Involvement of the sphingolipid intracellular signaling pathway in the effects of triiodothyronine (T3) on dendritic cell (DC) activation

GIUSIANO L; ALAMINO VA; FOZZATTI L; SOLER MF; GIGENA N; MONTESINOS MM; PELLIZAS CG

Río de Janeiro

Congreso; XVI Congreso Latinoamericano de la Sociedad Latinoamericana de Tiroides; 2017

Sociedad latinoamericana de tiroides (LATS)

Introduction: We reported that mice dendritic cells (DCs), the main antigen presenting cells, express TRb1 with a preferential cytoplasmic localization and that physiological levels of triiodothyronine (T3, 5nM) promotes their maturation and their ability to direct adaptive responses towards a Th1-type profile in vitro, as well as cytotoxic and antitumoral effects in vivo. Moreover, we previously demostrated that the underlying mechanism involved PI3K-independent Akt.Besides, "Sphingolipids" revealed as key elements in signal transduction cascades, regulate events related to death and survival of cells. The major bioactive sphingolipids include: ceramide (Cer), sphingosine (Sph), ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P). Noteworthy, there is evidence that they are involved in the non-classical Akt activation. In this sense, C1P through Akt phosphorylation was reported as mediator of the effect of thyroid hormones on the proliferation and activation of T-cells. Objectives: We aim to further disclose the molecular mechanism underlying the effects of T3 on DC functioning, in particular in the ?non-classical? activation of Akt. For this purpose, we characterized the intracellular pathway mediated by Sphingolipids in DCs and initiated the studies of its involvement in T3-mediated DC activation.Methods: Mice (C57BL/6) bone-marrow derived DCs were treated or not with chemical inhibitors of the sphingolipid pathway (SKI: Sphingosine kinases,SphK1/SphK2; GW 4869: neutral sphingomyelinase, nSMAse; Imipramine: acid sphingomyelinase, aSMAse and NVP 231: ceramide kinase, CK) at reported concentrations for 30 min before T3 (5nM) addition for 18 h. After treatment, supernatants were collected for determination of IL-12 secreted by ELISA and IL-12 produced by flow cytometry (IL-12 is a sensitive marker of T3 action on DCs). In turn, cells were processed for the determination of the mRNA levels of the involved enzymes by RT-PCR. Statistics: Student ?t? test, ANOVA/SNK.Results: In this study we demonstrate that DCs express mRNA for the enzymes SphK1, SphK2, CK, aSMAse and nSMAse; which are regulating the delicate balance between intracellular levels of interconvertible sphingolipids by their production or degradation. Furthermore, we found that IL-12 levels of T3-treated DCs in the presence of the inhibitors SKI, GW 4869, Imipramine and NVP 231 was significantly attenuated; both by ELISA and flow citometry. Conclusions and Discussion: Results suggest that S1P and C1P are involved in the maturation of DCs stimulated with T3 in agreement with growth-stimulation and antiapoptotic properties of S1P and C1P in the same way as opposite effects of Cer and Sph.These findings provide evidence for active involvement of the sphingolipid signaling pathway in the effects registered by T3 treatment to DCs. Considering the therapeutic impact we reported for T3-treated DCs, these results provide molecular tools to manipulate the immunogenic potential of DCs.