Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia

Abstract

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q ₁₀ (N5/C ₁₀) _, which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C ₁₀ protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.

Author Summary

Necroptosis is a pro-inflammatory mode of programmed cell death that is marked by the intentional disruption of host membranes and the release of pro-inflammatory cytosolic components into the milieu. Until just recently necroptosis was not appreciated to play a role during infectious disease. Herein, we demonstrate that alveolar macrophages exposed to the nosocomial pathogen Serratia marcescens undergo necroptosis and this leads to enhanced disease severity. We subsequently demonstrate that necroptosis is the principle mode of cell death experienced by macrophages following their exposure to bacteria that produce pore-forming toxins (PFTs). We dissect the molecular mechanisms by which PFTs induce necroptosis and demonstrate that loss of ion homeostasis at the cell membrane and mitochondrial damage result in ATP depletion and ROS generation that together are responsible. Finally, we demonstrate that inhibition of necroptosis by various means is protective against hemorrhagic pneumonia caused by S. marcescens.