Extracellular vesicles released by triiodothyronine-stimulated dendritic cells induce a pro-inflammatory profile of allogenic murine splenocytes

NEGRETTI BORGA, DANA MARÍA; ALAMINO, VANINA ALEJANDRA; SOLER, MARÍA FLORENCIA; BRAVO MIANA, ROCÍO M.; BLANCO, ANTONELLA; DONADIO, ANA CAROLINA; MONTESINOS, MARÍA DEL MAR; PELLIZAS, CLAUDIA G

Congreso; 19th International Congress of Endocrinology (ICE); 2021

ISE, FELAEN and FASEN.

Introduction: The relationship between the neuroendocrine and immune systems is essential for homeostatic maintenance. We reported that physiological levels of triiodothyronine (T3) activate mice dendritic cells (DCs) through thyroid hormone receptors (TRβ), inducing Th1 and Th17 proinflammatory responses and restraining tolerogenic signals. These results were exploited with success in an antitumor DC-based vaccination protocol in vivo. Besides, we described that in vitro, nano-sized extracellular vesicles (EVs) released by T3-stimulated DCs (T3-DCs) activate syngeneic DCs, contributing to paracrine DC communication. However, the immune modulatory role of T3-DCs derived EVs on T cells is still unknown.Objectives: To evaluate the immune response induced by T3-DC released EVs in allogenic splenocytes.Methods: Bone marrow DCs obtained from C57BL/6 WT mice were stimulated (or not, control: C) with T3 (10nM) for 18h. DC-secreted EVs (DCs-EVs) were isolated by differential ultracentrifugation of the supernatant (2,000g: 2K; 10,000g: 10K; and 100,000g: 100K). Allogenic splenocytes were obtained from BALB/c mice and stimulated with DC-EVs for 6 days. Proliferation, intracellular and secreted cytokine production were analyzed by flow cytometry and ELISA assays, respectively. Statistical analysis: Sidak´s multiple comparisons test. P<0.05 was considered statisticallysignificant. Results: We demonstrated that all DC-EVs fractions (T3 and C) evaluated trigger splenocyte proliferation. Both, T3-100K and T3-2K EVs increase CD8 splenocyte subpopulation (vs C-100K and C-2K, respectively). Moreover, the secretion of IFN-γ (p<0.001) and IL-17 (p<0.01) were augment in the splenocytes after T3-2K stimulus (vs C-2K). Similarly, T3-10K upregulated IL17 secretion (p<0.01; vs C-10K). Conclusion: Results showed that DC-EVs induce splenocyte proliferation in vitro. T3-DC released EVs can activate naïve splenocytes, with fraction dependence. After T3-100K and T3-2K EVs exposure, CD8 subpopulation increase. Besides, the pro-inflammatory cytokines IFN-γ and IL17 secretion augment after T3-2K or T3-10K stimulus. Thus, EVs are involved in the extracellular mechanism triggered by T3-DCs to spread their pro-inflammatory adaptive immune signature. These results reinforce previous findings reporting that T3-DCs are driving cytotoxic and pro-inflammatory responses, with Th1 and Th17 polarization. Further ongoing studies will enlighten this issue, providing valuable tools to manipulate the immunogenic potential of T3-DCs and their released EVs.