normal colon provide a signaling axis through HIF-1•Microbial-derived butyrate depletes O2 and activates HIF-1•Microbiota-derived butyrate is barrier-protective in the mucosa
Decreased short-chain fatty acid (SCFA) production by the microbiota has been correlated with colonic inflammation and disease. Kelly et al. show that microbial-derived SCFAs, particularly butyrate, stimulate epithelial metabolism and deplete intracellular O2, resulting in stabilization of the transcription factor HIF-1 and increased epithelial barrier function.
Epithelial barrier dysfunction is a significant factor in many allergic diseases, including eosinophilic esophagitis (EoE). Infiltrating leukocytes and tissue adaptations increase metabolic demands ...and decrease oxygen availability at barrier surfaces. Understanding of how these processes impact barrier is limited, particularly in allergy. Here, we identified a regulatory axis whereby the oxygen-sensing transcription factor HIF-1α orchestrated epithelial barrier integrity, selectively controlling tight junction CLDN1 (claudin-1). Prolonged experimental hypoxia or HIF1A knockdown suppressed HIF-1α-dependent claudin-1 expression and epithelial barrier function, as documented in 3D organotypic epithelial cultures. L2-IL5OXA mice with EoE-relevant allergic inflammation displayed localized eosinophil oxygen metabolism, tissue hypoxia, and impaired claudin-1 barrier via repression of HIF-1α/claudin-1 signaling, which was restored by transgenic expression of esophageal epithelial-targeted stabilized HIF-1α. EoE patient biopsy analysis identified a repressed HIF-1α/claudin-1 axis, which was restored via pharmacologic HIF-1α stabilization ex vivo. Collectively, these studies reveal HIF-1α's critical role in maintaining barrier and highlight the HIF-1α/claudin-1 axis as a potential therapeutic target for EoE.
BACKGROUND:Clostridium difficile infection has been associated with negative outcomes in the general population and in pregnant patients. Fecal microbiota transplant has become the standard for ...treatment of recurrent as well as refractory C difficile infection.
CASE:We present a case of a 28-year-old pregnant woman who presented with recurrent C difficile infection despite treatment with vancomycin and fidaxomicin and underwent a successful fecal microbiota transplant through colonoscopy at 18 weeks of gestation. She no longer required antibiotics for the remainder of her pregnancy to treat C difficile and had a term vaginal delivery at 39 weeks of gestation.
CONCLUSION:Our pregnant patient tolerated and responded to a fecal microbiota transplant for treatment of recurrent C difficile infection. Future large-scale studies are needed to determine the efficacy, safety, and long-term effects of manipulating the microbiome in pregnant patients and the neonates.
Abstract
We have demonstrated that neuropeptide Y (NPY) can regulate pro-inflammatory signaling in the gut via cross-talk with the pro-inflammatory cytokine tumor necrosis factor (TNF). Here, we ...investigated if selective blocking of NPY receptors, NPY1R or NPY2R, using small molecule non-peptide antagonists (BIBP-3222 for NPY1R and BIIE-0246 for NPY2R) in the colon could attenuate intestinal inflammation by lowering TNF levels (BIBP - N-(1R)-4-(Aminoiminomethyl)amino-1-(4-hydroxyphenyl)methylaminocarbonylbutyl-α-phenylbenzeneacetamide; BIIE - N-(1S)-4-(Aminoiminomethyl)amino-1-2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethylaminocarbonylbutyl-1-2-4-(6,11-dihydro-6-oxo-5H-dibenzb,eazepin-11-yl)-1-piperazinyl-2-oxoethyl-cyclopentaneacetamide). Colitis was induced using dextran sodium sulfate in drinking water for 7 days, or by adoptive T-cell transfer in RAG-/- mice. Colonic biopsies from healthy subjects (n = 10) and IBD patients (n = 34, UC = 20, CD = 14) were cultured ex vivo in presence or absence of NPY antagonists (100 µM, 20 h), and cytokine release into culture supernatants was measured by ELISA. Intracolonic administration of BIBP (but not BIIE) significantly reduced clinical, endoscopic, and histological scores, and serum TNF, interleukin (IL)-6, and IL-12p70 in DSS colitis; it also significantly attenuated histological damage and serum IL-6 in T-cell colitis (P < .05). Intracolonic administration of BIBP significantly reduced TNF and interferon (IFN)-γ release from UC biopsies, whereas BIIE downregulated only IFN-γ (P < .05). BIBP significantly reduced TNF and interferon (IFN)-γ release from UC biopsies, whereas BIIE downregulated only IFN-γ (P < .05). Our data suggest a promising therapeutic value for NPY1R inhibition in alleviating intestinal inflammation in UC, possibly as enemas to IBD patients.
Reactive oxygen species (ROS) are potent signaling molecules with critical roles in cellular pathology and homeostasis. They are produced in all cell types via a diverse array of cellular machinery, ...giving rise to an equally diverse repertoire of molecular effects. These range from cytotoxic killing of microbes to alteration of the cellular transcriptional response to stress. Despite their importance, research into ROS has been difficult given their inherent instability and transient signaling properties. Herein we describe methods for the use of the redox-sensitive probe hydro-Cy3 for the detection and quantification of ROS both in vitro and in vivo.
Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are ...orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine–creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2–dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.
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