Tempol alleviates autoimmune uveitis by targeting the oxidative stress-mediated cGAS-STING-NF-kB signaling pathway to restore M1/M2 macrophage polarization balance

Purpose: To evaluate whether Tempol alleviates autoimmune uveitis by attenuating oxidative stress-associated mitochondrial injury, reducing cytosolic mitochondrial DNA (mtDNA) accumulation, and suppressing activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-nuclear factor kappa-B (cGAS-STING-NF-κB) pathway, thereby limiting M1 macrophage polarization.

Methods: Peripheral blood samples were collected from uveitis patients and healthy controls, and an experimental autoimmune uveitis (EAU) rat model was established. Rats were assigned to normal control (NC), EAU, EAU with cGAS shRNA knocking down (sh-cGAS), EAU with empty vector (Vector), EAU with STING inhibitor H151 (H-151), and EAU with Tempol (Tempol) groups. Oxidative stress, mitochondrial status, cytosolic mtDNA, activation of the cGAS-STING pathway, and macrophage polarization were evaluated by Western blotting, Q-PCR, flow cytometry, Seahorse XF metabolic analysis, transmission electron microscopy, and Other methods.

Results: Compared with controls, uveitis patients and EAU rats exhibited increased Reactive Oxygen Species (ROS), loss of mitochondrial membrane potential, impaired mitochondrial respiration, and elevated cytosolic mtDNA, accompanied by activation of the cGAS-STING-NF-κB axis (increased cGAS, STING, p-TBK1, p-p65, and IFN-β) along with enhanced downstream pro-inflammatory output (increased TNF-α and iNOS), and a shift toward M1 macrophage polarization. Tempol reduced oxidative stress, improved mitochondrial function, ultrastructure, and Mitophagy, decreased cytosolic mtDNA, and attenuated cGAS-STING-NF-κB activation. Likewise, sh-cGAS or H-151 suppressed pathway activation, restored antioxidant defense (Nrf2/Keap1/HO-1), improved mitophagy-related quality control (increased PINK1/Parkin with reduced p62), reduced M1 macrophage polarization, and alleviated ocular inflammatory injury.

Conclusion: Tempol mitigates autoimmune uveitis by improving mitochondrial homeostasis, reducing cytosolic mtDNA, decreasing cGAS-STING-NF-κB activation, and enhancing mitophagy-related quality control, accompanied by reduced M1 macrophage polarization and ocular tissue injury. Findings from cGAS knockdown and STING inhibition provide mechanistic support for this oxidative stress-mtDNA-cGAS-STING axis as a therapeutic target in immune-mediated uveitis.