Mimicry of microbially-derived butyrate reveals templates for potent intestinal epithelial HIF stabilizers.

Microbiota-derived short-chain fatty acids, including butyrate (BA), have multiple beneficial health effects. In the colon, BA concentrations range from 10 to 20 mM and up to 95% is utilized as energy by the mucosa. BA plays a key role in epithelial-barrier regulation and anti-inflammation, and regulates cell growth and differentiation, at least in part, due to its direct influence on stabilization of the transcription factor hypoxia-inducible factor (HIF). It remains unclear whether BA is the optimal metabolite for such a response. In this study, we explored metabolite mimicry as an attractive strategy for the biological response to HIF. We discovered that 4-mercapto butyrate (MBA) stabilizes HIF more potently and has a longer biological half-life than BA in intestinal epithelial cells (IECs). We validated the MBA-mediated HIF transcriptional activity through the induction of classic HIF gene targets in IECs and enhanced epithelial barrier formation in vitro. In-vivo studies with MBA revealed systemic HIF stabilization in mice, which was more potent than its parent BA metabolite. Mechanistically, we found that MBA enhances oxygen consumption and that the sulfhydryl group is essential for HIF stabilization, but exclusively as a four-carbon SCFA. These findings reveal a combined biochemical mechanism for HIF stabilization and provide a foundation for the discovery of potent metabolite-like scaffolds.

The TNFΔARE Mouse as a Model of Intestinal Fibrosis.

Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease-related intestinal fibrosis. This study investigates whether the TNFΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNFΔARE mice as well as in cohorts of numerous ages. At >24 weeks of age, TNFΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNFΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

The TNF ΔARE mouse as a model of intestinal fibrosis.

Background & Aims: Crohn's disease (CD) is a highly morbid chronic inflammatory disease. The majority of CD patients also develop fibrostenosing complications. Despite this, there are no medical therapies for intestinal fibrosis. This is in part due to lack of high-fidelity biomimetic models to enhance understanding and drug development. There is a need to develop in vivo models of inflammatory bowel disease-related intestinal fibrosis. We sought to determine if the TNF ΔARE mouse, a model of ileal inflammation, may also develop intestinal fibrosis. Methods: Several clinically relevant outcomes were studied including features of structural fibrosis, histological fibrosis, and gene expression. These include the use of a luminal casting technique we developed, traditional histological outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNF ΔARE mice as well as in cohorts of numerous ages. Results: At ages of 24+ weeks, TNF ΔARE mice develop structural, histological, and genetic changes of ileal fibrosis. Genetic expression profiles have changes as early as six weeks, followed by histological changes occurring as early as 14-15 weeks, and overt structural fibrosis delayed until after 24 weeks. Discussion: The TNF ΔARE mouse is a viable and highly tractable model of intestinal fibrosis. This model and the techniques employed can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination.

The intestinal mucosa exists in a state of "physiologic hypoxia," where oxygen tensions are markedly lower than those in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain homeostasis in this austere environment through oxygen-sensitive transcription factors, including hypoxia-inducible factors (HIFs). Using an unbiased chromatin immunoprecipitation (ChIP) screen for HIF-1 targets, we identify autophagy as a major pathway induced by hypoxia in IECs. One important function of autophagy is to defend against intracellular pathogens, termed "xenophagy." Analysis reveals that HIF is a central regulator of autophagy and that in vitro infection of IECs with Salmonella Typhimurium results in induction of HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Work in vivo demonstrates that IEC-specific deletion of HIF compromises xenophagy and exacerbates bacterial dissemination. These results reveal that the interaction between hypoxia, HIF, and xenophagy is an essential innate immune component for the control of intracellular pathogens.

Contact-dependent, polarized acidification response during neutrophil-epithelial interactions.

Neutrophil (PMN) infiltration during active inflammation imprints changes in the local tissue environment. Such responses are often accompanied by significant extracellular acidosis that result in predictable transcriptional responses. In this study, we explore the mechanisms involved in inflammatory acidification as a result of PMN-intestinal epithelial cell (IEC) interactions. Using recently developed tools, we revealed that PMN transepithelial migration (TEM)-associated inflammatory acidosis is dependent on the total number of PMNs present during TEM and is polarized toward the apical surface. Extending these studies, we demonstrate that physical separation of the PMNs and IECs prevented acidification, whereas inhibition of PMN TEM using neutralizing antibodies enhanced extracellular acidification. Utilizing pharmaceutical inhibitors, we demonstrate that the acidification response is independent of myeloperoxidase and dependent on reactive oxygen species generated during PMN TEM. In conclusion, inflammatory acidosis represents a polarized PMN-IEC-dependent response by an as yet to be fully determined mechanism.

Mucosal acidosis elicits a unique molecular signature in epithelia and intestinal tissue mediated by GPR31-induced CREB phosphorylation.

Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.

The HIF target ATG9A is essential for epithelial barrier function and tight junction biogenesis.

Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed "physiologic hypoxia." An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α- or HIF-2α-specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1-targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown (KD) of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A KD cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein. These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

Adaptation to inflammatory acidity through neutrophil-derived adenosine regulation of SLC26A3.

Acute intestinal inflammation includes the early accumulation of neutrophils (PMN). Based on recent evidence that PMN infiltration "imprints" changes in the local tissue environment through local oxygen depletion and the release of adenine nucleotides, we hypothesized that the interaction between transmigrating PMN and intestinal epithelial cells (IECs) results in inflammatory acidification of the tissue. Using newly developed tools, we revealed that active PMN transepithelial migration (TEM) significantly acidifies the local microenvironment, a decrease of nearly 2 pH units. Using unbiased approaches, we sought to define acid-adaptive pathways elicited by PMN TEM. Given the significant amount of adenosine (Ado) generated during PMN TEM, we profiled the influence of Ado on IECs gene expression by microarray and identified the induction of SLC26A3, the major apical Cl-/HCO3- exchanger in IECs. Utilizing loss- and gain-of-function approaches, as well as murine and human colonoids, we demonstrate that Ado-induced SLC26A3 promotes an adaptive IECs phenotype that buffers local pH during active inflammation. Extending these studies, chronic murine colitis models were used to demonstrate that SLC26A3 expression rebounds during chronic DSS-induced inflammation. In conclusion, Ado signaling during PMN TEM induces an adaptive tissue response to inflammatory acidification through the induction of SLC26A3 expression, thereby promoting pH homeostasis.

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.