Sphingosine Kinase 1 is involved in Triiodothyronine effects in murine dendritic cells and the driven adaptive immunity

NEGRETTI BORGA, DM; BLANCO A; TEIXEIRA MP; ALAMINO VA; SOLER MF; PUENTES EN; DONADIO AC; CLARK CJ; HANNUN Y; MONTESINOS MM; PELLIZAS CG

Curitiba

Congreso; XIX LATS Congress; 2023

Latin American Thyroid Society (LATS)

Triiodothyronine (T3) is the biologically active thyroid hormone (TH), essential for homeostatic control. Dendritic cells (DCs) are highly phagocytic, specialized antigen presenting cells. They orchestrate the adaptive immune response through T-cell activation, leading to the production of pro-inflammatory cytokines. DCs additionally control immunity through the generation of regulatory T cells. Our research group reported that mice DCs express TH receptor β1 (TRβ1) and that physiological levels of T3 promotes DCs’ maturation, survival, and ability to direct pro-inflammatory responses with Th1, Th17 and cytotoxic profiles, restraining tolerogenic signals. These findings were successfully exploited in T3-activated DC-based antitumor vaccines against melanoma and colon carcinoma in mice. T3 effects in DCs are mainly triggered by non-genomic mechanisms involving TRβ1, Akt and NF-kB. In turn, Sphingolipids and their synthetic enzymes have been involved in inflammation, aging and cancer. Sphingosine-1-phosphate (S1P) is produced by Sphingosine Kinase 1 (SK1) and 2. Although this pathway is involved in many pro-inflammatory conditions, little is known about its role in innate immune cells. Moreover, the putative role of this pathway in the reported pro-inflammatory effects induced by T3 in DCs, is of high interest. The aim of this study was to evaluate the role of SK1 in T3-stimulated DCs, and the driven adaptive immunity. DCs differentiated from bone marrow precursors from Wild-Type (WT) and SK1 Knockout (KO) C57BL/6 mice were stimulated with T3 (10 nM, T3-DC) for 18h. Immature DCs (iDC) from WT mice were incubated with PF-543 (SK1 inhibitor, 100 nM), and 30 min later with the T3 stimuli (PF-T3-DC). After 30 min, p-Akt and total Akt were analyzed by Western Blot. Allogenic splenocytes isolated from BALB/c mice were co-cultured with T3-DC or PF-T3-DC (exposed to T3 for 18h), for 3 days. Viability and proliferation were evaluated by FACS. Cytokines were measured by FACS and ELISA. Statistical significance of differences between means was determined by Two-way ANOVA/Tukey test, and paired t test (p<0.05, statistically significant). Results showed that intracellular IL-12 production was increased in T3-DC from SK1-KO vs WT mice (p<0.0001). Accordingly, IL-12 secretion was higher in PF-T3-DC vs T3-DC (p<0.005). Of note, DC viability was not affectedmodified by PF-543. In turn, SK1 inhibition reduced p-Akt in T3-DC (p<0.005). Splenocytes proliferation, as well as IFN-γ and IL-17 production and secretion, markers of pro-inflammatory adaptive responses, were modified in the co-culture with PF-T3-DC (vs T3-DC, p<0.05). Our results revealed for the first time that the Sphingolipid intracellular pathway is involved in T3-DC activation. The immunomodulation exerted by SK1 on T3-DC and the driven adaptive response provide the first insights into a novel role of Sphingolipids in the immune-endocrine crosstalk, which will be intimately unveiled by further research.