Balázs Koscsó

Crosstalk between Muscularis Macrophages and Enteric Neurons Regulates Gastrointestinal Motility

Intestinal peristalsis is a dynamic physiologic process influenced by dietary and microbial changes. It is tightly regulated by complex cellular interactions; however, our understanding of these controls is incomplete. A distinct population of macrophages is distributed in the intestinal muscularis externa. We demonstrate that, in the steady state, muscularis macrophages regulate peristaltic activity of the colon. They change the pattern of smooth muscle contractions by secreting bone morphogenetic protein 2 (BMP2), which activates BMP receptor (BMPR) expressed by enteric neurons. Enteric neurons, in turn, secrete colony stimulatory factor 1 (CSF1), a growth factor required for macrophage development. Finally, stimuli from microbial commensals regulate BMP2 expression by macrophages and CSF1 expression by enteric neurons. Our findings identify a plastic, microbiota-driven crosstalk between muscularis macrophages and enteric neurons that controls gastrointestinal motility. PAPERFLICK:

A2B Adenosine Receptors Protect against Sepsis-Induced Mortality by Dampening Excessive Inflammation

Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule, and its levels are elevated in sepsis. Adenosine signals through G-protein–coupled receptors and can regulate the host’s response to sepsis. In this study, we studied the role of A2B adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A2B receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A2B knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-κB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A2B receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog activation and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A2B receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A2B receptor-deficient mice. These results indicate that A2B receptor activation may offer a new therapeutic approach for the management of sepsis.

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host’s response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5′-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.

Adenosine Augments IL-10 Production by Microglial Cells through an A2B Adenosine Receptor-Mediated Process

Microglia are activated by pathogen-associated molecular patterns and produce proinflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR, and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. In this study, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of proinflammatory cytokines. Because the order of potency of selective AR agonists in inducing IL-10 production was NECA > IB-MECA > CCPA ≥ CGS21680, and the A2BAR antagonist MRS1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered short hairpin RNA blocked the enhancing effect of adenosine on IL-10 production, confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia.

Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death

Activation‐induced cell death (AICD) is initiated by T‐cell receptor (TCR) restimulation of already activated and expanded peripheral T cells and is mediated through Fas/Fas ligand (FasL) interactions. Adenosine is a purine nucleoside signaling molecule, and its immunomodulatory effects are mediated by 4 G‐protein‐coupled receptors: A1,A2A, A2B, and A3. In this study, we investigated the role of A2A receptors in regulating CD4+ T lymphocyte AICD. Our results showed that the selective A2A receptor agonist CGS21680 (EC50 = 15.2–32.6 nM) rescued mouse CD4+ hybridomas and human Jurkat cells from AICD and that this effect was reversed by the selective A2A receptor antagonist ZM241385 (EC50 = 2.3 nM). CGS21680 decreased phosphatidylserine exposure on the membrane, as well as the cleavage of caspase‐3, caspase‐8 and poly(ADP‐ribose) polymerase indicating that A2A receptor stimulation blocks the extrinsic apoptotic pathway. In addition, CGS21680 attenuated both Fas and FasL mRNA expression. This decrease in FasL expression was associated with decreased activation of the transcription factor systems NF‐κB, NF‐ATp, early growth response (Egr)‐1, and Egr‐3. The antiapoptotic effect of A2A receptor stimulation was mediated by protein kinase A. Together, these results demonstrate that A2A receptor activation suppresses the AICD of peripheral T cells.—Himer, L., Csóka, B., Selmeczy, Z., Koscsó, B., Pócza, T., Pacher, P., Németh, Z. H., Deitch, E. A., Vizi, E. S., Cronstein, B. N., Haskó, G. Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation‐induced cell death. FASEB J. 24, 2631–2640 (2010). www.fasebj.org

Adenosine augments IL‐10‐induced STAT3 signaling in M2c macrophages

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.

A2B adenosine receptors prevent insulin resistance by inhibiting adipose tissue inflammation via maintaining alternative macrophage activation

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 regulatory factor 4, and peroxisome proliferator–activated receptor-γ, was decreased in adipose 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.

A2B Adenosine Receptor Induces Protective Antihelminth Type 2 Immune Responses

The type 2 immune response evoked by intestinal nematode parasites contributes to worm expulsion and tolerance to associated tissue damage. We investigated whether this host response is affected by blocking signaling by the putative endogenous danger signal adenosine, which can be released during inflammation and host cell damage. Specific blockade of the A2B adenosine receptor (A2BAR) inhibited worm elimination and the development of innate and adaptive components of the type 2 primary and memory response. Infected mice lacking A2BAR exhibited decreased M2 macrophage and eosinophil recruitment and reduced IL-4 and IL-13 cytokine production. Additionally, shortly after infection, upregulation of the alarmin IL-33, which drives type 2 immunity, and activation of innate lymphoid type 2 (ILC2) cells was inhibited, while exogenous IL-33 restored ILC2 cell activation and type 2 cytokine expression. Thus, adenosine acts as a danger-associated molecular pattern (DAMP) that initiates helminth-induced type 2 immune responses through A2BAR.

Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing

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.—Csóka, B., Németh, Z. H., Törő, G., Idzko, M., Zech, A., Koscsó, B., Spolarics, Z., Antonioli, L., Cseri, K., Erdélyi, K., Pacher, P., Haskó, G. Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing. FASEB J. 29, 3626‐3637 (2015). www.fasebj.org

Investigational A3 adenosine receptor targeting agents

Introduction : Adenosine is an endogenous nucleoside that accumulates in the extracellular space in response to metabolic stress and cell damage. Extracellular adenosine is a signaling molecule that signals by activating four GPCRs: the A1, A2A, A2B and A3 receptors. Since the discovery of A3 adenosine receptors, accumulating evidence has identified these receptors as potential targets for therapeutic intervention. Areas covered : A3 adenosine receptors are expressed on the surface of most immune cell types, including neutrophils, macrophages, dendritic cells, lymphocytes and mast cells. A3 adenosine receptor activation on immune cells governs a broad array of immune cell functions, which include cytokine production, degranulation, chemotaxis, cytotoxicity, apoptosis and proliferation. In accordance with their multitudinous immunoregulatory actions, targeting A3 adenosine receptors has been shown to impact the course of a wide spectrum of immune-related diseases, such as asthma, rheumatoid arthritis, cancer, ischemia and inflammatory disorders. Expert opinion : Given the existence of both preclinical and early clinical data supporting the utility of A3 adenosine receptor ligands in treating immune-related diseases, further development of A3 adenosine receptor ligands is anticipated.