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.

Microbial-derived indoles inhibit neutrophil myeloperoxidase to diminish bystander tissue damage.

During episodes of acute inflammation, polymorphonuclear leukocytes (PMNs) are actively recruited to sites of inflammation or injury where they provide anti-microbial and wound-healing functions. One enzyme crucial for fulfilling these functions is myeloperoxidase (MPO), which generates hypochlorous acid from Cl- and hydrogen peroxide. The potential exists, however, that uncontrolled the extracellular generation of hypochlorous acid by MPO can cause bystander tissue damage and inhibit the healing response. Previous work suggests that the microbiota-derived tryptophan metabolites 1H-indole and related molecules ("indoles") are protective during intestinal inflammation, although their precise mechanism of action is unclear. In the present work, we serendipitously discovered that indoles are potent and selective inhibitors of MPO. Using both primary human PMNs and recombinant human MPO in a cell-free system, we revealed that indoles inhibit MPO at physiologic concentrations. Particularly, indoles block the chlorinating activity of MPO, a reliable marker for MPO-associated tissue damage, as measured by coulometric-coupled HPLC. Further, we observed direct interaction between indoles and MPO using the established biochemical techniques microscale thermophoresis and STD-NMR. Utilizing a murine colitis model, we demonstrate that indoles inhibit bystander tissue damage, reflected in decreased colon 3-chlorotyrosine and pro-inflammatory chemokine expression in vivo. Taken together, these results identify microbiota-derived indoles that acts as endogenous immunomodulatory compounds through their actions on MPO, suggesting a symbiotic association between the gut microbiota and host innate immune system. Such findings offer exciting new targets for future pharmacological intervention.

Hypoxanthine is a checkpoint stress metabolite in colonic epithelial energy modulation and barrier function.

Intestinal epithelial cells form a selectively permeable barrier to protect colon tissues from luminal microbiota and antigens and to mediate nutrient, fluid, and waste flux in the intestinal tract. Dysregulation of the epithelial cell barrier coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically depleting state of physiological hypoxia. However, studies that systematically examine energy flux and adenylate metabolism during intestinal epithelial barrier development and restoration after disruption are lacking. Here, to delineate barrier-related energy flux, we developed an HPLC-based profiling method to track changes in energy flux and adenylate metabolites during barrier development and restoration. Cultured epithelia exhibited pooling of phosphocreatine and maintained ATP during barrier development. EDTA-induced epithelial barrier disruption revealed that hypoxanthine levels correlated with barrier resistance. Further studies uncovered that hypoxanthine supplementation improves barrier function and wound healing and that hypoxanthine appears to do so by increasing intracellular ATP, which improved cytoskeletal G- to F-actin polymerization. Hypoxanthine supplementation increased the adenylate energy charge in the murine colon, indicating potential to regulate adenylate energy charge-mediated metabolism in intestinal epithelial cells. Moreover, experiments in a murine colitis model disclosed that hypoxanthine loss during active inflammation correlates with markers of disease severity. In summary, our results indicate that hypoxanthine modulates energy metabolism in intestinal epithelial cells and is critical for intestinal barrier function.

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.

Microbial-Derived Butyrate Promotes Epithelial Barrier Function through IL-10 Receptor-Dependent Repression of Claudin-2.

Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which microbial-derived butyrate promotes barrier through IL-10RA-dependent repression of claudin-2.

Exposure of neutralizing epitopes in the carboxyl-terminal domain of TcdB is altered by a proximal hypervariable region.

The sequence, activity, and antigenicity of TcdB varies between different strains of Clostridium difficile. As a result, ribotype-specific forms of TcdB exhibit different toxicities and are not strongly cross-neutralized. Using a combination of biochemical and immunological approaches, we compared two important variants of TcdB (TcdB012 and TcdB027) to identify the mechanisms through which sequence differences alter epitopes and activity of the toxin. These analyses led to the discovery of a critical variation in the 1753-1851 (B2') region of TcdB, which affects the exposure of neutralizing epitopes in the toxin. Sequence comparisons found that the B2' region exhibits only 77% identity and is the most variable sequence between the two forms of TcdB. A combination of biochemical, analytical, and mutagenesis experiments revealed that the B2' region promotes protein-protein interactions. These interactions appear to shield neutralizing epitopes that would otherwise be exposed in the toxin, an event found to be less prominent in TcdB012 due to sequence differences in the 1773-1780 and 1791-1798 regions of the B2' domain. When the carboxyl-terminal domains of TcdB012 and TcdB027 are swapped, neutralization experiments suggest that the amino terminus of TcdB interacts with the B2' region and impacts the exposure of neutralizing epitopes in the carboxyl terminus. Collectively, these data suggest that variations in the B2' region affect protein-protein interactions within TcdB and that these interactions influence the exposure of neutralizing epitopes.

Memory B Cells Encode Neutralizing Antibody Specific for Toxin B from the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 but with Superior Neutralization of VPI 10463 Toxin B.

Secreted toxin B (TcdB) substantially contributes to the pathology observed during Clostridium difficile infection. To be successfully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encoding memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vaccination against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD) fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI 10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 induced strong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferred to naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Further experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible with Bmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells (PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven antibody titers, and the CTD2 antibody was neutralizing in vitro, but its protection against lethal challenge with TcdB2 was limited to delaying time to death. Therefore, CTD from different C. difficile strains may be a good immunogen for stimulating B cell memory that encodes in vitro neutralizing Ab but may be limited by variable protection against intoxication in vivo.

Clostridium difficile 027/BI/NAP1 encodes a hypertoxic and antigenically variable form of TcdB.

The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB003). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB027) was at least four fold more lethal than TcdB003. TcdB027 caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB003 and TcdB027 also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB027 and TcdB003 identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB027, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB027 is a more potent toxin than TcdB003 as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD.

TcdB from hypervirulent Clostridium difficile exhibits increased efficiency of autoprocessing.

TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdB(HV) ) was more efficient at autoprocessing than TcdB from a historical strain (TcdB(HIST) ). TcdB(HV) and TcdB(HIST) were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdB(HV) . Using an activity-based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdB(HIST) was identified, while only a processed form of TcdB(HV) could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdB(HV) , indicating a preferential and more efficient engagement of intramolecular substrate than TcdB(HIST) . Furthermore, a peptide-based inhibitor (Ac-GSL-AOMK) was found to block the cytotoxicity of TcdB(HIST) at a lower concentration than required to inhibit TcdB(HV) . These findings suggest that TcdB(HV) may cause increased cytotoxicity due to more efficient autoprocessing.

Fundamental role for the creatine kinase pathway in protection from murine colitis.

Inflammatory diseases of the digestive tract, including inflammatory bowel disease (IBD), cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in IBD patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CKB/CKMit (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length and histology). In a broad cytokine profiling, CKdKO mice expressed near absent IFN-γ levels. We identified losses in IFN-γ production from CD4 + and CD8 + T cells isolated from CKdKO mice. Addback of IFN-γ during DSS treatment resulted in partial protection for CKdKO mice. We identified basal stabilization of the transcription factor hypoxia-inducible factor (HIF) in CKdKO splenocytes and pharmacological stabilization of HIF resulted in reduced IFN-γ production by control splenocytes. Thus, the loss of IFN-γ production by CD4 + and CD8 + T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.

Fundamental role for the creatine kinase pathway in protection from murine colitis.

Inflammatory diseases of the digestive tract, including inflammatory bowel disease, cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in inflammatory bowel disease patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CK brain type/CK mitochondrial form (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length, and histology). In a broad cytokine profiling, CKdKO mice expressed near absent interferon gamma (IFN-γ) levels. We identified losses in IFN-γ production from CD4+ and CD8+ T cells isolated from CKdKO mice. Addback of IFN-γ during DSS treatment resulted in partial protection for CKdKO mice. Extensions of these studies identified basal stabilization of the transcription factor hypoxia-inducible factor in CKdKO splenocytes and pharmacological stabilization of hypoxia-inducible factor resulted in reduced IFN-γ production by control splenocytes. Thus, the loss of IFN-γ production by CD4+ and CD8+ T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.

Variations in TcdB activity and the hypervirulence of emerging strains of Clostridium difficile.

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdB(HV)) was more cytotoxic in vitro than TcdB from historical strains (TcdB(HIST)). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdB(HV) and TcdB(HIST). Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdB(HV) were found to be substantially broader and more pronounced than those caused by TcdB(HIST). The increased toxicity of TcdB(HV) was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdB(HIST). The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdB(HV) undergoes acid-induced conformational changes at a pH much higher than that of TcdB(HIST). Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2RγC-/- NODSIRPα) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Bile acids modulate colonic MAdCAM-1 expression in a murine model of combined cholestasis and colitis.

Primary sclerosing cholangitis (PSC) is a progressive fibrosing cholestatic liver disease that is strongly associated with inflammatory bowel disease (IBD). PSC-associated IBD (PSC-IBD) displays a unique phenotype characterized by right-side predominant colon inflammation and increased risk of colorectal cancer compared to non-PSC-IBD. The frequent association and unique phenotype of PSC-IBD suggest distinctive underlying disease mechanisms from other chronic liver diseases or IBD alone. Multidrug resistance protein 2 knockout (Mdr2-/-) mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. As a novel model of PSC-IBD, we treated Mdr2-/- mice with dextran sulfate sodium (DSS) to chemically induce colitis (Mdr2-/-/DSS). Mdr2-/- mice demonstrate alterations in fecal bile acid composition and enhanced colitis susceptibility with increased colonic adhesion molecule expression, particularly mucosal addressin-cell adhesion molecule 1 (MAdCAM-1). In vitro, ursodeoxycholic acid (UDCA) co-treatment resulted in a dose dependent attenuation of TNF-α-induced endothelial MAdCAM-1 expression. In the combined Mdr2-/-/DSS model, UDCA supplementation attenuated colitis severity and downregulated intestinal MAdCAM-1 expression. These findings suggest a potential mechanistic role for alterations in bile acid signaling in modulating MAdCAM-1 expression and colitis susceptibility in cholestasis-associated colitis. Together, our findings provide a novel model and new insight into the pathogenesis and potential treatment of PSC-IBD.

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.

Tissue metabolism and the inflammatory bowel diseases.

The intestinal mucosa provides a selective barrier between the anaerobic lumen and a highly metabolic lamina propria. A number of recent studies indicate that acute inflammation of the mucosa can result in tissue hypoxia and associated shifts in tissue metabolism. The activation of hypoxia-inducible factor (HIF) under these conditions has been demonstrated to function as an endogenous molecular cue to promote resolution of inflammation, particularly through the orchestration of barrier repair toward homeostasis. Given the central role of oxygen in tissue metabolism, ongoing studies have defined metabolic endpoints of HIF stabilization as important biomarkers of disease activity. Such findings make HIF and HIF-associated metabolic pathways particularly attractive therapeutic targets in inflammatory bowel disease (IBD). Here, we review the recent literature related to tissue metabolism in IBD.

HPLC-Based Metabolomic Analysis of Normal and Inflamed Gut.

The idiopathic inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, are multifactorial chronic conditions that result in numerous perturbations of metabolism in the gastrointestinal mucosa. Thus, methodologies for the qualitative and quantitative analysis of small molecule metabolites in mucosal tissues are important for further elucidation of mechanisms driving inflammation and the metabolic consequences of inflammation. High-performance liquid chromatography (HPLC) is a ubiquitous analytical technique that can be adapted for both targeted and non-targeted metabolomic analysis. Here, protocols for reversed-phase (RP) HPLC-based methods using two different detection modalities are presented. Ultraviolet detection is used for the analysis of adenine nucleotide metabolites, whereas electrochemical detection is used for the analysis of multiple amino acid metabolites. These methodologies provide platforms for further characterization of the metabolic changes that occur during gastrointestinal inflammation.

NMR-Based Metabolomic Analysis of Normal and Inflamed Gut.

Crohn's disease and ulcerative colitis, the two major forms of idiopathic inflammatory bowel disease (IBD), are thought to occur through a loss of intestinal barrier leading to an inappropriate immune response toward intestinal microbiota. While genome-wide association studies (GWAS) have provided much information about susceptibility loci associated with these diseases, the etiology of IBD is still unknown. Metabolomic analysis allows for the comprehensive measurement of multiple small molecule metabolites in biological samples. During the past decade, metabolomic techniques have yielded novel and potentially important findings, revealing insight into metabolic perturbations associated with these diseases. This chapter provides metabolomic methodologies describing a nuclear magnetic resonance (NMR)-based non-targeted approach that has been utilized to make important contributions toward a better understanding of IBD.

Metabolic regulation of intestinal epithelial barrier during inflammation.

The gastrointestinal mucosa has proven to be an interesting tissue for which to investigate disease-related metabolism. In this review, we outline some evidence that implicates metabolic signaling as important features of barrier in the healthy and disease. Studies from cultured cell systems, animal models and human patients have revealed that metabolites generated within the inflammatory microenvironment are central to barrier regulation. These studies have revealed a prominent role for hypoxia and hypoxia-inducible factor (HIF) at key steps in adenine nucleotide metabolism and within the creatine kinase pathway. Results from animal models of intestinal inflammation have demonstrated an almost uniformly beneficial influence of HIF stabilization on disease outcomes and barrier function. Studies underway to elucidate the contribution of immune responses will provide additional insight into how metabolic changes contribute to the complexity of the gastrointestinal tract and how such information might be harnessed for therapeutic benefit.