Surfactant protein-A enhances respiratory syncytial virus clearance in vivo

Abstract

To determine the role of surfactant protein-A(SP-A) in antiviral host defense, mice lacking SP-A (SP-A-/-) were produced by targeted gene inactivation. SP-A-/- and control mice (SP-A+/+) were infected with respiratory syncytial virus (RSV) by intratracheal instillation. Pulmonary infiltration after infection was more severe in SP-A-/- than in SP-A+/+ mice and was associated with increased RSV plaque-forming units in lung homogenates. Pulmonary infiltration with polymorphonuclear leukocytes was greater in the SP-A-/- mice. Levels of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 were enhanced in lungs of SP-A-/- mice. After RSV infection, superoxide and hydrogen peroxide generation was deficient in macrophages from SP-A-/- mice, demonstrating a critical role of SP-A in oxidant production associated with RSV infection. Coadministration of RSV with exogenous SP-A reduced viral titers and inflammatory cells in the lung of SP-A-/- mice. These findings demonstrate that SP-A plays an important host defense role against RSV in vivo.

Figure 1

Pulmonary pathology after RSV infection. Photomicrographs of lung parenchyma in (a) SP-A^–/– and (b) SP-A^+/+ mice were prepared 7 days after intratracheal administration of 10⁷ pfu RSV. Histological sections were stained with hematoxylin and eosin. Infiltration was observed surrounding the terminal bronchi and pulmonary vessels in the SP-A^–/– and SP-A^+/+ mice. Higher magnification demonstrates infiltration with mononuclear cells and polymorphonuclear leukocytes in SP-A^–/– mice (c). Cells from BAL consisted of mononuclear cells (arrowhead) and polymorphonuclear leukocytes (arrow) (d). a and b: ×10; c and d: ×40. BAL, bronchoalveolar lavage; pfu, plaque-forming units; RSV, respiratory syncytial virus; SP-A, surfactant protein-A.

Figure 2

Increased total cell counts in BAL fluid from SP-A^–/– mice. Lung cells were recovered by BAL, stained with trypan blue, and counted under light microscopy. (a) The low dose of RSV (10⁶ pfu) caused less migration of inflammatory cells in the lungs from both genotypes; however, an earlier influx of cells was found in SP-A^–/– (filled bars) BAL fluid than in SP-A^+/+ (hatched bars) mice. (b) SP-A^–/– mice infected with 10⁷ pfu RSV had significantly greater total cell counts in BAL fluid at all time points. Data are mean ± SEM with n = 5 mice per group (a) and n = 9 mice per group (b). *P < 0.05 compared with SP-A^+/+ mice.

Figure 3

Increased neutrophils in BAL fluid from SP-A^–/– mice after RSV infection. Cytospin preparations of BAL fluid were stained with Diff-Quik to identify macrophages, lymphocytes, and polymorphonuclear leukocytes. (a) The percentage of neutrophils in BAL fluid was significantly greater 3, 5, and 7 days after administration of 10⁷ pfu RSV to SP-A^–/– (filled bars) compared with SP-A^+/+ (hatched bars) mice. (b) Percentages of lymphocytes in BAL fluid were not different between the two groups. Data are mean ± SEM with n = 9 mice per group. *P < 0.05 compared with SP-A^+/+ mice. PMNs, polymorphonuclear leukocytes.

Figure 4

Increased RSV in lung homogenates from SP-A^–/– mice. RSV titers were determined by quantitative plaque assays of lung homogenates. Viral titers were significantly greater 3, 5, and 7 days after administration of both 10⁶ (a) and 10⁷ (b) pfu RSV in SP-A^–/– (filled bars) compared with SP-A^+/+ (hatched bars) mice. Data are mean ± SEM with n = 6 mice per group (a) and n = 10 mice per group (b). *P < 0.05 compared with SP-A^+/+ mice.

Figure 5

Increased tumor necrosis factor-α (TNF) and interleukin-6 (IL-6) in lung homogenates from SP-A^–/– mice after RSV infection. Concentrations of TNF-α, interleukin-1β (IL-1), IL-6, and macrophage inflammatory protein-2 (MIP-2) were assessed in lung homogenates from SP-A^–/– (filled bars) and SP-A^+/+ (hatched bars) mice. Increased concentrations of TNF-α and IL-6 were found in lung homogenates from the SP-A^–/– mice at 24 h. Although macrophage inflammatory MIP-2 was increased in SP-A^–/– at 24 h, differences did not reach statistical significance. Data are expressed as picograms per milliliter and represent mean ± SEM with n = 10 mice per group. *P < 0.05 compared with SP-A^+/+ mice.

Figure 6

SP-A enhanced RSV clearance from the lung of SP-A^–/– mice. RSV titers were determined by quantitative plaque assays of lung homogenates 3 days after infection with 10⁷ pfu of RSV. Viral titers in the lung were significantly reduced in SP-A^–/– mice treated with SP-A (100 μg) (cross-hatched bar) compared with untreated SP-A^–/– mice (filled bar) (a). Treatment of SP-A^–/– mice with SP-A reduced viral titers to the wild-type level. Viral titers were similar in SP-A–treated (open bar) and untreated wild-type mice (hatched bar) (b). Data are mean ± SEM. *P < 0.05 compared with SP-A^–/– mice. WT, wild-type.

Figure 7

SP-A decreased inflammatory cells in BAL fluid from SP-A^–/– mice. After RSV infection (10⁷ pfu), lung cells were recovered by BAL, stained with trypan blue, and counted under light microscopy. One and 3 days after RSV infection, total cell counts in BAL fluid were significantly reduced in SP-A^–/– mice treated with SP-A (100 μg) (cross-hatched bars) compared with untreated SP-A^–/– mice (filled bars). Treatment of SP-A^–/– mice reduced BAL cell counts to the wild-type level (hatched bars). Total cell counts in BAL fluid were similar in SP-A–treated (open bars) and untreated wild-type mice (hatched bars). Data are mean ± SEM. *P < 0.05 compared with SP-A^+/+ mice.