Macrophage-derived Exosomes Regulate Neuronal ACSL6 in Experimental Temporomandibular Disorder Pain

Background/Aim: Temporomandibular disorders (TMD) are frequently accompanied by persistent pain, but the underlying mechanisms remain incompletely understood. Macrophage-neuron communication in the trigeminal ganglion (TG) may contribute to TMD pain. This study investigated whether macrophage-derived exosomes (EXOs) promote neuronal inflammation and pain by regulating acyl-CoA synthetase long-chain family member 6 (ACSL6). Materials and Methods: A mouse model of TMD pain was established by intra-articular injection of complete Freund’s adjuvant (CFA) into the temporomandibular joint. Pain behavior, TG histopathology, inflammatory mediator expression, and ACSL6 levels were evaluated. The effects of clodronate liposome-mediated macrophage depletion and GW4869-mediated inhibition of EXO release were examined in vivo. In vitro, conditioned medium (CM) and EXOs derived from tumor necrosis factor-alpha-activated RAW264.7 cells were applied to primary TG neurons and PC12 cells. GW4869 treatment of RAW264.7 cells, ACSL6 overexpression, and docosahexaenoic acid (DHA) supplementation were used for further functional evaluation. Results: CFA reduced the head withdrawal threshold and increased TG inflammation, macrophage activation, and expression of interleukin-6, cyclooxygenase-2, and substance P, while decreasing ACSL6 expression. Macrophage depletion and systemic GW4869 treatment alleviated mechanical hypersensitivity, reduced inflammatory mediator expression, and partially restored ACSL6 levels. Activated macrophage-derived CM and EXOs decreased neuronal ACSL6 expression and enhanced inflammatory responses. Inhibition of EXO release by GW4869 attenuated these effects in primary TG neurons and PC12 cells. ACSL6 overexpression and DHA supplementation independently reduced macrophage-induced neuronal inflammatory responses. Conclusion: Macrophage-derived EXOs contribute to CFA-induced TMD pain and neuronal inflammation and are associated with reduced neuronal ACSL6 expression. ACSL6 may represent a functionally relevant downstream regulator, whereas DHA exerts an independent protective anti-inflammatory effect.