Amanda Bayless

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.

Fundamental role for HIF-1α in constitutive expression of human β defensin-1

Antimicrobial peptides are secreted by the intestinal epithelium to defend from microbial threats. The role of human β defensin-1 (hBD-1) is notable because its gene (beta-defensin 1 (DEFB1)) is constitutively expressed and its antimicrobial activity is potentiated in the low-oxygen environment that characterizes the intestinal mucosa. Hypoxia-inducible factor (HIF) is stabilized even in healthy intestinal mucosa, and we identified that epithelial HIF-1α maintains expression of murine defensins. Extension to a human model revealed that basal HIF-1α is critical for the constitutive expression of hBD-1. Chromatin immunoprecipitation identified HIF-1α binding to a hypoxia response element in the DEFB1 promoter whose importance was confirmed by site-directed mutagenesis. We used 94 human intestinal samples to identify a strong expression correlation between DEFB1 and the canonical HIF-1α target GLUT1. These findings indicate that basal HIF-1α is critical for constitutive expression of enteric DEFB1 and support targeting epithelial HIF for restoration and maintenance of intestinal integrity.

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

Significance Intestinal epithelial barrier dysregulation is a hallmark of inflammatory bowel diseases (IBDs). A central role for hypoxic signaling has been defined in barrier modulation during inflammation. We demonstrate that genes involved in creatine metabolism, the creatine kinases (CKs), are coordinately regulated by hypoxia-inducible transcription factors (HIFs) and that such regulation is critical to barrier function. Inhibition of the CK pathway abrogates apical junction assembly and barrier integrity. Dietary creatine supplementation profoundly attenuates the pathogenic course of mucosal inflammation in mouse colitis models. Moreover, we demonstrate altered expression of mitochondrial and cytosolic CK enzymes in IBD patient tissue. These findings highlight the fundamental contribution of creatine metabolism to intestinal mucosal function, homeostasis, and disease resolution. 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.

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.

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

This study demonstrates a critical link between hypoxia-inducible factor (HIF) and claudin-1 (CLDN1). HIF1β-deficient intestinal epithelial cells develop abnormal tight junction (TJ) structure and have striking barrier defects. CLDN1 is an HIF target gene, and overexpression of CLDN1 in HIF1β-deficient cells restores TJ structure and function.

Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses.

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

There is interest in understanding posttranslational 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‐1a, 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.—Curtis, V. F., Ehrentraut, S. F., Campbell, E. L., Glover, L. E., Bayless, A., Kelly, C. J., Kominsky, D. J., Colgan, S. P., Stabilization of HIF through inhibition of Cullin‐2 neddylation is protective in mucosal inflammatory responses. FASEB J. 29, 208–215 (2015). www.fasebj.org

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.

IFN-γ-Mediated Induction of an Apical IL-10 Receptor on Polarized Intestinal Epithelia: P-225

ease (IBD) patients could not be orally tolerized against keyhole limpet hemocyanin (KLH) [1]. Our aim is to test in vitro the antigen up-take ability of the same antigen in isolated IECs from IBD patients and non-inflammatory controls. 1. Kraus TA et al. Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease. Gastroenterology (2004) 126: 1771-1778. METHODS: Surgical specimens were obtained from intestinal resections performed in Mount Sinai Medical Center. Five ulcerative colitis (with inflamed tissue (UC I) and four out of them also with non-inflamed tissue (UC NI)), and five Crohn’s disease (CD) patients and 8 normal mucosal samples (NL) were included. Briefly, the protocol of IECs isolation consisted in intense cleaning of surgical pieces with PBS; the mucosa was stripped from the submucosa in small pieces (3-5 mm). The pieces were incubated in DTT (1 mM in RPMI medium) for 15 min followed by 2 incubations of 30 min each in 25 mL of Dispase II (1.5 mg/mL in RPMI) at 37 C. After washing and centrifugation, IECs were recovered from the supernatant and were counted and adjusted to a final concentration of 106 cells/ mL in RPMI (plus antibiotics and Fungizone). 105 cells/well were plated in 96 wells plates in a total volume of 200 ll/well of RPMI with or without 100 lg of KLH. At different time points (0.5, 1, 2, 4 and 18 h) IECs were surface stained for flow cytometry analysis with CD45 (as a marker of leukocytes) and Ep-CAM (as a marker of IECs) antibodies, fixed and permeabilized and intra-cellular stained with an anti-KLH antibody. In 4 UC inflamed, 4 CD and 7 NL samples from same patients MHCII expression was analyzed by flow cytometry. In other set of experiments, after overnight (ON) incubation with or without KLH, MHCII expression was analyzed by flow cytometry in Ep-CAM þ cells from 3 NL and 3 UC inflamed samples. Comparisons between groups were statistically analyzed by Mann-Whitney test for unpaired samples. RESULTS: As showed in Table 1, the proportion of leukocytes and IECs in mucosa reflected the inflammatory state of the tissue with increased proportion of leukocytes and decreased levels of IECs in IBD samples. IECs from UC inflamed showed an increased earlier up-take of KLH compared to CD, however CD IECs had larger overnight uptake of KLH than UC inflamed (Fig. 1). The percentage of IECs that are MHCII þ is significantly higher in inflamed UC patients compared to NL, although KLH incubation does not have any effect on% of IECs expressing MHCIIþ (Fig. 2). CONCLUSION(S): The different kinetics of KLH up-take by IECs from CD and UC patients can affect the way through which KLH can be presented to immune cells. Although KLH presence does not seem to be important to IECs expression of MHCII molecules, differences in MHCII expression suggests a difference in the ability of IECs from both types of IBD to stimulate different immune cells subsets.

Transmigrating Neutrophils Shape The Mucosal Microenvironment During Colitis: P-232 YI

BACKGROUND: Neutrophil (PMN) accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. PMN transepithelial migration is orchestrated through a complex series of cell-cell interactions involving bi-directional signaling molecules. Thus we hypothesized that migrating PMN influence the transcriptional profile of epithelia as they transmigrate, priming them for either resolution or chronic inflammation – a concept we term ‘transcriptional imprinting’ . Employing a novel approach to a PMN-transepithelial migration model, we attempted to ascertain the influence of transmigration on epithelial gene expression by microarray analysis. METHODS: DNA Microarray was used to identify transcriptional changes in intestinal epithelial cells post-PMN transmigration. Real-time oxygen sensing was performed with an SDR-OxoDish (PreSens). Wild-type C57/B6 mice, ODD-Luciferase (HIF reporter) mice and gp-null (NADPH oxidase deficient) mice were subjected to chemically-induced colitis (TNBS). RESULTS: Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Real-time oxygen measurements effectively demonstrated that activated PMN rapidly depleted microenvironmental oxygen. Subsequent studies indicated that activated PMNs are sufficient to induce hypoxia-inducible factor (HIF) stabilization and activity in epithelial cells. Intestinal epithelial hypoxia induced by activated PMNs during transmigration was found to be dependent on the PMN respiratory burst. HIF is known to influence the expression of barrier-protective genes in the epithelium, initiate glycolytic metabolism and attenuates the clinical course/disease parameters in murine colitis. To ascertain the clinical relevance of transmigrating PMN and the induction of epithelial hypoxia, we stained biopsies from UC patients for evidence of Glut1 expression. PMN transmigration increased crypt epithelial Glut1 expression (see Figure). We investigated the relative contribution of PMN to ‘inflammatory hypoxia’ in vivo, utilizing a colitis model. Antibody-mediated depletion of PMNs worsened the course of colitis, exhibited reduced tissue hypoxia visualized with Hypoxyprobe-1 staining and attenuated the induction of hypoxia-dependent genes. Patients with chronic granulomatous disease (CGD) lack functional NADPH oxidase and develop IBD-like symptoms. We demonstrated that murine gp91 mice mirror human CGD, develop a severe colitis, with exaggerated PMN infiltration. However, these infiltrating PMN are incapable of inducing a hypoxic microenvironment. Finally, pharmacological intervention with a HIF-stabilizing compound (PHD inhibitor), resulted in recapitulation of mucosal HIF-signaling concomitant with abrogation of colitis-severity in the gp91 mice. CONCLUSION(S): In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils significantly modulates the host response to inflammation. Moreover, the respiratory burst contributes fundamentally to localized oxygen depletion and resultant microenvironmental hypoxia. We propose that this microenvironment established by infiltrating PMN is protective during colitis.