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NOD1 PROTECTS NON-MYELOID CELLS FROM SHIGELLA FLEXNERI-INDUCED CELL DEATH BY PREVENTING MITOCHONDRIAL DAMAGE
LA Carneiro1, LH Travassos2, I Tattoli1, JG Magalhães2, DJ Philpott2, SE Girardin1
1Department of Laboratory Medicine and Pathobiology; 2Department of Immunology, University of Toronto, Toronto, Ontario
In contrast to macrophages that undergo apoptosis within a few hours following Shigella flexneri infection, non-myeloid cells seem to be protected against S. flexneri-induced cell death. In these cells, Nod1 plays a crucial role in the detection of cytosolic S. flexneri and initiation of inflammatory/immune responses. Thus, we hypothesized that Nod1 could be involved in the resistance of non-myeloid cells to S. flexneri-induced cell death.
We analyzed the cell death induced by S. flexneri in macrophages and mouse embryonic fibroblasts (MEFs) from wild-type and Nod1-deficient cells by flow cytometry (annexin-V and propidium iodide staining), TUNEL assay and eletronic microscopy. Mitochondrial membrane potential was analyzed using the cationic probe DiOC6. Western blots and selective inhibitors of several signaling pathways were used to dissect the mechanisms involved in the cell death observed in Nod1-deficient cells.
We show here that, while no difference in the cell death rates was observed between wild-type and Nod1-deficient macrophages after S. flexneri infection, Nod1-deficient MEFs were more susceptible to infection than wild-type controls, with significantly higher rates of cell death. Furthermore, infected MEFs exhibited features of necrosis (membrane permeabilization, vacuolization, mitochondrial swelling and loss of mitochondrial potential), which contrasts with the apoptotic pattern that occurs in macrophages. By blocking cyclophilin D, a key mediator of mitochondrial permeability transition, we observed that the cell death in the Nod1-deficient MEFs decreased to the levels observed in wild-type MEFs. In addition, MAPKs inhibitors also reduced cell death rates in Nod1-deficient cells, while NF-kB and PI3K inhibitors increased cell death rates in wild-type MEFs to levels similar to those observed in Nod1-deficient cells.
In conclusion, we demonstrated that Nod1 plays a key role in the protection of non-myeloid cells from necrotic-like cell death following S. flexneri infection, by preventing mitochondrial permeability transition.