Ox-mtDNA can amplify pyroptosis through direct engagement of the NLRP3 inflammasome, in addition to DNA-recognition receptors such as TLR9. Among the DAMPs released by pyroptotic cytolysis, and mitochondrial membrane depolarization, is ROS-oxidized mitochondrial DNA (ox-mtDNA) which is then recognized by pattern recognition receptors (PRRs). Increased cell-free DNA levels have been reported in chronic inflammatory disorders, where there is also increased reactive oxygen species (ROS) induced during pyroptotic events, which not only further contribute to inflammasome activation but to the release of cell-free DAMPs into the extracellular space. Elucidating those signals is important for understanding why the inflammasome is active in MDS.
Upon pyroptosis execution, cells expel their intracellular contents, including DAMPs, into the extracellular space triggering a feed-forward process that propagates inflammasome and innate immune activation to neighboring cells. Constitutive inflammasome activation prevents HPSCs from differentiating, causing cytopenias and contributing to expansion of the malignant clone. S100A9 engages Toll-like receptor (TLR)-4 to initiate pyroptosis in hematopoietic stem and progenitor cells (HSPCs) through the Nod-like receptor 3 (NLRP3) inflammasome complex, leading to the induction of clinically evident ineffective hematopoiesis. We and others demonstrated that the danger-associated molecular pattern (DAMP) protein S100A9 plays a critical role in the pathogenesis of MDS by creating an inflammatory microenvironment. Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure diseases typified by chronic BM inflammation, ineffective hematopoiesis, and peripheral blood (PB) cytopenias. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA.
The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death.
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