Local delivery of GM-CSF protects mice from lethal pneumococcal pneumonia

Abstract

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.

Figures

Figure 1. Effect of lung-specific overexpression of GM-CSF on pulmonary leukocyte recruitment

Mice were treated with either control vector or Ad-GM-CSF (1×108 pfu/mouse) for 1, 3, 5, 7, 10, 14, 21, or 28 days. At indicated time points, mice were subjected to bronchoalveolar lavage and lungs were removed for isolation of lung mononuclear phagocyte subsets. GM-CSF levels were determined in BAL fluids by ELISA (A). Recruitment profiles of resident alveolar macrophages (B) or newly recruited alveolar exudate macrophages in BAL fluids (C), or resident or recruited lung macrophages (D, E), as well as BAL fluid neutrophils (F), and lymphocytes (G) were determined. Values are shown as mean ± SEM for n=5-9 mice per time point and treatment group. * (**, ***) indicates significant difference of p<0.05 (p<0.01, p<0.001) relative to control vector treated mice.

Figure 2. Pulmonary PU.1 expression and STAT 5 phosphorylation in the lungs of mice transiently overexpressing GM-CSF

Mice were treated with either control vector or Ad-GM-CSF (1×108pfu/mouse) for 3, 10, 14, or 28 days, as indicated. Subsequently, mice were sacrificed and immunoblot analysis of PU.1 expression or STAT5 phosphorylation was performed in whole lung lysates (A, B) or in lysates of alveolar macrophages 14 days after adenoviral transfer of GM-CSF (C). β-actin was used to control for similar protein loading of the gel (A, C). (B) Fold change of STAT5 phosphorylation relative to unphosphorylated STAT5 in Ad-GM-CSF versus control vector pre-treated mice. The western blots shown are representative of 3 independently performed experiments.

Figure 3. Survival and bacterial pathogen elimination in Ad-GM-CSF treated mice challenged with S. pneumoniae

Mice were pretreated with either control vector or Ad-GM-CSF (1×108 pfu/mouse). At day 14 mice were infected with serotype 19 S. pneumoniae (~ 1.3×107 CFU/mouse; n=10 mice per group) (A) or were infected with S. pneumoniae (~1.1×107 CFU/mouse; n=10 mice per group) at day 28 (B). The time point of pneumococcal infection is indicated as day 0 (A, B) and corresponds to day 14 (A) or 28 (B) post control vector or Ad-GM-CSF treatment. Survival was monitored during an observation period of 10 days. In separate experiments, bacterial loads were determined in bronchoalveolar lavage fluids and lung parenchymal tissue of mice pretreated for 14 or 28 days with Ad-GM-CSF or control vector prior to infection with serotype 19 S. pneumoniae (~1×107 CFU/mouse), as indicated (C, D). Values are shown as mean ± SEM for n=5-9 mice per time point and treatment group. * (**, ***) indicates significant difference of p<0.05 (p<0.01, p<0.001) relative to control vector treated mice.

Figure 4. Alveolar leukocyte recruitment profiles in Ad-GM-CSF pretreated mice subsequently infected with S. pneumoniae

Mice were pretreated with either control vector or Ad-GM-CSF (1×108 pfu/mouse) for 14 days (A-C) or 28 days (D-F) prior to infection with serotype 19 S. pneumoniae (1×107 CFU/mouse). At indicated time points, mice of the respective treatment groups were subjected to bronchoalveolar lavage for determination of resident alveolar macrophages (A, D), or newly recruited exudate macrophages (B, E), or neutrophils (C, F). (G, H) Histopathological assessment of interstitial lung inflammation in hematoxylin/eosin (HE)-stained lung tissue sections of mice either pretreated with control vector or Ad-GM-CSF vector for 7 days (G) or 14 days (H) followed by infection with serotype 19 S. pneumoniae (1×107 CFU/mouse) for 7 days. Values are shown as mean ± SEM for n=5-9 mice per time point and treatment group. * (**, ***) indicates significant differences of p<0.05 (p<0.01, p<0.001) relative to control vector treated mice.

Figure 5. Apoptosis and necrosis induction in Ad-GM-CSF treated mice subsequently infected with S. pneumoniae

Mice were pretreated with either control vector or Ad-GM-CSF (1×108 pfu/mouse) for 14 days (A, B) or 28 days (C, D, E, F) prior to infection with serotype 19 S. pneumoniae (1×107 CFU/mouse). At 24 hours post-infection, mice of the respective treatment groups were subjected to bronchoalveolar lavage and percentage of apoptosis (annexin V-positive; A, C, E) and secondary necrosis (annexin V-positive and propidium iodide-positive; B, D, F) was determined by flow cytometry. Values are shown as mean ± SEM for n=6 mice per time point and treatment group. * indicates significant difference (p<0.05) relative to control vector treated mice. The shown experiment is representative of three independent determinations.

Figure 6. Prophylactic and therapeutic treatment of S. pneumoniae-infected mice with recombinant murine GM-CSF

For prophylactic GM-CSF treatment, mice were treated orotracheally with either vehicle (white bars) or recombinant murine GM-CSF (10 μg/mouse, black bars) for 3, 6, or 12 hours prior to infection with serotype 19 S. pneumoniae (5×106 CFU/mouse) (A, B). For therapeutic GM-CSF treatment, mice were infected with serotype 19 S. pneumoniae (5×106 CFU/mouse) followed by therapeutic application of either vehicle or recombinant murine GM-CSF (20 μg/mouse) applied at 1.5, 3, 6, or 12 hours post-infection (C, D). At 24 hours post-infection, mice were subjected to bronchoalveolar lavage and bacterial loads (A, C), or newly recruited exudate macrophages (B, D) were quantified in BAL fluids. Values are shown as mean ± SEM for n=5-9 mice per time point and treatment group. * (**) indicates significant difference of p<0.05 (p<0.01) relative to vehicle treated mice.

Figure 7. Effect of intra-alveolar therapeutic GM-CSF application on iNOS expression in flow-sorted alveolar macrophages after S. pneumoniae infection

Mice were infected with serotype 19 S. pneumoniae (5×106 CFU/mouse) followed by intra-alveolar delivery of recombinant mGM-CSF (20 μg/mouse) or vehicle at 3 hours post-infection. At 6 hours post-infection, mice were subjected to bronchoalveolar lavage, and flow-sorted alveolar macrophages and neutrophils were subjected to real-time RT-PCR (A, B) or western blot analysis of iNOS mRNA or protein expression (C), respectively. (A) Fold change in iNOS mRNA levels in S. pneumoniae-infected mice treated with GM-CSF relative to S. pneumoniae-infected, vehicle treated mice. (B) Fold change in iNOS mRNA levels of S. pneumoniae-infected vehicle-versus GM-CSF-treated mice relative to uninfected mice. (C) iNOS protein expression in flow-sorted alveolar macrophages of mice treated with GM-CSF (black bars) or vehicle (white bars) at 3 h after infection with S. pneumoniae for 24 h. iNOS protein is expressed as fold change relative to GAPDH. Data in A-C are shown as mean±SEM of n=3 mice per treatment group. (D) Pneumococcal killing by alveolar macrophages infected with serotype 19 S. pneumoniae (MOI 50) for 30 minutes, followed by incubation with or without GM-CSF for the indicated time points. Values are shown as mean ± SEM of triplicate determinations, and the experiment was repeated twice.

Figure 8. Apoptosis and secondary necrosis in alveolar macrophages of S. pneumoniae-infected mice after therapeutic treatment with GM-CSF

Mice were infected with serotype 19 S. pneumoniae (5×106 CFU/mouse) followed by intratracheal treatment with either vehicle (white bars) or recombinant murine GM-CSF (20 μg/mouse, black bars) at 3 hours post-infection. At 24 hours and 48 hours post pneumococcal challenge, mice were subjected to bronchoalveolar lavage and the percentage of apoptotic (A) and secondary necrotic (B) alveolar macrophages was determined by flow cytometry. (C) Immunoblot analysis of caspase 3 and cleaved caspase 3 expression in BAL cells of S. pneumoniae-infected mice treated with vehicle or GM-CSF at 3 h post-infection, as indicated. β-actin was used to control for similar protein loading of the gel (C). The given western blot is representative of 3 independent experiments. Values are shown as mean ± SEM for n=3 mice per time point and treatment group and experiments were repeated twice with similar results. * indicates significant difference of p<0.05 relative to vehicle treated mice.