The influence of CD40 ligation and interferon-γ on functional properties of human monocyte-derived dendritic cells activated with polyinosinic-polycytidylic acid

Abstract

Background/Aim. Ligation of a Toll-like receptor (TLR) by specific TLR agonists is a powerful tool for maturation induction of monocyte-derived dendritic cells (MoDCs). Studies so far have shown that the treatment of dendritic cells (DCs) with a TLR3 ligand, polyinosinic-polycytidylic acid [Poly(I:C)], may be an appropriate activation agent for obtaining mature MoDCs, competent to prime effective immune responses. However, little is known about how subsequent interaction of MoDCs with T cell-derived stimuli, such as CD40 or interferon-? (IFN-?), modulates MoDC functions. Therefore, this problem was the main objective of this study. Methods. Immature MoDCs were prepared by cultivation of monocytes from peripheral blood mononuclear cells with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 5 days. After that, maturation was induced by the treatment of these cells with Poly(I:C) for 2 days. At day 6, immature MoDCs and Poly(I:C)-activated MoDCs were incubated either with CD40 ligand (L)-transfected J558 cells or IFN-? for additional 24 hours. Cytokine production was measured by ELISA and FlowCytomix Human T helper Th1/Th2 11plex. Allostimulatory capability of MoDCs was tested using an allogeneic mixed leukocyte reaction (MLR) assay. Results. Immature MoDCs showed a moderate potential for stimulation of proliferation of CD4+ T cells, which was enhanced by the treatment with Poly(I:C). Ligation of CD40 or treatment with IFN-? of immature or Poly(I:C)-treated MoDCs significantly up-regulated their allostimulatory activity. MoDCs matured in the presence of Poly(I:C) up-regulated the production of IL- 12 and IL-10, which was followed by increased levels of IFN- ? and decreased levels of IL-5 in co-cultures with allogeneic CD4+ T cells. Ligation of CD40 on immature MoDCs upregulated the production of IL-12 and IL-23 which was accompanied by increased secretion of IFN-? in co-culture. Stimulation of CD40 on Poly(I:C)-treated MoDCs significantly enhanced the production of IL-12, IL-23 and IL-10. However, such treated MoDCs decreased the production of IFN-? and IL-10 and up-regulated the secretion of IL-17. Immature MoDCs treated with IFN-? up-regulated IL-12, but lowered the production of IL-5 and IL-17 by CD4+ T cells. Treatment of Poly(I:C)-activated MoDCs with IFN-? down-regulated the production of IL-12 and up-regulated IL- 10 by these cells and increased/decreased the levels of IL-10/ IFN-?, respectively, in co-culture with CD4+ T cells. Conclusion. Treatment with Poly(I:C) or ligation of CD40 on immature MoDCs induces maturation of these cells into a phenotype that supports Th1 response. Activation of CD40 on Poly(I:C)-treated MoDCs shifts the immune response towards Th17. Treatment of immature MoDCs with IFN-? down-regulated Th2 and Th17 responses. However, addition of IFN-? to Poly(I:C)-activated MoDCs down-regulated Th1 response and promote T regulatory mechanisms. Each of these results may have functional and therapeutic implications.Uvod/Cilj. Poliinosinsko-policitidilinska kiselina [Polyinosinic-polycytidylic acid - Poli (I:C)] stimulise funkcionalno i fenotipsko sazrevanje dendriticnih celija (DC). Medjutim, malo je podataka o modulaciji funkcije DC tokom interakcije sa T-limfocitima posredovanoj receptorom CD40 i interferonom-? (IFN-?), sto je bio cilj ovog istrazivanja. Metode. Nezrele DC dobijene su kultivacijom monocita (Mo) iz periferne krvi u prisustvu faktora stimulacije granulocitno- makrofagnih kolonija (Granulocyte-Macrophage Colony- Stimulating Factor - GM-CSF) i interleukina (IL)-4 tokom pet dana. Sazrevanje je indukovano dvodnevnom inkubacijom MoDC sa Poli(I:C). Poslednja 24 casa, nezrele i zrele MoDC kultivisane su sa celijama J558 koje su transfektovane ligandom CD40 ili u prisustvu IFN-?. Produkcija citokina odredjivana je ELISA metodom, a alostimulatorna sposobnost u mesanoj leukocitnoj kulturi. Rezultati. Stimulacija nezrelih MoDC sa Poli(I:C) povecala je sekreciju IL-12, njihovu alostimulatornu sposobnost i produkciju IFN-? u kokulturi sa CD4+ T limfocitima. Slicni rezultati dobijeni su povezivanjem CD40 molekula ili tretiranjem nezrelih MoDC sa IFN- ?. Medjutim, stimulacija CD40 molekula na MoDC koje su aktivisane sa Poli(I:C) povecala je produkciju IL-12, IL-23 i IL-10 sto je pospesilo produkciju IL-17, a snizilo produkciju IFN-? i IL-10 u MoDC/CD4+ kokulturi. Suprotno tome, IFN-? snizio je produkciju IL-12, a povecao produkciju IL- 10 od strane MoDC aktivisanih sa Poli(I:C), sto je bilo povezano sa snizenjem IFN-?, a porastom nivoa IL-10 u celijskoj kokulturi. Zakljucak. Poli(I:C), IFN-? i povezivanje CD40 molekula su aktivatori sazrevanja MoDC i stimulatori Th1 imunog odgovora. Ligacija CD40 molekula na MoDC aktivisanim sa Poli(I:C) usmerava u pravcu Th17, a inhibira Th1 imuni odgovor. U istom modelu IFN-? inhibira Th1 odgovor, a stimulise imunoregulatorne mehanizme. Svaki od dobijenih rezultata moze imati specificne funkcijske ili terapeutske implikacije. PR Projekat Ministarstva nauke Republike Srbije, br. 175102