Search

The macrophage is a major phagocytic cell type, and its impaired function is a primary cause of immune paralysis, organ injury, and death in sepsis. An incomplete understanding of the endogenous molecules that regulate macrophage bactericidal activity is a major barrier for developing effective therapies for sepsis. Using an in vitro killing assay, we report here that the endogenous purine ATP augments the killing of sepsis-causing bacteria by macrophages through P2X4 receptors (P2X4Rs). Using newly developed transgenic mice expressing a bioluminescent ATP probe on the cell surface, we found that extracellular ATP levels increase during sepsis, indicating that ATP may contribute to bacterial killing in vivo. Studies with P2X4R-deficient mice subjected to sepsis confirm the role of extracellular ATP acting on P2X4Rs in killing bacteria and protecting against organ injury and death. Results with adoptive transfer of macrophages, myeloid-specific P2X4R-deficient mice, and P2rx4 tdTomato reporter mice indicate that macrophages are essential for the antibacterial, antiinflammatory, and organ protective effects of P2X4Rs in sepsis. Pharmacological targeting of P2X4Rs with the allosteric activator ivermectin protects against bacterial dissemination and mortality in sepsis. We propose that P2X4Rs represent a promising target for drug development to control bacterial growth in sepsis and other infections.

Group 2 innate lymphoid cells (ILC2s) represent a rapid source of type 2 cytokines, such as IL-5 and IL-13, and play an important role in orchestrating type 2 immune response. Adenosine is an endogenous purine nucleoside, a catabolite of ATP that binds and activates ≥1 of 4 transmembrane G protein-coupled cell-surface adenosine receptors (ARs)-A 1 , A 2A , A 2B , and A 3 . Here, we studied the role of ARs in the regulation of cytokine production by ILC2s. We found that A 2B ARs suppress the production of both IL-5 and IL-13 by ILC2s, whereas A 2A ARs augment IL-5 production and fail to affect IL-13 release. Combined stimulation of all ARs led to the suppression of both IL-5 and IL-13 production, which indicated that A 2B ARs dominate A 2A ARs. Both pre- and post-transcriptional processes may be involved in the AR modulation of ILC2 IL-5 and IL-13 production. Thus, we identify adenosine as a novel negative regulator of ILC2 activation.-Csóka, B., Németh, Z. H., Duerr, C. U., Fritz, J. H., Pacher, P., Haskó, G. Adenosine receptors differentially regulate type 2 cytokine production by IL-33-activated bone marrow cells, ILC2s, and macrophages.

Extracellular ATP binds to and signals through P2X7 receptors (P2X7Rs) to modulate immune function in both inflammasome-dependent and -independent manners. In this study, P2X7(-/-) mice, the pharmacological agonists ATP-magnesium salt (Mg-ATP; 100 mg/kg, EC50 ≈ 1.32 mM) and benzoylbenzoyl-ATP (Bz-ATP; 10 mg/kg, EC50 ≈ 285 μM), and antagonist oxidized ATP (oxi-ATP; 40 mg/kg, IC50 ≈ 100 μM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture-induced polymicrobial sepsis in mice. Our results with P2X7(-/-) bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloid-specific P2X7(-/-) mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 ≈ 0.25 μM) and pannexin channel inhibitor probenecid (10 mg/kg, IC50 ≈ 11.7 μM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial burden. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the treatment of sepsis., (© FASEB.)

The alternatively activated macrophage phenotype induced by IL-10 is called M2c. Adenosine is an endogenous purine nucleoside that accumulates in the extracellular space in response to metabolic disturbances, hypoxia, inflammation, physical damage, or apoptosis. As adenosine is known to regulate classically activated M1 and IL4- and IL-13-activated M2a macrophages, the goal of the present study was to explore its effects on M2c macrophages. We found that adenosine augmented the IL-10-induced expression of TIMP-1 and arginase-1 by the mouse macrophage cell line RAW 264.7 and by mouse BMDMs. The effects of AR stimulation on IL-10-induced TIMP-1 or arginase-1 expression were lacking in A2BAR KO macrophages. The role of A2BAR on TIMP-1 production of RAW 264.7 cells was confirmed with specific agonist BAY606583 and antagonist PSB0788. AR stimulation augmented IL-10-induced STAT3 phosphorylation in macrophages, and pharmacological inhibition or silencing of STAT3 using siRNA reduced the stimulatory effect of AR stimulation on TIMP-1 production. In contrast to its stimulatory effect on IL-10-induced STAT3 activation, adenosine inhibited IL-6-induced STAT3 phosphorylation and SAA3 expression. In conclusion, adenosine enhances IL-10-induced STAT3 signaling and M2c macrophage activation.

Adenosine has been implicated in suppressing the proinflammatory responses of classically activated macrophages induced by Th1 cytokines. Alternative macrophage activation is induced by the Th2 cytokines interleukin (IL)-4 and IL-13; however, the role of adenosine in governing alternative macrophage activation is unknown. We show here that adenosine treatment of IL-4- or IL-13-activated macrophages augments the expression of alternative macrophage markers arginase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and macrophage galactose-type C-type lectin-1. The stimulatory effect of adenosine required primarily A(2B) receptors because the nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased both arginase activity (EC(50)=261.8 nM) and TIMP-1 production (EC(50)=80.67 nM), and both pharmacologic and genetic blockade of A(2B) receptors prevented the effect of NECA. A(2A) receptors also contributed to the adenosine augmentation of IL-4-induced TIMP-1 release, as both adenosine and NECA were less efficacious in augmenting TIMP-1 release by A(2A) receptor-deficient than control macrophages. Of the transcription factors known to drive alternative macrophage activation, CCAAT-enhancer-binding protein β was required, while cAMP response element-binding protein and signal transducer and activator of transcription 6 were dispensable in mediating the effect of adenosine. We propose that adenosine receptor activation suppresses inflammation and promotes tissue restitution, in part, by promoting alternative macrophage activation.

We recently showed that A(2A) adenosine receptor activation by endogenous adenosine contributes to interleukin-10 (IL-10) production in polymicrobial sepsis. Here we investigated the molecular mechanisms underpinning this interaction between adenosine receptor signaling and infection by exposing macrophages to Escherichia coli. We demonstrated using receptor knockout mice that A(2A) receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production by E coli-challenged macrophages, whereas A(2B) receptors have a minor role. The stimulatory effect of adenosine on E coli-induced IL-10 production did not require toll-like receptor 4 (TLR4) or MyD88, but was blocked by p38 inhibition. Using shRNA we demonstrated that TRAF6 impairs the potentiating effect of adenosine. Measuring IL-10 mRNA abundance and transfection with an IL-10 promoter-luciferase construct indicated that E coli and adenosine synergistically activate IL-10 transcription. Sequential deletion analysis and site-directed mutagenesis of the IL-10 promoter revealed that a region harboring C/EBP binding elements was responsible for the stimulatory effect of adenosine on E coli-induced IL-10 promoter activity. Adenosine augmented E coli-induced nuclear accumulation and DNA binding of C/EBPbeta. C/EBPbeta-deficient macrophages failed to produce IL-10 in response to adenosine and E coli. Our results suggest that the A(2A) receptor-C/EBPbeta axis is critical for IL-10 production after bacterial infection.

Adenosine receptor ligands have anti-inflammatory effects and modulate immune responses by up-regulating IL-10 production by immunostimulated macrophages. The adenosine receptor family comprises G protein-coupled heptahelical transmembrane receptors classified into four types: A1, A2A, A2B, and A3. Our understanding of the signaling mechanisms leading to enhanced IL-10 production following adenosine receptor occupancy on macrophages is limited. In this study, we demonstrate that adenosine receptor occupancy increases IL-10 production by LPS-stimulated macrophages without affecting IL-10 promoter activity and IL-10 mRNA levels, indicating a posttranscriptional mechanism. Transfection experiments with reporter constructs containing sequences corresponding to the AU-rich 3'-untranslated region (UTR) of IL-10 mRNA confirmed that adenosine receptor activation acts by relieving the translational repressive effect of the IL-10 3'-UTR. By contrast, adenosine receptor activation failed to liberate the translational arrest conferred by the 3'-UTR of TNF-alpha mRNA. The IL-10 3'-UTR formed specific complexes with proteins present in cytoplasmic extracts of RAW 264.7 cells. Adenosine enhanced binding of proteins to a region of the IL-10 3'-UTR containing the GUAUUUAUU nonamer. The stimulatory effect of adenosine on IL-10 production was mediated through the A(2B) receptor, because the order of potency of selective agonists was 5'-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) = 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680). Also, the selective A2B antagonist, alloxazine, prevented the effect of adenosine. Collectively, these studies identify a novel pathway in which activation of a G protein-coupled receptor augments translation of an anti-inflammatory gene.

Adenosine is an endogenously released autocoid that has potent receptor-mediated modulatory effects on macrophage function. The intracellular pathways mediating these effects are incompletely understood. Since adenosine receptor occupancy has been associated with activation of the cAMP-PKA system as well as of p38 MAPK and p42/44 MAPK, all of which can activate the CREB transcription factor system, we hypothesized that adenosine would activate CREB in macrophages. Using RAW 264.7 macrophages, we found that extracellular adenosine enhanced CREB transcriptional activity and increased phosphorylation of nuclear CREB. On the other hand, adenosine failed to alter CREB DNA binding. Adenosine stimulated both p38 and p42/44 MAPK activation. The p38 MAPK pathway inhibitor SB203580 but not the p42/44 MAPK pathway blocker PD98059 decreased adenosine-induced CREB activation, indicating that p38 MAPK but not p42/44 MAPK is an upstream mediator of CREB activation. Thus, some of the immunomodulatory effects of adenosine in macrophages may be explained by its augmenting effect on CREB activation.

Adenosine is released into the extracellular space from nerve terminals and cells subjected to ischemic stress. This nucleoside modulates a plethora of cellular functions via occupancy of specific receptors. Adenosine is also an important endogenous regulator of macrophage function, because it suppresses the production of a number of proinflammatory cytokines by these cells. However, the mechanisms of this anti-inflammatory effect have not been well characterized. We hypothesized that adenosine may exert some of its anti-inflammatory effects by decreasing activation of the transcription factor nuclear factor-kappaB (NF-kappaB), because gene expression of most of the proinflammatory cytokines inhibited by adenosine is dependent on NF-kappaB activation. Using bacterial lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, we found that adenosine as well as adenosine receptor agonists decreased the production of tumor necrosis factor (TNF)-alpha, a typical NF-kappaB-regulated cytokine. This effect of adenosine was not due to an action on the process of TNF-alpha release, because adenosine suppressed also the intracellular levels of TNF-alpha. However, cDNA microarray analysis revealed that mRNA levels of neither TNF-alpha nor other cytokines were altered by adenosine in either LPS-activated or quiescent macrophages. In addition, although LPS induced expression of a number of other, noncytokine genes, including the adenosine A2b receptor, adenosine did not affect the expression of these genes. Furthermore, adenosine as well as adenosine receptor agonists failed to decrease LPS-induced NF-kappaB DNA binding, NF-kappaB promoter activity, p65 nuclear translocation, and inhibitory kappaB degradation. Together, our results suggest that the anti-inflammatory effects of adenosine are independent of NF-kappaB.

In this study, we examined the effect of dopamine on the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated J774.1 macrophages and mouse peritoneal macrophages. Treatment of J774.1 cells with dopamine (0.01-100 microM) decreased the release of IL-12 p40, in a concentration-dependent manner. The attenuating effect of dopamine on IL-12 p40 production appeared to be pretranslational, because dopamine decreased mRNA accumulation of IL-12 p40. The inhibitory effect of dopamine on IL-12 p40 production by J774.1 macrophages was not mediated by dopamine receptors, because dopamine receptor antagonists were unable to reverse the dopamine-induced suppression of IL-12 p40 production. Since the beta-adrenoceptor antagonist propranolol completely prevented the inhibitory effect of dopamine on IL-12 p40 production, the suppressive effect of dopamine on IL-12 p40 production by J774.1 cells is mediated by beta-adrenoceptors. In contrast to J774.1 cells, propranolol only partially reversed the inhibitory effect of dopamine on IL-12 production by peritoneal macrophages. Furthermore, dopamine stimulated the production of the anti-inflammatory cytokine IL-10 in both J774.1 cells and peritoneal macrophages. While the stimulatory effect of dopamine on IL-10 production by J774.1 cells was beta-adrenoceptor-mediated, dopamine increased IL-10 production by peritoneal macrophages via both beta-adrenoceptor-dependent and independent mechanisms. These results indicate that dopamine has multiple anti-inflammatory effects mediated by both beta-adrenoceptor dependent and independent mechanisms.

Obesity causes increased classical and decreased alternative macrophage activation, which in turn cause insulin resistance in target organs. Because A2B adenosine receptors (ARs) are important regulators of macrophage activation, we examined the role of A2B ARs in adipose tissue inflammation and insulin resistance. A2B AR deletion impaired glucose and lipid metabolism in mice fed chow but not a high-fat diet, which was paralleled by dysregulation of the adipokine system, and increased classical macrophage activation and inhibited alternative macrophage activation. The expression of alternative macrophage activation--specific transcriptions factors, including CCAAT/enhancer-binding protein-β, interferon b regulatory factor 4, and peroxisome proliferator--activated receptor-γ, was decreased in adipose g tissue of A2B AR-deficient mice. Furthermore, in in vitro studies, we found that stimulation of A2B ARs suppressed free fatty acid-induced deleterious inflammatory and metabolic activation of macrophages. Moreover, AR activation upregulated the interleukin-4--induced expression of CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator--activated receptor-γ in macrophages. Altogether, our results indicate that therapeutic strategies targeting A2B ARs hold promise for preventing adipose tissue inflammation and insulin resistance. [ABSTRACT FROM AUTHOR]

Adenosine is a biologically active molecule that is formed at sites of metabolic stress associated with trauma and inflammation, and its systemic level reaches high concentrations in sepsis. We have recently shown that inactivation of A2A adenosine receptors decreases bacterial burden as well as IL-10, IL-6, and MIP-2 production in mice that were made septic by cecal ligation and puncture (CLP). Macrophages are important in both elimination of pathogens and cytokine production in sepsis. Therefore, in the present study, we questioned whether macrophages are responsible for the decreased bacterial load and cytokine production in A2A receptor-inactivated septic mice. We showed that A2A KO and WT peritoneal macrophages obtained from septic animals were equally effective in phagocytosing opsonized E. coli. IL-10 production induced by opsonized E. coli was decreased in macrophages obtained from septic A2A KO mice as compared to WT counterparts. In contrast, the release of IL-6 and MIP-2 induced by opsonized E. coli was higher in septic A2A KO macrophages than WT macrophages. These results suggest that peritoneal macrophages are not responsible for the decreased bacterial load and diminished MIP-2 and IL-6 production that are observed in septic A2A KO mice. In contrast, peritoneal macrophages may contribute to the suppressive effect of A2A receptor inactivation on IL-10 production during sepsis. [ABSTRACT FROM AUTHOR]

Adenosine A2A receptor (A2AR)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2ARinteracting partners have in macrophages. Here, we aimed to identify A2AR-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A2AR as the "bait" and a macrophage expression library as the "prey." We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A2AR-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPM-) cells. We also demonstrated that the A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate that A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes. [ABSTRACT FROM AUTHOR]