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.

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

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

Genetic and phylogenetic analysis of avian extraintestinal and intestinal Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates of animals and man are known to carry specific virulence associated genes. The intestinal tract, it is primarily colonized by various strains of commensal E. coli but it may include ExPEC as well. Here we aimed to assess possible genetic and evolutionary linkages between extraintestinal pathogenic and intestinal (commensal) E. coli of poultry. For that purpose we analysed 71 ExPEC isolates, and 40 intestinal isolates assumed to be commensal E. coli (IntEC), from dead chickens and turkey poults for 26 virulence related genes. Although the two groups shared several virulence determinants the genes pic, papC, and cdtIV were exclusively present in ExPEC and further five genes (colV, iss, kpsM, tsh and iutA), were significantly more frequent among ExPEC. Phylogenetic backgrounds of ExPEC and of IntEC isolates indicated significant differences. A 40% of ExPEC belonged to phylogroup A primarily containing strains of serogroup O78. Phylogroup D contained ExPEC strains of serogroups O53 (2 strains) and O115 (5 strains) characterized by the cdt-IV genes, suggesting the existence of new clones of avian ExPEC in phylogenetic group D. On the other hand, a 42.5% of IntEC belonged to phylogroup B1 with diverse serogroups. Our data provide insight into the clonal evolution of avian ExPEC especially in phylogenetic groups A and D, resulting avian ExPEC with similarities to human ExPEC