Mucosal surfaces, such as the intestine, are lined by epithelial cells. The epithelium is uniquely positioned to serve as a conduit between the extensive mucosal immune system and the external environment. In their normal state, mucosal surfaces are exposed at the lumenal surface to high concentrations of foreign antigens, whereas at the same time, intimately associated with the immune system through subepithelial lymphoid tissue.1 Consequently, the epithelium forms an important barrier, preventing the free mixing of lumenal antigenic material with the lamina propria that houses the mucosal immune system.2 Like many aspects of biology, this view has changed over the past decade. The epithelium is now viewed as an active player in normal homeostatic mechanisms of mucosal immunity, and in some instances, the epithelium may be the central conductor in orchestrating mucosal immune responses in a variety of diseases, particularly inflammatory bowel disease (IBD).
It is widely understood that the gastrointestinal (GI) tract functions in a state of “low-grade inflammation,” wherein tissues of the GI tract remain “primed” for rapid responses to acute stresses. Such inflammatory priming results from the constant processing of luminal antigenic material during the development of oral tolerance and the activation of the mucosal immune system by antigens or microbes that may penetrate the barrier. Significant evidence indicates that effective mucosal responses are derived from microenvironmental cues generated by complex interactions at local tissue sites. Here, we review some of these microenvironmental signals and potential therapeutic implications therein.
OXYGEN METABOLISM IN THE MICROENVIRONMENT AS IT RELATES TO THE IMMUNE RESPONSE
There is significant recent interest in defining the contribution of oxygen metabolism to the microenvironment of the mucosa in IBD.3,4 The intestine, in fact, provides a fascinating oxygenation profile. Under normal physiological conditions, the intestinal mucosa experiences profound fluctuations in blood flow and metabolism. Less than 5% of total blood volume is present in the gut during fasting; however, after ingestion of a meal, approximately 30% of total blood volume is redirected to the GI tract. Such changes in blood flow can result in marked shifts in local pO2. Notably, a steep oxygen gradient exists from the anaerobic lumen across the epithelium into a highly vascularized and metabolically active subepithelium. From this perspective, it is perhaps not surprising that the epithelium has evolved a number of features to cope with such metabolic shifts. Studies comparing functional responses between epithelial cells from different tissues have revealed that intestinal epithelial cells seem to be uniquely resistant to hypoxia and that an extremely low level of oxygenation within the normal intestinal epithelial barrier (so-called physiological hypoxia) may be a regulatory adaptation mechanism to this steep oxygen gradient.5
Sites of mucosal inflammation are characterized by profound changes in tissue metabolism, including local depletion of nutrients, imbalances in tissue oxygen supply and demand, and the generation of large quantities of reactive nitrogen and oxygen intermediates.6 In part, these changes can be attributed to recruitment of inflammatory cells, including myeloid cells such as neutrophils (polymorphonuclear cells [PMNs] and monocytes (Fig. 1). PMNs are recruited by chemical signals, such as the chemokine interleukin 8, complement factor C5a, N-formylated peptides, platelet-activating factor, and leukotriene B4, which are generated at sites of active inflammation as part of the innate host immune response to microorganisms. In transit, these cells expend tremendous amounts of energy. For instance, large amounts of adenosine 5′-triphosphate (ATP) are needed for the high actin turnover required for cell migration.7 Once at the sites of inflammation, the nutrient, energy, and oxygen demands of the PMNs increase to accomplish the processes of phagocytosis and microbial killing. It has long been known that PMNs are primarily glycolytic cells, with few mitochondria and little energy produced from respiration.8 A predominantly glycolytic metabolism ensures that PMN can function at the low oxygen concentrations (even anoxia) associated with inflammatory lesions.
Activated PMNs recognize and engulf pathogens and activate the release of antibacterial peptides, proteases, and reactive oxygen species (ROS; superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorous acid) into the vacuole, which together kill the invading microbes (Fig. 1).9 ROS are produced by phagocytes in a powerful oxidative burst, driven by a rapid increase in oxygen uptake and glucose consumption, which in turn triggers further generation of ROS. When activated, it is estimated that PMN can consume up to 10 times more O2 than any other cell in the body. Notably, the PMN oxidative burst is not hindered by even relatively low O2 (as low as 4.5% O2),10 which is important, because it means that ROS can be generated in the relatively low O2 environments of inflamed intestinal mucosa.6
In stark contrast to the innate immune response, adaptive immune responses are characterized by a combination of recruitment and high rates of local T-cell and B-cell proliferation. As a result, adaptive immune responses have markedly different demands for glucose, oxygen, and ATP within this microenvironment.11,12 In the past decade, we have begun to understand the nature of interactions between microenvironmental metabolic changes and the generation of recruitment signals and molecular mechanisms of leukocyte migration into these areas. These metabolic changes that occur as a result of the recruitment and activation of leukocytes during inflammation provide information about the potential sources of hypoxia at the intestinal epithelial barrier (Fig. 1).
IBD is a particularly interesting disease for studying the metabolic changes associated with inflammation, principally the development of hypoxia within inflammatory lesions.6 Some microvascular abnormalities that occur in patients with IBD have been associated with abnormal blood flow to the intestine, including increased production of tissue vasoconstrictor molecules and a reduced generation of nitric oxide by endothelial cells13 and vascular endothelial growth factor-dependent angiogenesis.14 In addition, studies of active inflammation in mouse models of IBD have shown the intestinal epithelial cell to be a primary target for hypoxia.15
Hypoxia in murine models of IBD was revealed using 2-nitroimidazole dyes, a class of compounds known to undergo intracellular metabolism depending on tissue oxygenation.16 Tissue staining with these nitroimidazole dyes revealed 2 profound observations. First, in the normal intestinal epithelial cells, especially in the colon, physiological hypoxia predominates. Whether such low oxygen levels function to regulate basal gene expression in intestinal epithelial cells is not known. Second, the inflammatory lesions seen in these mouse models are profoundly hypoxic or even anoxic, similar to that seen in some large tumors and penetrate deep into the mucosal tissue. It is likely that there are multiple contributing factors, such as vasculitis, vasoconstriction, edema, and increased O2 consumption (Fig. 1), which predispose the inflamed intestinal epithelia to decreased oxygen delivery and hypoxia.15 While these 2-niroimidazole compounds have not been used to image inflammatory lesions, they have shown significant clinical utility in tumor imaging and in the identification of stroke regions within the brain of patients.17 As opposed to other imaging techniques, these compounds have the advantages that they image only viable tissue and are not active within apoptotic or necrotic regions.18 Likewise, studies are underway to use these compounds as adjunct radiosensitizers for enhancing chemotherapy targeting.19
The discovery of hypoxia-inducible factor (HIF) in 199320 laid the groundwork for decades of fascinating work in far reaching areas of cancer biology, metabolism, and development. Our work demonstrating inflammation-related hypoxia also revealed the expression of HIF-1 in inflammatory lesions.15 Many cell types, including intestinal epithelial cells,21 express both HIF-1 and HIF-2 isoforms of this transcriptional regulator, and murine knockout studies suggest that these proteins have nonredundant roles.22 Some have suggested that distinct transcriptional responses mediated by HIF-1 and HIF-2 may be integrated in ways that support particular adaptations to hypoxia. For example, the transcriptional responses that coordinate the glycolytic pathways include more than 11 target genes and seem to be more selective for the HIF-1α than for the HIF-2α isoform.22 Studies addressing the selectivity of the 2 isoforms for erythropoietin induction have suggested a more important role for the HIF-2α isoform.22 Currently, this specificity is not well understood. Some have suggested that binding of HIF-1α or HIF-2α to other transcription factors at the site of DNA binding could determine such specificity,22 but these studies have not been conclusive.
Several studies have shown that HIF-1 triggers the transcription of many genes that enable intestinal epithelial cells to act as an effective barrier.5,23–25 Originally shown by microarray analysis of hypoxic intestinal epithelial cells,24 these studies have been validated in animal models of intestinal inflammation15,26–30 and in human intestinal inflammation tissues.31–33 The functional proteins encoded by hypoxia-induced HIF-dependent messenger RNAs (mRNAs) localize primarily to the most luminal aspect of polarized epithelia. Molecular studies of these hypoxia-elicited pathway(s) have shown a dependence on HIF-mediated transcriptional responses. Notably, epithelial barrier protective pathways driven by HIF tend not to be the classical regulators of barrier function, such as the tight junction proteins occludin or claudin. Rather, the HIF-dependent pathways are predisposed to regulate overall tissue integrity, ranging from increased mucin production,34 including molecules that modify mucins, such as, intestinal trefoil factor,5 to xenobiotic clearance by P-glycoprotein,23 to nucleotide metabolism (by ecto-5′-nucleotidase and CD73)24,25 and nucleotide signaling through the adenosine A2B receptor.25
As an extension of the original studies identifying HIF induction within the intestinal mucosa, Karhausen et al15 generated transgenic mice expressing either mutant Hif1-a (causing constitutive repression of Hif1a) or mutant von Hippel–Lindau (causing constitutive overexpression of HIF) targeted to the intestinal epithelial cells. Loss of epithelial HIF-1α resulted in a more severe colitic phenotype compared with wild-type animals with increased weight loss, decreased colon length, and increased epithelial permeability, whereas constitutively active intestinal epithelial HIF was protective for these parameters. These findings may be somewhat model dependent because epithelial HIF-based signaling has also been shown to promote inflammation in another study.30 However, the findings confirmed that intestinal epithelial cells can adapt to hypoxia and that HIF may contribute to this adaptation.
Nonepithelial cell types within the GI mucosa have also been studied for HIF expression and response to hypoxia. Activated T cells increased expression of HIF-1α, which prevents them from undergoing activation-induced cell death in hypoxic settings. T-cell survival in hypoxia is, at least in part, mediated by the vasoactive peptide adrenomedullin.35 Other studies using chimeric mice bearing HIF-1α–deficient T and B cells have revealed lineage-specific defects that result in increased autoimmunity, including autoantibodies, increased rheumatoid factor expression, and kidney damage.12 Most recently, it was shown that diminished O2 in the colitic microenvironment triggers HIF-dependent regulation of T-cell differentiation Indeed, Clambey et al36 showed that one of the key transcriptional differentiation factors, FoxP3, is a HIF target gene and that HIF-1 was required for optimal regulatory T cell (Treg) abundance and function. HIF-1α–deficient Tregs failed to control T cell–mediated colitis, providing an important role for microenvironmental O2 in productive mucosal immune responses.
HIF function has also been studied in some detail in myeloid cells. Cre-LoxP–based elimination of HIF-1α in cells of the myeloid lineage (lysozyme M promoter) have revealed multiple features that importantly implicate metabolic control of myeloid function.37 In particular, these studies have shown that PMN and macrophage bacterial killing capacities are severely limited in the absence of HIF-1α, as HIF-1α is central to production of antimicrobial peptides and granule proteases. These findings are explained, at least in part, by the inability of myeloid cells to mount appropriate metabolic responses to the diminished O2 characteristic of infectious sites.37 Finally, compelling evidence has revealed that HIF-1α transcriptionally controls the critical integrin important in all myeloid cell adhesion and transmigration (namely, the β2 integrin [CD18]).38,39 Such findings are important for our current understanding of the role of functional PMN in IBD. A recent study, for example, used PMN depletion to document a central role for PMN in the resolution of inflammation in several murine IBD models.40
MICROENVIRONMENTAL CONTROL OF NUCLEAR FACTOR κB IN IBD
The oxygen-dependent regulation of the microenvironment may not be restricted to the HIF pathway and may provide a means to better understand how hypoxia contributes to other aspects of inflammation. For instance, nuclear factor κB (NF-κB) may interact with the HIF pathway and is activated during inflammation. NF-κB consists of either homodimers or heterodimers, which, on activation, translocate to the nucleus and bind with the transcriptional coactivator CBP/p300 to initiate transcription or repression of various genes. The activity of NF-κB is regulated by the inhibitory IκB proteins.41 The best-studied complex is IκBα bound to the NF-κB p50–p65 dimer.41 The interaction with IκBα inhibits NF-κB from binding to DNA and maintains the complex in the cytoplasm. On activation by various extracellular signals, IκB kinase (IKK) is activated, resulting in phosphorylation42 and polyubiquitylation of IκBα43. The polyubiquitinated IκBα is then selectively degraded by the S26 proteasome. Once dissociated from IκBα, NF-κB rapidly enters the nucleus and activates gene expression.
It is now appreciated that NF-κB–dependent pathways are also regulated by the enzymes that promote the degradation of HIF-α subunits (namely HIF prolyl hydroxylases [PHDs]) and that the usefulness of PHD inhibitors in murine colitis models also target the NF-κB pathway.26 Indeed, hypoxia has been shown to activate NF-κB, and this activation seems, at least in part, to be dependent on PHD-mediated hydroxylation44,45 of IKKβ.38 In normoxia, IKKβ activity is held in check through LXXLAP-dependent hydroxylation by PHD1 and PHD2.45 It is notable that conditional deletion of the NF-κB pathway in intestinal epithelial cells in mice leads to an increased susceptibility to colitis,46 similar to that of the mice expressing homozygous mutant HIF-1α.15 This implicates epithelial NF-κB in a prominently protective role in colitis, probably through the expression of antiapoptotic genes in intestinal epithelial cells and through enhanced epithelial barrier function. Some studies have suggested that both the HIF and NF-κB pathways may also be influenced by mediators found within inflammatory sites, including microbial products, cytokines, and even intact bacteria.37 NF-κB is a classic transcriptional regulator activated by a spectrum of agonists, the activation of which drives a complex series of receptor-mediated signaling pathways. Recent studies indicate that transcription of HIF-1α is activated by NF-κB–mediated signaling.47 Inflammation-associated upregulation of HIF-1α mRNA occurs in an NF-κB-dependent manner.47 It also seems that increased NF-κB activity in hypoxia can be regulated by HIF-1,48 and thus, a cross-regulatory loop may exist between these 2 pathways and may involve other transcriptional regulators that bear nonredundant PHD sensitivity, including activating transcription factor-4 and Notch,49,50 both critical regulators of cell fate. Given that intestinal epithelial cells are situated in an environment with constant exposure to potentially inflammatory stimuli, the cross-regulation of HIF and NF-κB may have profound implications for intestinal epithelial cell function and survival under both homeostatic and disease conditions.
ADENOSINE METABOLISM IN THE INFLAMMATORY MICROENVIRONMENT
Nucleosides such as adenosine (Ado) influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides liberated at local sites of inflammation are metabolized through regulated PHD by a series of ecto-nucleotidases. The formation of extracellular Ado within the microenvironment from ATP is accomplished primarily through ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73), found on the surface of a variety of cell types (Fig. 2). Once generated, Ado is available to bind and activate 1 of 4 G-protein–coupled Ado receptors (A1AR, A2AAR, A2BAR, or A3AR).
Individual Ado receptors are widely expressed within the GI tract, and their function has been studied recently. Although less is known about A1AR in GI disease models, the A2AAR and A2BAR receptors have been studied in some detail.51–53 The A2AAR is critical for A2AAR signaling in T-cell–mediated regulation of colitis. Moreover, treatment with a specific A2AAR agonist attenuated the production of proinflammatory cytokines and attenuation of colitis.54 Recent studies have performed “head-on” comparisons between A2AAR −/− and A2BAR −/− in murine colitis models and revealed a relative importance for the A2BAR in murine colitis.55 Studies in A3AR −/− mice have proven quite interesting and potentially revealed a specific role for A3AR in the resolution of GI inflammation. For example, Butler et al56 have recently shown that A3AR−/− mice show overall decreased pathology in acute dextran-sodium sulfate colitis, but these animals failed to resolve inflammation associated with chronic inflammation in this model. Likewise, the adenosine A3 receptor agonist, N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide, has proven to be protective in at least 2 murine models of colitis.57 In humans, A3AR expression negatively correlates with acute inflammatory score, Crohn's Disease Activity Index, and disease chronicity,58 strongly implicating a need for further investigation of this receptor subtype.
CD73 is the terminal enzyme in the generation of extracellular Ado and is therefore considered the pacemaker for Ado actions within the microenvironment (Fig. 2).59 Original characterization of the Cd73 −/− mouse revealed that of the tissues surveyed, the colon showed the highest level of enzyme activity, a somewhat surprising result, as a number of previous studies suggested that the kidney likely carried the highest activity of any tissue. Instead, it was found that the colon expresses nearly twice as much activity as the kidney, with the rank order of tissue activity as follows: colon > kidney = brain > liver > lung > heart >> muscle.60 During experimental colitis induced by the hapten trinitrobenzene sulfonic acid, Cd73−/− mice develop a more severe phenotype.61 Cytokine profiling revealed similar increases in both interferon gamma and tumor necrosis factor alpha mRNA in colitic animals, independent of genotype. However, interleukin-10 mRNA increased in wild-type mice on day 3 after trinitrobenzene sulfonic acid administration, whereas Cd73 −/− mice mounted no interleukin-10 response. This interleukin-10 response was restored in the Cd73 −/− mice by exogenous interferon αA.61 More recently, it was shown that CD73 is central to both the magnitude and the resolution of murine DSS colitis.62
As CD39 hydrolyzes nucleotides to generate adenosine monophosphate (Fig. 2), it has become a point of interest in IBD. In 1 study, the authors hypothesized that CD39 might protect against IBD.63 They studied these possibilities in a mouse model of colitis using mice with global CD39 deletion and also tested whether human genetic polymorphisms in the CD39 gene might influence susceptibility to Crohn's disease. Mice deficient for CD39 were highly susceptible to chemically induced colitis, with heterozygote mice showing an intermediate phenotype. Moreover, they identified a common single nucleotide polymorphism that tags CD39 mRNA expression levels in man. The single nucleotide polymorphism tagging low levels of CD39 expression was associated with increased susceptibility to Crohn's disease in a case–control cohort comprised of 1748 patients and 2936 controls. These data indicate that CD39 deficiency exacerbates murine colitis and suggests that CD39 polymorphisms are associated with IBD in humans.63 Other studies have identified CD39 as a specific marker for Tregs and implicate CD39-dependent ATP/adenosine diphosphate breakdown in autocrine enhancement of the anti-inflammatory functions of this group of T cells.64
PRORESOLVING LIPIDS IN THE MICROENVIRONMENT OF IBD
Resolution of inflammation and return to tissue homeostasis is an exceptionally well-coordinated process. There is much recent interest in the identification of pro-resolving molecules generated within the inflammatory microenvironment. Significant progress has been made in this regard, especially pro-resolving lipids generated during the resolution phase of ongoing inflammation that actively stimulate restoration of tissue homeostasis.65
Particular attention has been paid to the resolvins (Fig. 2).66 Resolvins are omega-3 fatty acid–derived mediators that are agonist dependent, temporally distinct, and carry novel mucosal-directed signals.67 The first resolvin, known today as resolvin E1 (RvE1), was identified in 1999 as a potent and active initiator of inflammatory resolution.68 Inordinate, unrestricted acute inflammation is now acknowledged as an instigating factor, which when unchecked, contributes to numerous chronic disease states, including IBD. As such, an understanding of the pharmacology of anti-inflammation and endogenous pro-resolution has been a significant venture.67
A recent microarray screen to identify RvE1-regulated genes in intestinal epithelial cells revealed 2 important findings. First, these studies revealed the previously unappreciated native expression of the RvE1 receptor ChemR23 on epithelial cells. A screen of various epithelial cell lines revealed prominent expression of ChemR23 on human intestinal epithelial cell lines (T84 and Caco-2). Unique was the pattern of expression on polarized epithelia as this analysis revealed that ChemR23 localizes predominantly to the apical membrane surface, which was somewhat unexpected given that most other G-protein–coupled receptors exhibit basolateral expression in polarized epithelia.69 Such membrane distribution of ChemR23 suggested that the localized generation of RvE1 during PMN–epithelial interactions could occur at the apical (lumenal) aspect of the tissue. This is an intriguing possibility given that the other known function for RvE1 on mucosal epithelia is to promote the termination and clearance of PMN after transmigration70 through well-characterized CD55-dependent mechanisms.71,72 Thus, the PMN–epithelial interactions that occur within the lumen of the intestine may initiate a pro-resolving signature for the epithelium during PMN transit through the mucosa.
Second, these microarray studies identified a prominent RvE1-dependent antimicrobial signature within the epithelium, including the induction of bactericidal permeability-increasing protein and the bactericidal permeability-increasing protein-like molecule PLUNC (palate, lung, nasal epithelium clone).73 Also notable was the induction of epithelial intestinal alkaline phosphate (ALPI) by RvE1. Surface-expressed ALPI was shown to retard Gram-negative bacterial growth and to potently neutralize lipoposachride through a mechanism involving dephosphorylation of 1,4′-bisphosphorylated glucosamine disaccharide of lipoposachride lipid A.74,75 This observation was translated to the murine DSS colitis model and revealed that induction of ALPI by RvE1 in vivo strongly correlated with the resolution phase of inflammation (Fig. 2). Moreover, inhibition of ALPI activity was shown to increase the severity of colitic disease and abrogated the protective influences of RvE1.73 Like those defining epithelial expression of BPI,76 these studies provide an example of the critical interface between inflammatory resolution and the importance of antimicrobial mechanisms.
The GI mucosa is an interesting tissue in which to investigate the microenvironment and tissue-based metabolism. In this review, we have outlined the evidence for mediators generated in the local microenvironment as important signaling mechanisms within the intestinal mucosa. Studies from cultured cell systems, animal models, and patient-derived materials have documented that the metabolism of nucleotides and lipids provides a significant component of the inflammatory microenvironment. Likewise, studies to date in animal models of intestinal inflammation have demonstrated an almost uniformly beneficial influence of oxygen metabolism and HIF stabilization on disease outcomes. Ongoing studies to define the differences and similarities between innate and adaptive immune responses will continue to teach us important lessons about the complexity of the GI tract. Such information will provide new insight into the pathogenesis of the disease and importantly will provide new targets as templates for the development of therapies for human disease.
1. Beagley KW, Husband AJ. Intraepithelial lymphocytes: origins, distribution, and function. Crit Rev Immunol. 1998;18:237–254.
2. McCole DF, Barrett KE. Varied role of the gut epithelium in mucosal homeostasis. Curr Opin Gastroenterol. 2007;23:647–654.
3. Colgan SP, Taylor CT. Hypoxia: an alarm signal during intestinal inflammation. Nat Rev Gastroenterol Hepatol. 2010;7:281–287.
4. Glover LE, Colgan SP. Hypoxia and metabolic factors that influence inflammatory bowel disease pathogenesis. Gastroenterology. 2011;140:1748–1755.
5. Furuta GT, Turner JR, Taylor CT, et al.. Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia. J Exp Med. 2001;193:1027–1034.
6. Taylor CT, Colgan SP. Hypoxia and gastrointestinal disease. J Mol Med (Berl). 2008;85:1295–1300.
7. Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 2003;112:453–465.
8. Borregaard N, Herlin T. Energy metabolism of human neutrophils during phagocytosis. J Clin Invest. 1982;70:550–557.
9. El-Benna J, Dang PM, Gougerot-Pocidalo MA. Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane. Semin Immunopathol. 2008;30:279–289.
10. Gabig TG, Bearman SI, Babior BM. Effects of oxygen tension and pH on the respiratory burst of human neutrophils. Blood. 1979;53:1133–1139.
11. Fox CJ, Hammerman PS, Thompson CB. Fuel feeds function: energy metabolism and the T-cell response. Nat Rev Immunol. 2005;5:844–852.
12. Sitkovsky M, Lukashev D. Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors. Nat Rev Immunol. 2005;5:712–721.
13. Hatoum OA, Heidemann J, Binion DG. The intestinal microvasculature as a therapeutic target in inflammatory bowel disease. Ann N Y Acad Sci. 2006;1072:78–97.
14. Danese S, Dejana E, Fiocchi C. Immune regulation by microvascular endothelial cells: directing innate and adaptive immunity, coagulation, and inflammation. J Immunol. 2007;178:6017–6022.
15. Karhausen JO, Futura GT, Tomaszewski JE, et al.. Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis. J Clin Invest 2004;114;1098–1106.
16. Evans SM, Hahn S, Pook DR, et al.. Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res. 2000;60:2018–2024.
17. Takasawa M, Moustafa RR, Baron JC. Applications of nitroimidazole in vivo hypoxia imaging in ischemic stroke. Stroke. 2008;39:1629–1637.
18. Kizaka-Kondoh S, Konse-Nagasawa H. Significance of nitroimidazole compounds and hypoxia-inducible factor-1 for imaging tumor hypoxia. Cancer Sci. 2009;100:1366–1373.
19. Overgaard J. Hypoxic radiosensitization: adored and ignored. J Clin Oncol 2007;25:4066–4074.
20. Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Nat Acad Sci (USA). 1993;90:4304–4308.
21. Mastrogiannaki M, Matak P, Keith B, et al.. HIF-2alpha, but not HIF-1alpha, promotes iron absorption in mice. J Clin Invest. 2009;119:1159–1166.
22. Ratcliffe PJ. HIF-1 and HIF-2: working alone or together in hypoxia? J Clin Invest. 2007;117:862–865.
23. Comerford KM, Wallace TJ, Karhausen J, et al.. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res. 2002;62:3387–3394.
24. Synnestvedt K, Furuta GT, Comerford KM, et al.. Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 (HIF-1) mediates permeability changes in intestinal epithelia. J Clin Invest. 2002;110:993–1002.
25. Eltzschig HK, Ibla JC, Furuta GT, et al.. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B
receptors. J Exp Med. 2003;198, 783–796.
26. Cummins EP, Seeballuck F, Keely SJ, et al.. The hydroxylase inhibitor dimethyloxalylglycine is protective in a murine model of colitis. Gastroenterology. 2008;134:156–165.
27. Han IO, Kim HS, Kim HC, et al.. Synergistic expression of inducible nitric oxide synthase by phorbol ester and interferon-gamma is mediated through NF-kappaB and ERK in microglial cells. J Neurosci Res. 2003;73:659–669.
28. Morote-Garcia JC, Rosenberger P, Nivillac NM, et al.. Hypoxia-inducible factor-dependent repression of equilibrative nucleoside transporter 2 attenuates mucosal inflammation during intestinal hypoxia. Gastroenterology. 2009;136:607–618.
29. Robinson A, Keely S, Karhausen J, et al.. Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition. Gastroenterology. 2008;134:145–155.
30. Shah YM, Ito S, Morimura K, et al.. Hypoxia-inducible factor augments experimental colitis through an MIF-dependent inflammatory signaling cascade. Gastroenterology. 134:2008;2036–2048, 2048 e1-3.
31. Giatromanolaki A, Sivridis E, Maltezos E, et al.. Hypoxia inducible factor 1alpha and 2alpha overexpression in inflammatory bowel disease. J Clin Pathol. 2003;56:209–213.
32. Mariani F, Sena P, Marzona L, et al.. Cyclooxygenase-2 and hypoxia-inducible factor-1alpha protein expression is related to inflammation, and up-regulated since the early steps of colorectal carcinogenesis. Cancer Lett. 2009;279:221–229.
33. Matthijsen RA, Derikx JP, Kuipers D, et al.. Enterocyte shedding and epithelial lining repair following ischemia of the human small intestine attenuate inflammation. PLoS One. 2009;4:e7045.
34. Louis NA, Hamilton KE, Canny G, et al.. Selective induction of mucin-3 by hypoxia in intestinal epithelia. J Cell Biochem. 99:2006;1616–1627.
35. Makino Y, Nakamura H, Ikeda E, et al.. Hypoxia-inducible factor regulates survival of antigen receptor-driven T cells. J Immunol. 2003;171:6534–6540.
36. Clambey ET, McNamee EN, Westrich JA, et al.. Hypoxia-inducible factor-1 alpha-dependent induction of FoxP3 drives regulatory T-cell abundance and function during inflammatory hypoxia of the mucosa. Proc Natl Acad Sci U S A. 2012;109:E2784–E2793.
37. Nizet V, Johnson RS. Interdependence of hypoxic and innate immune responses. Nat Rev Immunol. 2009;9:609–617.
38. Kong T, Eltzschig HK, Karhausen J, et al.. Leukocyte adhesion during hypoxia is mediated by HIF-1-dependent induction of beta2 integrin gene expression. Proc Natl Acad Sci U S A. 2004;101:10440–10445.
39. Kong T, Scully M, Shelley CS, et al.. Identification of Pur alpha as a new hypoxia response factor responsible for coordinated induction of the beta 2 integrin family. J Immunol. 2007;179:1934–1941.
40. Kuhl AA, Kakirman H, Janotta M, et al.. Aggravation of different types of experimental colitis by depletion or adhesion blockade of neutrophils. Gastroenterology. 2007;133:1882–1892.
41. Chen Z, Hagler J, Palombella VJ, et al.. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev. 1995;9:1586–1597.
42. Luo JL, Kamata H, Karin M. IKK/NF-kappaB signaling: balancing life and death—a new approach to cancer therapy. J Clin Invest. 2005;115:2625–2632.
43. Chen ZJ. Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol. 2005;7:758–765.
44. Cockman ME, Lancaster DE, Stolze IP, et al.. Posttranslational hydroxylation of ankyrin repeats in IkappaB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc Natl Acad Sci U S A. 2006;103:14767–14772.
45. Cummins EP, Berra E, Comerford KM, et al.. Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB activity. Proc Natl Acad Sci U S A. 2006;103:18154–18159.
46. Zaph C, Troy AE, Taylor BC, et al.. Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasis. Nature. 2007;446:552–556.
47. Rius J, Guma M, Schachtrup C, et al.. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature. 2008;453:807–811.
48. Taylor CT. Interdependent roles for hypoxia inducible factor and nuclear factor-kappaB in hypoxic inflammation. J Physiol. 2008;586:4055–4059.
49. Coleman ML, McDonough MA, Hewitson KS, et al.. Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor. J Biol Chem. 2007;282:24027–24038.
50. Koditz J, Nesper J, Wottawa M, et al.. Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor. Blood. 2007;110:3610–3617.
51. Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 2001;414:916–920.
52. Rosenberger P, Schwab JM, Mirakaj V, et al.. Hypoxia-inducible factor-dependent induction of netrin-1 dampens inflammation caused by hypoxia. Nat Immunol. 2009;10:195–202.
53. Eckle T, Grenz A, Laucher S, et al.. A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice. J Clin Invest. 2008;118:3301–3315.
54. Naganuma M, Wiznerowicz EB, Lappas CM, et al.. Cutting edge: critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis. J Immunol. 2006;177:2765–2769.
55. Frick JS, MacManus CF, Scully M, et al.. Contribution of adenosine A2B receptors to inflammatory parameters of experimental colitis. J Immunol. 2009;182:4957–4964.
56. Butler M, Sanmugalingam D, Burton VJ, et al.. Impairment of adenosine a3 receptor activity disrupts neutrophil migratory capacity and impacts innate immune function in vivo. Eur J Immunol. 2012;42:3358–3568.
57. Mabley J, Soriano F, Pacher P, et al.. The adenosine A3 receptor agonist, N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, is protective in two murine models of colitis. Eur J Pharmacol. 2003;466:323–329.
58. Rybaczyk L, Rozmiarek A, Circle K, et al.. New bioinformatics approach to analyze gene expressions and signaling pathways reveals unique purine gene dysregulation profiles that distinguish between CD and UC. Inflamm Bowel Dis. 2009;15:971–984.
59. Colgan SP, Eltzschig HK. Adenosine and hypoxia-inducible factor signaling in intestinal injury and recovery. Annu Rev Physiol. 2012;74:153–175.
60. Thompson LF, Eltzschig HK, Ibla JC, et al.. Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia. J Exp Med. 2004;200:1395–1405.
61. Louis NA, Robinson AM, MacManus CF, et al.. Control of IFN-alphaA by CD73: implications for mucosal inflammation. J Immunol. 2008;180:4246–4255.
62. Bynoe MS, Waickman AT, Mahamed DA, et al.. CD73 is critical for the resolution of murine colonic inflammation. J Biomed Biotechnol. 2012;260983:1–13.
63. Friedman DJ, Kunzli BM, Yi AR, et al.. From the cover: CD39 deletion exacerbates experimental murine colitis and human polymorphisms increase susceptibility to inflammatory bowel disease. Proc Natl Acad Sci U S A. 2009;106:16788–16793.
64. Deaglio S, Dwyer KM, Gao W, et al.. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007;204:1257–1265.
65. Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008;8:349–361.
66. Campbell EL, Serhan CN, Colgan SP. Antimicrobial aspects of inflammatory resolution in the mucosa: a role for proresolving mediators. J Immunol. 2011;187:3475–3481.
67. Serhan CN, Chiang N. Endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus. Br J Pharmacol. 2008;153(suppl 1):S200–S215.
68. Serhan CN, Colgan SP. Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing. J Exp Med. 2000;192:1197–1204.
69. Wozniak M, Keefer JR, Saunders C, et al.. Differential targeting and retention of G protein-coupled receptors in polarized epithelial cells. J Recept Signal Transduct Res. 1997;17:373–383.
70. Campbell EL, Louis NA, Tomassetti SE, et al.. Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution. FASEB J. 2007;21:3162–3170.
71. Lawrence DW, Bruyninckx WJ, Louis NA, et al.. Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia. J Exp Med. 2003;198:999–1010.
72. Louis NA, Hamilton KE, Kong T, et al.. HIF-dependent induction of apical CD55 coordinates epithelial clearance of neutrophils. FASEB J. 2005;19:950–959.
73. Campbell EL, MacManus CF, Kominsky DJ, et al.. Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification. Proc Natl Acad Sci U S A. 2010;107:14298–14303.
74. Mata-Haro V, Cekic C, Martin M, et al.. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science. 2007;316:1628–1632.
75. Moyle PM, Toth I. Self-adjuvanting lipopeptide vaccines. Curr Med Chem. 2008;15:506–516.
76. Canny G, Levy O, Furuta GT, et al.. Lipid mediator-induced expression of bactericidal/permeability-increasing protein (BPI) in human mucosal epithelia. Proc Natl Acad Sci U S A. 2002;99:3902–3907.