Modelling the Gastrointestinal Carriage of Klebsiella pneumoniae Infections.

Klebsiella pneumoniae is a leading cause of nosocomial and community acquired infections, making K. pneumoniae the pathogen that is associated with the second largest number of deaths attributed to any antibiotic resistant infection. K. pneumoniae colonizes the nasopharynx and the gastrointestinal tract in an asymptomatic manner without dissemination to other tissues. Importantly, gastrointestinal colonization is a requisite for infection. Our understanding of K. pneumoniae colonization is still based on interrogating mouse models in which animals are pretreated with antibiotics to disturb the colonization resistance imposed by the gut microbiome. In these models, infections disseminate to other tissues. Here, we report a murine model to allow for the study of the gastrointestinal colonization of K. pneumoniae without tissue dissemination. Hypervirulent and antibiotic resistant strains stably colonize the gastrointestinal tract of in an inbred mouse population without antibiotic treatment. The small intestine is the primary site of colonization and is followed by a transition to the colon over time, without dissemination to other tissues. Our model recapitulates the disease dynamics of the metastatic K. pneumoniae strains that are able to disseminate from the gastrointestinal tract to other sterile sites. Colonization is associated with mild to moderate histopathology, no significant inflammation, and no effect on the richness of the microbiome. Our model sums up the clinical scenario in which antibiotic treatment disturbs the colonization of K. pneumoniae and results in dissemination to other tissues. Finally, we establish that the capsule polysaccharide is necessary for the colonization of the large intestine, whereas the type VI secretion system contributes to colonization across the gastrointestinal tract. IMPORTANCE Klebsiella pneumoniae is one of the pathogens that is sweeping the world in the antibiotic resistance pandemic. Klebsiella colonizes the nasopharynx and the gut of healthy subjects in an asymptomatic manner, making gut colonization a requisite for infection. This makes it essential to understand the gastrointestinal carriage in preventing Klebsiella infections. Current research models rely on the perturbation of the gut microbiome by antibiotics, resulting in an invasive infection. Here, we report a new model of K. pneumoniae gut colonization that recapitulates key features of the asymptomatic human gastrointestinal tract colonization. In our model, there is no need to disturb the microbiota to achieve stable colonization, and there is no dissemination to other tissues. Our model sums up the clinical scenario in which antibiotic treatment triggers invasive infection. We envision that our model will be an excellent platform upon which to investigate factors enhancing colonization and invasive infections and to test therapeutics to eliminate Klebsiella asymptomatic colonization.

A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.

Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated 'gut-like' DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/- (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These 'gut-like' DCs extended transepithelial dendrites across the intact epithelium of enteroids. 'Gut-like' DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in 'gut-like' DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.

Loss of interleukin-10 receptor disrupts intestinal epithelial cell proliferation and skews differentiation towards the goblet cell fate.

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of ß-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.

Microbiota-derived butyrate dynamically regulates intestinal homeostasis through regulation of actin-associated protein synaptopodin.

The intestinal mucosa exists in dynamic balance with trillions of luminal microbes. Disruption of the intestinal epithelial barrier, commonly observed in mucosal inflammation and diseases such as inflammatory bowel diseases (IBDs), is often associated with dysbiosis, particularly decreases in species producing short-chain fatty acids (SCFAs), such as butyrate. It remains unclear to what extent microbiota-derived factors contribute to the overall maintenance of intestinal homeostasis. Initial studies revealed that butyrate selectively promotes epithelial barrier function and wound healing. We aimed to define the specific mechanism(s) through which butyrate contributes to these epithelial responses. Guided by an unbiased profiling approach, we identified the dominant regulation of the actin-binding protein synaptopodin (SYNPO). Extensions of this work revealed a role for SYNPO in intestinal epithelial barrier function and wound healing. SYNPO was localized to the intestinal epithelial tight junction and within F-actin stress fibers where it is critical for barrier integrity and cell motility. Butyrate, but not other SCFAs, induced SYNPO in epithelial cell lines and murine colonic enteroids through mechanisms possibly involving histone deacetylase inhibition. Moreover, depletion of the microbiota abrogated expression of SYNPO in the mouse colon, which was rescued with butyrate repletion. Studies in Synpo-deficient mice demonstrated exacerbated disease susceptibility and increased intestinal permeability in a dextran sulfate sodium colitis model. These findings establish a critical role for the microbiota and their products, specifically butyrate, in the regulated expression of SYNPO for intestinal homeostasis and reveal a direct mechanistic link between microbiota-derived butyrate and barrier restoration.

Epithelial HIF-1α/claudin-1 axis regulates barrier dysfunction in eosinophilic esophagitis.

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.

The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells.

The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs.

Control and dysregulation of redox signalling in the gastrointestinal tract.

Redox signalling in the gastrointestinal mucosa is held in an intricate balance. Potent microbicidal mechanisms can be used by infiltrating immune cells, such as neutrophils, to protect compromised mucosae from microbial infection through the generation of reactive oxygen species. Unchecked, collateral damage to the surrounding tissue from neutrophil-derived reactive oxygen species can be detrimental; thus, maintenance and restitution of a breached intestinal mucosal barrier are paramount to host survival. Redox reactions and redox signalling have been studied for decades with a primary focus on contributions to disease processes. Within the past decade, an upsurge of exciting findings have implicated subtoxic levels of oxidative stress in processes such as maintenance of mucosal homeostasis, the control of protective inflammation and even regulation of tissue wound healing. Resident gut microbial communities have been shown to trigger redox signalling within the mucosa, which expresses similar but distinct enzymes to phagocytes. At the fulcrum of this delicate balance is the colonic mucosal epithelium, and emerging evidence suggests that precise control of redox signalling by these barrier-forming cells may dictate the outcome of an inflammatory event. This Review will address both the spectrum and intensity of redox activity pertaining to host-immune and host-microbiota crosstalk during homeostasis and disease processes in the gastrointestinal tract.

Neutrophils as sources of dinucleotide polyphosphates and metabolism by epithelial ENPP1 to influence barrier function via adenosine signaling.

Extracellular adenosine signaling is established as a protective component in mucosal inflammatory responses. The sources of extracellular adenosine include enzymatic processing from nucleotides, such as ATP and AMP, that can be liberated from a variety of cell types, including infiltrating leukocytes. Here we demonstrate that activated human neutrophils are a source of diadenosine triphosphate (Ap3A), providing an additional source of nucleotides during inflammation. Profiling murine enteroids and intestinal epithelial cell lines revealed that intestinal epithelia prominently express apical and lateral ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), a member of the ENPP family of enzymes that metabolize diadenosine phosphates, especially Ap3A. Extensions of these studies demonstrated that intestinal epithelia metabolize Ap3A to ADP and AMP, which are further metabolized to adenosine and made available to activate surface adenosine receptors. Using loss and gain of ENPP1 approaches, we revealed that ENPP1 coordinates epithelial barrier formation and promotes epithelial wound healing responses. These studies demonstrate the cooperative metabolism between Ap3A and ENPP1 function to provide a significant source of adenosine, subserving its role in inflammatory resolution.

Microbiota-Derived Indole Metabolites Promote Human and Murine Intestinal Homeostasis through Regulation of Interleukin-10 Receptor.

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease.

Neutrophils as Components of Mucosal Homeostasis.

Inflammatory responses in the intestinal mucosa inevitably result in the recruitment of neutrophils (polymorphonuclear leukocytes [PMNs]). Epithelial cells that line the mucosa play an integral role in the recruitment, maintenance, and clearance of PMNs at sites of inflammation. The consequences of such PMN-epithelial interactions often determine tissue responses and, ultimately, organ function. For this reason, there is significant interest in understanding how PMNs function in the mucosa during inflammation. Recent studies have shown that PMNs play a more significant role in molding of the immune response than previously thought. Here, we review the recent literature regarding the contribution of PMNs to the development and resolution of inflammation, with an emphasis on the role of the tissue microenvironment and pathways for promoting epithelial restitution. These studies highlight the complex nature of inflammatory pathways and provide important insight into the difficulties of treating mucosal inflammation.

MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-ß Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Oxygen metabolism and innate immune responses in the gut.

Epithelial cells of the mucosa provide a first line of defense to prevent the inappropriate translocation of luminal antigens, and therefore contribute significantly to nonspecific innate immunity. In the gastrointestinal (GI) tract, barrier is provided by multiple components of the mucosa, including mucus production, epithelial junctional complexes, and the production of antimicrobial molecules. In recent years, it is better appreciated that tissue oxygen metabolism is key to homeostasis in the mucosa. The intestine, for example, maintains a low baseline Po2 level due to high rates of metabolism, countercurrent blood flow, and the presence of a steep oxygen gradient across the luminal aspect of tissue surface. As a result, hypoxia and hypoxia-inducible factor (HIF)-dependent signaling exists even in the healthy, unperturbed intestinal mucosa. In a number of examples, HIF has been demonstrated both to promote barrier function during homeostasis and to promote resolution of active inflammation. Hypoxia-elicited factors that contribute to innate responses in the mucosa include the transcriptional regulation of mucin genes, junction proteins, and autophagic flux. Here, we review current literature related to hypoxia and innate immunity in health and during mucosal inflammation.

Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation.

Recent work has revealed a central role for neddylation (the conjugation of a Nedd8-moiety to Cullin proteins) in the fine tuning of the NF-κB response (via Cullin-1). In the present study, we investigated the contribution of Cullin-1 neddylation and NF-κB signaling to mucosal inflammatory responses in vitro and in vivo. Initial in vitro studies using cultured intestinal epithelial cells revealed that the neddylation inhibitor MLN4924 prominently induces the deneddylation of Cullin-1. Parallel western blot, luciferase reporter and gene target assays identified MLN4924 as a potent inhibitor of intestinal epithelial NF-κB. Subsequent studies revealed that MLN4924 potently induces epithelial apoptosis but only in the presence of additional inflammatory stimuli. In vivo administration of MLN4924 (3 mg/kg/d) in a TNBS-induce colitis model significantly accentuated disease severity. Indeed, MLN4924 resulted in worsened clinical scores and increased mortality early in the inflammatory response. Histologic analysis of the colon revealed that neddylation inhibition results in increased tissue damage and significantly increased mucosal apoptosis as determined by TUNEL and cleaved caspase-3 staining, particularly prominent within the epithelium. Extensions of these studies revealed that ongoing inflammation is associated with significant loss of deneddylase-1 (SENP8) expresssion. These studies reveal that intact Cullin-1 neddylation is central to resolution of acute inflammation.

Neutrophils and the inflammatory tissue microenvironment in the mucosa.

The interaction of neutrophils (PMNs) and epithelial cells are requisite lines of communication during mucosal inflammatory responses. Consequences of such interactions often determine endpoint organ function, and for this reason, much interest has developed around defining the constituents of the tissue microenvironment of inflammatory lesions. Physiologic in vitro and in vivo models have aided in the discovery of components that define the basic inflammatory machinery that mold the inflammatory tissue microenvironment. Here, we will review the recent literature related to the contribution of PMNs to molding of the tissue microenvironment, with an emphasis on the gastrointestinal (GI) tract. We focus on endogenous pathways for promoting tissue homeostasis and the molecular determinants of neutrophil-epithelial cell interactions during ongoing inflammation. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

Imaging Inflammatory Hypoxia in the Murine Gut.

The inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, result in chronic inflammation to the gastrointestinal tract. In ulcerative colitis, inflammation tends to be more superficial and restricted to the colon; contrastingly, Crohn's disease presents as patchy, more penetrative inflammation that can occur throughout the gastrointestinal tract. Other differences between these diseases include the nature of their respective immune responses-Crohn's disease presents as a Th1 and ulcerative colitis as a Th2-type inflammation. During any inflammatory episode, metabolic demand on the tissue increases accompanying the influx of inflammatory cells, increasing the demand for ATP and oxygen. When availability of oxygen is limiting, tissues become hypoxic, which results in adaptive pathways to enable survival of hypoxic episodes. The primary pathway activated is the HIF (hypoxia inducible factor) transcription factor, which regulates adaptive pathways including genes controlling glycolytic metabolism and angiogenesis. In adequately oxygenated tissues (i.e. normoxia), the HIF protein is constantly produced, but oxygen-dependent enzymes called prolyl-hydroxylases utilize available oxygen to hydroxylate HIF on proline residues, targeting it for ubiquitination and subsequent degradation. Here we describe methods for inducing, visualizing, and quantifying in vivo "inflammatory hypoxia," using the murine gut as a model system.

Hypoxia and Mucosal Inflammation.

Sites of inflammation are defined by significant changes in metabolic activity. Recent studies have suggested that O2 metabolism and hypoxia play a prominent role in inflammation so-called "inflammatory hypoxia," which results from a combination of recruited inflammatory cells (e.g., neutrophils and monocytes), the local proliferation of multiple cell types, and the activation of multiple O2-consuming enzymes during inflammation. These shifts in energy supply and demand result in localized regions of hypoxia and have revealed the important function off the transcription factor HIF (hypoxia-inducible factor) in the regulation of key target genes that promote inflammatory resolution. Analysis of these pathways has provided multiple opportunities for understanding basic mechanisms of inflammation and has defined new targets for intervention. Here, we review recent work addressing tissue hypoxia and metabolic control of inflammation and immunity.

Hypercapnia Suppresses the HIF-dependent Adaptive Response to Hypoxia.

Molecular oxygen and carbon dioxide are the primary gaseous substrate and product of oxidative metabolism, respectively. Hypoxia (low oxygen) and hypercapnia (high carbon dioxide) are co-incidental features of the tissue microenvironment in a range of pathophysiologic states, including acute and chronic respiratory diseases. The hypoxia-inducible factor (HIF) is the master regulator of the transcriptional response to hypoxia; however, little is known about the impact of hypercapnia on gene transcription. Because of the relationship between hypoxia and hypercapnia, we investigated the effect of hypercapnia on the HIF pathway. Hypercapnia suppressed HIF-α protein stability and HIF target gene expression both in mice and cultured cells in a manner that was at least in part independent of the canonical O2-dependent HIF degradation pathway. The suppressive effects of hypercapnia on HIF-α protein stability could be mimicked by reducing intracellular pH at a constant level of partial pressure of CO2 Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase that blocks lysosomal degradation, prevented the hypercapnic suppression of HIF-α protein. Based on these results, we hypothesize that hypercapnia counter-regulates activation of the HIF pathway by reducing intracellular pH and promoting lysosomal degradation of HIF-α subunits. Therefore, hypercapnia may play a key role in the pathophysiology of diseases where HIF is implicated.

Hypoxia-recruited angiogenic neutrophils.

In this issue of Blood, Massena et al identify a novel CD49d+CXCR4highVEGFR1high population of neutrophils that specifically migrate to sites of hypoxia and enhance angiogenesis.

HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity.

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 HIF1ß 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 HIF1ß-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1ß-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.

Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function.

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.