Blocking STING Pathway Halts Mitochondrial Damage Driving Sepsis Lung Injury
Researchers uncover how the STING immune pathway triggers mitochondrial fragmentation and DNA release, fueling deadly lung inflammation in sepsis.
Summary
Scientists have mapped a previously unknown chain of events where the STING immune sensor, when activated by bacterial toxins, drives mitochondrial fragmentation and releases mitochondrial DNA—amplifying dangerous lung inflammation in sepsis. Using human COVID-19 patient data, mouse models of LPS-induced lung injury, and human macrophage cell lines, researchers found that STING coordinates a destructive complex between two proteins (N-GSDMD and Drp1) on mitochondrial membranes. This complex punches holes in mitochondria, releasing DNA that re-activates STING in a vicious feedback loop. Blocking STING genetically or with the drug disulfiram (an existing anti-alcoholism medication) interrupted this loop, reduced inflammation, and protected lung tissue—suggesting a potential new treatment angle for sepsis-induced acute lung injury.
Detailed Summary
Sepsis-induced acute respiratory distress syndrome (ARDS) carries a hospital mortality rate approaching 46% in severe cases, yet the molecular mechanisms that amplify lung inflammation remain incompletely understood. This study, published in Cellular and Molecular Life Sciences, provides a detailed mechanistic account of how the cGAS/STING innate immune pathway hijacks mitochondrial biology in macrophages to sustain and amplify inflammatory injury during acute lung injury (ALI).
The researchers began with single-cell RNA sequencing (scRNA-seq) analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients (GEO dataset GSE145926) and LPS-treated mice (GSE276682), isolating macrophage populations. Using a previously validated pyroptosis-related gene score (PScore), they found striking co-upregulation of STING (TMEM173) and GSDMD expression in macrophages from both COVID-19 ARDS patients and LPS-challenged mice, with a strong positive correlation between STING pathway activity and pyroptosis scores. Notably, STING correlated with GSDMD but not with GSDMB, pointing to pathway specificity.
In a time-course mouse model of intratracheal LPS challenge (n=6 per timepoint), lung injury scores worsened progressively, peaking at 12 hours by histology. Crucially, mitochondrial DNA (mtDNA) in BALF rose as early as 6 hours—before peak histological injury—identifying mtDNA release as an early upstream driver rather than a downstream consequence. Western blot data showed STING/TBK1/IRF3 pathway activation beginning at 6 hours, while NLRP3/Caspase-1/GSDMD cleavage peaked at 24 hours, and pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-β were significantly elevated in lung tissue at 24 hours.
In THP-1 human macrophage cells, the cleaved N-terminal fragment of GSDMD (N-GSDMD) translocated specifically to mitochondria at 4 hours post-LPS (1 µg/mL), before later redistributing to the plasma membrane under stronger stimulation (LPS + ATP). Flow cytometry confirmed a significant increase in N-GSDMD-positive mitochondria following LPS treatment. The GSDMD inhibitor disulfiram (DSF), an FDA-approved drug used for alcohol dependence, dose-dependently reduced mitochondrial N-GSDMD accumulation, blocked cytosolic mtDNA release (measured by four mtDNA loci: ND-1, D-loop, COXIII, Cytochrome B), suppressed STING/TBK1/IRF3 signaling, and lowered IL-1β, IL-6, and IFN-β mRNA levels. PicoGreen and MitoTracker co-staining visually confirmed that DSF pretreatment prevented mtDNA escape from mitochondria.
The study further demonstrated that STING deficiency attenuated mitochondrial calcium uptake, which in turn reduced recruitment of the mitochondrial fission protein Drp1 to the outer mitochondrial membrane. STING was found to mediate direct interaction between Drp1 and N-GSDMD on the mitochondrial membrane, and this N-GSDMD/Drp1 complex mutually amplified pore assembly, driving mitochondrial membrane rupture and further mtDNA release—establishing a positive feedback loop through STING re-activation. Genetic STING knockout and pharmacological STING inhibition (using C-176) both interrupted this cascade and reduced lung injury severity in vivo. Collectively, the findings define a STING→mitochondrial calcium→Drp1/N-GSDMD→mtDNA→STING feedback loop as a central driver of macrophage-mediated lung inflammation in sepsis.
Key Findings
- MtDNA in BALF rose significantly at 6 hours post-LPS challenge—preceding peak histological lung injury at 12 hours—identifying it as an early upstream driver of ALI
- STING (TMEM173) expression showed strong positive correlation with GSDMD but not GSDMB in scRNA-seq from COVID-19 ARDS patient macrophages (GSE145926)
- N-GSDMD translocated to mitochondria peaking at 4 hours post-LPS (1 µg/mL) in THP-1 macrophages, before plasma membrane redistribution under stronger stimulation (LPS + ATP)
- Disulfiram (DSF) dose-dependently suppressed mitochondrial N-GSDMD accumulation, blocked cytosolic mtDNA release across four mtDNA loci (ND-1, D-loop, COXIII, Cytochrome B), and reduced IL-1β, IL-6, and IFN-β mRNA levels in LPS-challenged macrophages
- STING pathway (TBK1/IRF3) was activated first at 6 hours while NLRP3/Caspase-1/GSDMD cleavage peaked later at 24 hours, establishing a sequential activation timeline
- STING deficiency reduced mitochondrial calcium uptake, attenuating Drp1 recruitment to mitochondria and disrupting the N-GSDMD/Drp1 pore-forming complex
- Both genetic STING knockout and pharmacological inhibition with C-176 reduced lung injury severity in mouse LPS-ALI models, confirming therapeutic target validity
Methodology
The study combined scRNA-seq analysis of publicly available GEO datasets (GSE145926 for COVID-19 ARDS; GSE276682 for murine LPS-ALI) with an intratracheal LPS mouse model using 6 animals per timepoint across a 0–24 hour time course. In vitro experiments used LPS-challenged THP-1 human macrophages with concentration-gradient (up to 10 µg/mL) and time-course designs, incorporating subcellular fractionation, flow cytometry, immunofluorescence, co-immunoprecipitation, and qPCR for four mtDNA markers. Genetic approaches included STING knockout models, and pharmacological tools included disulfiram (GSDMD inhibitor) and C-176 (STING inhibitor); statistical methods included standard group comparisons with quantification from western blot densitometry and fluorescence imaging.
Study Limitations
The study is primarily mechanistic and preclinical, relying on mouse LPS models and THP-1 cell lines, which may not fully recapitulate the complexity of human sepsis-induced ARDS. The scRNA-seq analyses are correlative, drawn from publicly available datasets without prospective validation in independent patient cohorts. The authors do not explicitly discuss conflicts of interest, and dose translation of disulfiram from in vitro/mouse models to humans requires further pharmacokinetic and safety validation in sepsis populations.
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