Mitochondrial DNA release contributes to neuropathic pain via a cGAS-STING-IRF3-CMPK2-associated immunometabolic feedback mechanism.

BACKGROUND: Innate immune-driven neuroinflammation in the spinal cord is a key mechanism underlying neuropathic pain (NP). Increasing evidence indicates that mitochondrial dysfunction and metabolic stress critically influence inflammatory responses. However, the mechanistic link between mitochondrial impairment and persistent neuroinflammation in NP remains incompletely understood.

METHODS: A peripheral nerve injury model was used to induce NP in mice. Mitochondrial integrity, mitochondrial DNA (mtDNA) release, and activation of the cGAS-STING-IRF3 pathway were examined in the spinal cord using immunofluorescence, molecular analyses, and single-cell RNA sequencing. Genetic silencing of CMPK2 was achieved by adeno-associated virus delivery, and pharmacological inhibition was performed using nordihydroguaiaretic acid (NDGA). Pain-related behaviors were assessed in vivo. Complementary in vitro experiments were conducted in BV2 cells and primary microglia to evaluate mitochondrial function and mtDNA-driven innate immune activation.

RESULTS: Peripheral nerve injury induced mitochondrial damage in the spinal cord, accompanied by cytosolic mtDNA release and activation of cGAS-STING-IRF3 signaling. IRF3 was observed to associate with the CMPK2 promoter and regulate CMPK2 transcription, consistent with a potential feedback mechanism that may exacerbate mitochondrial stress, enhance mtDNA release, and sustain innate immune activation. Single-cell RNA sequencing and immunofluorescence analyses revealed that CMPK2 was expressed in multiple spinal cord cell types, with microglia representing a major population contributing to CMPK2 upregulation in the spinal dorsal horn after nerve injury. Genetic silencing or pharmacological inhibition of CMPK2 was associated with reduced cGAS-STING signaling, improved mitochondrial homeostasis, decreased microglial activation, and attenuation of NP-like behaviors in vivo. Consistently, CMPK2 knockdown in microglia attenuated mtDNA-induced innate immune activation and improved mitochondrial function in vitro.

CONCLUSIONS: These findings support a model in which an mtDNA-cGAS-STING-IRF3-CMPK2-associated immunometabolic feedback mechanism operates within the spinal cord microenvironment, with notable microglial involvement, linking mitochondrial dysfunction to sustained neuroinflammation and NP. Targeting mitochondrial immunometabolism may represent a potential therapeutic strategy for chronic inflammatory conditions characterized by persistent innate immune activation.