Brittelle Bowers

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-κB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.

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.

An Endogenously Anti-Inflammatory Role for Methylation in Mucosal Inflammation Identified through Metabolite Profiling

Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in inflammatory bowel diseases are accompanied by striking shifts in tissue metabolism. In this paper, we examined global metabolic consequences of mucosal inflammation using both in vitro and in vivo models of disease. Initial analysis of the metabolic signature elicited by inflammation in epithelial models and in colonic tissue isolated from murine colitis demonstrated that levels of specific metabolites associated with cellular methylation reactions are significantly altered by model inflammatory systems. Furthermore, expression of enzymes central to all cellular methylation, S-adenosylmethionine synthetase and S-adenosylhomocysteine hydrolase, are increased in response to inflammation. Subsequent studies showed that DNA methylation is substantially increased during inflammation and that epithelial NF-κB activity is significantly inhibited following treatment with a reversible S-adenosylhomocysteine hydrolase inhibitor, DZ2002. Finally, these studies demonstrated that inhibition of cellular methylation in a murine model of colitis results in disease exacerbation while folate supplementation to promote methylation partially ameliorates the severity of murine colitis. Taken together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.

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.

Defective leukocyte GM-CSF receptor (CD116) expression and function in inflammatory bowel disease.

BACKGROUND & AIMS Inflammatory bowel disease (IBD) refers to 2 chronic inflammatory diseases of the intestine, ie, ulcerative colitis and Crohns disease. IBD results from environmental factors (eg, bacterial antigens) triggering a dysregulated immune response in genetically predisposed hosts. Although the basis of IBD is incompletely understood, a number of recent studies have implicated defective innate immune responses in the pathogenesis of IBD. In this regard, there is much interest in therapies that activate innate immunity (eg, recombinant granulocyte-macrophage colony-stimulating factor). METHODS In this study, we screened expression and function of circulating leukocyte granulocyte-macrophage colony-stimulating factor receptor (CD116) messenger RNA and surface protein in 52 IBD patients and 52 healthy controls. RESULTS Our results show that both granulocyte and monocyte CD116 levels, but not CD114 or interleukin-3Rα, were significantly decreased in IBD compared to control (P<.001) and disease controls (irritable bowel syndrome; P<.001; rheumatoid arthritis; P<.025). IBD-associated CD116 repression was more prominent in patients with ulcerative colitis compared to Crohns disease (P<.05), was independent of disease activity (P>.05), and was not influenced by current medications (P>.05). Receiver operating characteristic curve analysis revealed that leukocyte CD116 expression is a sensitive (85%) and specific (92%) biomarker for IBD. Moreover, granulocyte CD116-mediated function (phosphorylation of signal transducers and activators of transcription 3) paralleled decreased expression of CD116 in IBD granulocytes compared to control (P<.001). CONCLUSIONS These studies identify defective expression and function of CD116 as a distinguishing feature of IBD and implicate an associated defect in innate immune responses toward granulocyte-macrophage colony-stimulating factor.

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 Attenuates Hypoxia-Inducible Factor (HIF) Activity in Intestinal Epithelial Cells through Transcriptional Repression of HIF-1β

Numerous studies have revealed that hypoxia and inflammation occur coincidentally in mucosal disorders, such as inflammatory bowel disease. During inflammation, epithelial-expressed hypoxia-inducible factor (HIF) serves an endogenously protective function. In this study, we sought to explore how mucosal immune responses influence HIF-dependent end points. Guided by a screen of relevant inflammatory mediators, we identified IFN-γ as a potent repressor of HIF-dependent transcription in human intestinal epithelial cells. Analysis of HIF levels revealed that HIF-1β, but not HIF-1α, is selectively repressed by IFN-γ in a JAK-dependent manner. Cloning and functional analysis of the HIF-1β promoter identified a prominent region for IFN-γ–dependent repression. Further studies revealed that colonic IFN-γ and HIF-1β levels were inversely correlated in a murine colitis model. Taken together, these studies demonstrated that intestinal epithelial HIF is attenuated by IFN-γ through transcriptional repression of HIF-1β. These observations are relevant to the pathophysiology of colitis (i.e., that loss of HIF signaling during active inflammation may exacerbate disease pathogenesis).

Creatine Kinase, a Novel HIF-2a Target, Regulates Apical Junction Complex Assembly in Intestinal Epithelial Cells: P-221

orphan nuclear receptor, plays an important role in the maintenance of gut epithelial homeostasis. We have shown that Pxr mice exhibit a distinctly leaky gut barrier pathology. The barrier defects in Pxr mice resulted from an enhanced epithelium-specific Tlr4 gene expression and signaling. These defects were found to be corrected in Pxr/Tlr4 double knock-out mice. Indeed, the role of PXR in maintaining gut epithelial barrier was found to be directly related to its ability to inversely regulate TLR4 expression. PXR is known to predominantly function as a classical ligand-dependent transcription factor. However, nuclear run-on and ChIP studies have shown that PXR has minimal effects on TLR4 gene transcription. However, we observed that PXR activation in vitro, decreased TLR4 mRNA stability and this effect was mediated by PXR binding to the TLR4 mRNA 30 untranslated region. CONCLUSION(S): Overall, our studies implicate epithelial PXR as a central regulator of TLR4 mediated control of gut immunity. These findings indicate that novel immune regulation strategies might involve PXR-RNA binding.

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.