Participação do sistema endocanabinóide na modulação da função imunológica de células-tronco da polpa dentária humana

Abstract

The endogenous cannabinoid signaling system (eCBS) is composed of endocannabinoid molecules that interact with G-protein coupled receptors CB1 and CB2, and it is present in many tissues and cell types, including central nervous system (CNS) cells, the immune system cells and mesenchymal stem cells (MSC). The presence of such receptors in both immune and stem cells may be indicative of the cellular signaling pathway through which the two systems communicate. Lately there has been shown a growth in treatments of various CNS-related diseases using cannabis derivatives such as cannabidiol, showing positive results. However, we still do not know the consequences of treatments involving eCBS in other cell types.Thus, there is a need to understand such communication, as well as the systemic and related side effects upon receptors stimulation, since it may influence the activation and response of human dental pulp stem cells (DPSC). Such knowledge also form the basis for a better understanding of cannabis medicinal effects, contributing to clarify its action for example in neurodegenerative diseases and also i the development of new medicines. Thus, the present study aims at evaluating the effects of cannabinoid receptor stimulatory and inhibitory agents uppon DPSC immune and proliferative responses. So, human DPSC were isolated from dental pulp and characterized to confirm their identity as stem cells. DPSC were then cultured in the presence or absence of an agonist (anandamide) and antagonists (AM251 and SR144528) for eCBS receptors, whether or not stimulated by TNF-α. For immunomodulation analysis, immunosuppressionrelated surface molecules (HLA-DR, PD-L1, PD-L2) were measured by flow cytometry. eCBS components mRNA expression was verified by Real time RTPCR as well as the expression for TGF-β and IL-6 cytokines. The proliferation rate of DPSCs under study was also evaluated. The characterization of the isolated cells confirmed their identity as stem cells. Cytometry for HLA-DR, PDL1 and PD-L2 markers indicated that DPSCs express these markers on their cell surface and that their expression responds differentially to eCBS stimulus as well as to the presence of TNF-α, which proves the role of such system in influencing the DPSCs immunomodulatory profile. Gene expression analysis for eCBS-related enzymes and receptors provided a deep and detailed understanding of the system's functioning in DPSCs, both in terms of its selfregulation and its response to the presence of TNF-α. Such results confirmed that eCBS is a present and functionally active system in DPSCs, directly involved with immunomodulation. Gene expression analysis for TGF-β and IL-6 cytokines showed that DPSCs express genes for these cytokines even when cultured without specific stimulation. It also showed that this expression is profoundly altered as a response to eCBS stimuli and TNF-α stimulation, again indicating the role of this system in coordinating the cellular response of DPSCs, especially concerning immunomodulation. Analysis of DPSC proliferation rate under eCBS and TNF-α stimulation showed that eCBS is not able to significantly affect DPSC proliferation, but TNF-α does. The combined analysis of these data indicates that eCBS present in DPSCs not only responds to immune stimulation, but also alters key immune response molecules, providing the necessary stimuli for DPSCs to act in the coordination of such environment.