Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

Mitochondrial ROS promotes susceptibility to infection via gasdermin D-mediated necroptosis.

Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.

Mitochondrial DNA release and sensing in innate immune responses.

Mitochondria are pleiotropic organelles central to an array of cellular pathways including metabolism, signal transduction, and programmed cell death. Mitochondria are also key drivers of mammalian immune responses, functioning as scaffolds for innate immune signaling, governing metabolic switches required for immune cell activation, and releasing agonists that promote inflammation. Mitochondrial DNA (mtDNA) is a potent immunostimulatory agonist, triggering pro-inflammatory and type I interferon responses in a host of mammalian cell types. Here we review recent advances in how mtDNA is detected by nucleic acid sensors of the innate immune system upon release into the cytoplasm and extracellular space. We also discuss how the interplay between mtDNA release and sensing impacts cellular innate immune endpoints relevant to health and disease.

Caspase-11 drives macrophage hyperinflammation in models of Polg-related mitochondrial disease.

Mitochondrial diseases (MtD) represent a significant public health challenge due to their heterogenous clinical presentation, often severe and progressive symptoms, and the lack of effective therapies. Environmental exposures, such bacterial and viral infection, can further compromise mitochondrial function and exacerbate the progression of MtD. Infections in MtD patients more frequently progress to sepsis, pneumonia, and other detrimental inflammatory endpoints. However, the underlying immune alterations that enhance immunopathology in MtD remain unclear, constituting a key gap in knowledge that complicates treatment and increases mortality in this population. Here we employ in vitro and in vivo approaches to clarify the molecular and cellular basis for innate immune hyperactivity in models of polymerase gamma (Polg)-related MtD. We reveal that type I interferon (IFN-I)-mediated upregulation of caspase-11 and guanylate-binding proteins (GBPs) increase macrophage sensing of the opportunistic microbe Pseudomonas aeruginosa (PA) in Polg mutant mice. Furthermore, we show that excessive macrophage cytokine secretion and pyroptotic cell death contribute to lung inflammation and morbidity after infection with PA. Our work sheds new light on innate immune dysregulation in MtD and reveals potential targets for limiting infection- and inflammation-related complications in Polg-related MtD.

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

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. © 2022 Wiley Periodicals LLC. Basic Protocol 1: siRNA-mediated knockdown of TFAM to induce mtDNA instability, cytosolic release, and activation of the cGAS-STING pathway Alternate Protocol: Pharmacological induction of mtDNA release and cGAS-STING activation using ABT-737 and Q-VD-OPH Basic Protocol 2: Isolation and quantitation of DNA from cytosolic, mitochondrial, and nuclear fractions Basic Protocol 3: Pharmacological inhibition of mtDNA replication and release.