No 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.

Mitochondrial DNA (mtDNA) is a potent agonist of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-Stimulator of Interferon Genes (STING)-type I interferon (IFN-I) pathway. However, the kinetics of mtDNA detection by cGAS or other nucleic acid sensors and the exact immunostimulatory features of mtDNA remain poorly defined. Here, we show that robust and sustained IFN-I responses downstream of mtDNA stress require the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) in a cell death independent manner. Cells experiencing persistent mtDNA release display robust ZBP1 expression, as well as a marked increase in the cytoplasmic pool of cGAS. Biochemical and microscopy analyses reveal that the receptor-interacting protein homotypic interaction motif (RHIMs) of ZBP1 bind the N-terminus of cGAS, leading to its entrapment in the cytoplasm. Moreover, we show that genetic and pharmacologic induction of mtDNA stress leads to the mitochondrial and cytoplasmic accumulation of Z-form DNA. Nuclease treatment or deletion of Z-DNA binding domains of ZBP1 reduces its interaction with cGAS and impairs cGAMP production downstream of mtDNA stress. Finally, we uncover that ZBP1 is a novel regulator of IFN-I-mediated disease pathology, working in tandem with the cGAS-STING pathway to sense mtDNA instability and sustain IFN-I signaling that contributes to cardiac remodeling and heart failure. These results provide new insight into the molecular mechanisms of mtDNA sensing by the innate immune system and reveal that ZBP1 is a cooperative partner for cGAS. Moreover, our findings highlight ZBP1 as a potential therapeutic target in heart failure and other disorders where mtDNA instability drives disease-promoting IFN-I and inflammatory responses.

Human genetic variants associated with mitochondrial dysfunction have been linked to chronic inflammatory diseases as well as susceptibility to infection. However, the mechanistic impact that these variants have on the immune system is poorly understood. We have discovered that macrophages harboring the common Parkinson’s disease associated variant, Lrrk2G2019S are more prone to cell death in response to both Mycobacterium tuberculosis (Mtb) infection and AIM2 inflammasome activation. Unexpectedly, the enhanced cell death in Lrrk2G2019S macrophages is driven by increased susceptibility to gasdermin D (GSDMD)-mediated mitochondrial pore formation, which releases accumulated mitochondrial ROS and pushes Lrrk2G2019S cells to undergo RIPK3-mediated necroptosis. Consistent with elevated necroptotic cell death, infection of Lrrk2G2019S mice with Mtb elicits dramatic hyperinflammation and exacerbated pathogenesis via enhanced neutrophil infiltration. Remarkably, expression of hLRRK2G2019S in Drosophila melanogaster recapitulates a similar phenotype, suggesting that Lrrk2G2019S plays an evolutionarily conserved role in regulating innate immunity. Our findings demonstrate that altered mitochondrial function can reprogram canonical innate immune and cell death pathways to elicit distinct immune outcomes, providing mechanistic insights into why mutations in LRRK2 are associated with susceptibility to chronic inflammatory and infectious diseases. Supported by grants from the Parkinson's Foundation (PF-FBS-1932), NIH (R01 AI125512), Michael J. Fox Foundation (grant 12185), and the Texas A&M Clinical Science and Translational Research (CSTR) Pilot Grant Program.