Jennifer Bohling

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

Neutrophil-derived TRAIL induces apoptosis of alveolar macrophages, limiting the spread of S. pneumoniae infection.

Cathepsin G and Neutrophil Elastase Play Critical and Nonredundant Roles in Lung-Protective Immunity against Streptococcus pneumoniae in Mice

ABSTRACT Neutrophil serine proteases cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3) have recently been shown to contribute to killing of Streptococcus pneumoniae in vitro. However, their relevance in lung-protective immunity against different serotypes of S. pneumoniae in vivo has not been determined so far. Here, we examined the effect of CG and CG/NE deficiency on the lung host defense against S. pneumoniae in mice. Despite similar neutrophil recruitment, both CG knockout (KO) mice and CG/NE double-KO mice infected with focal pneumonia-inducing serotype 19 S. pneumoniae demonstrated a severely impaired bacterial clearance, which was accompanied by lack of CG and NE but not PR3 proteolytic activity in recruited neutrophils, as determined using fluorescence resonance energy transfer (FRET) substrates. Moreover, both CG and CG/NE KO mice but not wild-type mice responded with increased lung permeability to infection with S. pneumoniae, resulting in severe respiratory distress and progressive mortality. Both neutrophil depletion and ablation of hematopoietic CG/NE in bone marrow chimeras abolished intra-alveolar CG and NE immunoreactivity and led to bacterial outgrowth in the lungs of mice, thereby identifying recruited neutrophils as the primary cellular source of intra-alveolar CG and NE. This is the first study showing a contribution of neutrophil-derived neutral serine proteases CG and NE to lung-protective immunity against focal pneumonia-inducing serotype 19 S. pneumoniae in mice. These data may be important for the development of novel intervention strategies to improve lung-protective immune mechanisms in critically ill patients suffering from severe pneumococcal pneumonia.

Local Delivery of GM-CSF Protects Mice from Lethal Pneumococcal Pneumonia

The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2–4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3–dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.

Cathepsin G and Neutrophil Elastase Contribute to Lung-Protective Immunity against Mycobacterial Infections in Mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

Efficacy Profiles of Daptomycin for Treatment of Invasive and Noninvasive Pulmonary Infections with Streptococcus pneumoniae

ABSTRACT Daptomycin is a novel lipopeptide antibiotic with excellent activity against Gram-positive bacterial pathogens, but its therapeutic value for the treatment of invasive pneumococcal disease compared to that for the treatment of pneumococcal pneumonia is incompletely defined. We investigated the efficacy of daptomycin in two models of Streptococcus pneumoniae-induced lung infection, i.e., pneumococcal pneumonia and septic pneumococcal disease. Mice were infected with a bioluminescent, invasive serotype 2 S. pneumoniae strain or a less virulent serotype 19 S. pneumoniae strain and were then given semitherapeutic or therapeutic daptomycin or ceftriaxone. Readouts included survival; bacterial loads; and septic disease progression, as determined by biophotonic imaging. Semitherapeutic daptomycin treatment fully protected the mice against the progression of septic disease induced by serotype 2 S. pneumoniae, while therapeutic treatment of the mice with daptomycin or ceftriaxone led to ∼70% or ∼60% survival, respectively. In contrast, mice infected with serotype 19 S. pneumoniae developed severe pneumonia and lung leakage even in the presence of increased intra-alveolar daptomycin levels, resulting in only 40% survival, whereas the ceftriaxone-treated mice had 100% survival. Together, although daptomycin demonstrates little efficacy in the treatment of pneumococcal pneumonia, daptomycin is highly effective in preventing S. pneumoniae-induced septic death, thus possibly offering a therapeutic option for patients with life-threatening septic pneumococcal disease.

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin

Rationale Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly. Objectives To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice. Methods Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis. Measurements and main results We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-β1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFβ1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFβ1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFβ1-exposed mice. Conclusions Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice.

Macrophage-Inducible C-Type Lectin Mincle-Expressing Dendritic Cells Contribute to Control of Splenic Mycobacterium bovis BCG Infection in Mice

ABSTRACT The macrophage-inducible C-type lectin Mincle has recently been identified to be a pattern recognition receptor sensing mycobacterial infection via recognition of the mycobacterial cell wall component trehalose-6′,6-dimycolate (TDM). However, its role in systemic mycobacterial infections has not been examined so far. Mincle-knockout (KO) mice were infected intravenously with Mycobacterium bovis BCG to mimic the systemic spread of mycobacteria under defined experimental conditions. After intravenous infection with M. bovis BCG, Mincle-KO mice responded with significantly higher numbers of mycobacterial CFU in spleen and liver, while reduced granuloma formation was observed only in the spleen. At the same time, reduced Th1 cytokine production and decreased numbers of gamma interferon-producing T cells were observed in the spleens of Mincle-KO mice relative to the numbers in the spleens of wild-type (WT) mice. The effect of adoptive transfer of defined WT leukocyte subsets generated from bone marrow cells of zDC+/DTR mice (which bear the human diphtheria toxin receptor [DTR] under the control of the classical dendritic cell-specific zinc finger transcription factor zDC) to specifically deplete Mincle-expressing classical dendritic cells (cDCs) but not macrophages after diphtheria toxin application on the numbers of splenic and hepatic CFU and T cell subsets was then determined. Adoptive transfer experiments revealed that Mincle-expressing splenic cDCs rather than Mincle-expressing macrophages contributed to the reconstitution of attenuated splenic antimycobacterial immune responses in Mincle-KO mice after intravenous challenge with BCG. Collectively, we show that expression of Mincle, particularly by cDCs, contributes to the control of splenic M. bovis BCG infection in mice.

Evaluation of Biophotonic Imaging To Estimate Bacterial Burden in Mice Infected with Highly Virulent Compared to Less Virulent Streptococcus pneumoniae Serotypes

ABSTRACT Bioluminescence imaging is an innovative, noninvasive tool to analyze infectious disease progression under real-life conditions in small laboratory animals. However, the relevance of bioluminescence imaging to monitor invasive compared to noninvasive bacterial infections of the lung has not been examined so far. In the current study, we systematically evaluated the importance of bioluminescence imaging to monitor pneumococcal disease progression by correlating biophotonic signals with lung bacterial loads in two mouse strains (BALB/c, C57BL/6) infected with either self-glowing, bioluminescent serotype 19 Streptococcus pneumoniae causing focal pneumonia or serotype 2 S. pneumoniae causing invasive pneumococcal disease. The best correlations between bioluminescence signals and lung CFU counts were observed in BALB/c mice compared to C57BL/6 mice just on day 3 after infection with invasive serotype 2 S. pneumoniae, while excellent correlations between photon counts and bacterial loads were observed in isolated lungs of BALB/c and C57BL/6 mice, irrespective of the employed pneumococcal serotype. Moreover, good correlations between biophotonic signals and CFU counts were also observed in mice upon infection with serotype 19 S. pneumoniae causing focal pneumonia in mice, again with best correlation values obtained for BALB/c mice at day 3 postinfection. Collectively, we show that the relevance of biophotonic imaging to monitor S. pneumoniae-induced lung infections in mice is largely influenced by the disease model under investigation. The provided data may be important for studies of infectious diseases.

FMS-Like Tyrosine Kinase 3 Ligand Treatment of Mice Aggravates Acute Lung Injury in Response to Streptococcus pneumoniae: Role of Pneumolysin

ABSTRACT FMS-like tyrosine kinase-3 ligand (Flt3L) is a dendritic cell (DC) growth and differentiation factor with potential in antitumor therapies and antibacterial immunization strategies. However, the effect of systemic Flt3L treatment on lung-protective immunity against bacterial infection is incompletely defined. Here, we examined the impact of deficient (in Flt3L knockout [KO] mice), normal (in wild-type [WT] mice), or increased Flt3L availability (in WT mice pretreated with Flt3L for 3, 5, or 7 days) on lung DC subset profiles and lung-protective immunity against the major lung-tropic pathogen, Streptococcus pneumoniae. Although in Flt3L-deficient mice the numbers of DCs positive for CD11b (CD11bpos DCs) and for CD103 (CD103pos DCs) were diminished, lung permeability, a marker of injury, was unaltered in response to S. pneumoniae. In contrast, WT mice pretreated with Flt3L particularly responded with increased numbers of CD11bpos DCs and with less pronounced numbers of CD103pos DCs and impaired bacterial clearance and with increased lung permeability following S. pneumoniae challenge. Notably, infection of Flt3L-pretreated mice with S. pneumoniae lacking the pore-forming toxin, pneumolysin (PLY), resulted in substantially less lung CD11bpos DCs activation and reduced lung permeability. Collectively, this study establishes that Flt3L treatment enhances the accumulation of proinflammatory activated lung CD11bpos DCs which contribute to acute lung injury in response to PLY released by S. pneumoniae.

Cathepsin G in Experimental Tuberculosis: Relevance for Antibacterial Protection and Potential for Immunotherapy

Neutrophil serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), have been implicated in the protective response against infections, including experimental mycobacterial infections. The goal of this study was to explore the role of CG in immunocompetent mice challenged aerogenically with Mycobacterium tuberculosis. We used genetically CG- or CG/NE-deficient mice to define the importance of these neutrophil serine proteases for antibacterial protection, granulomatous response, and survival. In addition, we explored the effect of intratracheally delivered liposomally encapsulated CG/NE as a therapeutic approach early during M. tuberculosis infection. Our data show that the presence of CG or CG/NE prolongs survival in M. tuberculosis–infected mice. However, CG is not directly involved in antibacterial defenses, and exogenous intratracheal administration of CG combined with NE does not reduce bacterial loads in the lungs of M. tuberculosis–infected mice.