The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei- MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
Department of Endodontics, School and Hospital of Stomatology, Wuhan University, HongShan District, LuoYu Road No. 237, Wuhan, 430079, China.
Cell Commun Signal. 2021 May 20;19(1):58. doi: 10.1186/s12964-021-00738-7.
Mitochondrial DNA (mtDNA) is a vital driver of inflammation when it leaks from damaged mitochondria into the cytosol. mtDNA stress may contribute to cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway activation in infectious diseases. Odontoblasts are the first cells challenged by cariogenic bacteria and involved in maintenance of the pulp immune and inflammatory responses to dentine-invading pathogens. In this study, we investigated that mtDNA as an important inflammatory driver participated in defending against bacterial invasion via cGAS-STING pathway in odontoblasts.
The normal tissues, caries tissues and pulpitis tissues were measured by western blotting and immunohistochemical staining. Pulpitis model was built in vitro to evaluated the effect of the cGAS-STING pathway in odontoblast-like cell line (mDPC6T) under inflammation. Western blot and real-time PCR were performed to detect the expression of cGAS-STING pathway and pro-inflammatory cytokines. The mitochondrial function was evaluated reactive oxygen species (ROS) generated by mitochondria using MitoSOX Red dye staining. Cytosolic DNA was assessed by immunofluorescent staining and real-time PCR in mDPC6T cells after LPS stimulation. Furthermore, mDPC6T cells were treated with ethidium bromide (EtBr) to deplete mtDNA or transfected with isolated mtDNA. The expression of cGAS-STING pathway and pro-inflammatory cytokines were measured.
The high expression of cGAS and STING in caries and pulpitis tissues in patients, which was associated with inflammatory progression. The cGAS-STING pathway was activated in inflamed mDPC6T. STING knockdown inhibited the nuclear import of p65 and IRF3 and restricted the secretion of the inflammatory cytokines CXCL10 and IL-6 induced by LPS. LPS caused mitochondrial damage in mDPC6T, which promoted mtDNA leakage into the cytosol. Depletion of mtDNA inhibited the cGAS-STING pathway and nuclear translocation of p65 and IRF3. Moreover, repletion of mtDNA rescued the inflammatory response, which was inhibited by STING knockdown.
Our study systematically identified a novel mechanism of LPS-induced odontoblast inflammation, which involved mtDNA leakage from damaged mitochondria into the cytosol stimulating the cGAS-STING pathway and the inflammatory cytokines IL-6 and CXCL10 secretion. The mtDNA-cGAS-STING axis could be a potent therapeutic target to prevent severe bacterial inflammation in pulpitis. Video Abstract.
当受损线粒体中的线粒体 DNA（mtDNA）漏入细胞质时，它是炎症的重要驱动因素。mtDNA 应激可能导致传染性疾病中环鸟苷酸-腺苷酸合酶（cGAS）刺激干扰素基因（STING）途径的激活。成牙本质细胞是受到致龋细菌挑战的第一类细胞，并且参与维持牙髓对入侵牙本质病原体的免疫和炎症反应。在这项研究中，我们研究了 mtDNA 作为一种重要的炎症驱动因素，通过 cGAS-STING 途径参与成牙本质细胞抵抗细菌入侵。
通过 Western blot 和免疫组织化学染色测量正常组织、龋组织和牙髓炎组织。体外建立牙髓炎模型，以评估炎症下成牙本质样细胞系（mDPC6T）中 cGAS-STING 途径的作用。通过 Western blot 和实时 PCR 检测 cGAS-STING 途径和促炎细胞因子的表达。使用 MitoSOX Red 染料染色评估线粒体产生的活性氧（ROS）来评估线粒体功能。用 LPS 刺激后，通过免疫荧光染色和实时 PCR 评估 mDPC6T 细胞中的细胞质 DNA。此外，用溴化乙锭（EtBr）处理 mDPC6T 细胞以耗尽 mtDNA 或转染分离的 mtDNA。测量 cGAS-STING 途径和促炎细胞因子的表达。
患者龋病和牙髓炎组织中 cGAS 和 STING 高表达，与炎症进展相关。炎症 mDPC6T 中激活了 cGAS-STING 途径。STING 敲低抑制了 LPS 诱导的 p65 和 IRF3 的核内导入，并限制了 CXCL10 和 IL-6 等炎症细胞因子的分泌。LPS 引起 mDPC6T 线粒体损伤，促进 mtDNA 漏入细胞质。耗尽 mtDNA 抑制了 cGAS-STING 途径和 p65 和 IRF3 的核内易位。此外，mtDNA 的补充挽救了炎症反应，该反应被 STING 敲低抑制。
我们的研究系统地确定了 LPS 诱导的成牙本质细胞炎症的新机制，即受损线粒体中的 mtDNA 漏入细胞质，刺激 cGAS-STING 途径和促炎细胞因子 IL-6 和 CXCL10 的分泌。mtDNA-cGAS-STING 轴可能是预防牙髓严重细菌炎症的有效治疗靶点。
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