SUMMARY Acute intestinal inflammation involves early accumulation of neutrophils (PMN) followed by either resolution or progression to chronic inflammation. Based on recent evidence mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMN influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMN rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). Utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMN 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.

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.epithelial junctions | energy metabolism | actomyosin | IBD

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 (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 (HRE) 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.

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.colitis | endotoxin | lipid mediator | mucosal | epithelia

OBJECTIVE Investigation of potential anti‐inflammatory mechanisms of IFN‐ in intestinal epithelial cells (IEC) in models of inflammatory bowel disease (IBD). METHODS Guided by microarray analysis, we utilized qPCR, western blot, and confocal microscopy to characterize epithelial IL‐10 receptor (IL‐10R) expression in vitro. Next, the functional consequences of receptor expression were examined. Finally, IL‐10R expression was investigated in a murine IBD model and in human IBD patient samples. RESULTS In vitro studies revealed that IFN‐ selectively induced the expression of IL‐10R1 on IEC. Further analysis revealed that IL‐10R1 was expressed predominantly on the apical membrane of polarized IEC. Receptor activation functionally induced canonical IL‐10 target gene expression in IEC, concomitant with enhanced barrier restitution. Colonic tissue isolated from murine colitis revealed that levels of IL‐10R1 and SOCS3 were increased in the epithelium and coincided with increased tissue IFN‐ and IL‐10 cytokines. Immunofluorescent staining revealed apical expression of the IL‐10R in colitic mouse tissue. Finally, diseased human tissue demonstrated increased IL‐10R1 transcript with apical IEC localization. CONCLUSIONS These results provide novel insight into an anti‐inflammatory role for IFN‐ in intestinal inflammation through the induction of a polarized IL‐10 receptor complex.

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-Ring-Ligases (CRL), which play a central role in the stabilization of NF-κB and HIF. In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cullin neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMEC), we examined inflammatory responses to LPS or TNF-α by assessing Cullin-neddylation status, NF-κB and HIF-1α stabilization and inflammatory cytokine secretion. HMEC with an intact neddylation pathway showed a time-dependent induction of Cullin-1 (Cul-1) neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α and increased NF-κB/HIF-α promoter activity in response to LPS. HMEC cells 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 pro-inflammatory cytokines. MLN4924 stabilized HIF and abrogated pro-inflammatory responses while maintaining anti-inflammatory IL-10 response in vivo, following LPS administration. These studies identify SENP8 as a proximal regulator of Cullin neddylation and provide an important role for SENP8 in fine-tuning of the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Cullins during inflammation.

Inflammatory bowel diseases are disorders of unknown etiology characterized by inappropriate inflammation. There is recent intense interest in understanding mechanisms of inflammatory resolution. Resolvin‐E1 (RvE1) has been demonstrated to promote resolution in numerous disease models. Given the importance of epithelial cells in coordinating mucosal inflammation, we hypothesized that RvE1 elicits an epithelial inflammatory resolution signature. Microarray profiling of epithelial cells exposed to RvE1, revealed transcriptional induction of numerous inflammatory response genes. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and enhanced epithelial ALPI enzymatic activity. ALPI was recently demonstrated to detoxify bacterial LPS. In our studies, both purified ALPI and "conditioned" LPS‐containing media derived from RvE1‐exposed epithelia were demonstrated to detoxify LPS. To define these features in vivo, we employed a murine DSS model of colitis. Compared to vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices. RvE1 treatment resulted in significantly increased ALPI expression in murine intestinal epithelial cells. Moreover, disease severity inversely correlated with ALPI expression. Taken together, these data implicate a previously unappreciated role for ALPI in RvE1 mediated inflammatory resolution.

Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in IBD are accompanied by striking shifts in tissue metabolism. Here we examined global metabolic consequences of mucosal inflammation employing 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, SAM synthetase and SAH 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 SAH 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. Together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.