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Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

Objectives: Community-acquired pneumonia is a very common infectious disease associated with significant morbidity and mortality. Streptococcus pneumoniae is the predominant pathogen in this disease, and pneumococcal resistance to multiple antibiotics is increasing. The recently purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci on contact. The aim of this study was to determine the therapeutic potential of Cpl-1 in a mouse model of severe pneumococcal pneumonia., Design: Controlled, in vivo laboratory study., Subjects: Female C57/Bl6 mice, 8-12 weeks old., Interventions: Mice were transnasally infected with pneumococci and therapeutically treated with Cpl-1 or amoxicillin by intraperitoneal injections starting 24 or 48 hours after infection., Measurements and Main Results: Judged from clinical appearance, decreased body weight, reduced dynamic lung compliance and Pao2/Fio2 ratio, and morphologic changes in the lungs, mice suffered from severe pneumonia at the onset of therapy. When treatment was commenced 24 hours after infection, 100% Cpl-1-treated and 86% amoxicillin-treated mice survived otherwise fatal pneumonia and showed rapid recovery. When treatment was started 48 hours after infection, mice had developed bacteremia, and three of seven (42%) Cpl-1-treated and five of seven (71%) amoxicillin-treated animals survived. Cpl-1 dramatically reduced pulmonary bacterial counts, and prevented bacteremia, systemic hypotension, and lactate increase when treatment commenced at 24 hours. In vivo, treatment with Cpl-1 or amoxicillin effectively reduced counts of penicillin-susceptible pneumococci. The inflammatory response in Cpl-1-and amoxicillin-treated mice was lower than in untreated mice, as determined by multiplex cytokine assay of lung and blood samples. In human epithelial cell cultures, lysed bacteria evoked less proinflammatory cytokine release and cell death, as compared with viable bacteria., Conclusions: Cpl-1 may provide a new therapeutic option in the treatment of pneumococcal pneumonia.

Objective: Severe pneumococcal pneumonia frequently causes respiratory failure. Both pathogen factors and an uncontrolled host response may contribute to acute lung injury by impairing microvascular barrier function. Phosphodiesterase 2 (PDE2) was examined as a potential target in pneumonia-induced lung microvascular hyperpermeability., Design: Controlled, in vitro, ex vivo, and in vivo laboratory study., Subjects: Female Balb/C and C57Bl/6 mice, 8-12 weeks old., Interventions: Human umbilical vein endothelial cells and isolated mouse lungs were challenged with the pneumococcal exotoxin pneumolysin in the presence or absence of the selective PDE2 inhibitors 9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6one (PDP) or hydroxy-PDP. Transcellular electrical resistance or human serum albumin leakage in bronchoalveolar lavage fluid was determined, respectively. In addition, we induced pneumococcal pneumonia in mice and treated with hydroxy-PDP via continuous subcutaneous application by osmotic pumps. Human serum albumin leakage in bronchoalveolar lavage fluid was measured 48 hours after transnasal infection, and lung specimens were analyzed by TaqMan real-time polymerase chain reaction and Western blot for PDE2 gene and protein expression., Measurements and Main Results: In isolated perfused mouse lungs and in human umbilical vein endothelial cell monolayers, selective inhibition of PDE2 markedly decreased pneumolysin-induced hyperpermeability. Furthermore, in murine pneumococcal pneumonia, pulmonary PDE2-mRNA and -protein expression was significantly increased, and pneumonia-induced vascular permeability was distinctively reduced by PDE2 inhibition., Conclusions: PDE2 inhibition diminished microvascular leakage in pneumococcal pneumonia, and pulmonary PDE2 upregulation may play a crucial role in this respect. Selective PDE2 inhibitors thus may offer a promising therapeutic approach in severe pneumococcal pneumonia.

Objective: Acute respiratory failure is a major complication of severe pneumococcal pneumonia, characterized by impairment of pulmonary microvascular barrier function and pulmonary hypertension. Both features can be evoked by pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae. We hypothesized that platelet-activating factor (PAF) and associated downstream signaling pathways play a role in the PLY-induced development of acute lung injury., Design: Controlled, ex vivo laboratory study., Subjects: Female Balb/C mice, 8-12 wks old., Interventions: Ventilated and blood-free-perfused lungs of wild-type and PAF receptor-deficient mice were challenged with recombinant PLY., Measurements and Main Results: Intravascular PLY, but not the pneumolysoid Pd-B (PLY with a Trp-Phe substitution at position 433), caused an impressive dose-dependent increase in pulmonary vascular resistance and increased PAF in lung homogenates, as detected by reversed-phase high-performance liquid chromatography coupled to tandem mass spectrometry. The pressor response was reduced in lungs of PAF receptor-deficient mice and after PAF receptor blockade by BN 50730. PLY and exogenous PAF increased thromboxane B2 in lung effluate, and thromboxane receptor inhibition by BM 13505 diminished the pressor response to PLY. Differential inhibition of intracellular signaling steps suggested significant contribution of phosphatidylcholine-specific phospholipase C and protein kinase C and of the Rho/Rho-kinase pathway to PLY-induced pulmonary vasoconstriction. Unrelated to the pulmonary arterial pressor response, microvascular leakage of PLY was diminished in lungs of PAF receptor-deficient mice as well., Conclusions: PAF significantly contributed to PLY-induced acute injury in murine lungs. The PAF-mediated pressor response to PLY depends on thromboxane and on the downstream effectors phosphatidylcholine-specific phospholipase C, protein kinase C, and Rho-kinase.

Interleukin (IL)-15 has critical impact on the homeostasis and activation of natural killer cells, natural killer T cells, gammadeltaT cells, and CD8(+)T cells, and contributes to antimicrobial defenses particularly at mucosal sites. The respiratory tract comprises a large mucosal surface and harbors significant amounts of lymphocytes, however the expression pattern of IL-15 in the lung and its role in local immune responses are largely unknown. We therefore analyzed the differential expression of IL-15 and the IL-15 receptor (IL-15R) complex in the lungs of mice and demonstrated substantial constitutive expression in bronchial and alveolar epithelial cells, alveolar macrophages, and vascular smooth muscle cells, implicating contribution to pulmonary immune cell homeostasis already under normal conditions. The induction of pneumococcal pneumonia but not the infection with Chlamydophila pneumoniae evoked a significant up-regulation of IL-15 on alveolar macrophages and bronchial epithelial cells, with the latter presenting de-novo expression of IL-15 on their basolateral surface and additional up-regulation of IL-15Ralpha. Moreover, transcriptome analysis as well as semi-quantitative PCR indicated at least partial transcriptional regulation in mice lungs. In conclusion IL-15 is suggested being of functional importance in the pulmonary immune response against pneumococcal pneumonia.

Objective: Acute respiratory failure is a significant complication of severe pneumococcal pneumonia. In a mouse model, we observed early-onset lung microvascular leakage after pulmonary infection with Streptococcus pneumoniae, and we hypothesized that the important virulence factor pneumolysin may be the direct causative agent., Design: Controlled, in vivo, ex vivo, and in vitro laboratory study., Setting: Laboratory., Subjects: Female mice, 8-12 wks old., Interventions: Ventilated and blood-free perfused murine lungs were challenged with recombinant pneumolysin via the airways as well as via the vascular bed. In addition, we analyzed the impact of pneumolysin on electrical cell impedance and hydraulic conductivity of human umbilical vein endothelial cell (HUVEC) and alveolar epithelial cell (A549) monolayers., Measurements and Main Results: Aerosolized pneumolysin dose-dependently increased capillary permeability with formation of severe lung edema but did not affect pulmonary vascular resistance. Intravascular pneumolysin caused an impressive dose-dependent increase in pulmonary vascular resistance and in lung microvascular permeability. By immunohistochemistry, pneumolysin was detected mainly in endothelial cells of pulmonary arterial vessels, which concomitantly displayed strong vasoconstriction. Moreover, pneumolysin increased permeability of HUVEC and A549 monolayers. Interestingly, immunofluorescence of endothelial cell monolayers exposed to pneumolysin showed gap formation and moderate stress fiber generation., Conclusions: Pneumolysin may play a central role for early-onset acute lung injury due to severe pneumococcal pneumonia by causing impairment of pulmonary microvascular barrier function and severe pulmonary hypertension.