Gasdermin-D tags mitochondria to serve as mobile propagators of inflammatory responses

Mitochondria are motile, multifunctional organelles that partake in a multitude of cellular processes including energy production, contributing to anti-viral innate immune signaling, and intracellular crosstalk with other cell organelles. Horizontal mitochondrial transfer, which entails the export of whole mitochondria or mitochondrial components from one cell to another, has hitherto been demonstrated to be a mechanism to increase the survival of, and dampen inflammation within recipient cells. Mitochondria often become dysfunctional during homeostasis (over the course of normal aging) or disease. It is unclear whether the horizontal transfer of damaged mitochondria can be utilized as a mechanism to conversely propagate/amplify pro-inflammatory signaling within recipient cells. Myeloid cells employ innate-immune signaling pathways to recognize damage or pathogen-associated molecular patterns (DAMPs or PAMPs). The NLRP3 (Nod-Like-Receptor Protein-3) Inflammasome and the STING (Stimulator of Interferon Genes) complexes are prominent innate-immune assemblies that can be activated following local mitochondrial dysfunction. NLRP3 senses mitochondria-derived reactive oxygen species (mitoROS), and the STING complex sensor protein cGAS (cyclic GMP-AMP Synthase) can detect mitochondria- derived DNA. Inflammasome assembly culminates in the Casp1-dependent processing of the cytokine Interleukin-1beta and the proteolytic activation of the pore-forming protein Gasdermin-D (GSDMD), which initiates a form of inflammatory cell death called pyroptosis. Recently-published work, as well as our preliminary data utilizing macrophages and microglia, suggest that NLRP3 Inflammasome activation results in the mitochondrial translocation of cleaved GSDMD, serving to permeabilize mitochondrial membranes, amplifying mitoROS release and consequent Inflammasome responses. Our preliminary data suggest that damaged, cl-GSDMD-laden mitochondria can be horizontally transferred from donor to recipient cells, serving as mobile, NLRP3 Inflammasome/STING-activating agents. Therefore, we seek to explore the mechanisms and consequences of this fundamental, hitherto uncharacterized mechanism of cellular communication that utilizes GSDMD-laden mitochondria to propagate ligand-independent inflammatory signaling. This proposal leverages the PI's expertise in innate-immune signaling with collaborative input from experts in mitochondrial transmission, extracellular vesicle biology, proteomics, and two-photon imaging techniques. We have acquired and/or generated multiple in-vitro and in-vivo tools that facilitate the pursuit of our aims. These include BioID-based approaches to characterize the GSDMD-interacting proteome in an unbiased manner, constructs/transgenic tools that will facilitate the real-time tracking of GSDMD mitochondrial translocation/ transmission in live cells, as well as systems/rigs to investigate this signaling pathway in-vivo. Project Number: 1R35GM162199-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Nikhil Panicker | Institution: CLEVELAND CLINIC LERNER COM-CWRU, CLEVELAND, OH | Award Amount: $431,750 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - D Study Section[MRAD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11267310