Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune‐related Pathways in Mammalian Cells

Abstract

Mitochondria have emerged as key drivers of mammalian innate immune responses, functioning as signaling hubs to trigger inflammation and orchestrating metabolic switches required for phagocyte activation. Mitochondria also contain damage-associated molecular patterns (DAMPs), molecules that share similarity with pathogen-associated molecular patterns (PAMPs) and can engage innate immune sensors to drive inflammation. The aberrant release of mitochondrial DAMPs during cellular stress and injury is an increasingly recognized trigger of inflammatory responses in human diseases. Mitochondrial DNA (mtDNA) is a particularly potent DAMP that engages multiple innate immune sensors, although mounting evidence suggests that cytosolic mtDNA is primarily detected via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. cGAS and STING are widely expressed in mammalian cells and serve as key regulators of type I interferon and cytokine expression in both infectious and inflammatory diseases. Despite growing roles for the mtDNA-cGAS-STING axis in human disease, assays to quantify mtDNA release into the cytosol and approaches to link mtDNA to cGAS-STING signaling are not standardized, which increases the possibility for experimental artifacts and misinterpretation of data. Here, we present a series of protocols for assaying the release of mtDNA into the cytosol and subsequent activation of innate immune signaling in mammalian cells. We highlight genetic and pharmacological approaches to induce and inhibit mtDNA release from mitochondria. We also describe immunofluorescence microscopy and cellular fractionation assays to visualize morphological changes in mtDNA and quantify mtDNA accumulation in the cytosol. Finally, we include protocols to examine mtDNA-dependent cGAS-STING activation by RT-qPCR and western blotting. These methods can be performed with standard laboratory equipment and are highly adaptable to a wide range of mammalian cell types. They will permit researchers working across the spectrum of biological and biomedical sciences to accurately and reproducibly measure cytosolic mtDNA release and resulting innate immune responses.

Protein engineering reveals that gasdermin A preferentially targets mitochondrial membranes over the plasma membrane during pyroptosis

“…Measurement of cytochrome c and mtDNA release into the cytosol was adapted from Bryant et al. ( 49 ). THP-1 monocytes were differentiated overnight and stimulated with nigericin as described previously.…”

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity

“…cDNA was then subjected to qPCR using PerfeCTa SYBR Green FastMix (84069, Quanta). Technical triplicates were run each biological sample, and average expression values were normalized against Rpl37 cDNA using the 2 -1′1′CT method (Bryant et al, 2022). For mtDNA abundance assessment, 10 ng/μL of template DNA was used for qPCR, and expression values of each replicate were normalized against nuclear-encoded Tert or RNA18S (Bryant et al, 2022).…”

“…Subcellular fractionation, DNA isolation and slot blotting 3 x 10 6 MEFs were seeded in 15 cm dishes overnight and treated with vehicle or 500 nM of Doxo for 24 hrs. Subcellular fractionation and DNA isolation were performed as previously described (Bryant et al, 2022). In brief, cells were lysed in digitonin lysis buffer (150 mM NaCl, 50 mM HEPES pH 7.4, and 5 μg/mL digitonin), incubated end over end for 10 min at 4 °C, and centrifuged at 950 x g for 5 min at 4 °C.…”

Systemic inflammatory response syndrome is triggered by mitochondrial damage (Review)

“…STING was initially identified as a key immune molecule that detects nuclear or cytoplasmic DNA fragments from pathogen-infected cells and triggers defensive immune responses ( 54 ). cGAS and STING are expressed in different types of cell, including cancer, immune and non-immune cells ( 55 ). However, increasing evidence has suggested that activation of the STING pathway can lead to both tissue inflammation and damage ( 55 , 56 ).…”

“…cGAS and STING are expressed in different types of cell, including cancer, immune and non-immune cells ( 55 ). However, increasing evidence has suggested that activation of the STING pathway can lead to both tissue inflammation and damage ( 55 , 56 ). mtDNA can promote the onset of inflammatory signaling responses via activating the cGAS/STING/interferon regulatory factor 3 (IRF3) pathway ( 57 , 58 ).…”