THE ROLE OF IL-4 ON THE PHENOTYPE AND FUNCTIONS OF MURINE BONE MARROW- DERIVED DENDRITIC CELLS

Abstract

[...]evaluation of the functional and phenotypic properties of in vitro generated BM-DCs with or without IL-4 can be crucial for the selection of relevant generation protocols for particular research objectives. Additionally, we tested the capacity of antigen-pulsed BM-DCs to prime naive T cells using an in vitro BMDC/T cell co-culture system. IL-4 presence during development stages exhibited typical characteristics for in vitro generated BM-DCs, immature phenotype, upregulation of cytokines in response to LPS-driven maturation, however, promoted nonspecific T cell signaling.

Objective. The involvement of IL-4 in the generation and maturation of murine bone marrowderived dendritic cells (BM-DCs) remains unclear, especially since granulocyte macrophage colonystimulating factor (GM-CSF) is considered the primary growth factor driving BM-DCs differentiation of murine precursors. Consequently, evaluation of the functional and phenotypic properties of in vitro generated BM-DCs with or without IL-4 can be crucial for the selection of relevant generation protocols for particular research objectives.

Materials and methods. We investigated the maturation of BM-DCs following stimulation with lipopolysaccharide (LPS) by assessing cytokine secretion and the expression of specific cell surface markers in BM-DCs generated with GM-CSF in the presence or absence of IL-4. Additionally, we tested the capacity of antigen-pulsed BM-DCs to prime naive T cells using an in vitro BMDC/T cell co-culture system. Cytokine production by Th1/Th2 cell subclasses was analyzed using a multiplex assay.

Results and discussion. BM-DCs generated with GM-CSF alone induced high levels of costimulatory molecules, similar to a mature phenotype. Cytokine production however, indicated the responsiveness to LPS-induced maturation. Additionally, cytokine secretion patterns in the co-culture system suggested that antigen-pulsed BM-DCs generated with GM-CSF alone could effectively prime naive T cells. IL-4 presence during development stages exhibited typical characteristics for in vitro generated BM-DCs, immature phenotype, upregulation of cytokines in response to LPS-driven maturation, however, promoted nonspecific T cell signaling.

Conclusions. Our findings indicate that while IL-4 presence in BM-DCs generation produces cells with the expected phenotype and maturation characteristics, it may alter the induction of antigenspecific responses in experimental setups designed to targeted T cell responses.

Funding: work carried out within the PCCDI - UEFISCDI project, number PN-III-P1-1.2PCCDI-2017-0529/62PCCDI/2018 (CONVAC), and the Nucleu Program, contract 25N/2023, project PN 23 44 01 01.

Obiective. Rolul IL-4 in diferentierea si maturarea celulelor dendritice derivate din máduva osoasá muriná (BM-DCs) rámáne neclar, mai ales avand in vedere са principalul factor de diferentiere din progenitori murini este GM-CSF (granulocyte-macrophage colony stimulating factor). In consecintä, evaluarea caracteristicilor fenotipice si functionale ale BM-DCs diferentiate in vitro cu sau fara IL-4 este esentialá pentru selectarea unui protocol din care sa rezulte BM-DCs cu caracteristici adecvate diferitelor obiective de cercetare.

Materiale si metode. Capacitatea de maturare a BM-DCs generate cu GM-CSF in prezenta sau absenta IL-4 s-a analizat dupa stimularea cu lipopolizaharid (LPS) prin evaluarea secretiei de citokine si a expresiei unor markeri specifici de suprafatá. In plus, utilizand un sistem de co-culturä in vitro, s-a evaluat capacitatea BM-DCs pulsate cu antigen de a activa limfocitele T naive. Productia de citokine secretate de cátre subclasele Th1/Th2 a fost analizatá printr-un test multiplex.

Rezultate si discutii. Diferentierea BM-DCs cu GM-CSF a indus nivele ridicate de expresie а moleculelor costimulatoare, expresie similará unui fenotip matur dar productia de citokine a crescut la stimularea cu LPS, indicánd capacitatea acestora de maturare. In plus, paternul de secretie al citokinelor in sistemul de co-culturá a sugerat са BM-DCs pulsate cu antigen activeaza limfocitele T naive. Prezenta IL-4 in procesul de diferentiere a dus la obtinerea de BM-DCs cu un fenotip imatur si la cresterea secretiei de citokine ca ráspuns la maturarea indusa de LPS. Cu toate acestea, in sistemul de co-culturä, prezenta IL-4 a determinat un ráspuns nespecific al limfocitelor T.

Concluzii. Rezultatele indica faptul са, desi IL-4 contribuie la diferentierea BM-DCs cu fenotip specific, receptive la agentii de maturare, in model de co-culturä cu limfocite T, prezenta IL-4 poate masca ráspunsul antigen-specific.

Sursa de finantare: lucrare desfásuratá in cadrul proiectului PCCDI - UEFISCDI, numar PN-II-P1-1.2-PCCDI-2017-0529/62PCCD1/2018 (CONVAC), si programului Nucleu, contract 25N/2023, proiect PN 23 44 01 01.