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Inflammatory diseases influence tissue metabolism, significantly altering the profile of extracellular adenine nucleotides. A number of studies have suggested that adenosine (Ado) may function as an endogenously generated anti-inflammatory molecule. Given the central role of intestinal epithelial cells to the development of colitis, we hypothesized that specific Ado receptors would contribute to disease resolution in mucosal inflammation as modeled by DSS (dextran sodium sulfate) colitis. Initial profiling studies revealed that murine intestinal epithelial cells express predominantly the adenosine A2B receptor (AA2BR) and to a lesser extent AA2AR. Guided by these results, we examined the contribution of AA2BR to colitis. Initial studies indicated that the severity of colitis was increased in Aa2br−/− mice relative to Aa2br+/+ controls, as reflected by increased weight loss, colonic shortening and disease activity indices. Likewise, enteral administration of the selective AA2BR inhibitor PSB1115 to Aa2br+/+ mice resulted in a similar increase in severity of DSS colitis. Cytokine profiling of colonic tissue revealed specific deficiencies in IL-10 in Aa2br−/− mice relative to controls. Extensions of these findings in cultured human intestinal epithelial cells revealed that stable adenosine analogs induce IL-10 mRNA and protein and that such increases can be blocked with PSB1115. Together, these studies indicate a central regulatory role for AA2BR-modulated IL-10 in the acute inflammatory phase of DSS colitis, thereby implicating AA2BR as an endogenously protective molecule expressed on intestinal epithelial cells.
The inflammatory bowel diseases (IBD), including both Crohn's disease and ulcerative colitis, are debilitating diseases of unknown etiology (1). The most recent evidence suggests that IBD likely results from dysregulated immune responses to luminal triggers (1). Human IBD has benefited immensely from the development of a number of investigational models to study disease pathogenesis (2). Studies in animal models of IBD, primarily murine colitis models, have identified both pathological disease markers as well as endogenously protective molecules which promote the resolution of ongoing inflammation (2). One such molecule of intense recent interest in IBD and other inflammatory diseases is the purine adenosine (3-5). A number of studies have indicated that adenosine and its analogs can ameliorate the course of a variety of inflammatory diseases, including IBD (3, 5). Depending on the target receptor (see below), adenosine has also been demonstrated to either not influence disease outcome (6) or exacerbate inflammation in some models (7).
While the source of interstitial adenosine in inflammation has been the basis of some debate, it is now appreciated that inhibition of adenosine kinase and the dephosphorylation of ATP and AMP by surface apyrases (e.g. CD39) and ecto-5′-nucleotidase (CD73), respectively, represent the major pathways of extracellular adenosine (8-10). Once liberated in the extracellular space, adenosine is either recycled (e.g. through dipyridamole-sensitive carriers) or interacts with cell surface Ado receptors (11). Presently, four subtypes of G protein-coupled Ado receptors exist, designated AA1R, AA2AR, AA2BR or AA3R and are classified according to utilization of pertussis toxin-sensitive (A1 and A3) or insensitive (A2A and A2B) pathways (11). Recent work has specifically implicated the AA2BR in anti-inflammatory responses, wherein activation of this receptor elicits potent inhibition of inflammatory signaling cascades mediated by NF-κB (12).
Here, we sought to investigate the role of AA2BR in a model of murine colitis. Initial studies identified high expression of AA2AR and AA2BR in colonic mucosa. Studies with Aa2ar−/− and Aa2br−/− mice identified significantly increased susceptibility of the Aa2br−/− genotype. At multiple levels, Aa2br−/− mice were unable to mount effective responses to DSS-induced colitis. Similar findings were observed in wild-type animals administered the AA2BR antagonist PSB1115. Cytokine profiling of mucosal tissue identified specific deficiencies in IL-10 by AA2BR-deficient mucosa generated, at least in part, by colonic epithelial cells.
Mice with homozygous deficiency for Aa2ar (Aa2ar−/−) or Aa2br (Aa2br−/−) on the C57BL/6 SVJ strain were generated and characterized as previously described (13) and maintained as homozygous crosses. Control C57BL/6 mice (Aa2br+/+) were matched for age, and gender in each experiment. All procedures involving animals were performed according to NIH guidelines for use of live animals and were approved by the Institutional Animal Care and Use Committee at the University of Colorado and the University of Tuebingen.
DSS colitis was induced with a modification of the technique of Okayasu, et al. (14). Colitis was induced on day 0 by the addition of 2.5% DSS (MW = 36,000-50,000, MP Biomedicals, Illkirch, France) solution in drinking water. Control animals received water alone. Only animals that showed an initial response to DSS treatment, defined by at least a 5 % weight loss relative to baseline weight, were included in the weight loss analysis for the DSS treatment groups. For these studies, 1 animal (of 53 induced) was excluded under these criteria.
In subsets of experiments, mice were treated with the AA2BR inhibitor, PSB1115 (Tocris, Bristol, UK) was prepared at a concentration of 100 μg/ml PBS and animals were dosed by gavage with PSB1115 at 1 mg/kg every other day starting on day −1. In DSS experiments, PSB1115 treatment was initiated on the day −1, 1 day prior to the induction of colitis. Mice were then treated every second day until harvest on day 6 (4 total doses). Control animals received corresponding volumes of PBS.
Macroscopic symptoms of inflammation (stool consistency, presence of blood in stool, presence of blood at the anus, presence of relieving posture and appearence of the fur) was assessed daily during the course of the experiment. The following scores were given to stool consistency: 0: formed stool, 1: formed and soft stool, 3: diarrhea. For blood in stool: 0 no presence of fecal blood, 3 presence of fecal blood. Anal blood score: 0: no blood, 1: inflamed anus, 2: slight bleeding, 3: bleeding and oedema. Relieving posture was scored with 0-3 points and unkempt fur was given 1 point. The scores were added and the disease activity index was calculated. Maximum possible score was 10.
Histological examination was performed on three samples of the distal colon. Samples were fixed in 10% formalin before staining with hematoxylin and eosin. All histological quantitation was performed in a blinded fashion, using a previously described scoring system (15) Severity of inflammation: rare inflammatory cells in the lamina propria (0); increased numbers of inflammatory cells (1); confluence of inflammatory cells extending into the submucosa (2); transmural extension of the inflammatory cell infiltrate (3). Extend of injury: nil mucosal damage (0); discrete lymphoepithelial lesions (1); surface mucosal erosion (2); and widespread mucosal ulceration and extension through deeper bowel wall structures (3). The scores of the two parameter were added and the mean was calculated. Maximum possible score was 3.
Where indicated, Th1 and Th2 cytokine levels were screened from colonic tissue derived from Aa2br+/+ or Aa2br−/− mice. Colon samples (50 mg) were homogenized for 10 sec in lysis buffer [150mM NaCL, 20mM Tris pH 7.5, 1mM EDTA, 1mM EGTA, 0.1% SDS, protease inhibitor cocktail (Roche), phosphatase inhibitor 1 and 2 (Sigma), 2mM PMSF] at 4°C. Samples were cleared by centrifugation and supernatants were assayed for indicated cytokines by chemiluminescence-based sandwich immunoassay (Meso Scale Diagnostics, Gaithersburg, MD) according to manufacturer's protocols. Plates were analyzed on a Sector 2400 Imager (Meso Scale Diagnostics, Gaithersburg, MD). Where indicated, phospho-Akt was measured in a similar manner using chemiluminescence-based sandwich immunoassay (Meso Scale Diagnostics, Gaithersburg, MD) according to manufacturer's protocols.
RNA was isolated for transcriptional analysis from colonic mucosal scrapings homogenized in TRIzol™ (Invitrogen Corp.) using a 550 sonic dismembrator (Fisher Scientific International Co.) followed by phenol-chloroform extraction, as previously described (16). Reverse transcription was done using iScriptTM cDNA synthesis kit using the manufacturer's instructions (BioRad Laboratories Inc., Hercules, California, USA).
Real-time PCR was employed using iQ™ SYBR mix (iCycler; Bio-Rad), as described previously (17). Primer sets were as follows: murine β-actin (sense primer, 5′-CTC TCC CTC ACG CCA TCC TG -3′ and antisense primer, 5′- TCA CGC ACG ATT TCC CTC TCA G -3′, 124 bp); murine AA2AR (sense primer, 5′- CCG AAT TCC ACT CCG GTA CA -3′and antisense primer, 5′- CAG TTG TTC CAG CCC AGC AT -3′, 120 bp); and murine AA2BR (sense primer, 5′- TCT TCC TCG CCT GCT TCG T -3′ and antisense primer 5′- CCA GTG ACC AAA CCT TTA TAC CTG A -3′, 121 bp). The final reaction volume (25μl) consisted of 12.5μl SYBR Green I (Bio-Rad), 2.5μl diluted cDNA and 200nM of each primer. Reaction cycle consisted of 95°C for 2 mins, followed by 45 cycles of 95°C for 15sec, 62°C for 20sec and 72°C for 35 sec, and a final extension at 72°C for 3 mins.
Caco-2 cells were grown on permeable 5 cm2 polycarbonate ring supports (Costar Corp., Cambridge, MA) to high confluence, using previously described protocols (18). Where indicated, Caco2 cells were exposed to the adenosine analog N-ethyl-carbamidoadenosine (NECA) for 48h. Media was harvested, filtered and assayed for IL-10 levels by electrochemiluminescence-based sandwich immunoassay (Meso Scale Diagnostics, Gaithersburg, MD) according to manufacturer's protocols. Plates were analyzed on a Sector 2400 Imager (Meso Scale Diagnostics, Gaithersburg, MD).
Statistical analysis was performed using Student's t-test or ANOVA, as indicated.
Extracellular Ado signaling has been widely implicated in adaptive responses to inflammation (19). Previous studies have revealed that AA2BR is abundantly expressed on human intestinal epithelial cells (20), and that such expression is regulated by hypoxia-inducible factor-1 (HIF-1) (17). We therefore sought to define the contribution of AA2BR to colitic disease. Initially, we examined adenosine receptor mRNA expression in mucosal scrapings (enriched in epithelial cells) from wild-type C57BL/6 mice. As shown in Figure 1A and 1B, this analysis revealed expression of both AA2AR and AA2BR in mucosal scrapings. Such findings are consistent with previous studies in human epithelia (20), and therefore compelled us to examine how the adenosine receptors contribute to mucosal inflammation, as modeled by DSS colitis.
Based on the finding that murine intestinal epithelia express AA2AR and AA2BR, we subjected Aa2ar−/− and Aa2br−/− mice to DSS protocols and compared their responses to littermate wild-type controls. Our previous studies revealed that neither Aa2ar−/− or Aa2br−/− animals manifest outward immunological defects when housed in specific pathogen-free conditions (13). Here, we compared the clinical course of DSS-induced colitis in Aa2ar−/− and Aa2br−/− mice.
Initial studies using 3.5% DSS revealed that Aa2br−/− mice became profoundly ill within 3 days of induction and resulted in high mortality (data not shown). For this reason, we decreased drinking water DSS concentrations to 2.5%. This comparison between Aa2ar−/− and Aa2br−/− is shown in Figure 1C and 1D, respectively. Weight loss is a reliable method to assess DSS colitis severity (14), and as shown in Figure 1C, Aa2br−/− mice exposed to DSS lost weight more rapidly and failed to regain weight over the 6 days following induction of colitis (Figure 1C, p<0.01 by ANOVA). By comparison, Aa2ar−/− animals remained healthy at 2.5% DSS with no significant differences observed in weight loss curves between Aa2ar−/− and littermate controls (p = not significant). Notably, Aa2ar−/− mice showed increased susceptibility to higher concentrations of DSS. Indeed, when DSS concentrations were increased from 2.5% to 4.5%, Aa2ar−/− showed significantly increased weight loss on days 2 – 6 (maximal difference 12±4% at day 5, p<0.05 by ANOVA) and increased colon contraction (31±5% decrease in colon length, p<0.05) compared to wild-type controls (n=4 per group). Thus, it would appear that both Aa2ar−/− and Aa2br−/− mice have increased susceptibility to DSS colitis, but that this phenotype is more severe in Aa2br−/−.
Additional endpoints including disease activity index over the course of colitis (Figure 2A) and colon contraction at harvest (Figure 2B) also revealed significantly increased disease severity in Aa2br−/− mice compared to controls. Histologically, Aa2br−/− mice showed a prominent loss of tissue architecture and fulminant leukocyte infiltration compared to Aa2br+/+ (Figures 2C and 2D). In this regard, the AA2BR has been broadly implicated in inflammation associated with murine myocardial ischemia (21, 22), ischemic preconditioning of the kidneys (23) and acute lung injury (24), wherein mechanisms have been tracked to inhibition of NF-κB through Ado-mediated cullin deneddylation (12). Thus, we determined whether colonic tissue from Aa2br−/− showed increased NF-κB activation. Initial studies to document direct NF-κB activity in colonic tissue proved technically difficult, and we therefore addressed signaling pathways upstream of NF-κB. In particular, activity levels of the phosphoinositide 3-kinase (PI3K)-Akt pathway, which critically regulates NF-κB (25) proved interesting. As shown in Figure 2E, levels of activated Akt (phospho-Akt) were significantly elevated over vehicle controls in both colitic Aa2br+/+ and in Aa2br−/− (p<0.01 for both). Notably, colitic Aa2br−/− mice showed increased phosho-Akt levels compared to their wild-type counterparts (p<0.025). No differences were noted between Aa2br−/− and Aa2br−/− in baseline phospho-Akt levels. Such findings strongly implicate a protective role for AA2BR in experimental colitis.
To rule out the possibility that increased susceptibility of Aa2br−/− mice to colitis could be attributable to a developmental etiology, we determined whether this phenotype could be mimicked by pharmacologic blockade of AA2BR using the selective inhibitor PSB1115 (26). For these purposes, Aa2br+/+ mice were administered PSB1115 (1 mg/kg) by gavage, a concentration we have previously shown to effectively inhibit AA2BR in a systemic fashion (13). Mice were then subjected to DSS colitis with or without PSB1115 administered by gavage beginning on day −1 and then every second day (for a total of 4 doses) until harvest on day 6 following the induction of colitis. Similar to Aa2br−/− mice, wild type PSB1115-treated mice exposed to DSS lost weight more rapidly and failed to show recovery weight gain during the course of the experiment (p < 0.05 by ANOVA), compared to mice receiving DSS alone (Figure 3A). Likewise, other disease markers including DAI (Figure 3B), colon length (Figure 3C) and histological scores (Figure 3D and 3E) reflected worsened disease in those animals receiving PSB1115. Such findings confirm that altered AA2BR activity likely explains our findings of increased susceptibility in Aa2br−/− animals.
In an attempt to gain specific insight into potential mechanisms of increased colitis susceptibility of Aa2br−/− mice, we profiled the expression of colon Th1 and Th2 immunoregulators following DSS colitis (day 6) in Aa2br−/− and Aa2br+/+ mice (n = 5 animals per group). This analysis was insightful inasmuch as it revealed that Aa2br−/− colons specifically express decreased amounts of IL-10. Indeed, as shown in Figure 4A and 4B, a comparative profile of a number of colitis-associated Th1 and Th2 immunomodulators revealed significant changes only in IL-10. Indeed, while protein levels of IFNγ, IL-1, IL-2, IL-12, TNFα, KC, IL-5 and IL-4 were not changed significantly in Aa2br−/− relative to controls, IL-10 was repressed by as much as 77±10% compared to Aa2br+/+ animals (p<0.01), suggesting a relatively selective reduction in IL-10 expression in Aa2br−/− mice.
We next extended these findings to a cultured human epithelia model. Here we examined whether cultured human intestinal epithelial cells (Caco2) respond to adenosine through induction of IL-10. Intestinal epithelial cells (IEC) are know to express high levels of AA2BR, particularly on the apical plasma membrane (20). As shown in Figure 5, we exposed intestinal epithelial cells to the stable adenosine analog NECA and examined IL-10 protein levels. Apical, but not basolateral, NECA induced a concentration-dependent increase in IL-10 protein (p<0.025 by ANOVA, Figure 5A). Maximal levels of soluble IL-10 were 21.5±4.2 pg/ml with the addition of NECA (1μM). To define the relative contribution of AA2BR to IL-10 induction, we employed PSB1115 in combination with NECA. As shown in Figure 5B, PSB1115 (1μM) attenuated NECA-induced IL-10 expression by 63±7% (p<0.025), thereby strongly implicating epithelial AA2BR in adenosine-mediated IL-10 production.
Extracellular Ado signaling has been widely implicated as an endogenous protective mechanism for a number of inflammatory diseases (19). The studies presented here indicate that AA2BR expression inversely correlates with colitis severity. Indeed, elimination of functional AA2BR, either by genetic mutation in the Aa2br−/− mice, or pharmacologic inhibition with PSB1115, results in an exacerbation of the acute inflammatory phase of DSS colitis. Additional markers of inflammation, including colonic shortening and histologic examination of colonic mucosal PMN infiltration, also support the finding of a more severe acute inflammatory response in the Aa2br−/− mice, and therein implicate a protective role for Ado signaling in mucosal inflammation.
To date, the AA2BR has not been defined in detail with regard to inflammatory models. One study addressed the role of AA2AR in murine diabetes models (27). In particular, they showed that the NECA-induced suppression of immune-mediated hyperglycemia could be reversed with the AA2BR antagonist MRS1754, implicating(27) a specific role for AA2BR in immune suppression. Other studies have detailed the role of Ado receptor signaling in multiple experimental models of colitis resulting from a variety of methods of induction of inflammation. Using a T cell transfer model, Naganuma, et al demonstrated that AA2AR appear to block the production of proinflammatory cytokines and that AA2AR agonists attenuate T cell-mediated colitis (28). Additional work has revealed a protective role for AA2AR in various other colitic models (29, 30). One recent study identified over-expression of AA2BR in experimental colitis (31), and two additional reports, one by Guzman, et al. Grady987
Gusing a rat colitis model (32) and another by Mabley et al. (33) studying two independent mouse models of colitis, have indicated a protective role for the AA3R. Taken together, these findings suggest a more universal role for Ado receptor signaling in the resolution of colitis.
Our rationale for focusing on AA2BR in colitis was three-fold. First, intestinal epithelial cells provide a first line of defense during colitis and coordinate mucosal inflammation at a number of levels (1). Since human intestinal epithelial cells prominently express AA2BR (20), we reasoned that adenosine signaling at the level of the epithelium may contribute to disease severity. An initial screen of murine colonic scrapings (enriched by more that 70% in epithelial cells (34)) revealed that mouse colonic epithelia appear to express nearly equivalent levels of AA2AR and AA2BR, and essentially no detectable AA1R or AA3R. In this regard, Selmeczy et al. showed that the AA2AR agonist CGS 21680 fails to ameliorate murine DSS colitis, thereby placing the AA2BR as an interesting target in colitis (6). Second, AA2BR expression, specifically on the intestinal epithelium, is dynamically regulated by inflammatory conditions found at colitic lesion sites(31). Moreover, metabolic changes associated with colitis, including hypoxia, have been shown to induce epithelial and endothelial AA2BR (17). Mechanisms of such regulation occur through transcription-dependent pathways involving hypoxia-inducible factor-1(17), a transcription factor expressed in intestinal epithelia and previously shown to be protective for colitis (16). Third, the recent generation of Aa2br−/− mice has provided opportunities to define the contribution of AA2BR in various models of inflammation. A primary endpoint to Ado receptor activation, including AA2BR, has been barrier function. In vitro and in vivo studies dating back more than a decade have implicated Ado and AA2BR as a mechanism to promote epithelial restitution and tight junction re-sealing (10, 35), as well as promoting endothelial barrier function (9, 36). These studies revealed that Ado signals to the tight junction via phosphorylation of the actin-binding protein vasodilator- stimulated phosphoprotein (37, 38). More recent studies have clearly defined a protective role for AA2BR in hypoxia and inflammation-based disease models, including vascular leak syndromes (13). Thus, given the importance of barrier function to mucosal inflammation, we hypothesized a role for AA2BR in a murine model of colitis.
Initial studies suggested that intestinal epithelial cells express mRNA for both AA2AR and AA2BR. Based on these findings, we employed the DSS colitis model to define the contribution of these receptors to disease outcome. Studies with Aa2ar−/− and Aa2br−/− mice under similar conditions (2.5% DSS) revealed that Aa2br−/− mice are profoundly more susceptible to DSS-based colitis. It should be noted that these studies have not defined precise details regarding the contribution of the Aa2ar genotype to DSS colitis, but our studies also suggest that Aa2ar−/− also show increased susceptibility to DSS colitis. Indeed, conditions which would likely result in more severe disease in Aa2ar−/− mice (e.g. 4.5% DSS) revealed profound disease with high mortality in Aa2br−/− mice and increased susceptibility of Aa2ar−/− compared to Aa2ar+/+. While we do not know the exact basis of these differences between Aa2ar−/− and Aa2br−/−, it could be explained by the distribution of these receptors within the colon. For example, while both AA2AR and AA2BR appear to be expressed on leukocyte populations, epithelial cells express primarily AA2BR and this receptor is induced during colitis (39). Since DSS colitis, in particular, functions as an epithelial irritant, it is possible that this phenotype is explained more by the expression of AA2BR on the epithelium. Based on these initial findings, we further detailed DSS colitis in the Aa2br−/− genotype. Multiple levels of analysis, including body weight loss, disease activity index, histology and colon length revealed significantly more disease in Aa2br−/− mice. Importantly, this increased disease severity is not likely to be related to a developmental issue of the Aa2br−/− genotype. In particular, these results are, at least in part, recapitulated by the systemic administration of the AA2BR antagonist PSB1115 in wild-type mice. Such results implicate a specific role for AA2BR in DSS-based colitis.
Profiling of cytokine levels from colons derived from Aa2br+/+ and Aa2br−/− animals revealed that the loss of AA2BR is closely linked to specific reductions in the levels of IL-10, suggesting a relationship between AA2BR activity and downstream regulation of IL-10 expression. These findings were further supported by the demonstration that the pharmacologic inhibition of AA2BR activity also leads to down-regulation of IL-10 protein expression in human intestinal epithelia. Clear evidence supports a role for IL-10 in colonic inflammation (1). IL-10-deficient mice are well established murine models of IBD and are known to develop spontaneous chronic enterocolitis (40). Furthermore, IL-10 levels have been demonstrated to increase both in colonic tissue in experimental models of colitis (41) and in serum from human patients with active ulcerative colitis and in the recovery phase of both ulcerative colitis and Crohn's Disease (42). Here we demonstrate a role for AA2BR-mediated IL-10 in response to colonic inflammation in the setting of DSS colitis in vivo and in response to exogenous adenosine in vitro. These studies are in line with recent work ecto-5′-nucleotidase (CD73) in murine colitis (34), wherein animals genetically deficient in the terminal enzyme for extracellular adenosine production (CD73) showed increased susceptibility to hapten-based colitis. Similar to our findings here, these animals lacked the capacity to make normal levels of IL-10 in response to colitis. We do not currently know exact mechanisms of adenosine-mediated IL-10 induction. The AA2BR is a Gαs protein-coupled receptor tightly linked elevations in intracellular cyclic AMP (11). It is possible, therefore, that adenosine could directly activate the IL-10 promoter, particularly since IL-10 has a well-established regulatory response to the transcription factor cyclic AMP response element binding protein (43, 44). Alternatively, induction of IL-10 could occur through alternative pathways. For example, Nemeth, et. al. has demonstrated that adenosine augments macrophage IL-10 through an AA2BR-mediated posttranscriptional pathway involving the 3′-untranslated region of the IL-10 gene (45). Of note, in the current studies, we have not precisely localized the source of IL-10 in the colon. Epithelial isolations are sufficiently contaminated to disallow specific conclusions. We presume, based on our data obtained from human epithelial cells, that IL-10 is derived from colonic epithelia but that other sources exist. Indeed, while IL-10 can be made by most any nucleated cells (46), including intestinal epithelial cells (47), leukocyte subpopulations are a major source of IL-10 (46). It is possible, therefore, that IL-10 could be derived from more than one source in these studies.
In contrast to the findings of the present studies, a recent study suggests an anti-protective role of A2BAR signaling in murine colitis (39). Consistent with our studies, the authors found that the A2BAR is the predominant adenosine receptor expressed in the colonic epithelia. However, when exposing mice to DSS induced colitis, they found that mice with genetic deletion of the A2BAR are protected and mucosal inflammation is attenuated. Why the result from this study (39) is different from the present studies is not clearly understood. In contrast to the study by Kolachala et al. (39) several previous studies support an anti-inflammatory and tissue protective role of A2BAR signaling in different organ systems. For example, gene-targeted mice for the A2BAR show enhanced vascular inflammation when exposed to endotoxin (48) or during acute vascular injury (49). Similarly, gene-targeted deletion of the A2BAR is associated with enhanced vascular leakage and inflammation during ambient hypoxia (50, 51). In addition, studies of murine myocardial ischemia (21, 22), ischemic preconditioning of the kidneys (23) or during acute lung injury (24) provide convincing evidence for an anti-inflammatory and tissue-protective role of A2BAR signaling. Potential explanations why the studies of Kolachala et al. (39) found a detrimental role of A2BAR signaling during murine colitis could include details in the colitis protocol, differences in murine strains with genetic deletion of the A2BAR or differences of the ambience, including differences in bacterial flora of the mice. However, it seems important to point out that in some of their studies - and in contrast to the overall theme of this paper - gene-targeted for the A2BAR mice experienced a proinflammatory phenotype (39). For example, A2BAR−/− mice showed increased susceptibility to systemic Salmonella infection. In fact, 90% of A2BAR−/− mice died within 10 days compared with 20% of wild-type mice following orally administered S typhimurium. Consistent with the mortality data, A2BAR−/− mice also showed signs of weight loss earlier than WT mice (39). Some additional comparisions between the individual mouse strains may be necessary to rectify some of these apparent discrepancies.
Taken together, our findings strongly implicate AA2BR as an endogenous mechanism of protection in a murine model of colitis. These studies identify IL-10 as a specific endpoint for regulated colitic responses and highlight the essential role of the epithelium in these responses. Further studies will be necessary to determine the underlying mechanisms of this response and whether AA2BR might be an attractive therapeutic target in mucosal diseases such as IBD.
This work was supported by National Institutes of Health grants DK50189, DE016191, HL60569 and by a grant from the Crohn's and Colitis Foundation of America.
The authors declare no financial interests in any of the work submitted here.