Pharmacology/Drug Development

Osteoarthritis is characterized by the loss of the articular cartilage, bone remodeling, pain, and disability. No pharmacological intervention can currently halt progression of osteoarthritis. Here, we show that blocking receptor tyrosine kinase-like orphan receptor 2 (ROR2) improves cartilage integrity and pain in osteoarthritis models by inhibiting yes-associated protein (YAP) signaling. ROR2 was up-regulated in the cartilage in response to inflammatory cytokines and mechanical stress. The main ligand for ROR2, WNT5A, and the targets YAP and connective tissue growth factor were up-regulated in osteoarthritis in humans. In vitro, ROR2 overexpression inhibited chondrocytic differentiation. Conversely, ROR2 blockade triggered chondrogenic differentiation of C3H10T/ cells and suppressed the expression of the cartilage-degrading enzymes a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. The chondrogenic effect of ROR2 blockade in the cartilage was independent of WNT signaling and was mediated by down-regulation of YAP signaling. ROR2 signaling induced G protein and Rho-dependent nuclear accumulation of YAP, and YAP inhibition was required but not sufficient for ROR2 blockade-induced chondrogenesis. ROR2 silencing protected mice from instability-induced osteoarthritis with improved structural outcomes, sustained pain relief, and without apparent side effects or organ toxicity. Last, ROR2 silencing in human articular chondrocytes transplanted in nude mice led to the formation of cartilage organoids with more and better differentiated extracellular matrix, suggesting that the anabolic effect of ROR2 blockade is conserved in humans. Thus, ROR2 blockade is efficacious and well tolerated in preclinical animal models of osteoarthritis.

Pain sensation is powerfully modulated by signal processing in the brain, and pain becomes chronic with the dysfunction of the pain modulatory system; however, the underlying mechanisms are unclear. We found that the metabotropic glutamate receptor 5 (mGluR5) in the periaqueductal gray (PAG), the key area of endogenous pain modulation, is persistently active in normal conditions to maintain an appropriate sensory perception. In the neuropathic pain condition, Homer1a, an activity-dependent immediate early gene product, disrupted the persistent mGluR5 activity resulting in chronic pain. Remarkably a single-time blockage of the mGluR5 resulted in chronic neuropathic pain-like symptoms even in the absence of nerve injury. The decline of mGluR5 activity induced the pain modulatory dysfunction with a profound reduction of excitability of PAG neurons. These findings uncover the role of the persistent mGluR5 activity in vivo and provide new insight into how pain becomes chronic with the maladaptive coping of the PAG to pain sensation.

Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3 × daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (-)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes' activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cell-specific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.