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Modulation of the function of human monocyte derived dentritic cells by combined use of the endosomal Toll-like receptors, Dectin-1receptor and proinflamatory cytokines

dc.contributor.advisorČolić, Miodrag
dc.contributor.otherBožić, Biljana
dc.contributor.otherVučević, Dragana
dc.creatorDragičević, Ana Ž.
dc.date.accessioned2016-01-05T11:45:24Z
dc.date.available2016-01-05T11:45:24Z
dc.date.issued2012-05-22
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=8
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:2129/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=
dc.identifier.urihttp://nardus.mpn.gov.rs/123456789/2024
dc.descriptionDendritske ćelije (DĆ), najpotentnije antigen-prezentujuće ćelije (APĆ), integrišu signale koje primaju sa različitih receptora u jedinstveni odgovor. Ključni značaj u ostvarivanju funkcija DĆ imaju receptori za prepoznavanje konzervisanih struktura, tzv. molekularnih obrazaca patogena (engl. Pattern Recognition Receptor, PRR). Aktivacija pojedinačnih PRR, posebno Toll-sličnih receptora (engl. Toll-like receptor, TLR) ili lektinskih receptora C-tipa poput dektina-1, dovodi do sazrevanja DĆ, dok je za razvoj efikasnog imunskog odgovora neophodna kooperacija više receptora. Dektin-1 receptor je, pored TLR, jedini receptor urođenog imuniteta čija aktivacija samostalno indukuje signalnu kaskadu koja dovodi do sazrevanja MoDĆ sa sposobnošću indukcije Th1 i Th17 odgovora. Imajući navedno u vidu, sledeći cilj ovog istraživanja je bio da se ispita efekat kombinovane primene Poly (I:C) i kurdlana, agonista dektin-1 receptora, na funkcionalne i fenotipske karakteristike MoDĆ. Za modulaciju sazrevanja i funkcija DĆ, pored stimulacije PRR, značajnu ulogu imaju i citokini prirodnog imuniteta koji se produkuju prilikom infekcije. Najnovija istraživanja ukazuju na ulogu TNF-α, jednog od najznačajnijih proinflamatornih citokina, u sazrevanju DĆ u ranim fazama infekcije kao i razvoju antigen-specifičnog odgovora. Naredni cilj našeg istraživanja bio je ispitivanje dozno- i vremenski- zavisnog efekta kombinovane primene TNF-α i Poly (I:C) na funkcionalne i fenotipske karakteristike MoDĆ. Poslednji deo istraživanja se odnosio na ispitivanje uticaja signala stečenog imuniteta koje DĆ dobijaju tokom interakcije sa T-limfocitima, uključujući signalizaciju preko CD40 receptora i receptora za IFN-γ na njihove karakteristike. Metode. Nezrele MoDĆ, dobijene kultivacijom humanih monocita, su stimulisane samim Poly (I:C) ili njegovom kombinacijom sa loksoribinom, kurdlanom ili TNF-α tokom 48h. Da bi ispitali uticaj signala stečenog imuniteta, MoDĆ stimulisane samim Poly (I:C) ili u kombinaciji sa TNF-α dalje su kultivisane u prisustvu ćelija J558 transfektovanim ligandom za CD40, solubilnog CD40L ili IFN-γ. U cilju ispitivanja uticaja kinetike aktivacije na kapacitet MoDĆ da polarizuju imunski odgovor, MoDĆ su stimulisane kombinacijama različitih koncentracija Poly (I:C) i TNF-α tokom 24h i 48h. Protočnom citofluorimetrijom su analizirane fenotipske karakteristike MoDĆ. Alostimulatorna sposobnost MoDĆ je određena testom mešane leukocitne reakcije. Produkcija citokina je određena ELISA metodom i testom za detekciju citokina pomoću imunofluorescentnih kuglica. Rezultati. Stimulacija MoDĆ optimalnom koncentracijom Poly (I:C) dovela je do povećanja ekspresije HLA-DR, CD86, CD40, CD54, CD83 i CCR7 molekula, povećanja produkcije IL-12, umerene produkcije IL-23 i niske produkcije IL-10. Ovako stimulisane MoDĆ dovode do povećane produkcije IFN-γ i umerene produkcije IL-17 tokom kokultivacije sa CD4+ T limfocitima. U preliminarnim eksperimentima nezrele MoDĆ su stimulisane različitim koncentracijama Poly (I:C) (5 μg/ml, 10 μg/ml, 25 μg/ml i 50 μg/ml), loksoribina (34 μg/ml i 85 μg/ml) i kurdlana (10 μg/ml , 50 μg/ml, 100 μg/ml i 200 μg/ml). Na osnovu fenotipskih i funkcionalnih karakteristika MoDĆ procenjeno je da je optimalna koncentracija za aktivaciju MoDĆ za Poly (I:C) 25 μg/ml, za loksoribin 85 μg/ml i za kurdlan 100 μg/ml, dok je koncentracija Poly (I:C) od 10 μg/ml i loksoribina od 34 μg/ml suboptimalna. Optimalne i suboptimalne koncentracije agonista su dalje korišćene za stimulaciju nezrelih MoDĆ u ovom istraživanju.sr
dc.descriptionDendritic cells (DCs) are the most potent antigen presenting cells (APCs) which receive and integrate multiple signals to initiate and direct a response appropriate to extracellular milleu. These APCs perform these functions mostly due to the expression of a wide variety of pattern recognition receptors (PRRs). PRRs discriminate self-tissues from infectious non-self tissues through molecular pattern (MPs) recognition. Although triggering of a single pattern recognition receptor (PRR), especially Toll-like receptors (TLRs) or C-type lectins, results in phenotypic changes in DCs, for functional maturation cooperativity between multiple PRRs is needed in order to achieve an effective immune response. Recent studies have shown that the ligation of Dectin-1, C-type lectin receptor, on MoDCs elicits their maturation. Dectin-1, a DC-associated C-type lectin, is the first of many PRRs which mediate their own signaling and induces the maturation of DCs capable of eliciting the generation of different T helper (Th) effectors. The next aim of this work was to study the response of MoDCs to the combined effect of Poly (I:C) and curdlan, selective Dectin-1 agonists. Tumor necrosis factor (TNF)-α is important for early DC maturation and as a bridge between initiation of the inflammatory cascade and generation of the antigen-specific response. To gain insight into this scientific problem we investigated the kinetics of maturation and the length of exposure of MoDCs to a pathogen (mimicked by Poly (I:C) in our study) in an inflamed tissue (mimicked by TNF-α) on phenotypic and functional characteristics of MoDCs. Finally, little is known about how subsequent interaction of MoDCs with T cell-derived stimuli, such as CD40 or interferon-γ (IFN-γ), modulates MoDC functions. Therefore, this problem was the last objective of this study. Methods. Immature MoDCs (iMoDCs), generated from human monocytes, were treated with Poly (I:C) alone or in combination with loxoribine, curdlan or TNF-α for 48h. To investigate the influence of T-cell derived stimuli, MoDCs cultivated for 24h with Poly (I:C) alone or in combination with TNF-α were incubated either with CD40 ligand (L)-transfected J558 cells, soluble CD40L or IFN-γ for additional 24h. To examine the influence of kinetics of activation on the Th polarizing capability of MoDCs, we stimulated MoDCs with different doses of Poly (I:C) in combination with TNF-α for 24h and 48h. Phenotypic characteristics of MoDCs were determined by flow cytometry. Allostimulatory capability of MoDCs was tested using a mixed leukocyte reaction assay. Cytokine production was measured by ELISA and FlowCytomix. Results. Optimal concentration of Poly (I:C) stimulated the maturation of MoDCs as judged by the up-regulation of HLA-DR, CD86, CD40, CD54, CD83 and CCR7 expression. Poly (I:C)-treated MoDCs were potent producers of interleukin (IL)-12, moderate producers of IL-23 and weak producers of IL-10, which was followed by high production of IFN-γ and moderate production of IL-17 by allogeneic CD4+ T cells. In preliminary experiments iMoDCs were treated with Poly (I:C) (5 μg/ml, 10 μg/ml, 25 μg/ml and 50 μg/ml), loxoribine (34 μg/ml and 85 μg/ml) or curdlan (10 μg/ml , 50 μg/ml, 100 μg/ml and 200 μg/ml). Based on phenotypic characteristics and functional capabilities of MoDCs, the concentrations of Poly (I:C) (25 μg/ml), loxoribine (85 μg/ml) and curdlan (100 μg/ml) were found to be optimal for activation of MoDCs, while the concentrations of Poly (I:C) (10 μg/ml) and loxoribine (34 μg/ml) were found to be suboptimal. For stimulation of iMoDCs we used these concentrations of the agonists. The combined treatment of MoDCs with suboptimal concentrations of TLR3 and TLR7 agonists resulted in slight potentiation of HLA-DR, CD86, CD83, CD54 and CD40 molecules and stimulation of IL-27, IL-23 and IL-10 secretion, compared to effects of single agonists. This was followed by up-regulated secretion of IFN-γ and IL-17 in the co-culture with allogeneic CD4+ T cells. When the suboptimal concentration of Poly (I:C) was combined with the optimal concentration of loxoribine, MoDCs down-regulated HLA-DR and up-regulated CD86 expression, enhanced the production of IL-12 and IL-23 and down-regulated the levels of IL-10 and IL-27, compared to the effects of single agonists. MoDCs pretreated in this way stimulated the production of IFN-γ and lowered the levels of IL-4 and IL-10 by CD4+ T cells. The treatment of MoDCs with optimal concentrations of both TLR agonists was followed by down-regulation of HLA-DR, CD83 and CD40 expression and augmented the production of IL-12, IL-27 and IL-10, whereas the level of IL-23 was significantly lower, compared to relevant controls. These MoDCs promoted the production of IFN-γ and inhibited the production of IL-4, IL-10 and IL-17 in co-culture, compared to the effect of corresponding controls. The combination of Poly (I:C) and curdlan induced phenotypic maturation of MoDCs with the capability to stimulate alloreactive response. Such treated MoDCs up-regulated the production IL-12, IL-23 and IL-10, compared to the effect of Poly (I:C), alone. The opposite effect was observed for IFN-γ production. When combined, these agonists primed MoDCs to further increase the production of IFN-γ by CD4+ T cells in co-culture, especially those of naïve (CD45RA+) phenotype, and IL-17 by memory (CD45RO+) CD4+ T cells.en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Биолошки факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/175102/RS//
dc.rightsAutorstvo-Nekomercijalno-Bez prerade 3.0 Srbija (CC BY-NC-ND 3.0)
dc.sourceУниверзитет у Београдуsr
dc.subjectCD40, CD4+ T-ćelijski odgovor, Dektin-1 receptor, humane dendritske ćelije monocitnog porekla, imunoterapija tumora, IFN-γ, TNF-α, Toll-sličan receptor 3, Toll-sličan receptor 7sr
dc.subjectCD40, Dectin-1 receptor, human monocyte-derived dendritic cells, IFN-γ, TNF-α, T helper immune response, Toll-like receptor 3, Toll-like receptor 7, tumor immunotherapyen
dc.titleModulacija funkcije humanih dendritskih ćelija kombinovanom primenom agonista endozomnih Toll-sličnih receptora, Dektin-1 receptora i proinflamatornih citokinasr
dc.titleModulation of the function of human monocyte derived dentritic cells by combined use of the endosomal Toll-like receptors, Dectin-1receptor and proinflamatory cytokinesen
dc.typePhD thesis
dcterms.abstractЧолић, Миодраг; Божић, Биљана; Вучевић, Драгана; Драгичевић, Aна Ж.; Модулација функције хуманих дендритских ћелија комбинованом применом агониста ендозомних Толл-сличних рецептора, Дектин-1 рецептора и проинфламаторних цитокина; Модулација функције хуманих дендритских ћелија комбинованом применом агониста ендозомних Толл-сличних рецептора, Дектин-1 рецептора и проинфламаторних цитокина;


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