Most studies treat MSC as potent inhibitors of the immune system. It is currently understood that such property of MSC is not fixed or constitutive, but influenced by the inflammatory environment. The immune system should act as a mediator, regulating not only the immunomodulatory properties of MSC but also their proliferation and differentiation, actively influencing the damage recovery process. Some inflammatory molecules, such as TNF-α and IFN-γ, seem to be involved [3;8;10;15;23;25].
MSC apparently has an immunoregulatory role and an antigen-presenting cell (APC) role, depending on the type, intensity and timing. At the beginning of the establishment of the inflammatory process MSC increases their antigen-presenting potential in order to fight a possible infection. However, as inflammation progresses, the environment modulates MSC to acquire their immunosuppressive potential, bringing the cellular and tissue environment to homeostasis. [5; 9; 10; 56–62].
Different types of MSC share immunomodulatory effects with great therapeutic potential, and in this sense the immunomodulatory capacity of dental pulp stem cells (DPSC) and SHED is no exception, showing sometimes superior capacity to traditional bone marrow mesenchymal stem cells (BMSC) [1; 51; 56].
About ECS, outside of the CNS, CB1 has also been identified in the liver, bone marrow, pancreas, lungs, vascular system, muscles, gastrointestinal tract, reproductive organs, and the immune system [45–47]. CB2 has also been detected in the immune system, mainly in B cells, natural killer cells, and monocytes, which have an active role in the modulation of cell migration and cytokine release [45–47]. Molecules involved in ECS appear to be produced from membrane phospholipid precursors, exerting as immunomodulatory actions, such as decreased production of pro-inflammatory cytokines, modulation of T-helper (Th) 1 and Th2 (TH1/TH2) cell responsiveness and [39;42;63–64].
MSC can express all the components of ECS. AEA, CB1, and CB2 are expressed and are involved in the physiological protection of intense inflammatory responses, with CB1 participating in the healing of inflamed tissues. Furthermore, CB1 is activated during osteogenic differentiation of BMSC and periodontal ligament stem cells, even in an inflammatory environment. Moreover, CB1 was observed to be an essential component in the survival of differentiated cells during acute stress [48–49].
The present study sought to evaluate the results within two situations: the presence of TNF-α and the role of ECS in the possible cellular phenotypic alterations. In general, TNF-α and CB1 and CB2 receptor antagonists, but especially the CB2 antagonist, were able to alter the immunological phenotype of ECS relative to the three markers analyzed. Since there was no significant difference in the cell viability test, any change occurring between the groups is from alteration in the immunological phenotype of SHED.
On HLA-DR expression, not only the presence of TNF-α together with the antagonists (mainly CB2-related) but the presence of the agonist led to increased HLA-DR expression in SHED. In this sense, anandamide may be activating other ECS receptors, such as G protein-coupled receptor 55 (GPR55) and Transient receptor potential (TRP) channels and their respective subfamilies . These results suggest that SHED become able to respond immunologically under these conditions. Cabeza et al. (2019) showed that these cells, under inflammatory conditions, are able to interact with T lymphocytes via HLA-DR, since HLA-DR is especially responsible for initiating adaptive immunity by presenting antigens to these lymphocytes (Perales and Brink, 2012). The results of the present study corroborate the possibility of SHED are functioning as APC or the possibility of inhibiting inflammatory processes via the presentation of specific antigens to T cells, depending on the degree of inflammation of the microenvironment. It is known that the presence of SHED in the inflammatory environment is able to lead to decreased proliferation of Th and increased Treg [9; 56–58], explaining the targeting/maintenance of an immunoregulatory profile.
According to Figs. 3A and 4A, it can be seen that TNF-α together with the antagonists were able to drive SHED towards an immunomodulatory profile both via PD-L1 and PD-L2. This result was most evident for PD-L1, in which blockade of CB1 and CB2 increased the levels of this marker, especially in the presence of TNF-α. Thus, the increase in PD-L1 and PD-L2 under these conditions corroborates the results of studies in which the presence of SHED in an environment of inflammation was able to lead to immunoregulation [9; 56–58] Specifically, the CB1 receptor antagonist showed only a slight increase in HLA-DR and PD-L1 levels when SHED were cultured in the presence of TNF-α; however, not significantly. The contribution of the CB2 antagonist, in the presence of TNF-α, on all three markers was evident, most notably for HLA-DR and PD-L1. However, the changes were only significant when both antagonists were used. In this sense, regarding CB1 and CB2 receptors, the literature is contradictory, with the consensus being only the participation of ECS in immunomodulation, but not the role of activation and blockade of each one of the receptors in specific [39; 42; 63–64]. Galve-Roperh et al. (2013) describe that CB2 activation is associated with chronic inflammation of the nervous system as well as different immune disorders. Montanari et al. (2019), in turn, describe a series of novel benzofuran-based compounds with neuroprotective and immunomodulatory properties for the treatment of Alzheimer's disease, from the change of pro-inflammatory M1 phenotype to neuroprotective M2 phenotype of microglia. Rossi (2013) reports that CB1 is known to promote inflammation while CB2 regulates its magnitude. Moreno et al. (2019) observed that CB2 ligands are responsible for the observed immunomodulatory effects.
As already known, ECS are present in both immune system cells and MSC, expressing both CB1 and CB2 receptors, suggesting a possible common route of immunomodulation, mainly via CB2 [48;50;64]. In a study with endothelial progenitor cells, the release of the endocannabinoids AEA and 2-AG by these cells was verified. In the presence of TNF-α, there was an increase in 2-AG. Moreover, after treatment of this mature cell line with endocannabinoids, a reduction in the induction (by TNF-α) of the pro-inflammatory adhesion molecule CD106 (VCAM) was observed . In addition, cannabidiol was also found to protect oligodendrocyte progenitor cells from apoptosis induced by lipopolysaccharides LPS- or INF-γ-stimulated inflammation. Cannabidiol treatment reversed caspase 3 induction, decreased reactive oxygen species production and endoplasmic reticulum stress-induced apoptosis, followed by decreased molecular effectors Bax and caspase 12 .
The CB1 receptor has been shown to have its expression increased during osteogenic MSC differentiation and to be essential for the survival of these differentiated cells . Tetrahydrocannabinol (THC) was able to activate the CB2 receptor, increasing BMSC immunoregulation on the release of inflammation-associated cytokines [TNF-α, interleukin (IL) -1β, IL-6, and IL-8]. Furthermore, hyperalgesia and allodynia were significantly reduced when MSC pretreated with THC was administered; consequently, pro-inflammatory cytokines were also observed to be significantly reduced, while IL-10 showed increased . Also, it was shown that CB2 activation in MSC led to increased IL-10 release and reduced LPS-induced pro-inflammatory cytokines IL-1β, IL-8, and IL-17 .
A study analyzing the molecular phenotype of Gingival Mesenchymal Stem Cells (GMSC), verified that treatment with cannabidiol prevented the expression of genes of the NALP3-inflammasome pathway, suppressing the levels of NALP3, CASP1, and IL-18, demonstrating in parallel the inhibition of apoptosis, accompanied by the suppression of Bax. Cannabidiol treatment was also able to reduce the expression of genes involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while stimulating the expression of genes involved in the inhibition of immune responses (CD47, CD55, CD276) (Libro et al., 2017). In another study, it was shown that pretreatment of GMSC with cannabidiol led to attenuation of the expression of genes implicated in the etiopathogenesis of Alzheimer's disease, showing that preconditioned GMSC have potential in the treatment of early-stage Alzheimer's disease .
The findings of the present work give strength to the hypothesis that SHED have an immunomodulatory role in an environment with the presence of inflammatory molecules, and may act directly in contact with cells of the immune system in coordinating the inflammatory process by inhibiting the adaptive response via PD-L1 and PD-L2; possibly occurring together with the HLA-DR and TCR interaction, leading -to lymphocyte tolerance [27–29; 34; 68].