Infiltrated Macrophages Die of Pneumolysin-Mediated Necroptosis following Pneumococcal Myocardial Invasion

Abstract

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.

Cell Invasion and Pyruvate Oxidase-Derived H 2 O 2 Are Critical for Streptococcus pneumoniae-Mediated Cardiomyocyte Killing

“…After crossing the vascular endothelium, S. pneumoniae damages the myocardium, creating bacterium-associated microlesions. Our published studies using immunofluorescence microscopy suggest that this begins with the interaction of a single pneumococcus with surrounding cardiomyocytes (15). A trypan blue exclusion assay showed that at a low multiplicity of infection (MOI), i.e., less than 10 bacteria per cardiomyocyte, S. pneumoniae was able to induce cell death in approximately 60% of HL-1 cardiomyocytes after 6 h of infection in vitro, without detachment (Fig.…”

“…S2). Pneumolysin is a pore-forming toxin produced by S. pneumoniae (24), and its role in cardiomyocyte killing has already been demonstrated, albeit at late stages of microlesion formation or when administered extracellularly (13,15). In contrast, SpxB is a metabolic enzyme that generates H 2 O 2 as a result of converting pyruvate to acetylphosphate.…”

“…This most likely reflects the events that occur in the heart following bacterial translocation into the myocardium from the bloodstream and serves as a basis of a working model in which cardiomyocytes might serve as a protective niche, following cardiac invasion, that allows S. pneumoniae to replicate while avoiding immune system or host defenses. Ultimately, cardiomyocyte killing occurs from inside the cell, and as documented for mature lesions, bacteria that emerge are able to kill immune cells with extracellular release of pneumolysin (15).…”

“…pneumoniae is thought to invade the heart through the coronary circulation, as most pneumococci detected within the myocardium are close to the vascular network (13,15). Interaction of S. pneumoniae with endothelial cells has been extensively studied as a part of S. pneumoniae translocation across the blood-brain barrier (BBB), which occurs during the development of pneumococcal meningitis (16).…”

Pharmacological Targeting of the Host–Pathogen Interaction: Alternatives to Classical Antibiotics to Combat Drug-Resistant Superbugs

“…Several bacterial pathogens with pore-forming toxins induce macrophage necroptosis, a pro-inflammatory mode of cell death regulated by receptor interacting protein kinases RIP1 and RIP3 and mediated by the effector mixed-lineage kinase domain-like protein MLKL; macrophages deficient in MLKL are consequently resistant to pore-forming toxin-induced cell death. Treatment of mice with necrostatin-5, an inhibitor of RIP1 and/or GW806742X, an inhibitor of MLKL, reduced severity of Serratia marcescens pneumonia in mice, protecting alveolar macrophages from cell death and reducing bacterial burden [33]; necrostatin-5 also reduced pneumolysin-mediated macrophage necroptosis and cardiac damage following pneumococcal bacteremia and myocardial invasion [34]. …”