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 Q10 (N5/C10), which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C10 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.

Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1−/−, Ripk3−/− and Mlkl−/− mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling.

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.

dStreptococcus pneumoniae (the pneumococcus) is capable of invading the heart. Herein we observed that pneumococcal invasion of the myocardium occurred soon after development of bacteremia and was continuous thereafter. Using immunofluorescence microscopy (IFM), we observed that S. pneumoniae replication within the heart preceded visual signs of tissue damage in cardiac tissue sections stained with hematoxylin and eosin. Different S. pneumoniae strains caused distinct cardiac pathologies: strain TIGR4, a serotype 4 isolate, caused discrete pneumococcus-filled microscopic lesions (microlesions), whereas strain D39, a serotype 2 isolate, was, in most instances, detectable only using IFM and was associated with foci of cardiomyocyte hydropic degeneration and immune cell infiltration. Both strains efficiently invaded the myocardium, but cardiac damage was entirely dependent on the pore-forming toxin pneumolysin only for D39. Early microlesions caused by TIGR4 and microlesions formed by a TIGR4 pneumolysin-deficient mutant were infiltrated with CD11b؉ and Ly6G-positive neutrophils and CD11b ؉ and F4/80-positive (F4/80 ؉ ) macrophages. We subsequently demonstrated that macrophages in TIGR4-infected hearts died as a result of pneumolysin-induced necroptosis. The effector of necroptosis, phosphorylated mixed-lineage kinase domain-like protein (MLKL), was detected in CD11b؉ and F4/80 ؉ cells associated with microlesions. Likewise, treatment of infected mice and THP-1 macrophages in vitro with the receptor-interacting protein 1 kinase (RIP1) inhibitor necrostatin-5 promoted the formation of purulent microlesions and blocked cell death, respectively. We conclude that pneumococci that have invaded the myocardium are an important cause of cardiac damage, pneumolysin contributes to cardiac damage in a bacterial strain-specific manner, and pneumolysin kills infiltrated macrophages via necroptosis, which alters the immune response.