Characterization of extracelular vesicles released by Triiodothyronine-stimulated mice dendritic cells (DCs): Rol in paracrine DC activation

NEGRETTI-BORGA D; BRAVO-MIANA R; SOLER MF; GIUSIANO L; DE PAUL AL; DONADIO, AC; MONTESINOS, MM; PELLIZAS CG

Mar del Plata

Congreso; LXIII Reunión anual de la Sociedad de Investigación Clínica (SAIC) y LXVI Reunión anual de la Sociedad de Inmunología (SAI); 2018

Sociedad de Investigación Clínica (SAIC) y Sociedad Argentina de Inmunología (SAI)

We provided evidence of triiodothyronine (T3) stimulation of micedendritic cells (DCs), driving pro-inflammatory and cytotoxic responses,exploited in an antitumor DC-based vaccination protocol.Extracellular Vesicles (EVs) exhibit a crucial role in cellular communicationand EVs secreted by DCs (EVs-DCs) may be involved inthe amplification of the immune response. Our aim was to characterizethe populations of EVs-DCs and assess their role in paracrineDC communication after T3 exposition. Immature bone marrow DCs(iDC) were obtained from WT C57BL/6 mice and stimulated (ornot) with T3 (5nM-18h, DCs-T3). Secreted EVs-DCs from iDCs andDCs-T3 (EVs-DCs-T3) were isolated by differential ultracentrifugationat 2,000g (2K, large EVs); 10,000g (10k, Microvesicles); and100,000g (100K, small EVs: sEVs). Morphological analysis of EVswas conducted by Transmission Electron Microscopy (TEM) anddynamic light scattering (DLS), and molecular characterization ofEVs by western blot analysis (CD63 and CD81). A functional assayevaluated the syngeneic DC profile induced by treatment with EVs-DCs-T3 (markers of DC maturation: MHCII, CD86, and CD40, FlowCytometry-FACS; and IL-12, FACS and ELISA). Statistical analysis:ANOVA-SNK. Data obtained from TEM analysis of the different fractionsfrom both, iDCs and DCs-T3, showed the presence of secretedEVs. Size profile analysis revealed a significant higher frequencyof 150-600 nm for 2K and 10K, whereas 100k exhibited more than90% (p<0.01) of EVs sized 30-150 nm (sEVs). DLS analysis showeda similar pattern. Besides, sEVs secreted by DCs-T3 significantlyincreased the expression of DC phenotypic maturation markers,and the production and secretion of IL-12. This study allowed themorphological characterization of EVs populations secreted by miceDCs that in turn induced the activation of syngeneic iDCs, endowingthese cells with a Th1-type driving phenotype that may be involvedin the adaptive response induced by T3 exposition to DCs.