Mitochondrial DNA leakage induces odontoblast inflammation via the cGAS-STING pathway

Abstract

Background 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. Methods 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. Results 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. Conclusion 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. Graphic abstract

Mitochondrial DNA leakage exacerbates odontoblast inflammation through gasdermin D-mediated pyroptosis

“…Moreover, we found that leaked cytosolic mtDNA acts as a DAMP to activate the NLRP3 inflammasome, causing pyroptosis and intensifying inflammation in odontoblast-like cells. From our previous [ 11 ] and present study, cytosolic mtDNA promotes pulp inflammation through the pyroptosis pathway and the STING pathway, suggesting that protecting mitochondria and reducing mtDNA leakage under inflammatory stimuli can delay the progression of inflammation. To further explore the relationship between these two signaling pathways, we found that the STING pathway was compensatorily activated after GSDMD knockdown.…”

“…Human dental pulp tissue was collected as described above [ 11 , 19 ] and separated into two groups: 16 healthy teeth and 16 pulpitis teeth. The healthy group included no history of dental pulp disease and no oral diseases, such as dental caries.…”

“…Cytosolic mtDNA participates in inflammation progression in various diseases, such as rheumatoid arthritis [ 10 ], lupus and atherogenesis [ 7 ]. Our previous studies indicate that lipopolysaccharide (LPS) induces odontoblast mitochondrial dysfunction and mtDNA leakage to activate robust inflammation [ 8 , 11 ]. Cytosolic mtDNA can directly activate various pattern recognition receptors (PRRs) to cause inflammation, such as TLR9 and inflammasomes [ 6 ].…”

Protective effect of Astragaloside IV on chronic intermittent hypoxia-induced vascular endothelial dysfunction through the calpain-1/SIRT1/AMPK signaling pathway

“…All images were analyzed by ImageJ software. Mitochondrial reactive oxygen species (mitoROS) were determined using MitoSOX as previously described ( Zhou et al, 2021 ). Briefly, the cells were incubated with 5 µM MitoSOX for 10 min at 37°C in the dark.…”

What role of the cGAS-STING pathway plays in chronic pain?

“…Compared with nDNA, mtDNA is more unstable and vulnerable to the effects of oxidative stress due to its proximity to mitochondrial reactive oxygen species (mtROS) and a lack of repair machinery. mtDNA stress may contribute to the cGAS-STING pathway activation and type 1 interferon responses in various pathological states, including infectious diseases, cancer, neurodegeneration, and other mitochondria-related illnesses (Zhou et al, 2021 ). Importantly, it has been established that mtDNA depletion inhibited the cGAS-STING pathway and nuclear translocation of p65 and IRF3 (Zhou et al, 2021 ).…”