Simon Keely

Mucosal Protection by Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibition

BACKGROUND & AIMS A number of recent studies have implicated tissue hypoxia in both acute and chronic inflammatory diseases, particularly as they relate to mucosal surfaces lined by epithelial cells. In this context, a protective role for the transcriptional regulator hypoxia-inducible factor (HIF) was shown through conditional deletion of epithelial HIF-1alpha in a murine model of colitis. Here, we hypothesized that pharmacologic activation of HIF would similarly provide a protective adaptation to murine colitic disease. METHODS For these purposes, we used a novel prolyl hydroxylase (PHD) inhibitor (FG-4497) that readily stabilizes HIF-1alpha and subsequently drives the expression downstream of HIF target genes (eg, erythropoietin). RESULTS Our results show that the FG-4497-mediated induction of HIF-1alpha provides an overall beneficial influence on clinical symptoms [weight loss, colon length, tissue tumor necrosis factor-alpha (TNFalpha)] in murine trinitrobenzene sulfonic acid (TNBS) colitis, most likely because of their barrier protective function and wound healing during severe tissue hypoxia at the site of inflammation. CONCLUSIONS Taken together these findings emphasize the role of epithelial HIF-1alpha during inflammatory diseases in the colon and may provide the basis for a therapeutic use of PHD inhibitors in inflammatory mucosal disease.

A new short-term mouse model of chronic obstructive pulmonary disease identifies a role for mast cell tryptase in pathogenesis.

BACKGROUND Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short timeframe. OBJECTIVES We sought to create an early-onset mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short time-frame. We also sought to use this model to better understand pathogenesis and the roles of macrophages and mast cells (MCs) in patients with COPD. METHODS Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathologic features of COPD was assessed. The roles of macrophages and MC tryptase in pathogenesis were evaluated by using depletion and in vitro studies and MC protease 6-deficient mice. RESULTS After just 8 weeks of smoke exposure, wild-type mice had chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart and increased susceptibility to respiratory tract infections also were observed. Macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced proinflammatory responses from cultured macrophages. CONCLUSION A short-term mouse model of cigarette smoke-induced COPD was developed in which the characteristic features of the disease were induced more rapidly than in existing models. The model can be used to better understand COPD pathogenesis, and we show a requirement for macrophages and tryptase-expressing MCs.

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.

Advances in oral nano-delivery systems for colon targeted drug delivery in inflammatory bowel disease: selective targeting to diseased versus healthy tissue.

UNLABELLED Colon targeted drug delivery is an active area of research for local diseases affecting the colon, as it improves the efficacy of therapeutics and enables localized treatment, which reduces systemic toxicity. Targeted delivery of therapeutics to the colon is particularly advantageous for the treatment of inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohns disease. Advances in oral drug delivery design have significantly improved the bioavailability of drugs to the colon; however in order for a drug to have therapeutic efficacy during disease, considerations must be made for the altered physiology of the gastrointestinal (GI) tract that is associated with GI inflammation. Nanotechnology has been used in oral dosage formulation design as strategies to further enhance uptake into diseased tissue within the colon. This review will describe some of the physiological challenges faced by orally administered delivery systems in IBD, the important developments in orally administered nano-delivery systems for colon targeting, and the future advances of this research. FROM THE CLINICAL EDITOR Inflammatory Bowel Disease (IBD) poses a significant problem for a large number of patients worldwide. Current medical therapy mostly aims at suppressing the active inflammatory episodes. In this review article, the authors described and discussed the various approaches current nano-delivery systems can offer in overcoming the limitations of conventional drug formulations.

Pulmonary-intestinal cross-talk in mucosal inflammatory disease.

Chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (IBDs) are chronic inflammatory diseases of mucosal tissues that affect the respiratory and gastrointestinal tracts, respectively. They share many similarities in epidemiological and clinical characteristics, as well as in inflammatory pathologies. Importantly, both conditions are accompanied by systemic comorbidities that are largely overlooked in both basic and clinical research. Therefore, consideration of these complications may maximize the efficacy of prevention and treatment approaches. Here, we examine both the intestinal involvement in COPD and the pulmonary manifestations of IBD. We also review the evidence for inflammatory organ cross-talk that may drive these associations, and discuss the current frontiers of research into these issues.

In Vitro and ex Vivo Intestinal Tissue Models to Measure Mucoadhesion of Poly (Methacrylate) and N-Trimethylated Chitosan Polymers

No HeadingPurpose.The adhesion of a range of polymers based on poly(2-(dimethylamino-ethyl) methacrylate (pDMAEMA) was assessed using human mucus-secreting and non mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12) and HT29 monolayers, as well as excised non-everted intestinal sacs from rats. Differentiation of mucoadhesion from bioadhesion was achieved by pre-treatment with the mucolytic agent, N-acetyl cysteine (NAC). Adherence of pDMAEMA polymers was compared to that obtained with the mucoadhesive, N-trimethylated chitosan (TMC).Methods.The quantity of adherent coumarin 343-conjugated polymers to HT29, E12, and intestinal sacs was measured by fluorescence. Confocal laser scanning microscopy (CLSM), light microscopy, and fluorescent microscopy were used to provide direct evidence. Measurements of transepithelial electrical resistance (TEER), permeability to FITC-dextran 4000 (FD-4), and the release of lactate dehydrogenase (LDH) were used to assess potential cytotoxicity of polymers.Results.Adherence of unquaternized and of 10%, 24%, and 32% methyl iodide-quaternized pDMAEMA polymers was measured in E12, HT29, and sacs. All pDMAEMA polymers showed significantly higher levels of adhesion to mucus (mucoadhesion) than to epithelium (bioadhesion). Colocalization of pDMAEMA with mucus was confirmed in E12 by microscopy. TMC showed equally high levels of mucoadhesion as unquaternized and 24% quaternized pDMAEMA, but displayed higher levels of bioadhesion. pDMAEMA-based polymers demonstrated lower levels of adherence to E12 and rat sacs in the presence of NAC, whereas adherence of TMC was unchanged. pDMAEMA significantly decreased the permeability of FD-4 across E12 monolayers and sacs and was less cytotoxic in E12 than in HT29. In contrast, TMC increased the permeability of FD-4 across E12 and sacs and was less cytotoxic in E12 than in HT29.Conclusions.Human mucus–producing E12 monolayers can be used to assess polymer mucoadhesion and give similar data to isolated rat intestinal sacs. pDMAEMA displayed similar levels of mucoadhesion and lower levels of bioadhesion than a chitosan derivative and it was not cytotoxic. pDMAEMA decreased FD-4 flux in the presence of mucus, whereas TMC increased it. The combination of mucus and methacrylate polymers appears to increase barrier function of the apical membrane.

Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis.

Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by trinitrobenzene sulfonic acid were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi, and clinical, immunological, and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared with vehicle controls. Treated groups were pyrexic but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α-deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies that the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.

Selective induction of integrin β1 by hypoxia-inducible factor: implications for wound healing

Because of localized vascular damage and increased tissue oxygen demand, wound healing occurs in a relatively hypoxic microenvironment. These features are particularly relevant to wound healing and fibrosis in chronic inflammatory conditions, such as Crohns disease and ulcerative colitis. In these studies, we sought to identify the contribution of hypoxia to mechanisms of wound repair in a model of the intestinal submucosa. Initial studies revealed that hypoxia promotes wound healing, as modeled by an increase in intestinal fibroblastmediated collagen gel contraction. Guided by results from transcriptional profiling, we identified the selective induction of fibroblast integrin β1 (ITGB1) by hypoxia. Further analysis revealed that hypoxia, as well as pharmacological activators of hypoxia‐inducible factor (HIF), induce fibroblast pi integrin mRNA, protein, and function by as much as 4‐fold. Cloning and analysis of the βi integrin gene promoter revealed a 10 ± 0.8‐fold increase in promoter activity in response to hypoxia, and subsequent studies identified a functional DNA binding region for HIF in the ITGB1 gene promoter. Mutational analysis of the HIF binding site within the ITGB1 promoter resulted in a significant loss of ITGB1 hypoxia‐inducibility. As proof of principle, studies in a murine model of colitis revealed a correlation between colitic disease severity and tissue ITGB1 expression (R2=0.80). Taken together, these results demonstrate that hypoxia induces fibroblast ITGB1 expression and function by transcriptional mechanisms dependent on HIF.— Keely, S., Glover, L. E., MacManus, C. F., Campbell, E. L., Scully, M. M., Furuta, G. T., Colgan, S. P. Selective induction of integrin β1 by hypoxia‐inducible factor: implications for wound healing. FASEBJ. 23, 1338–1346 (2009)

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.

Activated fluid transport regulates bacterial-epithelial interactions and significantly shifts the murine colonic microbiome.

Within the intestinal mucosa, epithelial cells serve multiple functions to partition the lumen from the lamina propria. As part of their natural function, intestinal epithelial cells actively transport electrolytes with passive water movement as a mechanism for mucosal hydration. Here, we hypothesized that electrogenic Cl- secretion, and associated mucosal hydration, influences bacterial-epithelial interactions and significantly influences the composition of the intestinal microbiota. An initial screen of different epithelial secretagogues identified lubiprostone as the most potent agonist for which to define these principles. In in vitro studies using cultured T84 cells, lubiprostone decreased E. coli translocation in a concentration-dependent manner (p < 0.001) and decreased S. typhimurium internalization and translocation by as much as 71 ± 6% (p < 0.01). Such decreases in bacterial translocation were abolished by inhibition of electrogenic Cl- secretion and water transport using the Na-K-Cl- antagonist bumetanide (p < 0.01). Extensions of these findings to microbiome analysis in vivo revealed that lubiprostone delivered orally to mice fundamentally shifted the intestinal microbiota, with notable changes within the Firmicutes and Bacteroidetes phyla of resident colonic bacteria. Such findings document a previously unappreciated role for epithelial Cl- secretion and water transport in influencing bacterial-epithelial interactions and suggest that active mucosal hydration functions as a primitive innate epithelial defense mechanism.