Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.

All Fiber is Not Fiber.

PURPOSE OF REVIEW: Epidemiologic studies and clinical trials have demonstrated the benefits of dietary fiber. This occurs through a combination of the physiochemical properties of fiber and through microbial fermentation that occurs in the colon which result in the production of short-chain fatty acids (SCFA). The purpose of this review is to highlight the physiochemical properties of fiber that result in the range of physiologic effects and to review the literature on the health benefits of acetate, propionate, and butyrate. RECENT FINDINGS: Of the variety of properties and functions exerted by dietary fibers, the fermentability and production of SCFA's are emphasized in this review. Studies done in both animal and humans reveal the anti-obesity, anti-inflammatory, and possible anti-neoplastic roles SCFAs exert at the mucosal level. Many clinical questions remain regarding the optimal dose, type, and method of delivery of fiber to exert the desired beneficial effects. It has the potential to be used in the management of clinical symptoms, prevention of disease, and improvement in human health. Further studies to address this novel use of fiber has the potential to make a large impact in clinical practice.

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.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.

IFN-γ-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis.

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.

Physiologic hypoxia and oxygen homeostasis in the healthy intestine. A Review in the Theme: Cellular Responses to Hypoxia.

In recent years, the intestinal mucosa has proven to be an intriguing organ to study tissue oxygenation. The highly vascularized lamina propria juxtaposed to an anaerobic lumen containing trillions of metabolically active microbes results in one of the most austere tissue microenvironments in the body. Studies to date have determined that a healthy mucosa contains a steep oxygen gradient along the length of the intestine and from the lumen to the serosa. Advances in technology have allowed multiple independent measures and indicate that, in the healthy mucosa of the small and large intestine, the lumen-apposed epithelia experience Po2 conditions of <10 mmHg, so-called physiologic hypoxia. This unique physiology results from a combination of factors, including countercurrent exchange blood flow, fluctuating oxygen demands, epithelial metabolism, and oxygen diffusion into the lumen. Such conditions result in the activation of a number of hypoxia-related signaling processes, including stabilization of the transcription factor hypoxia-inducible factor. Here, we review the principles of mucosal oxygen delivery, metabolism, and end-point functional responses that result from this unique oxygenation profile.

Central role for endothelial human deneddylase-1/SENP8 in fine-tuning the vascular inflammatory response.

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-α by assessing Cul neddylation status, NF-κB and HIF-1α stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α, and increased NF-κB/HIF-α promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity, and reduced secretion of TNF-α-elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.

The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis.

OBJECTIVE: Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE. DESIGN: The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot-Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies. RESULTS: ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies. CONCLUSIONS: The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Women in Nutrition Science: A Biographical Review.

PURPOSE OF THE REVIEW: The field of nutrition is in debt to a cadre of women who led the field through its formative years. This review highlights the contributions of these women that are gleaned through analysis of biographical articles published in The Journal of Nutrition. RECENT FINDINGS: Forces emerged during the development of nutrition science, such as departments of home economics and the majority-female field of dietetics, that suggest women might be well represented in biographical articles in the field of nutrition. However, just 29 women have been the subject of biographical articles in The Journal of Nutrition representing 14.3% of the 202 biographical articles published to date - a percentage lower than scientific journals overall. This review explores these biographies to identify factors that facilitated and hindered careers and to highlight the manifold scientific contributions of women in nutrition science. This review looks toward the past to provide perspective and inspiration for those working in the field of nutrition today.

Enteral ventilation via anus: You can hold your breath.

Okabe et al.1 present data from animal models of acute hypoxia showing that oxygen-carrying liquid applied to the distal gut improves oxygenation and prolongs survival. This opens the possibility of recruiting the distal gut to aid when mechanical ventilation of the lungs is inadequate.

Evidence for dendritic cell-dependent CD4(+) T helper-1 type responses to commensal bacteria in normal human intestinal lamina propria.

Reactivity of lamina propria (LP) T cells to commensal bacteria has been demonstrated in animal models of inflammatory bowel disease (IBD) and in humans with IBD, but few studies have evaluated the function of such cells in normal individuals. LP mononuclear cells (LPMC) were disaggregated from healthy human intestinal tissue and cultured with heat-killed commensal and pathogenic bacteria. CD3(+)CD4(+) IFN-gamma-producing (Th1) cells reactive to commensal bacteria were demonstrated at frequencies ranging from 0.05 to 2.28% in LPMC. Bacteria-specific Th1 responses were inhibited by anti-HLA-DR antibodies and chloroquine exposure, were enriched in LP relative to peripheral blood, and expressed effector memory cell markers. Bacteria-specific CD4(+) T cell proliferation in vitro was dependent on the presence of LP dendritic cells (DCs), which produced pro-inflammatory cytokines upon bacterial exposure. These results suggest that bacteria-reactive DCs and CD4(+) T cells in normal LP have substantial pro-inflammatory potential that is revealed upon disaggregation in vitro.

Oral vitamin B12 supplement is delivered to the distal gut, altering the corrinoid profile and selectively depleting Bacteroides in C57BL/6 mice.

Vitamin B12 is a critical nutrient for humans as well as microbes. Due to saturable uptake, high dose oral B12 supplements are largely unabsorbed and reach the distal gut where they are available to interact with the microbiota. The aim of this study was to determine if oral B12 supplementation in mice alters 1) the concentration of B12 and related corrinoids in the distal gut, 2) the fecal microbiome, 3) short chain fatty acids (SCFA), and 4) susceptibility to experimental colitis. C57BL/6 mice (up to 24 animals/group) were supplemented with oral 3.94 µg/ml cyanocobalamin (B12), a dose selected to approximate a single 5 mg supplement for a human. Active vitamin B12 (cobalamin), and four B12-analogues ([ADE]CN-Cba, [2Me-ADE]CN-Cba, [2MeS-ADE]CN-Cba, CN-Cbi) were analyzed in cecal and fecal contents using liquid chromatography/mass spectrometry (LC/MS), in parallel with evaluation of fecal microbiota, cecal SCFA, and susceptibility to dextran sodium sulfate (DSS) colitis. At baseline, active B12 was a minor constituent of overall cecal (0.86%) and fecal (0.44%) corrinoid. Oral B12 supplementation increased active B12 at distal sites by >130-fold (cecal B12 increased from 0.08 to 10.60 ng/mg, fecal B12 increased from 0.06 to 7.81 ng/ml) and reduced microbe-derived fecal corrinoid analogues ([ADE]CN-Cba, [2Me-ADE]CN-Cba, [2MeS-ADE]CN-Cba). Oral B12 had no effect on cecal SCFA. Microbial diversity was unaffected by this intervention, however a selective decrease in Bacteroides was observed with B12 treatment. Lastly, no difference in markers of DSS-induced colitis were detected with B12 treatment.

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.

An adjuvant-free mouse model of tree nut allergy using hazelnut as a model tree nut.

BACKGROUND: Tree nut allergy, a major group of food allergy, is often linked to fatal or near-fatal systemic anaphylaxis. Currently, an adjuvant-free mouse model to study tree nut hypersensitivity is unavailable. Here we tested the hypothesis that transdermal exposure to hazelnut, a model tree nut, without the use of an adjuvant is sufficient to sensitize mice for immediate hypersensitivity reaction to oral hazelnut challenge. METHODS: BALB/c mice were repeatedly exposed to hazelnut protein via the transdermal route and systemic allergic and anaphylactic responses were studied. RESULTS: Transdermal exposure to hazelnut protein elicited robust systemic IgE response in a dose-dependent manner with immunological memory. Oral challenge of transdermally sensitized mice with hazelnut protein resulted in immediate (30 min after the challenge) clinical signs of systemic anaphylaxis as measured by significant clinical scores and drop in rectal temperature. Clinical hypersensitivity reaction was associated with severe pathological changes in the small intestine. Hazelnut-allergic but not control mice exhibited in vivo activation of GATA-3 and hazelnut-driven recall IL-4, IL-5 and IL-13 response by splenocytes, thus elucidating the underlying mechanism of hazelnut allergy development in this model. CONCLUSIONS: These data suggest that (1) transdermal exposure to hazelnut protein is sufficient to activate the key immune pathways necessary for sensitizing mice for clinical immediate hypersensitivity reactions and (2) this mouse model may be useful for further basic and applied studies on tree nut allergy, especially because it does not depend on an adjuvant for eliciting immediate hypersensitivity reactions to nut protein.

Breathless in the Gut: Implications of Luminal O2 for Microbial Pathogenicity.

Salmonella employs a variety of strategies to survive and colonize the colon. In this issue of Cell Host & Microbe, Rivera-Chávez et al. (2016) identify a new mechanism whereby antibiotic-mediated depletion of anaerobes (e.g., Clostridia) and associated decreases in butyrate result in increased tissue oxygen and increased aerobic expansion of Salmonella.

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.