Toll-Like Receptor 4 Agonistic Antibody Promotes Innate Immunity against Severe Pneumonia Induced by Coinfection with Influenza Virus and Streptococcus pneumoniae

ABSTRACTCoinfection with bacteria is a major cause of mortality during influenza epidemics. Recently, Toll-like receptor (TLR) agonists were shown to have immunomodulatory functions. In the present study, we investigated the effectiveness and mechanisms of the new TLR4 agonistic monoclonal antibody UT12 against secondary pneumococcal pneumonia induced by coinfection with influenza virus in a mouse model. Mice were intranasally inoculated withStreptococcus pneumoniae2 days after influenza virus inoculation. UT12 was intraperitoneally administered 2 h before each inoculation. Survival rates were significantly increased and body weight loss was significantly decreased by UT12 administration. Additionally, the production of inflammatory mediators was significantly suppressed by the administration of UT12. In a histopathological study, pneumonia in UT12-treated mice was very mild compared to that in control mice. UT12 increased antimicrobial defense through the acceleration of macrophage recruitment into the lower respiratory tract induced by c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-κB) pathway-dependent monocyte chemoattractant protein 1 (MCP-1) production. Collectively, these findings indicate that UT12 promoted pulmonary innate immunity and may reduce the severity of severe pneumonia induced by coinfection with influenza virus andS. pneumoniae. This immunomodulatory effect of UT12 improves the prognosis of secondary pneumococcal pneumonia and makes UT12 an attractive candidate for treating severe infectious diseases.

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Comparison of Immunogenicity and Protection of Two Pneumococcal Protein Vaccines Based on PsaA and PspA

Infection and Immunity ◽

10.1128/iai.00916-17 ◽

2018 ◽

Vol 86 (6) ◽

Cited By ~ 5

Author(s):

Jinfei Yu ◽

Bo Li ◽

Xiaorui Chen ◽

Jingcai Lu ◽

Dandan Wang ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Disease ◽

Bacterial Load ◽

Survival Rates ◽

Mucosal Vaccine ◽

High Morbidity ◽

Content Type ◽

Single Protein ◽

Specific Igg ◽

Children Under 5

ABSTRACTStreptococcus pneumoniaeis a major cause of invasive pneumococcal disease, septicemia, and meningitis that can result in high morbidity rates in children under 5 years old. The current polysaccharide-based vaccines can provide type-specific immunity, but a broad-spectrum vaccine would provide greater coverage. Therefore, developing pneumococcal-protein-based vaccines that can extend to more serum types is highly important. In this study, we vaccinated mice via the subcutaneous (s.c.) route with a systemic vaccine that is a mixture of fusion protein PsaA-PspA23 and a single protein, PspA4, with aluminum hydroxide as an adjuvant. As a comparison, mice were immunized intranasally with a mucosal vaccine that is a mixture of PspA2-PA-BLP (where PA is protein anchor and BLP is bacterium-like particle) and PspA4-PA-BLP, via the intranasal (i.n.) route. The two immunization processes were followed by challenge withStreptococcus pneumoniaebacteria from two different PspA families. Specific IgG titers in the serum and specific IgA titers in the mucosa were determined following immunizations. Bacterial loads and survival rates after challenge were compared. Both the systemic vaccine and the mucosal vaccine induced a significant increase of IgG against PspAs. Only the mucosal vaccine also induced specific IgA in the mucosa. The two vaccines provided protection, but each vaccine showed an advantage. The systemic vaccine induced higher levels of serum antibodies, whereas the mucosal vaccine limited the bacterial load in the lung and blood. Therefore, coimmunizations with the two types of vaccines may be implemented in the future.

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Allergic Airway Disease Prevents Lethal Synergy of Influenza A Virus-Streptococcus pneumoniaeCoinfection

mBio ◽

10.1128/mbio.01335-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

Sean Roberts ◽

Sharon L. Salmon ◽

Donald J. Steiner ◽

Clare M. Williams ◽

Dennis W. Metzger ◽

...

Keyword(s):

Influenza Virus ◽

Streptococcus Pneumoniae ◽

Influenza Infection ◽

Airway Disease ◽

Allergic Airway Disease ◽

Bacterial Clearance ◽

Content Type ◽

Ifn Γ ◽

The Impact ◽

First Time

ABSTRACTFatal outcomes following influenza infection are often associated with secondary bacterial infections. Allergic airway disease (AAD) is known to influence severe complications from respiratory infections, and yet the mechanistic effect of AAD on influenza virus-Streptococcus pneumoniaecoinfection has not been investigated previously. We examined the impact of AAD on host susceptibility to viral-bacterial coinfections. We report that AAD improved survival during coinfection when viral-bacterial challenge occurred 1 week after AAD. Counterintuitively, mice with AAD had significantly deceased proinflammatory responses during infection. Specifically, both CD4+and CD8+T cell interferon gamma (IFN-γ) responses were suppressed following AAD. Resistance to coinfection was also associated with strong transforming growth factor β1 (TGF-β1) expression and increased bacterial clearance. Treatment of AAD mice with IFN-γ or genetic deletion of TGF-β receptor II expression reversed the protective effects of AAD. Using a novel triple-challenge model system, we show for the first time that AAD can provide protection against influenza virus-S. pneumoniaecoinfection through the production of TGF-β that suppresses the influenza virus-induced IFN-γ response, thereby preserving antibacterial immunity.IMPORTANCEAsthma has become one of the most common chronic diseases and has been identified as a risk factor for developing influenza. However, the impact of asthma on postinfluenza secondary bacterial infection is currently not known. Here, we developed a novel triple-challenge model of allergic airway disease, primary influenza infection, and secondaryStreptococcus pneumoniaeinfection to investigate the impact of asthma on susceptibility to viral-bacterial coinfections. We report for the first time that mice recovering from acute allergic airway disease are highly resistant to influenza-pneumococcal coinfection and that this resistance is due to inhibition of influenza virus-mediated impairment of bacterial clearance. Further characterization of allergic airway disease-associated resistance against postinfluenza secondary bacterial infection may aid in the development of prophylactic and/or therapeutic treatment against coinfection.

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Enhanced Influenza Virus-Like Particle Vaccines Containing the Extracellular Domain of Matrix Protein 2 and a Toll-Like Receptor Ligand

Clinical and Vaccine Immunology ◽

10.1128/cvi.00153-12 ◽

2012 ◽

Vol 19 (8) ◽

pp. 1119-1125 ◽

Cited By ~ 37

Author(s):

Bao-Zhong Wang ◽

Harvinder S. Gill ◽

Sang-Moo Kang ◽

Li Wang ◽

Ying-Chun Wang ◽

...

Keyword(s):

Influenza Virus ◽

Fusion Protein ◽

Influenza A ◽

Matrix Protein ◽

Protective Efficacy ◽

Extracellular Domain ◽

Toll Like Receptor ◽

Influenza A Viruses ◽

Live Virus ◽

Content Type

ABSTRACTThe extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region ofSalmonella entericaserovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.

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A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01210-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6064-6072 ◽

Cited By ~ 21

Author(s):

Rémi Porte ◽

Delphine Fougeron ◽

Natalia Muñoz-Wolf ◽

Julien Tabareau ◽

Anne-France Georgel ◽

...

Keyword(s):

Influenza Virus ◽

Combination Treatment ◽

Intranasal Administration ◽

Pathogenic Bacteria ◽

Bacterial Load ◽

Pneumococcal Infection ◽

Toll Like Receptor ◽

Content Type ◽

Toll Like Receptor 5 ◽

Stimulation Of

ABSTRACTProphylactic intranasal administration of the Toll-like receptor 5 (TLR5) agonist flagellin protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might improve the therapeutic index of antibiotics for the treatment ofStreptococcus pneumoniaerespiratory infections in mice. Intranasal administration of flagellin was combined with either oral administration of amoxicillin or intraperitoneal injection of trimethoprim-sulfamethoxazole to treatS. pneumoniae-infected animals. Compared with standalone treatments, the combination of antibiotic and flagellin resulted in a lower bacterial load in the lungs and greater protection againstS. pneumoniaedissemination and was associated with an early increase in neutrophil infiltration in the airways. The antibiotic-flagellin combination treatment was, however, not associated with any exacerbation of inflammation. Moreover, combination treatment was more efficacious than standalone antibiotic treatments in the context of post-influenza virus pneumococcal infection. Lastly, TLR5 signaling was shown to be mandatory for the efficacy of the combined antibacterial therapy. This report is the first to show that combining antibiotic treatment with the stimulation of mucosal innate immunity is a potent antibacterial strategy against pneumonia.

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Impact of theglpQ2Gene on Virulence in a Streptococcus pneumoniae Serotype 19A Sequence Type 320 Strain

Infection and Immunity ◽

10.1128/iai.02357-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 682-692 ◽

Cited By ~ 1

Author(s):

Yi-Ping Chuang ◽

Zih-Rong Peng ◽

Shun-Fu Tseng ◽

Yu-Chun Lin ◽

Huey-Kang Sytwu ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Parent Strain ◽

Severe Pneumonia ◽

Exponential Phase ◽

Sequence Type ◽

Infection Model ◽

Respiratory Pathogens ◽

Content Type ◽

Human Lung Epithelial Cell ◽

Lung Colonization

Glycerophosphodiester phosphodiesterase (GlpQ) metabolizes glycerophosphorylcholine from the lung epithelium to produce free choline, which is transformed into phosphorylcholine and presented on the surfaces of many respiratory pathogens. Two orthologs ofglpQgenes are found inStreptococcus pneumoniae:glpQ, with a membrane motif, is widespread in pneumococci, whereasglpQ2, which shares high similarity withglpQinHaemophilus influenzaeandMycoplasma pneumoniae, is present only inS. pneumoniaeserotype 3, 6B, 19A, and 19F strains. Recently, serotype 19A has emerged as an epidemiological etiology associated with invasive pneumococcal diseases. Thus, we investigated the pathophysiological role ofglpQ2in a serotype 19A sequence type 320 (19AST320) strain, which was the prevalent sequence type in 19A associated with severe pneumonia and invasive pneumococcal disease in pediatric patients. Mutations inglpQ2reduced phosphorylcholine expression and the anchorage of choline-binding proteins to the pneumococcal surface during the exponential phase, where the mutants exhibited reduced autolysis and lower natural transformation abilities than the parent strain. The deletion ofglpQ2also decreased the adherence and cytotoxicity to human lung epithelial cell lines, whereas these functions were indistinguishable from those of the wild type in complementation strains. In a murine respiratory tract infection model,glpQ2was important for nasopharynx and lung colonization. Furthermore, infection with aglpQ2mutant decreased the severity of pneumonia compared with the parent strain, andglpQ2gene complementation restored the inflammation level. Therefore,glpQ2enhances surface phosphorylcholine expression inS. pneumoniae19AST320 during the exponential phase, which contributes to the severity of pneumonia by promoting adherence and host cell cytotoxicity.

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Purified Streptococcus pneumoniae Endopeptidase O (PepO) Enhances Particle Uptake by Macrophages in a Toll-Like Receptor 2- and miR-155-Dependent Manner

Infection and Immunity ◽

10.1128/iai.01012-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 10

Author(s):

Hua Yao ◽

Hong Zhang ◽

Kai Lan ◽

Hong Wang ◽

Yufeng Su ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Host Defense ◽

Defense Response ◽

Dependent Manner ◽

Toll Like Receptor ◽

Content Type ◽

Host Defense Response ◽

Toll Like Receptor 2 ◽

In Cells

ABSTRACT Insights into the host-microbial virulence factor interaction, especially the immune signaling mechanisms, could provide novel prevention and treatment options for pneumococcal diseases. Streptococcus pneumoniae endopeptidase O (PepO) is a newly discovered and ubiquitously expressed pneumococcal virulence protein. A PepO-mutant strain showed impaired adherence to and invasion of host cells compared with the isogenic wild-type strain. It is still unknown whether PepO is involved in the host defense response to pneumococcal infection. Here, we demonstrated that PepO could enhance phagocytosis of Streptococcus pneumoniae and Staphylococcus aureus by peritoneal exudate macrophages (PEMs). Further studies showed that PepO stimulation upregulated the expression of microRNA-155 (miR-155) in PEMs in a time- and dose-dependent manner. PepO-induced enhanced phagocytosis was decreased in cells transfected with an inhibitor of miR-155, while it was increased in cells transfected with a mimic of miR-155. We also revealed that PepO-induced upregulation of miR-155 in PEMs was mediated by Toll-like receptor 2 (TLR2)–NF-κB signaling and that the increased expression of miR-155 downregulated expression of SHIP1. Taken together, these results indicate that PepO induces upregulation of miR-155 in PEMs, contributing to enhanced phagocytosis and host defense response to pneumococci and Staphylococcus aureus.

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Parasite-Derived Neurotrophic Factor/trans-Sialidase of Trypanosoma cruzi Links Neurotrophic Signaling to Cardiac Innate Immune Response

Infection and Immunity ◽

10.1128/iai.02098-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3687-3696 ◽

Cited By ~ 6

Author(s):

Ryan Salvador ◽

Daniel Aridgides ◽

Mercio PereiraPerrin

Keyword(s):

Innate Immunity ◽

Trypanosoma Cruzi ◽

Neurotrophic Factor ◽

Neutralizing Antibodies ◽

Tissue Growth ◽

Content Type ◽

Monocyte Chemoattractant Protein 1 ◽

Neurotrophic Signaling ◽

Neurotrophic Receptors ◽

Dose Dependent Increase

ABSTRACTThe Chagas' disease parasiteTrypanosoma cruzielicits a potent inflammatory response in acutely infected hearts that keeps parasitism in check and triggers cardiac abnormalities. A most-studied mechanism underlying innate immunity inT. cruziinfection is Toll-like receptor (TLR) activation by lipids and other parasite molecules. However, yet-to-be-identified pathways should exist. Here, we show thatT. cruzistrongly upregulates monocyte chemoattractant protein 1 (MCP-1)/CCL2 and fractalkine (FKN)/CX3CL1 in cellular and mouse models of heart infection. Mechanistically, upregulation of MCP-1 and FKN stems from the interaction of parasite-derived neurotrophic factor (PDNF)/trans-sialidase with neurotrophic receptors TrkA and TrkC, as assessed by pharmacological inhibition, neutralizing antibodies, and gene silencing studies. Administration of a single dose of intravenous PDNF to naive mice results in a dose-dependent increase in MCP-1 and FKN in the heart and liver with pulse-like kinetics that peak at 3 h postinjection. Intravenous PDNF also augments MCP-1 and FKN in TLR signaling-deficient MyD88-knockout mice, underscoring the MyD88-independent action of PDNF. Although single PDNF injections do not increase MCP-1 and FKN receptors, multiple PDNF injections at short intervals up the levels of receptor transcripts in the heart and liver, suggesting that sustained PDNF triggers cell recruitment at infection sites. Thus, given that MCP-1 and FKN are chemokines essential to the recruitment of immune cells to combat inflammation triggers and to enhance tissue repair, our findings uncover a new mechanism in innate immunity againstT. cruziinfection mediated by Trk signaling akin to an endogenous inflammatory and fibrotic pathway resulting from cardiomyocyte-TrkA recognition by matricellular connective tissue growth factor (CTGF/CCN2).

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Lethal Synergism of 2009 Pandemic H1N1 Influenza Virus and Streptococcus pneumoniae Coinfection Is Associated with Loss of Murine Lung Repair Responses

mBio ◽

10.1128/mbio.00172-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 74

Author(s):

John C. Kash ◽

Kathie-Anne Walters ◽

A. Sally Davis ◽

Aline Sandouk ◽

Louis M. Schwartzman ◽

...

Keyword(s):

Weight Loss ◽

Influenza Virus ◽

Streptococcus Pneumoniae ◽

Bacterial Infections ◽

H1n1 Virus ◽

Severe Disease ◽

Significant Loss ◽

Pandemic H1n1 ◽

Content Type ◽

Lung Repair

ABSTRACTSecondary bacterial infections increase disease severity of influenza virus infections and contribute greatly to increased morbidity and mortality during pandemics. To study secondary bacterial infection following influenza virus infection, mice were inoculated with sublethal doses of 2009 seasonal H1N1 virus (NIH50) or pandemic H1N1 virus (Mex09) followed by inoculation withStreptococcus pneumoniae48 h later. Disease was characterized by assessment of weight loss and survival, titration of virus and bacteria by quantitative reverse transcription-PCR (qRT-PCR), histopathology, expression microarray, and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09 plusS. pneumoniaeshowed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium, and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50 plusS. pneumoniaeshowed increased weight loss, 100% survival, and slightly enhanced lung pathology. Mex09-S. pneumoniaecoinfection also resulted in increasedS. pneumoniaereplication in lung and bacteremia late in infection. Global gene expression profiling revealed that Mex09-S. pneumoniaecoinfection did not induce significantly more severe inflammatory responses but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09-S. pneumoniae-infected mice. This study demonstrates that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in more severe disease and loss of lung repair responses than did seasonal influenza viral and bacterial coinfection. Moreover, this study provides novel insights into influenza virus and bacterial coinfection by showing correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses.IMPORTANCESecondary bacterial pneumonias lead to increased disease severity and have resulted in a significant percentage of deaths during influenza pandemics. To understand the biological basis for the interaction of bacterial and viral infections, mice were infected with sublethal doses of 2009 seasonal H1N1 and pandemic H1N1 viruses followed by infection withStreptococcus pneumoniae48 h later. Only infection with 2009 pandemic H1N1 virus andS. pneumoniaeresulted in severe disease with a 100% fatality rate. Analysis of the host response to infection during lethal coinfection showed a significant loss of responses associated with lung repair that was not observed in any of the other experimental groups. This group of mice also showed enhanced bacterial replication in the lung. This study reveals that the extent of lung damage during viral infection influences the severity of secondary bacterial infections and may help explain some differences in mortality during influenza pandemics.

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Pulmonary Immunostimulation with MALP-2 in Influenza Virus-Infected Mice Increases Survival after Pneumococcal Superinfection

Infection and Immunity ◽

10.1128/iai.00948-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4617-4629 ◽

Cited By ~ 17

Author(s):

Katrin Reppe ◽

Peter Radünzel ◽

Kristina Dietert ◽

Thomas Tschernig ◽

Thorsten Wolff ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Survival Rates ◽

Pneumococcal Infection ◽

Influenza Virus Infection ◽

High Morbidity ◽

Bacterial Number ◽

Content Type ◽

Local Immunosuppression

Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, withStreptococcus pneumoniaebeing the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged withS. pneumoniaeon day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection.

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2473 Cardiac injury due to Streptococcus pneumoniae invasion during severe pneumococcal pneumonia in a novel nonhuman primate model

Journal of Clinical and Translational Science ◽

10.1017/cts.2018.51 ◽

2018 ◽

Vol 2 (S1) ◽

pp. 6-6

Author(s):

Luis F. Reyes ◽

Cecilia A. Hinojosa ◽

Nilam J. Soni ◽

Julio Noda ◽

Vicki T. Winter ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Pneumonia ◽

Troponin I ◽

Cardiac Injury ◽

Severe Pneumonia ◽

Cardiac Events ◽

Cardiac Damage ◽

Nonhuman Primate Model ◽

Primate Model ◽

Adverse Cardiac Events

OBJECTIVES/SPECIFIC AIMS: The aims of this study are (1) to develop and characterize a novel nonhuman primate model of pneumococcal pneumonia that mimics human disease; and (2) determine whether Streptococcus pneumoniae can: (a) translocate to the heart, (b) cause adverse cardiac events, (c) induce cardiomyocyte death, and (d) lead to scar formation during severe pneumonia in baboons. METHODS/STUDY POPULATION: Six adult baboons (Papio cynocephalus) were surgically tethered to a monitoring system to continuously assess their heart rate, temperature, and electrocardiogram (ECG). A baseline transthoracic echocardiogram, 12-lead ECG, serum troponin-I levels, brain natriuretic peptide, and heart-type fatty acid binding protein (HFABP) levels were obtained before infection and at the end of the experiment to determine cardiovascular damage during pneumococcal pneumonia. Animals were challenged with 108 colony-forming units of S. pneumoniae in the right middle lobe using flexible bronchoscopy. Three baboons were rescued with ampicillin therapy (80 mg/kg/d) after the development of pneumonia. Cardiac damage was confirmed by examination of tissue sections using immunohistochemistry as well as electron and fluorescence microscopy. Western-blots and tissue staining were used to determine the presence of necroptosis (RIP3 and pMLKL) and apoptosis (Caspase-3) in the cardiac tissue. Cytokine and chemokine levels in the heart tissue were determined using Luminex technology. RESULTS/ANTICIPATED RESULTS: Four males (57%) and three (43%) females were challenged. The median age of all baboons was 11 (IQR, 10-19) years old, which corresponds to a middle-aged human. Infected baboons consistently developed severe pneumonia. All animals developed systemic inflammatory response syndrome with tachycardia, tachypnea, fever, and leukocytosis. Infection was characterized by initial leukocytosis followed by severe leukopenia on day 3 postinoculation. Non-specific ischemic alterations by ECG (ST segment and T-wave flattering) and in the premortem echocardiogram were observed. The median (IQR) levels of troponin I and HFABP at the end of the experiment were 3550 ng/mL (1717–5383) and 916.9 ng/mL (520.8–1323), respectively. Severe cardiomyopathy was observed using TEM and H&E stains in animals with severe pneumonia. Necroptosis was detected in cardiomyocytes of infected animals by the presence of pMLKL and RIP3 in cardiac tissues. Signs of cardiac remodeling indicated by disorganized collagen deposition was present in rescued animals but not in the other animals. DISCUSSION/SIGNIFICANCE OF IMPACT: We confirmed that baboons experience cardiac injury during severe pneumococcal pneumonia that is characterized by myocardial invasion, activation of necroptosis, and tissue remodeling in animals rescued by antimicrobial therapy. Cardiac damage by invading pneumococci may explain why adverse cardiac events that occur during and after pneumococcal pneumonia in adult human patients.

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