Lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled GM-CSF in a postinfluenza pneumococcal pneumonia model

Abstract

Inhaled granulocyte/macrophage colony-stimulating factor (GM-CSF) shows promise as a therapeutic to treat viral and bacterial pneumonia, but no mouse model of inhaled GM-CSF has been described. We sought to 1) develop a mouse model of aerosolized recombinant mouse GM-CSF administration and 2) investigate the protection conferred by inhaled GM-CSF during influenza A virus (IAV) infection against secondary bacterial infection with pneumococcus. To assess lower respiratory tract delivery of aerosolized therapeutics, mice were exposed to aerosolized fluorescein (FITC)-labeled dextran noninvasively via an aerosolization tower or invasively using a rodent ventilator. The efficiency of delivery to the lower respiratory tracts of mice was 0.01% noninvasively compared with 0.3% invasively. The airway pharmacokinetics of inhaled GM-CSF fit a two-compartment model with a terminal phase half-life of 1.3 h. To test if lower respiratory tract levels were sufficient for biological effect, mice were infected intranasally with IAV, treated with aerosolized recombinant mouse GM-CSF, and then secondarily infected with Streptococcus pneumoniae. Inhaled GM-CSF conferred a significant survival benefit to mice against secondary challenge with S. pneumoniae (P < 0.05). Inhaled GM-CSF did not reduce airway or lung parenchymal bacterial growth but significantly reduced the incidence of S. pneumoniae bacteremia (P < 0.01). However, GM-CSF overexpression during influenza virus infection did not affect lung epithelial permeability to FITC-dextran ingress into the bloodstream. Therefore, the mechanism of protection conferred by inhaled GM-CSF appears to be locally mediated improved lung antibacterial resistance to systemic bacteremia during IAV infection.

Keywords: GM-CSF; aerosol; inhaled; pharmacokinetics; pneumonia.

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.

A: Scireq inExpose aerosolization tower system wherein aerosolized drugs can be administered simultaneously to up to 12 mice. B: mice are held in place, with their noses exposed to the aerosol, using a soft restraint system that prevents repeated sedation. C: FITC-dextran (mol wt 10,000) was diluted in PBS at 200, 2,000, and 20,000 μg/mL, aerosolized, and administered to mice (n = 12–18 mice per group). At 0–400 s during aerosolization, mice were removed from the tower, the lower respiratory tract was immediately sampled via tracheostomy and bronchoalveolar lavage, and the amount of FITC-dextran was quantified by spectrophotometry. D: lower respiratory tract recovery (delivery) in the mouse was low, with %recovery [recovery (%) = measured (μg)/theoretical delivery (μg) × 100] stabilizing at ~0.01%. E: optimal exposure time was determined by administration of a fixed amount of FITC-labeled dextran (5 μg) to mice (n = 4–6 per group) over various exposure times and aerosol solution concentrations shown in Table 1. Mice treated with FITC-dextran over 22.5 s had the highest mean recovery (3.71 ± 0.21 ng) that was significantly (*P < 0.05) increased from all durations of exposure >45 s; yet the 22.5-s duration group had the lowest coefficient of variance (5.5%).

Fig. 2.

A: lower respiratory tract recovery of FITC-dextran after tracheostomy and aerosol administration using the Scireq flexiVent rodent ventilator. During mechanical ventilation, various concentrations (20, 200, and 2,000 μg/mL) of aerosols were generated and administered over a fixed time period (120 s) to mice (n = 5–14 mice per group). B: recovered FITC-dextran correlated with the concentration administered (r = 0.969, P < 0.001) via the flexiVent, and recovery (%) was similar across all aerosol concentrations. C: FITC-dextran delivery/recovery was significantly reduced in younger mice, regardless of aerosol concentration. *P < 0.05, **P < 0.01, ***P < 0.001. D: size effect in C was also observed when delivery/recovery was correlated with body weight (BW) in adult mice.

Fig. 3.

A: lower respiratory tract delivery/recovery of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) following aerosolization and administration of recombinant mouse GM-CSF (rmGM-CSF, 5 μg) to mice (n = 4–6 per group) over various exposure times and aerosol concentrations via the inExpose system. Immediately after aerosol exposure, bronchoalveolar lavage (BAL) fluid was recovered, and mouse GM-CSF levels were measured by ELISA. High-concentration aerosols with short exposure times (analyzed as a categorical variable) led to increased GM-CSF deposition in the lower respiratory tract compared with longer exposure times (*P < 0.05). B: analysis of exposure time as a continuous variable showed a significant negative correlation between exposure time and GM-CSF recovery. C: to assay the airway pharmacokinetics, aerosolized rmGM-CSF (5 μg, 100 μg/mL in PBS) was administered to mice (n = 5–6 per group) over 22.5 s, BAL fluid was recovered 0–4 h after exposure, and mouse GM-CSF levels were measured by ELISA. Airway pharmacokinetics of inhaled GM-CSF demonstrated a 2-compartment model with an initial (distribution) phase [half-life (t1/2) = 0.88 h] followed by a less-steep (terminal) phase (t1/2 = 1.3 h).

Fig. 4.

A and B: effect of administration of recombinant mouse granulocyte/macrophage colony-stimulating factor [rmGM-CSF (Rx)] during influenza A virus (IAV) infection and secondary bacterial pneumonia with Streptococcus pneumoniae was examined in terms of body weight loss/recovery and survival. rmGM-CSF (5 μg) was administered twice daily via inhalation using the inExpose system (iGM-CSF), or 1 ng was administered via intraperitoneal injection at the same time points (ip GM-CSF), whereas control mice only received inhaled PBS (iPBS). Numbers of mice per group surviving or still at risk are listed below days postinjection (DPI) in B. Inhaled rmGM-CSF conferred a significant survival (*P < 0.05) compared with the iPBS control group. C: to determine whether iGM-CSF enhanced bacterial clearance in various organ compartments, mice were harvested 18 h after secondary infection with S. pneumoniae, and bronchoalveolar lavage (BAL) fluid, lung homogenates, and liver homogenates were grown on blood agar plates. Inhaled GM-CSF did not affect bacterial counts in airways or lung parenchyma but lowered bacterial growth from liver homogenates (**P < 0.01), suggesting less systemic bacteremia.

Fig. 5.

Effect of granulocyte/macrophage colony-stimulating factor (GM-CSF) overexpression during influenza A virus (IAV) infection on lung epithelial permeability was examined using FITC-dextran. Airway GM-CSF-overexpressing transgenic (DTGM) or littermate control (LM) mice were infected with IAV, and doxycycline was administered in drinking water starting at 3 days postinfection. At 10 days postinfection, mice were tracheotomized, received FITC-dextran via the tracheostomy needle, and were ventilated for 15 min. Serum FITC-dextran levels were measured by spectrophotometry in uninfected LM (△) and DTGM (▲) mice and at 10 days postinfection in LM (○) and DTGM (●) mice. GM-CSF overexpression in the absence of IAV infection significantly increased lung permeability (*P < 0.05). While IAV infection increased lung permeability compared with uninfected LM mice (**P < 0.01), in IAV-infected animals (○ and ●), GM-CSF overexpression did not affect serum levels of FITC-dextran.