Pneumococcal Pneumonia In Mice Research Articles

Targeted delivery of flagellin by nebulization offers optimized respiratory immunity and defense against pneumococcal pneumonia.

Novel therapeutic strategies are urgently needed to combat pneumonia caused by Streptococcus pneumoniae strains resistant to standard-of-care antibiotics. Previous studies have shown that targeted stimulation of lung innate immune defenses through intranasal administration of the Toll-like receptor 5 agonist flagellin improves the treatment of pneumonia when combined with antibiotics. To promote translation to the clinic application, this study assessed the direct delivery of flagellin to the airways through nebulization using a vibrating mesh nebulizer in mice. Intranasal delivery achieved approximately 40% lung deposition of the administered flagellin dose, whereas nebulization yielded less than 1%. Despite these differences, nebulized flagellin induced transient activation of lung innate immunity characterized by cytokine/chemokine production and neutrophil infiltration into airways analogous to intranasal administration. Furthermore, inhalation by nebulization resulted in an accelerated resolution of systemic pro-inflammatory responses. Lastly, adjunct therapy combining nebulized flagellin and amoxicillin proved effective against antibiotic-resistant pneumococcal pneumonia in mice. We posit that flagellin aerosol therapy represents a safe and promising approach to address bacterial pneumonia within the context of antimicrobial resistance.

Biological effects of corticosteroids on pneumococcal pneumonia in Mice—translational significance

BackgroundStreptococcus pneumoniae is the most common bacterial cause of community acquired pneumonia and the acute respiratory distress syndrome (ARDS). Some clinical trials have demonstrated a beneficial effect of corticosteroid therapy in community acquired pneumonia, COVID-19, and ARDS, but the mechanisms of this benefit remain unclear. The primary objective of this study was to investigate the effects of corticosteroids on the pulmonary biology of pneumococcal pneumonia in a mouse model. A secondary objective was to identify shared transcriptomic features of pneumococcal pneumonia and steroid treatment in the mouse model and clinical samples.MethodsWe carried out comprehensive physiologic, biochemical, and histological analyses in mice to identify the mechanisms of lung injury in Streptococcus pneumoniae with and without adjunctive steroid therapy. We also studied lower respiratory tract gene expression from a cohort of 15 mechanically ventilated patients (10 with Streptococcus pneumoniae and 5 controls) to compare with the transcriptional studies in the mice.ResultsIn mice with pneumonia, dexamethasone in combination with ceftriaxone reduced (1) pulmonary edema formation, (2) alveolar protein permeability, (3) proinflammatory cytokine release, (4) histopathologic lung injury score, and (5) hypoxemia but did not increase bacterial burden. Transcriptomic analyses identified effects of steroid therapy in mice that were also observed in the clinical samples.ConclusionsIn combination with appropriate antibiotic therapy in mice, treatment of pneumococcal pneumonia with steroid therapy reduced hypoxemia, pulmonary edema, lung permeability, and histologic criteria of lung injury, and also altered inflammatory responses at the protein and gene expression level. The transcriptional studies in patients suggest that the mouse model replicates some of the features of pneumonia in patients with Streptococcus pneumoniae and steroid treatment. Overall, these studies provide evidence for the mechanisms that may explain the beneficial effects of glucocorticoid therapy in patients with community acquired pneumonia from Streptococcus Pneumoniae.

Neutrophil reduction attenuates the severity of lung injury in the early phase of pneumococcal pneumonia in mice.

Neutrophils are the first leukocytes to be recruited to sites of inflammation in response to chemotactic factors released by activated macrophages and pulmonary epithelial and endothelial cells in bacterial pneumonia, a common cause of acute respiratory distress syndrome (ARDS). Although neutrophilic inflammation facilitates the elimination of pathogens, neutrophils also may cause bystander tissue injury. Even though the presence of neutrophils in alveolar spaces is a key feature of acute lung injury and ARDS especially from pneumonia, their contribution to the pathogenesis of lung injury is uncertain. The goal of this study was to elucidate the role of neutrophils in a clinically relevant model of bacterial pneumonia. We investigated the effect of reducing neutrophils in a mouse model of pneumococcal pneumonia treated with antibiotics. Neutrophils were reduced with anti-lymphocyte antigen 6 complex locus G6D (Ly6G) monoclonal antibody 24 h before and immediately preceding infection. Mice were inoculated intranasally with Streptococcus pneumoniae and received ceftriaxone 12 h after bacterial inoculation. Neutrophil reduction in mice treated with ceftriaxone attenuated hypoxemia, alveolar permeability, epithelial injury, pulmonary edema, and inflammatory biomarker release induced by bacterial pneumonia, even though bacterial loads in the distal air spaces of the lung were modestly increased as compared with antibiotic treatment alone. Thus, when appropriate antibiotics are administered, lung injury in the early phase of bacterial pneumonia is mediated in part by neutrophils. In the early phase of bacterial pneumonia, neutrophils contribute to the severity of lung injury, although they also participate in host defense.NEW & NOTEWORTHY Neutrophil accumulation is a key feature of ARDS, but their contribution to the pathogenesis is still uncertain. We investigated the effect of reducing neutrophils in a clinically relevant mouse model of pneumococcal pneumonia treated with antibiotics. When appropriate antibiotics were administered, neutrophil reduction with Ly6G antibody markedly attenuated lung injury and improved oxygenation. In the early phase of bacterial pneumonia, neutrophils contribute to the severity of lung injury, although they also participate in host defense.

Impact of the new membrane-targeting lipoglycopeptide antibiotic MCC5145 on the treatment of bacteremic pneumococcal pneumonia in mice.

Bacteremic Streptococcus pneumoniae pneumonia is one of the most severe forms of invasive pneumococcal disease (IPD) and with particularly high case-fatality rates among the elderly and individuals with comorbidities, exacerbated by rising antibiotic resistance and time to initiation of therapy. Here, we examined the efficacy of the preclinical "vancapticin" glycopeptide MCC5145 against fulminant infection by S. pneumoniae serotype 2 strain D39 in a bioluminescent, neutropenic mouse model of bacteremic pneumonia. MCC5145 is a semisynthetic vancomycin derivative chemically modified at the C-terminus with a membrane-targeting motif designed to preferentially bind the anionic bacterial surface. We show that similar to vancomycin, subcutaneous administration of MCC5145 to mice 1 day after intranasal infection with a bioluminescent derivative of S. pneumoniae D39 elicited time and concentration-dependent reduction in total flux in the lungs and blood. Together, our finding supports the further development of MCC5145 as a potential new treatment option for pneumonia and/or bacteremic pneumonia in clinical settings, particularly for immunocompromised individuals. IMPORTANCE S. pneumoniae (the pneumococcus) causes severe community acquired lung and blood infection, especially among the elderly and people with underlying medical conditions and/or weakened immune systems. The rising incidence of antibiotic resistance and delays between diagnosis of infection and commencement of effective therapy make treatment difficult and result in high mortality rates. In this work, we show that a new derivative (MCC5145) of an existing antibiotic (vancomycin) rapidly eradicated lethal pneumococcal challenge from the lungs and blood of mice with a suppressed immune system. Our findings support that MCC5145 is a promising option for the treatment of lung and blood infections caused by the pneumococcus at point-of-care settings, particularly for the elderly and individuals with a weakened immune system.

S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

A fusion protein comprising pneumococcal surface protein A and a pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia.

PspA and pneumolysin are two important vaccine candidates, able to elicit protection in different models of pneumococcal infection. The high immunogenic potential of PspA, combined with a possible adjuvant effect of pneumolysin derivatives (due to their ability to interact with TLR-4) could greatly improve the immunogenicity and coverage of a protein-based pneumococcal vaccine. A chimeric protein including the N-terminal region of PspA in fusion with the pneumolysin derivative, PlD1, has been shown to induce high antibody levels against each protein, and protect mice against invasive challenge. The aim of the present study was to investigate the cellular response induced by such vaccine, and to evaluate protection in a murine model of lobar pneumococcal pneumonia. Pneumococcal pneumonia was induced in BALB/c mice by nasal instillation of a high dose of a serotype 14 strain with low virulence. Airway inflammation was confirmed by total and differential cell counts in BAL and by histological analysis of the lungs, and bacterial loads were measured 7 days after challenge. Cytokine levels were determined in the bronchoalveolar fluid (BALF) of mice immunized with rPspA-PlD1 fusion after challenge, by flow cytometry and ELISA. After challenge, the mice developed lung inflammation with no invasion of other sites, as demonstrated by histological analysis. We detected significant production of TNF-α and IL-6 in the BALF, which correlated with protection against pneumonia in the group immunized with rPspA-PlD1. In conclusion, we found that the rPspA-PlD1fusion is protective against pneumococcal pneumonia in mice, and protection is correlated with an early and controlled local inflammatory response. These results are in agreement with previous data demonstrating the efficacy of the fusion protein against pneumococcal sepsis and reinforce the potential of the rPspA-PlD1 protein chimera as a promising vaccine strategy to prevent pneumococcal disease.

Inhibition of the lipoxin A4 and resolvin D1 receptor impairs host response to acute lung injury caused by pneumococcal pneumonia in mice.

Resolution of the acute respiratory distress syndrome (ARDS) from pneumonia requires repair of the injured lung endothelium and alveolar epithelium, removal of neutrophils from the distal airspaces of the lung, and clearance of the pathogen. Previous studies have demonstrated the importance of specialized proresolving mediators (SPMs) in the regulation of host responses during inflammation. Although ARDS is commonly caused by Streptococcus pneumoniae, the role of lipoxin A4 (LXA4) and resolvin D1 (RvD1) in pneumococcal pneumonia is not well understood. In the present experimental study, we tested the hypothesis that endogenous SPMs play a role in the resolution of lung injury in a clinically relevant model of bacterial pneumonia. Blockade of formyl peptide receptor 2 (ALX/FPR2), the receptor for LXA4 and RvD1, with the peptide WRW4 resulted in more pulmonary edema, greater protein accumulation in the air spaces, and increased bacteria accumulation in the air spaces and the blood. Inhibition of this receptor was also associated with decreased levels of proinflammatory cytokines. Even in the presence of antibiotic treatment, WRW4 inhibited the resolution of lung injury. In summary, these experiments demonstrated two novel findings: LXA4 and RvD1 contribute to the resolution of lung injury due to pneumococcal pneumonia, and the mechanism of their benefit likely includes augmenting bacterial clearance and reducing pulmonary edema via the restoration of lung alveolar-capillary barrier permeability.

Overexpression of Macrophage-Inducible C-Type Lectin Mincle Aggravates Proinflammatory Responses to Streptococcus pneumoniae with Fatal Outcome in Mice.

Macrophage-inducible C-type lectin (Mincle)-dependent sensing of pathogens triggers proinflammatory immune responses in professional phagocytes that contribute to protecting the host against pathogen invasion. In this study, we examined whether overexpression of Mincle that is designed to improve early pathogen sensing by professional phagocytes would improve lung-protective immunity against Streptococcus pneumoniae in mice. Proteomic profiling of alveolar macrophages of Mincle transgenic (tg) mice stimulated with the Mincle-specific pneumococcal ligand glucosyl-diacylglycerol (Glc-DAG) revealed increased Nlrp3 inflammasome activation and downstream IL-1β cytokine release that was not observed in Glc-DAG-stimulated Mincle knockout or Nlrp3 knockout macrophages. Along this line, Mincle tg mice also responded with a stronger Nlrp3 expression and early proinflammatory cytokine release after challenge with S. pneumoniae, ultimately leading to fatal pneumonia in the Mincle tg mice. Importantly, Nlrp3 inhibitor treatment of Mincle tg mice significantly mitigated the observed hyperinflammatory response to pneumococcal challenge. Together, we show that overexpression of the pattern recognition receptor Mincle triggers increased Glc-DAG-dependent Nlrp3 inflammasome activation in professional phagocytes leading to fatal pneumococcal pneumonia in mice that is amenable to Nlrp3 inhibitor treatment. These data show that ectopic expression of the Mincle receptor confers increased susceptibility rather than resistance to S. pneumoniae in mice, thus highlighting the importance of an inducible Mincle receptor expression in response to microbial challenge.

Neutralizing Complement C5a Protects Mice with Pneumococcal Pulmonary Sepsis

Community-acquired pneumonia and associated sepsis cause high mortality despite antibiotic treatment. Uncontrolled inflammatory host responses contribute to the unfavorable outcome by driving lung and extrapulmonary organ failure. The complement fragment C5a holds significant proinflammatory functions and is associated with tissue damage in various inflammatory conditions. The authors hypothesized that C5a concentrations are increased in pneumonia and C5a neutralization promotes barrier stabilization in the lung and is protective in pneumococcal pulmonary sepsis. The authors investigated regulation of C5a in pneumonia in a prospective patient cohort and in experimental pneumonia. Two complementary models of murine pneumococcal pneumonia were applied. Female mice were treated with NOX-D19, a C5a-neutralizing L-RNA-aptamer. Lung, liver, and kidney injury and the inflammatory response were assessed by measuring pulmonary permeability (primary outcome), pulmonary and blood leukocytes, cytokine concentrations in lung and blood, and bacterial load in lung, spleen, and blood, and performing histologic analyses of tissue damage, apoptosis, and fibrin deposition (n = 5 to 13). In hospitalized patients with pneumonia (n = 395), higher serum C5a concentrations were observed compared to healthy subjects (n = 24; 6.3 nmol/l [3.9 to 10.0] vs. 4.5 nmol/l [3.8 to 6.6], median [25 to 75% interquartile range]; difference: 1.4 [95% CI, 0.1 to 2.9]; P = 0.029). Neutralization of C5a in mice resulted in lower pulmonary permeability in pneumococcal pneumonia (1.38 ± 0.89 vs. 3.29 ± 2.34, mean ± SD; difference: 1.90 [95% CI, 0.15 to 3.66]; P = 0.035; n = 10 or 11) or combined severe pneumonia and mechanical ventilation (2.56 ± 1.17 vs. 7.31 ± 5.22; difference: 4.76 [95% CI, 1.22 to 8.30]; P = 0.011; n = 9 or 10). Further, C5a neutralization led to lower blood granulocyte colony-stimulating factor concentrations and protected against sepsis-associated liver injury. Systemic C5a is elevated in pneumonia patients. Neutralizing C5a protected against lung and liver injury in pneumococcal pneumonia in mice. Early neutralization of C5a might be a promising adjunctive treatment strategy to improve outcome in community-acquired pneumonia.

The Annexin A1/FPR2 pathway controls the inflammatory response and bacterial dissemination in experimental pneumococcal pneumonia.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia leading to high mortality rates. Inflammation triggered by pneumococcal infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Annexin A1 (AnxA1) mainly acts through Formyl Peptide Receptor 2 (FPR2) inducing the resolution of inflammation. Here, we have evaluated the role of AnxA1 and FPR2 during pneumococcal pneumonia in mice. For that, AnxA1, Fpr2/3 knockout (KO) mice and wild-type (WT) controls were infected intranasally with S pneumoniae. AnxA1 and Fpr2/3 KO mice were highly susceptible to infection, displaying uncontrolled inflammation, increased bacterial dissemination, and pulmonary dysfunction compared to WT animals. Mechanistically, the absence of AnxA1 resulted in the loss of lung barrier integrity and increased neutrophil activation upon S pneumoniae stimulation. Importantly, treatment of WT or AnxA1 KO-infected mice with Ac2-26 decreased inflammation, lung damage, and bacterial burden in the airways by increasing macrophage phagocytosis. Conversely, Ac2-26 peptide was ineffective to afford protection in Fpr2/3 KO mice during infection. Altogether, these findings show that AnxA1, via FPR2, controls inflammation and bacterial dissemination during pneumococcal pneumonia by promoting host defenses, suggesting AnxA1-based peptides as a novel therapeutic strategy to control pneumococcal pneumonia.

Treatment with broadly neutralizing influenza antibodies reduces severity of secondary pneumococcal pneumonia in mice.

Secondary bacterial pneumonia is a frequent complication of influenza, associated with high morbidity and mortality. We hypothesized that treatment with neutralizing influenza A antibody AT10_002 protects against severe secondary pneumococcal infection in a mouse model of influenza A infection. Influenza A (H3N2) virus–infected male C57Bl6 mice were treated intravenously with either AT10_002 or a control 2 days postinfection. Seven days later, both groups were infected with Streptococcus pneumoniae and killed 18 hours later. Mice receiving AT10_002 showed less loss of bodyweight compared with controls (+1% vs −12%, P < .001), lower viral loads in bronchoalveolar lavage fluids (BALFs) (7 vs 194 RNA copies per µL; P < .001), and reduced bacterial outgrowth in lung homogenates (3.3 × 101 vs 2.5 × 105 colony‐forming units per mg; P < .001). The treatment group showed lower pulmonary wet weights, lower cell counts, and lower protein levels in BALF compared with controls. Treatment with AT10_002 was associated with lower levels of tumor necrosis factor‐α, interleukin (IL)‐6, cytokine‐induced neutrophil chemoattractant (KC), and interferon‐γ in BALF and lower IL‐6 and KC in lung homogenates. Treatment with anti‐influenza antibody AT10_002 is associated with reduced weight loss, viral load, bacterial outgrowth, and lung injury in a murine model of secondary pneumococcal pneumonia following influenza infection.

A Novel PspA Protein Vaccine Intranasal Delivered by Bacterium-Like Particles Provides Broad Protection Against Pneumococcal Pneumonia in Mice

ABSTRACTBackground: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine.Method: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs.Result: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families.Discussion and conclusion: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.

Peptidoglycan Recognition Protein 3 Does Not Alter the Outcome of Pneumococcal Pneumonia in Mice.

Pneumococci frequently cause community-acquired pneumonia, a disease with high mortality rates, particularly in young children and in the elderly. Endogenous antimicrobial peptides and proteins such as PGLYRP3 may contribute to the progression and outcome of this disease. Since increasing antibiotic resistant strains occur all over the world, these endogenous antimicrobial molecules are interesting new targets for future therapies. In this study, the expression pattern of PGLYRP3 was analyzed in alveolar epithelial cells, alveolar macrophages and neutrophils. Additionally, the function of PGLYRP3 during Streptococcus pneumoniae-induced pneumonia was investigated in a murine pneumococcal pneumonia model using PGLYRP3KO mice. PGLYRP3 is expressed in all selected cell types but pneumococcus-dependent induction of PGLYRP3 was observed only in neutrophils and alveolar macrophages. Interestingly, there were no significant differences in the bacterial loads within the lungs, the blood or the spleens, in the cytokine response, the composition of immune cells and the histopathology between wild type and PGLYRP3KO mice. Finally, we could neither observe significant differences in the clinical symptoms nor in the overall survival. Collectively, PGLYRP3 seems to be dispensable for the antibacterial defense during pneumococcal pneumonia.

Corrigendum to "Epicutaneous immunization with phosphorylcholine conjugated to bovine serum albumin (PC-BSA) and TLR9 ligand CpG alleviates pneumococcal pneumonia in mice" [Pharmacol. Rep. 66 (2014) 570-575

Corrigendum to "Epicutaneous immunization with phosphorylcholine conjugated to bovine serum albumin (PC-BSA) and TLR9 ligand CpG alleviates pneumococcal pneumonia in mice" [Pharmacol. Rep. 66 (2014) 570-575

The Effect of Intravenous Peramivir, Compared with Oral Oseltamivir, on the Outcome of Post-Influenza Pneumococcal Pneumonia in Mice

Pneumococcal pneumonia often occurs secondary to influenza infection and accounts for a large proportion of the morbidity and mortality associated with seasonal and pandemic influenza outbreaks. Peramivir is a novel, intravenous neuraminidase inhibitor that exhibits potent antiviral activity against influenza A and B viruses. We investigated the efficacy of peramivir for modulating the severity of secondary pneumococcal pneumonia. CBA/JNCrlj mice, infected with influenza virus and superinfected with Streptococcus pneumoniae, were treated with either intravenous peramivir (single or multiple doses of 60 mg/kg/day) or oral oseltamivir at doses of 10 or 40 mg/kg/day in divided doses. The survival rate, viable bacterial count and virus titre in the lungs, as well as cytokine/chemokine concentration and histopathological findings were compared between both groups. The median duration of survival of coinfected mice was significantly prolonged by treatment with multiple doses of peramivir, relative to mice treated with oseltamivir at either dose. Viable bacterial counts and virus titres in the lungs were significantly reduced by intravenous peramivir treatment compared with no treatment or oral oseltamivir treatment. The production of inflammatory cytokines/chemokines was also significantly suppressed by multiple dosing of peramivir compared with oseltamivir. Increased survival appeared to be mediated by decreased inflammation, manifested as lower levels of inflammatory cells and proinflammatory cytokines in the lungs and less severe histopathological findings. The lungs of mice treated with multiple doses of peramivir showed mild inflammatory changes compared to oseltamivir. This study demonstrated that a multiple-dose regimen of intravenous peramivir was more efficacious than a single peramivir dose or multiple doses of oseltamivir for improving outcomes in pneumococcal pneumonia following influenza virus infection in mice.

Epicutaneous immunization with phosphorylcholine conjugated to bovine serum albumin (PC-BSA) and TLR9 ligand CpG alleviates pneumococcal pneumonia in mice

BackgroundEpicutaneous (EC) immunization is a potential new strategy of a needle-free and self-administrable immunization by using a topically applied patch to deliver vaccine. We have previously shown that EC immunization with various protein antigens inhibits both Th1- and Tc1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that maneuver of EC immunization with an antigen and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. MethodsAnimal model of pneumococcal pneumonia was used to study efficacy of EC induced immunopotentiation. ResultsCurrent work showed that EC immunization with phosphorylcholine conjugated to bovine serum albumin (PC-BSA) and CpG prior to Streptococcus pneumoniae infection results in smaller decrease of body weight when compared to PBS treated mice. Consistent with the behavioral observations and body weight, smaller numbers of bacteria were quantitated in lung homogenates of mice patched with PC-BSA and CpG prior inoculation with S. pneumonia when compared to mice patched with PBS alone.In vitro experiments showed that lymph node cells and spleen cells from mice EC immunized with PC-BSA plus CpG produced high levels of IFN-γ and IL-17A when compared to PBS or PC-BSA or CpG treated mice. ConclusionThis work shows that EC immunization with PC-BSA plus TLR9 ligand CpG may be a potential tool to boost immunity to S. pneumoniae.

Adjunctive Corticosteroid Therapy Improves Lung Immunopathology and Survival During Severe Secondary Pneumococcal Pneumonia in Mice

Secondary bacterial pneumonia is a significant cause of morbidity and mortality during influenza, despite routine use of standard antibiotics. Antibiotic-induced immunopathology associated with bacterial cell wall lysis has been suggested to contribute to these poor outcomes. Using Streptococcus pneumoniae in a well-established murine model of secondary bacterial pneumonia (SBP) following influenza, we stratified disease severity based on pneumococcal load in the lungs via in vivo bioluminescence imaging. Ampicillin treatment cured mice with mild pneumonia but was ineffective against severely pneumonic mice, despite effective bacterial killing. Adjunctive dexamethasone therapy improved ampicillin-induced immunopathology and improved outcomes in mice with severe SBP. However, early dexamethasone therapy during primary influenza infection impaired lung adaptive immunity as manifest by increased viral titers, with an associated loss of its protective functions in SBP. These data support adjunctive clinical use of corticosteroids in severe cases of community-acquired pneumonia.

Prevotella intermedia Induces Severe Bacteremic Pneumococcal Pneumonia in Mice with Upregulated Platelet-Activating Factor Receptor Expression

Streptococcus pneumoniae is the leading cause of respiratory infection worldwide. Although oral hygiene has been considered a risk factor for developing pneumonia, the relationship between oral bacteria and pneumococcal infection is unknown. In this study, we examined the synergic effects of Prevotella intermedia, a major periodontopathic bacterium, on pneumococcal pneumonia. The synergic effects of the supernatant of P. intermedia (PiSup) on pneumococcal pneumonia were investigated in mice, and the stimulation of pneumococcal adhesion to human alveolar (A549) cells by PiSup was assessed. The effects of PiSup on platelet-activating factor receptor (PAFR) transcript levels in vitro and in vivo were analyzed by quantitative real-time PCR, and the differences between the effects of pneumococcal infection induced by various periodontopathic bacterial species were verified in mice. Mice inoculated with S. pneumoniae plus PiSup exhibited a significantly lower survival rate, higher bacterial loads in the lungs, spleen, and blood, and higher inflammatory cytokine levels in the bronchoalveolar lavage fluid (macrophage inflammatory protein 2 and tumor necrosis factor alpha) than those infected without PiSup. In A549 cells, PiSup increased pneumococcal adhesion and PAFR transcript levels. PiSup also increased lung PAFR transcript levels in mice. Similar effects were not observed in the supernatants of Porphyromonas gingivalis or Fusobacterium nucleatum. Thus, P. intermedia has the potential to induce severe bacteremic pneumococcal pneumonia with enhanced pneumococcal adhesion to lower airway cells.

TNF-related apoptosis-inducing ligand (TRAIL) exerts therapeutic efficacy for the treatment of pneumococcal pneumonia in mice

Apoptotic death of alveolar macrophages observed during lung infection with Streptococcus pneumoniae is thought to limit overwhelming lung inflammation in response to bacterial challenge. However, the underlying apoptotic death mechanism has not been defined. Here, we examined the role of the TNF superfamily member TNF-related apoptosis-inducing ligand (TRAIL) in S. pneumoniae-induced macrophage apoptosis, and investigated the potential benefit of TRAIL-based therapy during pneumococcal pneumonia in mice. Compared with WT mice, Trail(-/-) mice demonstrated significantly decreased lung bacterial clearance and survival in response to S. pneumoniae, which was accompanied by significantly reduced apoptosis and caspase 3 cleavage but rather increased necrosis in alveolar macrophages. In WT mice, neutrophils were identified as a major source of intraalveolar released TRAIL, and their depletion led to a shift from apoptosis toward necrosis as the dominant mechanism of alveolar macrophage cell death in pneumococcal pneumonia. Therapeutic application of TRAIL or agonistic anti-DR5 mAb (MD5-1) dramatically improved survival of S. pneumoniae-infected WT mice. Most importantly, neutropenic mice lacking neutrophil-derived TRAIL were protected from lethal pneumonia by MD5-1 therapy. We have identified a previously unrecognized mechanism by which neutrophil-derived TRAIL induces apoptosis of DR5-expressing macrophages, thus promoting early bacterial killing in pneumococcal pneumonia. TRAIL-based therapy in neutropenic hosts may represent a novel antibacterial treatment option.

Role of p38 mitogen-activated protein kinase in posttraumatic immunosuppression in mice

Patients with multiple injuries surviving the initial insult are highly susceptible to secondary pneumonia, frequently progressing into sepsis and multiorgan failure. However, the underlying mechanisms of posttraumatic immunosuppression are poorly understood. We hypothesized that dysregulated p38 mitogen-activated protein kinase (MAPK) signaling accounts for impaired lung protective immunity in a model of trauma/hemorrhage (T/H) and subsequent pneumococcal pneumonia in mice. C57BL6/N mice were subjected to trauma by midline laparotomy, and T/H was induced by midline laparotomy followed by cannulation of femoral arteries and veins to induce hemorrhage. Subsequently, mice were infected with Streptococcus pneumoniae. In selected experiments, mice were treated with a p38 MAPK inhibitor or vehicle control immediately after induction of T/H. Mice subjected to T/H showed significantly increased p38 MAPK activation in their lungs, which was accompanied by a reduced Escherichia coli phagocytosis by macrophages from T/H mice in vitro and an impaired pneumococcal killing activity of T/H mice in vivo, overall resulting in increased mortality of T/H mice after infection with S. pneumoniae. Application of p38 MAPK inhibitor BIRB796 immediately after T/H induction improved the bacterial phagocytosis activity of macrophages from T/H mice in vitro and lung pneumococcal killing in vivo but did not improve the survival of T/H mice challenged with S. pneumoniae. T/H triggers sustained p38 MAPK activation in the lungs of mice, which attenuates lung macrophage antibacterial activities and renders mice more susceptible to pneumococcal pneumonia. However, no major role for dysregulated p38 MAPK to affect survival of T/H mice after pneumococcal challenge was detected, suggesting that dysregulated p38 MAPK activity may possibly play only a limited role in posttraumatic immunosuppression in mice.