Activating the cholinergic system a novel opportunity for treating osteoarthritis

Purpose: Beyond its role on the autonomic nervous system, the main parasympathetic neuromediator acetylcholine (Ach) has anti-inflammatory properties through the activation of the nicotinic alpha-7 receptor (Chrna7, homopentamer of 5 alpha-7 subunits) expressed by non-neuronal cells. Little is known on its role in joints in general and in OA in particular. We determined 1) the presence of neuronal cholinergic fibers in human and murine joints tissues 2) whether chondrocytes and osteoblasts have the ability to produce Ach (non-neuronal production) 3) the expression and the biological role of Chrna7 in chondrocytes and osteoblasts 4) and the in vivo involvement of Chrna7 using medial meniscus destabilization model of osteoarthritis (OA-DMM). Methods: In order to explore the presence of neuronal cholinergic fibers in joints, we took advantage of a recent sophisticated method that uses a whole joint immunolabeling protocol: after 3Disco clearing, in toto 3D immunofluorescence was performed to evaluate the colocalization of peripherin (nerve marker) and choline acetyltransferase (ChAT, cholinergic fiber marker) in human OA bone and cartilage samples, obtained from OA patients undergoing knee arthroplasty, and from 6-day-old C57Bl6 mice. Acquisitions were performed using an ultramicroscope and analyzed using IMARIS. Primary cultures of murine osteoblasts and chondrocytes were obtained from 6-day-old C57Bl6 mice calvaria and cartilage knees and femoral heads respectively. Primary cultures of human OA chondrocytes were obtained from human OA cartilage obtained from OA patients undergoing knee arthroplasty. At confluence, RNA was extracted to determine by PCR the expression of the molecular actors of production, transport and degradation of Ach as well as the nicotinic subunits alpha 1 to 7, alpha 9 and beta 1 to 4. In vitro, WT and KO for Chrna7 (Chrna7 - / -) chondrocytes and WT osteoblasts were treated with IL1β. In order to study the role of nicotinic receptors in cell activation, murine chondrocytes and osteoblasts were pretreated with nicotine at 1; 10 or 100 μM. After 24 hours, we collected mRNA and medium to quantify the expression of cytokines (IL6, IL8-Kc) and metalloproteinases (MMP3) along with RANK-ligand and osteoprotegerin expression for osteoblasts using quantitative PCR. DMM was performed on the right knees of 12-week-old WT (n=4) and KO Chrna7 - / - mice (n=4 per group). After 9 weeks, histological analysis of the knees was performed using safranin and light green coloration to determine the OARSI score on femoral and tibial compartments (from 0 to 12). Results: Cholinergic fibers were present in subchondral bone of all 3 OA joints explants analyzed coming from 3 different patients (Figure 1). No cholinergic nerves were found in cartilage. We also confirmed the presence of cholinergic fibers in murine bone, located into the cortical bone. Human OA and murine chondrocytes as well as murine osteoblasts express the whole system needed for the production (carnityl acetyltransferase), the transport (vesicular transporter of Ach) and the degradation of Ach (acetylcholine transferase). All these cells express nicotinic subunits alpha 4, 5, 6, 7 and Beta 4. IL1β stimulation results in a significant increased expression of IL6, IL8 / Kc and MMP3 by WT and Chrna7-/- chondrocytes. Nicotine pretreatment has anti-inflammatory and anti-catabolic properties characterized by a significant decrease in the expression of IL6, IL8 / Kc and MMP3 induced by IL1β (decreased of 62%, 16 % and 64% of IL6, IL8/kc and MMP3 with nicotine 10μM pretreatment, n=6; p<0.05). Interestingly, nicotine had no effect on Chrna7 - / - chondrocytes (n=4) suggesting a predominant role of this receptor in the effects mediated by nicotine in chondrocytes. Compared to IL1β stimulation, RANKL expression was decreased of 22% and 57% and OPG expression was increased of 59% and 90% in murine osteoblasts treated by nicotine 10μM and 100μM respectively suggesting a role of nicotine in bone remodeling. Finally, after DMM, Chrna7 -/- mice displayed more OA lesions than their WT counterparts with a mean±SD OARSI score of 9.4±2.5 for Chrna7 -/- and 7.1±2.2 for WT (Figure 2). Conclusions: Neuronal and non-neuronal cholinergic system are present in murine and human joints. The activation of nicotinic receptors displays anti-inflammatory, anti-catabolic and antiresorptive properties on bone and cartilage. This protective effect is mediated, at least in part, by the alpha 7 nicotinic receptor. We show here for the first time that activating the cholinergic system could be a novel way for treating OA.Figure 2Representative Safranin O/Light Green stained sections of Chrna7 -/- and WT knee mice, 9 weeks after DMM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)