**4. Discussion**

In this study, we show for the first time the effects of THC treatment on the function of RASF and PBMC. Firstly, we determined the expression of the main target receptors for THC, but also for endocannabinoids produced in the joint [28], CB1 and CB2, in and on RASF, and found CB1 in intracellular compartments, whereas CB2 was located at the plasma membrane. The latter was also upregulated by TNF. However, we demonstrated that THC elevates intracellular calcium and PoPo3 uptake by a TRPA1, rather than a CB receptor-dependent mechanism. RASF IL-6, IL-8 and MMP-3 production was reduced by THC only at the highest concentration investigated, and this effect was not antagonized by either TRPA1 or CB1 antagonists, suggesting a receptor-independent effect. In addition, we showed that THC had only a minor influence on PBMC (alone or in co-culture with RASF) cytokine and immunoglobulin production.

We detected CB1 protein intracellularly in RASF, which is line with our previous results [45]. In that study, we utilized a different antibody but it also detected CB1 protein at the nuclear membrane, suggesting that signals initiated by this receptor are spatially confined, as already confirmed by other intracellular G-protein-coupled receptors (GPCRs) [50]. Another study showed that membrane GPCRs might "communicate" with its fraction of intracellular receptors by β-arrestin eliciting a combined response [51]. This might also be the case in RASF, since we found little but detectable cell surface expression of CB1. CB2 was exclusively located at the plasma membrane and its levels were regulated by TNF. In a previous study, we already demonstrated CB2 upregulation in response to TNF, but these experiments were conducted under hypoxic conditions and CB2 was detected by cell-based ELISA rather than flow cytometry [45].

In a next step, we investigated the effects of THC on intracellular calcium since it has been shown that CB1, CB2 and TRPA1 are able to modulate calcium levels [52–54]. We found that THC via TRPA1 activation elevates calcium levels and we already demonstrated that only TNF pre-stimulated RASF responded to TRPA1 agonism [54], which was confirmed in this study. In contrast to cannabidiol (CBD), THC is specific for TRPA1 and does not engage in significant off-target effects. Although both THC and CBD bind to TRPA1, CBD increased intracellular calcium levels mainly by disrupting mitochondrial calcium homeostasis with little TRPA1 contribution [47].

In previous studies, we revealed that elevations in intracellular calcium are coupled to the uptake of the cationic dye PoPo3 [47,54]. Therefore, we also investigated this uptake in response to THC and we found that intracellular PoPo3 levels increased. PoPo3 was identified as a surrogate marker for drug uptake [47], since its uptake is likely controlled by organic cation transporters that are also responsible for the uptake of several therapeutic compounds and drugs [47]. This finding might be relevant in RA therapy, since a combination of THC with an antiarthritic compound might have synergistic effects and, due to increased cellular uptake, lower drug concentrations might be necessary to elicit similar effects. In fact, this has already been demonstrated with CBD, and, in their study, the authors showed a higher efficacy of the chemotherapeutic drug doxorubicin when combined with CBD [55].

TRPA1 was identified as target receptor of THC and this channel is usually expressed at high levels in sensory nerve fibers where activation mediates the release of pain transmitters, calcitonin gene-related peptide and substance P [56]. While TRP channels are also ionotropic target receptors for endocannabinoids [57], they are also signaling partners with G-protein-coupled receptors [58]. After the identification of TRPA1 as a target receptor for THC, we investigated IL-6, IL-8, and MMP-3 production and proliferation of RASF in response to THC. We found that THC reduced cytokine levels only at the highest concentration and this effect was not antagonized by a TRPA1 inhibitor. This suggests an off-target effect, since the CB1 antagonist rimonabant did not reverse the reduction induced by THC. CB2 was also not involved, as we previously screened several CB2 agonists and antagonists (data not shown) for their ability to modulate cytokine expression in SF and found no effect. This is in line with results from Fechtner et al., who showed that CB2 ligands had negligible

effects but deletion of the receptor had a proinflammatory impact on SF [32]. In addition, cell viability was impaired at high THC concentrations and we also observed extensive cell death, suggesting a cytotoxic effect by THC. Furthermore, given the affinity of THC at CB1 and CB2 [59], it is unlikely that only high micromolar concentrations of THC would affect cytokine production. Similar to THC, we already showed a reduction in cytokine production and cell viability by CBD [47]. At high CBD concentrations, this cannabinoid elicited the assembly of the mitochondrial permeability transition pore, which entailed cell death [47], and such a mechanism might also be employed by THC. Concomitantly with the reduction in cytokine levels, we also found reduced proliferation of RASF in response to THC, which demonstrates that cell death at least contributes to reduced cytokine levels.

In RA, lymphocytes and macrophages activate SF in synovial tissue, promoting them from a bystander to an active participant in the inflammatory process [60] and, therefore, we investigated the impact of THC on healthy PBMC alone or in co-culture with RASF. While IL-6 was increased in PBMC co-culture compared to monoculture, IL-10 and TNF levels were lower in co-culture. While IL-6 is mainly produced by RASF, IL-10 and TNF are exclusively produced by PBMC, and RASF have an inhibitory influence on production of these cytokines. A functional repression of B lymphocytes by RASF has also been confirmed in a study by Storch et al. [61]. We found that THC reduced TNF production by PBMC only in co-culture, suggesting a combined inhibitory influence of RASF and THC, while IL-10 was reduced in monoculture. Many studies showed an inhibitory effect of THC on lymphocyte function, but these studies did not investigate cytokine production [62–65]. In PBMC monoculture, THC had a significant effect on immunoglobulin G production without an activating stimulus (e.g., CpG). This suggests that THC in high concentrations is able to support T or B cell activation directly. In fact, it has been shown that T cells express TRPA1, and activation increased T cell activation and calcium influx [66]. This is in contrast to published findings that CB2 mediates the immunosuppressive function of cannabinoids and THC via several different mechanisms [67–69], but necessary THC concentrations to activate CB2 are much lower than those needed for TRPA1 activation. In line with this, THC in high concentrations has been found to modulate cytokine levels independent of cannabinoid receptors [34]. In general, it is ye<sup>t</sup> unclear whether immunoglobulin production is enhanced or suppressed by cannabinoids. In mice, it has been shown that antigen-specific immunoglobulin M (IgM) and G (IgG) production are not affected by CB1 and CB2 deficiency [70]. Immunoglobulin E (IgE) production was enhanced by THC in a non-cannabinoid-receptor-dependent fashion [71], and class switching from IgM to IgE was supported by CB2 activation in isolated murine B cells [72]. Due to these differential effects of cannabinoids on immune cells, it is difficult to predict whether THC has a stimulatory or inhibitory effect on the immune response in general. In fact, THC impairs the differentiation of monocyte-derived dendritic cells [65], regulates micro RNA relevant for T cell differentiation [73] or inhibits STAT3 phosphorylation [74], and none of these effects might regulate cytokine production in our experimental setting. In addition, THC has several cannabinoid-receptor-independent effects [34,64] and these might be different in every cell type. Single-cell transcriptomic analyses of PBMC revealed that THC does not suppress immune reactions in general but interferes with pro- and anti-inflammatory innate and adaptive pathways of the immune system [75], which might be counterbalanced in our setting.
