Antiinflamacijska i imunomodulacijska svojstva sitagliptina, inhibitora dipeptidil peptidaze-4, u modelima interakcije humanih antigen-prezentujućih ćelija i T limfocita in vitro/

Abstract

Dipeptidil peptidaza 4 (DPP4), takođe poznata i kao CD26, je transmembranski glikoprotein tipa II, molekulske mase 110 kD. DPP4/CD26 je široko eksprimiran u različitim organima (bubrezi, moždano tkivo, pluća, jetra, gastrointestinalni trakt, kostna srž), kao i na površini različitih tipova ćelija kao što su endotelne, epitelne, stromalne, matične ćelije, kao i ćelije imunskog sistema (limfociti, monociti, NK ćelije). Danas je u upotrebi, relativno nova, grupa oralnih antidijabetičnih lekova, koji predstavljaju selektivne DPP4 inhibitore (DPP4i), često jednim imenom nazvani gliptini. Usled široke ekspresije DPP4/CD26 molekula, poslednjih godina veliki broj istraživanja ukazuje na druge efekte DPP4 inhibitora, nezavisno od njihovog hipoglikemijskog delovanja. Postojeća saznanja, dobijena u kliničkim studijama, in vitro istraživanjima i in vivo eksperimentima na životinjama, sugerišu i potencijalni antiinflamacijski i imunomodulacijski efekat ove grupe lekova, međutim tačani mehanizmi koji se nalaze u osnovi ovih procesa nisu dovoljno istraženi. Ciljevi istraživanja ovog projekta su bili da se ispita citotoksičnost sitagliptina na modelima kultura humanih mononuklearnih ćelija periferne krvi (PBMC) i dendritiskih ćelija diferentovanih iz monocita (MoDC fenotipske i funkcionalne karakteristike MoDC diferentovanih u prisustvu sitagliptina, kao i ekspresija signalnih molekula p65 nuklearnog faktora kapa B (NF-kappa-B) i p38 mitogenom aktiviranih protein (MAP) kinaza u MoDCs tretiranim sitagliptinom. U istaživanju su korištene MoDCs dobrovoljnih davalaca, korištenjem standardnog protokola (kultivacija ćelija sa granulocitno-monocitnim faktorom rasta u kombinaciji sa interleukinom-4 - GM-CSF/IL-4), tokom 4 dana. Tretman sa lipoplisasaridom (LPS) i interferonom-γ (IFN-γ), tokom 24h, je korišten za indukciju sazrevanja MoDCs. Citotoksičnost sitagliptina je procenjivana pomoću testova apoptoze, nekroze i metaboličke aktivnosti (MTT). Fenotipske i funkcionalne karakteristike MoDC ispitivane su metodom protočne citometrije, testom alogene mešane leukocitne reakcije i analizom devet različitih citokina. Western blot metoda je korištena za procenu aktivacije signalnih molekula. Ćelijske kulture su tretirane sa različitim necitotoksičnim koncentracijama sitagliptina na osnovu rezultata preliminarnih eksperimenata (najviše 500μg/mL) na početku diferencijacije (sitagliptin 0d protokol) ili nakon diferencijacije MoDC (sitagliptin 4d protokol). Sitagliptin je inhibirao diferencijaciju i sazrevanje MoDC, što je procenjeno na osnovu snižene ekspresije markera CD40, CD83, CD86, NLRP3, HLA-DR, nepromenjene ekspresije CD14 i inhibicije produkcije IL-1β, IL-12p70, IL-27, IL-23. Nasuprot tome sitagliptin je doveo do povećanja ekspresije CD26, tolerogenih markera (ILT4, IDO) i povećanje produkcije imunoregulatornih citokina (IL-10 i TGF-β). Svi rezultati su bili izraženiji kada je sitagliptin dodat na početku diferencijacije (sitagliptin 0d protokol). MoDC tretirane sitagliptinom su pokazale slabiji kapacitet alostimulacije T ćelija u MoDC/T ćelijskim ko-kulturama. Primena sitagliptina je dovela do inhibicije Th1 i Th17 odgovora, a stimulacije Th2 i T regulatornih (Treg) ćelija. Tolerogena svojstva MoDC tretiranih sitagliptinom dodatno su potvrđena povećanjem udela CD4+CD25+CD127- FoxP3+ Treg i Tr1 ćelija (CD4+IL-10+FoxP3-) u MoDC/T ćelijskim ko-kulturama. Diferencijacija IL-10+ i TGF-β+ Tregs, zavisila je od korištenog protokola sitagliptina. Western blot analiza je pokazala da sitagliptin inhibira ekspresiju p65NF-kB i p38MAPK, tokom sazrevanja MoDC. Dobijeni rezultati ukazuju da sitagliptin indukuje diferencijaciju tolerogenih DC, što doprinosi boljem razumevanju imunomodulacijskog delovanja DPP-4i i otvara mogućnosti za dalja istraživanja potencijalne primene ove grupe lekova u terapiji autoimunskih bolesti ili hroničnih inflamacijskih bolesti.

DPP-4, also known as CD26, is a type II transmembrane glycoprotein with a molecular weight of 110 kD. DPP-4/CD26 is expressed in various organs (kidney, brain tissue, lung, liver, gastrointestinal tract, bone marrow), as well as on the surfaces of many cell types such as endothelial, epithelial, stromal, stem cells, and immune system cells. (lymphocytes, monocytes, and NK cells). Selective DPP-4 inhibitors (DPP-4i), often known as gliptins, are a relatively new oral antidiabetic agents. Due to the wide expression of the DPP-4/CD26 molecule, a great number of studies, in recent years, have found that DPP-4 inhibitors have additional effects which occur independently of their hypoglycemic action. Existing evidence, obtained in clinical and experimental studies, suggest a potential anti-inflammatory and immunomodulatory effect of this group of drugs, however, the exact mechanisms underlying these processes have not been sufficiently investigated. The objectives of this project were to investigate: the cytotoxicity of sitagliptin on a model of peripheral blood mononuclear cells (PBMC) and dendritic cells differentiated from peripheral blood monocytes (MoDC); the phenotypic and functional characteristics of MoDC differentiated in the presence of sitagliptin; and to investigate the expression of signaling molecules p65 NF-kappa-B and p38 MAP kinase in sitagliptin-treated MoDCs. MoDCs were generated for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. The cytotoxicity of sitagliptin was determined using apoptosis/necrosis and MTT assays. The morphological and functional properties of MoDC were investigated using flow cytometry, the allogeneic mixed leukocyte reaction test, and cytokine analysis. Western blot analysis has been used to assess the activation of signaling molecules. Sitagliptin was added at the highest non-cytotoxic concentration (500 μg/mL) either at the beginning (sita 0d protocol) or after MoDCs differentiation (sita 4d protocol). Sitagliptin suppressed differentiation and maturation of MoDCs, by lowering expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retaining CD14 expression, and inhibiting production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, CD26 expression, tolerogenic DC markers (ILT4 and IDO1), and immunoregulatory cytokine production (IL-10 and TGF-β) were elevated. Overall, the sitagliptin 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture, and reduced Th1 and Th17 responses, but promoted Th2 and Treg responses. Tolerogenic features of sitagliptin-treated MoDCs were further validated by an increase in the frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell coculture. IL-10+ and TGF-β+ Tregs were differentiated depending on the used sitagliptin protocol. Western blot study demonstrated that sitagliptin suppressed p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. The obtained results indicate that sitagliptin induces differentiation of tolerogenic DCs, which advances our knowledge of the immunomodulatory action of DPP-4i and provides new possibilities for investigation towards the potential use of this class of medications in the management of chronic inflammation or autoimmune diseases.