Uticaj selektivnog agoniste A3 receptora adenozina, piklidenozona, na fenotipske i funkcionalne karakteristike humanih ćelija periferne krvi, in vitro/

Abstract

A3 receptor je široko eksprimiran u različitim tkivima, kao i na površini imunskih ćelija (neutrofila, monocita, eozinofila, makrofaga, dendritskih ćelija). Humani A3 je G proteinski receptor sa C terminalnim regionom okrenutim intraćelijski, i 7 transmembranskih regiona, sa serinskim i treoninskim ostacima kao mjestima fosforilacije. S obzirom na ekspresiju receptora u fiziološkim i patološkim uslovima, istraženi su različiti agonisti, antagonisti i modulatori A3 receptora. U poslednje dvije decenije, A3 adenozinski receptor biva prepoznat kao potencijalna terapijska meta u borbi protiv inflamacije i tumora. Osim kao terapijski cilj, A3 receptor biva prepoznat kao biološki marker bolesti, zbog prekomjerne ekspresije u ćelijama imunskog sistema i tumorskim ćelijama, u odnosu na zdrave ćelije. Piklidenozon (CF101, IB MECA), selektivni agonist A3 receptora, se nalazi u kliničkim ispitivanjima za liječenje psorijaze. Iako podaci iz in vitro i in vivo studija sugerišu da piklidenozon posjeduje antiinflamacijska i imunomodulacijska svojstva, tačni mehanizmi u osnovi ovog procesa su još uvijek nepoznati. Ciljevi istaživanja ove doktorske disertacije su bili da se ispita citotoksičnost piklidenozona na modelima neutrofilnih granulocita i mononuklearnih ćelija periferne krvi (PBMCs, engl. peripheral blood mononuclear cell) zdravih davaoca, efekat necitotoksičnih doza lijeka na funkcionalne i fenotipske karakteristike neutrofilnih granulocita, kao i efekat lijeka na proliferaciju i produkciju proinflamacijiskih i Th polarišućih citokina od strane PBMCs. U istraživanju su korišteni neutrofilni granulociti i PBMCs zdravih dobrovoljnih davalaca. Citotoksičnost piklidenozona i njegov uticaj na proliferaciju PBMCs određeni su metodom protočne citometrije i MTT testom (od engl. 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolijum bromid test).. Produkcija slobodnih kiseoničkih vrsta (ROS, od engl. reactive oxygen species) od strane neutrofila analizirana je metodom hemiluminiscencije pomoću luminola nakon stimulacije sa fMLP (od engl. N-formylmethionyl-leucyl-phenylalanine) i PMA (od engl. phorbol 12-myristate 13-acetate), dok je intenzitet NEToze (od engl. neutrophil extracellular trap formation) procijenjen metodom Sytox Green fluorescencije na fluorometru kod PMA- i CAI- (od engl. calcium ionophore) stimulisanih neutrofila. Dodatno, u cilju ispitivanja mogućih mehanizama djelovanja piklidenozona, sprovedeni su posebni eksperimenti sa pretretmanom antagonistom adenozinskog A3 receptora, MRS1220, u esejima koji su se odnosili na produkciju ROS-a i intenzitet NEToze. Fenotipske karakteristike neutrofila određene su protočnom citofluorometrijom nakon stimulacije fMLP i fMLP + TNF-α (od engl. tumor necrosis factor alpha). ELISA metodom (od engl. enzyme-linked immunosorbent assay) analizirana je produkcija citokina i enzima azurofilnih granula. Takođe, piklidenozon je smanjio produkciju neutrofilne elastaze (NE, od engl. neutrophil elastase) i mijeloperoksidaze (MPO, od engl. myeloperoxidase) kod opZY- (od engl. opsonized zymosan), fMLP- i TNF-α–stimulisanih neutrofila, kao i produkciju IL-8 (od engl. interleukin 8) kod opZY- i fMLP-stimulisanih neutrofila. Piklidenozon je u svim primijenjenim dozama (250, 500 i 1000 nM) inhibirao produkciju reaktivnih kiseoničkih vrsta kod fMLP- i PMA-stimulisanih neutrofila, dok je MRS1220 (100 nM), antagonist A3 receptora, poništio ovaj efekat kod fMLP-stimulisanih neutrofila. U svim primijenjenim dozama, lijek je smanjio intenzitet NEToze kod PMA- i CAI-stimulisanih neutrofila, a ovaj efekat je neutralisan pretretmanom sa MRS1220 (100 nM). Takođe, piklidenozon je smanjio produkciju NE i mijeloperoksidaze MPO kod opsonizovanim zimozanom (opZY, od engl. opsonized zymosan), fMLP- i TNF-α–stimulisanih neutrofila, kao i produkciju IL-8 kod opZY- i fMLP-stimulisanih neutrofila. Piklidenozon je modulirao fenotipske karakteristike neutrofilnih granulocita, što je pokazano smanjenom ekspresijom CD11b, CD18, CD66b, CD32, CD89 i CD16 kod fMLP/TNF-α–stimulisanih neutrofila, kao i povećanom ekspresijom CD181 i CD182 kod fMLP-stimulisanih neutrofila. Osim toga, lijek je inhibirao proliferaciju PBMCs i smanjio produkciju proinflamacijskih citokina TNF-α, IL-23 i IL-36. U najvećoj primijenjenoj dozi (1000 nM) suprimirao je produkciju IFN-γ i IL-5, citokina koje uglavnom produkuju Th1 i Th2 limfociti, dok je u svim primijenjenim dozama smanjio produkciju IL-17, karakterističnog za Th17 ćelije. Ovi rezultati sugerišu da piklidenozon modulira funkcionalne i fenotipske karakteristike ćelija periferne krvi in vitro. Dobijeni podaci doprinose boljem razumijevanju imunomodulacijskog djelovanja agonista adenozinskog A3 receptora , piklidenozona, na humane neutrofilne granulocite i mononuklearne ćelije periferne krvi. Nadalje, rezultati ovog istraživanja mogu poslužiti kao osnova za buduća istraživanja usmjerena na ispitivanje potencijalnog proširenja indikacija za primjenu ovog lijeka u terapiji.

The A3 adenosine receptor is highly expressed in various tissues and on the surface of immune cells, including neutrophils, monocytes, eosinophils, macrophages, and dendritic cells. It is a G protein-coupled receptor (GPCR) with a C-terminal region facing intracellularly and seven transmembrane domains, where serine and threonine residues serve as phosphorylation sites. Given its involvement in physiological and pathological processes, numerous A3 receptor agonists, antagonists, and modulators have been investigated. Over the past two decades, the A3 receptor has been recognized as a potential therapeutic target in inflammation and cancer. Additionally, its overexpression in immune and tumor cells compared to healthy cells highlights its potential as a biological marker of disease. Piclidenoson (CF101, IB-MECA), a selective A3 receptor agonist, is currently undergoing clinical trials for the treatment of psoriasis. Although in vitro and in vivo studies suggest its anti-inflammatory and immunomodulatory properties, the precise mechanisms underlying these effects remain unclear. This doctoral dissertation aimed to examine the cytotoxicity of piclidenoson on neutrophil granulocytes and peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, as well as its effects on the functional and phenotypic characteristics of neutrophils, PBMC proliferation, and the production of pro-inflammatory and Th cytokines. Neutrophil granulocytes and peripheral blood mononuclear cells (PBMCs) from healthy voluntary donors were used in the research. The cytotoxicity of piclidenoson and its effect on PBMC proliferation were determined using flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). The production of reactive oxygen species (ROS) was assessed using luminol-enhanced chemiluminescence in neutrophils stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA), while the intensity of neutrophil extracellular trap formation (NETosis) was measured by the Sytox Green fluorescence method in neutrophils stimulated with PMA and calcium ionophore (CAI). To further investigate the potential mechanisms of action of piclidenoson, additional experiments were conducted using pretreatment with the adenosine A3 receptor antagonist MRS1220, specifically in assays evaluating ROS production and the extent of NETosis. Phenotypic characteristics of neutrophils were analyzed by flow cytometry in cells stimulated with fMLP alone or in combination with tumor necrosis factor-alpha (TNFα). Cytokine and azurophilic granule enzyme production were quantified using the enzyme-linked immunosorbent assay (ELISA). Piclidenoson inhibited ROS production at all tested concentrations (250, 500, and 1000 nM) in fMLP- and PMA-stimulated neutrophils, while the A3 receptor antagonist MRS 1220 (100 nM) reversed this inhibitory effect in fMLP-stimulated neutrophils. At all tested concentrations, piclidenoson also reduced the intensity of NETosis in PMA- and CAI-stimulated neutrophils, an effect that was abolished by pretreatment with MRS 1220. Furthermore, it decreased the production of neutrophil elastase (NE) and myeloperoxidase (MPO) in neutrophils stimulated with opsonized zymosan (opZY), fMLP, and TNFα, as well as IL-8 production in opZY- and fMLP-stimulated neutrophils. The drug modulated the phenotypic characteristics of neutrophil granulocytes by reducing the expression of CD11b, CD18, CD66b, CD32, CD89, and CD16 in fMLP/TNFα-stimulated neutrophils, while increasing the expression of CD181 and CD182 in fMLP-stimulated neutrophils. In PBMC cultures, piclidenoson inhibited proliferation and reduced the production of pro-inflammatory cytokines TNFα, IL-23, and IL-36. At the highest tested concentration (1000 nM), it suppressed the production of IL-5 and IFN-γ, cytokines characteristic of the Th1 and Th2 profiles, respectively, while at all tested concentrations, it reduced IL-17 production, which is associated with Th17 cells. These findings suggest that piclidenoson modulates both the functional and phenotypic characteristics of human peripheral blood cells in vitro. The results contribute to the existing knowledge on the immunomodulatory effects of A3 adenosine receptor agonists, particularly piclidenoson, on neutrophils and PBMCs. The observed in vitro effects provide a basis for future research that could explore the potential for expanding the therapeutic indications of this drug.