Adenozin által okozott T limfocita diszfunkció szepszisben = The role of adenosine in septic T lymphocyte dysfunction

A projekt során in vitro Th1/Th2 polarizációs rendszer, Th1 és Th2 irányba differenciáltnak tekinthető hibridóma sejtek, illetve in vivo immunizálással nyert Th1 és Th2 sejtek felhasználásával, valamint A2A receptor specifikus agonista CGS21680, A2A specifikus antagonista ZM241385 és A2A KO állatok alkalmazásával kimutattuk, hogy az A2A receptorok aktivációjának erős gátló hatása van mind a Th1, mind a Th2 sejtekre a limfocita aktiváció korai és késői szakasza során. Eredményeink alapján a hatás hátterében a sejtek proliferációjára kifejtett gátlás és a citokin termelésre mRNS szinten kifejtett közvetlen gátló hatás áll. Emellett az adenozin fontos szerepet játszik a T limfociták számára antigént bemutató makrofágok működésének, különösen azok citokin termelésének szabályozásában. A szabályozás ebben az esetben is már transzkripciós szinten érvényesül. Végezetül kimutattuk, hogy az adenozin T limfocitákra gyakorolt hatása kiemelt jelentőséggel bírhat bizonyos autoimmun hátterű betegségekben, például az 1-es típusú diabéteszben, ahol a limfociták citokin termelésének gátlásán keresztül kivédheti a béta-sejtek pusztulását. Az adenozinnak ezért a hatásáért elsősorban az A2B receptorok lehetnek felelősek, de az A1 és A3 receptorok is hozzájárulhatnak. A további előrehaladás az adenozin immunoszuppresszív hatásainak megértésében segíthet a gyulladásos, autoimmun és neoplasztikus betegségeket célzó terápiás megközelítések kifejlesztésében. | During this project, we demonstrated that A2A receptor activation has strong inhibitory actions on both Th1 and Th2 cells during early and late stages of lymphocyte activation using in vitro Th1/Th2 polarizing system, hybridomas differentiated towards Th1 and Th2 direction, and Th1 and Th2 cells gained from in vivo immunized mice as well as A2A receptor specific agonist CGS21680, A2A specific antagonist ZM241385 and A2A KO mice. Based on our results the inhibition of cell proliferation and the direct inhibitory effect on cytokine production at mRNA level are in the background of these effects of adenosine. Besides, adenosine has an important role in the regulation of function, especially cytokine production, of macrophages presenting antigens to T lymphocytes. In this case, the regulation also prevails at transcriptional level. Finally, we demonstrated that the effect of adenosine on lymphocytes may have special importance in certain autoimmune diseases such as diabetes type 1, where adenosine may prevent beta cell death through the inhibition of cytokine production of lymphocytes. Principally A2B receptors may be responsible for this effect of adenosine, but A1 and A3 receptors may contribute to this. Further advances in understanding the immunosuppressive effects of adenosine may help develop therapeutic approaches to target inflammatory, autoimmune, and neoplastic disorders

Makrofág adenozin receptorok szepszisben = Macrophage adenosine receptors in sepsis

Csoportunk korábbi kísérletek során kimutatta, hogy az adenozin növeli a hőinaktivált Escherichia coli kezelés által indukált IL-10 termelést egér makrofág sejtekben és a Raw 264.7 egér makrofág sejtvonalban is. Munkánk során arra kerestük a választ, hogy mely toll-szerű receptorok (TLR) játszanak szerepet a folyamatban. Kísérleteink során egérből származó intraperitoneális (IP) makrofágokat illetve Raw 264.7 egér makrofág sejtvonalat kezeltünk különböző TLR agonistákkal adenozin jelenlétében illetve hiányában. Eredményeink alapján az IP makrofágoknál a HKLM illetve az LPS, míg a Raw 264.7 sejteknél a PAM3CSK4, az LPS, illetve az ODN1826 kezelés esetében detektáltuk a legnagyobb mennyiségű IL-10 felszabadulást adenozin jelenlétében. Ezek az adatok a TLR2, a TLR4, a TLR1/2, a TLR9 szerepét valószínűsítik. A továbbiakban az adenozin receptor stimulációnak T sejtek aktiváció által indukált sejthalálában (AICD) játszott szerepét vizsgáltuk. Eredményeink szerint az A2A receptor agonista CGS216820-val végzett kezelések csökkentették az AICD során kulcs szerepet játszó Fas receptor illetve liganduma (FasL) kifejeződését, illetve a caspase-3 és PARP apoptotikus fehérjék hasítását.

The Endocannabinoid System and Plant-Derived Cannabinoids in Diabetes and Diabetic Complications

Oxidative stress and inflammation play critical roles in the development of diabetes and its complications. Recent studies provided compelling evidence that the newly discovered lipid signaling system (ie, the endocannabinoid system) may significantly influence reactive oxygen species production, inflammation, and subsequent tissue injury, in addition to its well-known metabolic effects and functions. The modulation of the activity of this system holds tremendous therapeutic potential in a wide range of diseases, ranging from cancer, pain, neurodegenerative, and cardiovascular diseases to obesity and metabolic syndrome, diabetes, and diabetic complications. This review focuses on the role of the endocannabinoid system in primary diabetes and its effects on various diabetic complications, such as diabetic cardiovascular dysfunction, nephropathy, retinopathy, and neuropathy, particularly highlighting the mechanisms beyond the metabolic consequences of the activation of the endocannabinoid system. The therapeutic potential of targeting the endocannabinoid system and certain plant-derived cannabinoids, such as cannabidiol and Δ9-tetrahydrocannabivarin, which are devoid of psychotropic effects and possess potent anti-inflammatory and/or antioxidant properties, in diabetes and diabetic complications is also discussed

Intesztinális gyulladás ozmotikus regulációja = Osmotic regulation of intestinal inflammation

A projekt során in vitro rendszerben kimutattuk az aktin citoszkeleton fontos szerepét az NF-kappaB aktiváció és a gyulladásos események, például az interleukin-8 és a GRO-a kemokinek génexpressziójának és termelődésének, szabályozásában intesztinális epitéliális sejtekben. In vivo kísérletekben kimutattuk, hogy az intravénásan adott, Na/H-pumpa és epitélsejt Na-csatornákat gátló amiloriddal és a hipertóniás sóoldattal (HTS) történő kezelés a vérzéses sokkból (T/HS) történő újjáélesztés során külön-külön mérsékeli a bélkárosodást, míg kombinációjuk további javulást nem eredményez. Ezzel szemben az amilorid és a HTS mind egyedileg, mind kombinációban alkalmazva csökkentik a T/HS-indukált pulmonáris permeabilitást és neutrofil szekvesztrációt, és együttes alkalmazásuk hatásosabb, mint az egyedi. Az amiloriddal kezelt T/HS állatokból nyert sokk utáni nyirok előaktiváló hatása a neutrofilekre szignifikánsan kisebb, mint a hordozóval kezelt T/HS patkányokból vett nyiroké. Ugyanakkor a sokk utáni nyirokhoz hasonlóan a T/HS utáni plazma gyulladáskeltő hatása csökken, ha amiloriddal kezelt állatokból származik. A bélpermeabilitás funkcionális vizsgálata azt mutatta, hogy a vérzéses sokkon keresztülment patkányok kezelése amiloriddal a bélpermeabilitás szignifikáns csökkenéséhez vezet. Ezek a fontos megfigyelések a bél-indukált tüdőkárosodástól való védelem új mechanizmusára hívják fel a figyelmet. | During this project in vitro we demonstrated the important role of actin cytoskeleton in the regulation of NF-kappaB activation and inflammatory events, for example gene expression and production of interleukin-8 and GRO-a, in intestinal epithelial cells. In vivo we demonstrated that intravenous injection of amiloride, an inhibitor of Na/H-exchange and epithelial Na-channels, and resuscitation with hypertonic saline (HTS), respectively, ameliorate gut injury after trauma-hemorrhagic shock (T/HS), while their combination is not more effective. Contrary, amiloride and HTS both alone and in combination decrease pulmonary permeability and neutrophil sequestration, and they are more effective in combination than alone. The priming effect of post-shock lymph obtained from amiloride-treated T/HS animals was significantly less than that of lymph taken from vehicle treated T/HS animals. Similar to post-shock lymph, the proinflammatory effect of T/HS post-shock plasma was diminished when taken from amiloride-treated animals. The functional study of gut permeability demonstrated that amiloride treatment of rats undergoing T/HS led to a significant decrease in gut permeability when compared with vehicle-treated T/HS-induced animals. Altogether, these are important observations, which suggest a novel mechanism of protection from gut-induced lung injury

Adenosine Metabolized From Extracellular ATP Ameliorates Organ Injury by Triggering A2BR Signaling

BACKGROUND: Trauma and a subsequent hemorrhagic shock (T/HS) result in insufficient oxygen delivery to tissues and multiple organ failure. Extracellular adenosine, which is a product of the extracellular degradation of adenosine 5\u27 triphosphate (ATP) by the membrane-embedded enzymes CD39 and CD73, is organ protective, as it participates in signaling pathways, which promote cell survival and suppress inflammation through adenosine receptors including the A METHODS: T/HS shock was induced by blood withdrawal from the femoral artery in wild-type, global knockout (CD39, CD73, A RESULTS: T/HS upregulated the expression of CD39, CD73, and the A CONCLUSION: In conclusion, the CD39-CD73-

Adenosine metabolized from extracellular ATP ameliorates organ injury by triggering A2BR signaling

BACKGROUND: Trauma and a subsequent hemorrhagic shock (T/HS) result in insufficient oxygen delivery to tissues and multiple organ failure. Extracellular adenosine, which is a product of the extracellular degradation of adenosine 5' triphosphate (ATP) by the membrane-embedded enzymes CD39 and CD73, is organ protective, as it participates in signaling pathways, which promote cell survival and suppress inflammation through adenosine receptors including the A2BR. The aim of this study was to evaluate the role of CD39 and CD73 delivering adenosine to A2BRs in regulating the host's response to T/HS. METHODS: T/HS shock was induced by blood withdrawal from the femoral artery in wild-type, global knockout (CD39, CD73, A2BR) and conditional knockout (intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl) mice. At 3 three hours after resuscitation, blood and tissue samples were collected to analyze organ injury. RESULTS: T/HS upregulated the expression of CD39, CD73, and the A2BR in organs. ATP and adenosine levels increased after T/HS in bronchoalveolar lavage fluid. CD39, CD73, and A2BR mimics/agonists alleviated lung and liver injury. Antagonists or the CD39, CD73, and A2BR knockout (KO) exacerbated lung injury, inflammatory cytokines, and chemokines as well as macrophage and neutrophil infiltration and accumulation in the lung. Agonists reduced the levels of the liver enzymes aspartate transferase and alanine transaminase in the blood, whereas antagonist administration or CD39, CD73, and A2BR KO enhanced enzyme levels. In addition, intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl mice showed increased intestinal injury compared to their wild-type VillinCre controls. CONCLUSION: In conclusion, the CD39-CD73-A2BR axis protects against T/HS-induced multiple organ failure

Role of A2A adenosine receptors in regulation of opsonized E. coli-induced macrophage function

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

CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes

Aims Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX). Methods and results Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure-volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1+/+), and these effects were markedly attenuated in CB1 knockouts (CB1−/−). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT-PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX ± AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation. Conclusion CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular disease

Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

Adenosine A(2A) receptor (A(2A)R)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A(2A)R targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A(2A)R and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A(2A)R-interacting partners have in macrophages. Here, we aimed to identify A(2A)R-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 A(2A)R 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 A(2A)R-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 A(2A)R is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A(2A)R in macrophages. Conversely, we demonstrate that A(2A)R activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A(2A)R-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes