**1. Introduction**

*Cannabis sativa* contains over 400 compounds, with tetrahydrocannabinol (THC) and cannabidiol (CBD) being the most thoroughly investigated [1]. THC binds to classical cannabinoid receptors (CB1 and CB2) with high affinity but also targets other receptors, enzymes and transporters at higher concentrations [2–4]. While CB1 is the most abundant G-protein-coupled receptor in the brain [5], CB2 is mainly located peripherally [6,7]. Antiinflammatory effects of cannabinoids occur directly via CB2 activation [8], but also by modulation of sympathetic nervous system activity [9]. THC, an agonist at CB1 and CB2, resembles the effect of endogenous cannabinoids (endocannabinoids), which are produced by a wide variety of cells [10]. Of note, endocannabinoids control the tone of nervous system activity, including the sympathetic nervous system [11]. THC mediates its effects via several mechanisms: (1) directly on cells that carry target receptors for THC (e.g., CB1 and CB2) [12], (2) indirectly by modulation of neurotransmitter release via CB1 on nerve terminals (e.g., acetylcholine and norepinephrine) in the periphery [13], and (3) indirectly by binding to fatty acid binding proteins, which releases endocannabinoids from these binding sites and increases their endogenous concentration [4]. THC has analgesic properties, but

**Citation:** Lowin, T.; Kok, C.; Smutny, S.; Pongratz, G. Impact of Δ9-Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells. *Biomedicines* **2022**, *10*, 1118. https://doi.org/10.3390/ biomedicines10051118

Academic Editor: Wesley M. Raup-Konsavage

Received: 8 April 2022 Accepted: 9 May 2022 Published: 11 May 2022

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also demonstrates antiedema and anti-inflammatory effects [14–18] in animal models of arthritis. However, it is still unclear what receptors and pathways are engaged by THC to elicit these effects.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder that is characterized by joint destruction, elevated cytokine burden and several comorbidities such as depression, cachexia, insulin resistance and fatigue [19–22]. Cannabinoids might reduce pain and inflammation in RA [15,16] but they might also have beneficial effects on RA comorbidities, since these are often mediated by alterations in sympathetic nervous system activity [23–26]. One major mediator of joint pathology in RA is synovial fibroblasts (SF), which not only produce cytokines and matrix metalloproteinases, but also actively engage in cartilage invasion [27]. Rheumatoid arthritis synovial fibroblasts (RASF) express cannabinoid receptors, and activation of CB1 or CB2 initiates MAP kinase signaling [28]. While CB1 mediates adhesion of RASF to extracellular matrix [29], CB2 activation was described as anti-inflammatory in several studies [30,31] but, in contrast, it was also identified as proinflammatory by initiating transforming growth factor beta-activated kinase 1 kinase signaling [32]. In addition, inhibitory effects of synthetic cannabinoids on cytokine production were cannabinoid-receptor-independent [31,33]. In peripheral blood mononuclear cells (PBMC), similar results regarding the effects of cannabinoids have been documented. Springs et al. showed a reduction in splenocyte cytokine production by THC, which was independent of cannabinoid receptors [34]. CB2, in general, affects many aspects of the immune response but, in most models of experimental arthritis in rodents, CB2 reduces inflammation and arthritis severity by inhibiting immune cell migration to sites of inflammation [35–39].

In this study, we investigate the effects of THC (0.1–25 μM) on RASF in respect to intracellular calcium levels, drug uptake, proliferation and cytokine production. In addition, we perform RASF/PBMC co-cultures and PBMC monocultures under the influence of THC (1 and 10 μM) and determine cytokine and immunoglobulin production. This study might help to pinpoint the effects of cannabis and THC on arthritic disease and establish a rationale for why medical cannabis might be an appropriate adjunct therapy in RA.

#### **2. Materials and Methods**
