Necroptotic Cell Death Promotes Adaptive Immunity Against Colonizing Pneumococci

Pore-forming toxin (PFT) induced necroptosis exacerbates pulmonary injury during bacterial pneumonia. However, its role during asymptomatic nasopharyngeal colonization and toward the development of protective immunity was unknown. Using a mouse model of Streptococcus pneumoniae (Spn) asymptomatic colonization, we determined that nasopharyngeal epithelial cells (nEC) died of pneumolysin (Ply)-dependent necroptosis. Mice deficient in MLKL, the necroptosis effector, or challenged with Ply-deficient Spn showed less nEC sloughing, increased neutrophil infiltration, and altered IL-1α, IL-33, CXCL2, IL-17, and IL-6 levels in nasal lavage fluid (NALF). Activated MLKL correlated with increased presence of CD11c+ antigen presenting cells in Spn-associated submucosa. Colonized MLKL KO mice and wildtype mice colonized with Ply-deficient Spn produced less antibody against the bacterial surface protein PspA, were delayed in bacterial clearance, and were more susceptible to a lethal secondary Spn challenge. We conclude that PFT-induced necroptosis is instrumental in the natural development of protective immunity against opportunistic PFT-producing bacterial pathogens

Severe Pneumococcal Pneumonia Causes Acute Cardiac Toxicity and Subsequent Cardiac Remodeling

Rationale: Up to one-third of patients hospitalized with pneumococcal pneumonia experience major adverse cardiac events (MACE) during or after pneumonia. In mice, Streptococcus pneumoniae caninvade themyocardium, induce cardiomyocyte death, and disrupt cardiac function following bacteremia, but it is unknown whether the same occurs in humans with severe pneumonia. Objectives: We sought to determine whether S. pneumoniae can (1) translocate the heart, (2) induce cardiomyocyte death, (3) causeMACE, and (4) induce cardiac scar formation after antibiotic treatment during severe pneumonia using a nonhuman primate (NHP) model. Methods: We examined cardiac tissue from six adult NHPs with severe pneumococcal pneumonia and three uninfected control animals. Three animals were rescued with antibiotics (convalescent animals). Electrocardiographic, echocardiographic, and serum biomarkers of cardiac damage were measured (troponin T, N-terminal pro-brain natriuretic peptide, and heart-type fatty acid binding protein). Histological examination included hematoxylin and eosin staining, immunofluorescence, immunohistochemistry, picrosirius red staining, and transmission electron microscopy. Immunoblots were used to assess the underlying mechanisms. Measurements and Main Results: Nonspecific ischemic alterations were detected by electrocardiography and echocardiography. Serum levels of troponin T and heart-type fatty acid binding protein were increased (P,0.05) after pneumococcal infection in both acutely ill and convalescent NHPs. S. pneumoniae was detected in the myocardium of all NHPs with acute severe pneumonia. Necroptosis and apoptosis were detected in the myocardium of both acutely ill and convalescent NHPs. Evidence of cardiac scar formation was observed only in convalescent animals by transmission electron microscopy and picrosirius red staining. Conclusions: S. pneumoniae invades the myocardium and induces cardiac injury with necroptosis and apoptosis, followed by cardiac scarring after antibiotic therapy, in anNHP model of severe pneumonia

A Non-Human Primate Model of Severe Pneumococcal Pneumonia.

RATIONALE:Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and infectious death in adults worldwide. A non-human primate model is needed to study the molecular mechanisms that underlie the development of severe pneumonia, identify diagnostic tools, explore potential therapeutic targets, and test clinical interventions during pneumococcal pneumonia. OBJECTIVE:To develop a non-human primate model of pneumococcal pneumonia. METHODS:Seven adult baboons (Papio cynocephalus) were surgically tethered to a continuous monitoring system that recorded heart rate, temperature, and electrocardiography. Animals were inoculated with 109 colony-forming units of S. pneumoniae using bronchoscopy. Three baboons were rescued with intravenous ampicillin therapy. Pneumonia was diagnosed using lung ultrasonography and ex vivo confirmation by histopathology and immunodetection of pneumococcal capsule. Organ failure, using serum biomarkers and quantification of bacteremia, was assessed daily. RESULTS:Challenged animals developed signs and symptoms of pneumonia 4 days after infection. Infection was characterized by the presence of cough, tachypnea, dyspnea, tachycardia and fever. All animals developed leukocytosis and bacteremia 24 hours after infection. A severe inflammatory reaction was detected by elevation of serum cytokines, including Interleukin (IL)1Ra, IL-6, and IL-8, after infection. Lung ultrasonography precisely detected the lobes with pneumonia that were later confirmed by pathological analysis. Lung pathology positively correlated with disease severity. Antimicrobial therapy rapidly reversed symptomology and reduced serum cytokines. CONCLUSIONS:We have developed a novel animal model for severe pneumococcal pneumonia that mimics the clinical presentation, inflammatory response, and infection kinetics seen in humans. This is a novel model to test vaccines and treatments, measure biomarkers to diagnose pneumonia, and predict outcomes

<i>Streptococcus pneumoniae</i> in the heart subvert the host response through biofilm-mediated resident macrophage killing

<div><p>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 <i>Streptococcus pneumoniae</i> 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 <i>in vivo</i> 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 <i>in vivo</i>. 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.</p></div