Objective The non‐neuronal cholinergic system represents non‐neuronal cells that have the biochemical machinery to synthetize de novo and/or respond to acetylcholine (ACh). We undertook this study to investigate this biochemical machinery in chondrocytes and its involvement in osteoarthritis (OA). Methods Expression of the biochemical machinery for ACh metabolism and nicotinic ACh receptors (nAChR), particularly α7‐nAChR, in human OA and murine chondrocytes was determined by polymerase chain reaction and ligand‐binding. We investigated the messenger RNA expression of the human duplicate α7‐nACh subunit, called CHRFAM7A, which is responsible for truncated α7‐nAChR. We assessed the effect of nAChR on chondrocytes activated by interleukin‐1β (IL‐1β) and the involvement of α7‐nAChR using chondrocytes from wild‐type (WT) and α7‐deficient Chrna7−/− mice. The role of α7‐nAChR in OA was explored after medial meniscectomy in WT and Chrna7−/− mice. Results Human and murine chondrocytes express the biochemical partners of the non‐neuronal cholinergic system and a functional α7‐nAChR at their cell surface (n = 5 experiments with 5 samples each). The expression of CHRFAM7A in human OA chondrocytes (n = 23 samples) correlated positively with matrix metalloproteinase 3 (MMP‐3) (r = 0.38, P < 0.05) and MMP‐13 (r = 0.48, P < 0.05) expression. Nicotine decreased the IL‐1β–induced IL‐6 and MMP expression, in a dose‐dependent manner, in WT chondrocytes but not in Chrna7−/− chondrocytes. Chrna7−/− mice that underwent meniscectomy (n = 7) displayed more severe OA cartilage damage (mean ± SD Osteoarthritis Research Society International [OARSI] score 4.46 ± 1.09) compared to WT mice that underwent meniscectomy (n = 9) (mean ± SD OARSI score 3.05 ± 0.9; P < 0.05). Conclusion The non‐neuronal cholinergic system is functionally expressed in chondrocytes. Stimulation of nAChR induces antiinflammatory and anticatabolic activity through α7‐nAChR, but the anticatabolic activity may be mitigated by truncated α7‐nAChR in human chondrocytes. In vivo experiments strongly suggest that α7‐nAChR has a protective role in OA.

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 IL1b. 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 mM. 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 (n¼4) and KO Chrna7 -/mice (n¼4 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 tran...