GM-CSF modulates pulmonary resistance to influenza A infection

Abstract

Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9×10(4) ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient (GM(-/-)) mice compared to GM(-/-) mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO(-/-) alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza.

Fig. 1

GM-CSF enhances host survival from influenza infection. The survival profile (A), rate of body weight loss (B) of WT, GM-CSF^−/− (GM^−/−), and SFTPC-GM^+/+ (SP-C-GM^+/+) mice, was determined after intranasal infection with 1 × 10⁴ ffc of PR8. Infected mice were weighed daily and assessed visually every 12 h for the presence of clinical symptoms of the infection. Mice displaying severe illness were euthanized immediately and counted as dead. The number of mice used and statistical parameters derived from survival curves are listed on Table 1. (C) Comparison of body weight of uninfected WT (n = 13), GM^−/− (n = 19), and SP-C-GM^+/+ (n = 18) mice. Data shown are mean ± SD. ^**p < 0.01. (D) ELISA assays assessed the amount of GM-CSF in lavage and post-lavage lung homogenates before, and at indicated intervals after infection with PR8. Data shown are mean ± SEM. N = 3–4 mice per time point. ^*p < 0.05, and ^**p < 0.001 compared to un-infected control.

Fig. 2

Histopathology of infected SP-C-GM^+/+ mice. Lung histopathology was evaluated at 7 (A) and 29 (B) days after infection of SP-C-GM^+/+ mice with 1.9 × 10³ ffc influenza PR8. Images were captured at 10× magnification. Interstitial thickening and macrophage aggregates at day 7 are indicated by closed and open arrows, respectively. At day 29, degeneration of alveolar structure (closed arrow) and large spaces containing desquamated cells (open arrow) are indicated.

Fig. 3

Conditional expression of GM-CSF in the lung. Tet-GM^+/+ mice were provided sterile water supplemented with 1 mg/mL doxycycline to induce expression of GM-CSF. (A) ELISA assays assessed expression of GM-CSF in lavage and post-lavage tissue homogenate at indicated time intervals. Data shown are means ± SD, n = 3–5 mice per time point. ^***p < 0.001 compared to the absence of doxycycline. (B) Flow cytometry reported the light scatter properties of cells in alveolar lavage before (0 days) or 6 and 12 days after addition of doxycycline in water. Gating distinguished the location of alveolar macrophages from surfactant debris at the bottom corner of the dot plots. Data shown are representative of 3–6 separate experiments per time point. (C) Representative cytospin evaluation of alveolar macrophages from tet-GM^+/+ mice before (left panel) and 6 days after administration of doxycycline (right panel).

Fig. 4

Effects of conditional expression of GM-CSF on differentiation and number of alveolar macrophages. Alveolar macrophages were isolated by lung lavage before (0 day) or after induction of GM-CSF in tet-GM^+/+ mice. (A) Alveolar macrophage differentiation was assessed by dual flow cytometric cell surface staining using CD11c and SP-R210 antibodies. The percentage of gated alveolar cells that express CD11c and SP-R210 before or 6 and 12 days after induction of GM-CSF is shown for each marker. (B) Alveolar cells were counted using a hemacytometer before or at 12 and 29 days after administration of doxycycline. Data shown are means ± SD; n = 3 on day 0, and n = 4 on days 6, and n = 3 on day 29. ^***p < 0.001.

Fig. 5

Effect of conditional expression of GM-CSF on survival from influenza infection. Survival curves (A) and body weight (B) of tet-GM^+/+ mice was assessed after intranasal infection with 1.9 × 10⁴ ffc of influenza PR8 of conditional mice placed on doxycycline 6, 3, or 2 days after, same day, and 3 days before infection. Mice displaying severe illness were euthanized immediately and counted as dead. Statistical parameters and number of mice used in this study are shown on Table 2.

Fig. 6

Lung histopathology of WT and transgenic mice infected with influenza. The histology of naive WT (A) and GM^−/− (D) mouse lungs was visualized after staining with anti-SP-B antibodies. Open arrows point to SP-B staining in alveolar type II epithelial cells in WT lungs (A) and surfactant aggregates accumulated in the alveolar lumen of GM^−/− lungs (D). Lung histopathology after infection with 1.9 × 10³ ffc of influenza PR8 was evaluated following H&E stained lung sections from WT (B and C), GM^−/− (E and F), and tet-GM^+/+ mice infected 3 days after induction of GM-CSF with doxycycline. WT mice B and C; the open arrows on panel B show lymphocytic infiltrates in peribronchial spaces lacking defined alveolar structure in WT mice 6 days after infection. Open arrows on panel C show alveolar and bronchiolar hemorrhage and closed arrows on the same panel point to vacuolated alveolar epithelium. GM^−/− mice E and F; open arrows on E and F show eosinophilic surfactant material filling alveolar spaces. The stars on panel F show bronchi with flattened epithelium with light blue eosinophilic material filling bronchial spaces. tet-GM^+/+ mice G–I; closed thin arrows on panels E, H, and I show organized lymphocytic inflammation in peribronchiolar interstitium. Thick stealth arrows on panel H point to sub mucosal lymphocytic infiltrates and bronchial epithelium with columnar morphology. The star on panel H indicates the presence of proteinaceous edema. Images H and I were captured at 40× magnifications. All other images are 20×. Representative images from 2 to 3 mice are shown.

Fig. 7

Role of SR-A and MARCO on host survival from influenza infection. Survival (A) and body weight change (B) after intranasal infection of WT, SR-A^−/−, and MARCO^−/− mice with PR8 influenza. WT and SR-A^−/− mice were infected with 1.9 × 10⁴ ffc of PR8. MARCO^−/− mice were infected with either 1.9 × 10⁴ or 1.9 × 10³ ffc of PR8 as indicated. Infected mice were weighed daily and assessed every 12 h for the presence of clinical symptoms of the infection. Mice displaying severe illness were euthanized immediately and counted as dead. Statistical parameters and number of mice used in this study are shown on Table 1. (C) The percentage of SP-R210-positive alveolar macrophages from WT and MARCO^−/− mice was assessed by flow cytometry. (D) The expression level of SP-R210 shown as mean fluorescence intensity in alveolar macrophages from WT and SR-A ^−/−, MARCO^−/−, SP-C-GM^+/+, GM^−/−, and 6 day induced tet-GM^+/+ mice was assessed by flow cytometry. Data shown are means ± SD, n = 3–6 per mouse. ^***p ≤ 0.001 compared to WT mice.