Surfactant protein C-deficient mice are susceptible to respiratory syncytial virus infection

Abstract

Patients with mutations in the pulmonary surfactant protein C (SP-C) gene develop interstitial lung disease and pulmonary exacerbations associated with viral infections including respiratory syncytial virus (RSV). Pulmonary infection with RSV caused more severe interstitial thickening, air space consolidation, and goblet cell hyperplasia in SP-C-deficient (Sftpc(-/-)) mice compared with SP-C replete mice. The RSV-induced pathology resolved more slowly in Sftpc(-/-) mice with lung inflammation persistent up to 30 days postinfection. Polymorphonuclear leukocyte and macrophage counts were increased in the bronchoalveolar lavage (BAL) fluid of Sftpc(-/-) mice. Viral titers and viral F and G protein mRNA were significantly increased in both Sftpc(-/-) and heterozygous Sftpc(+/-) mice compared with controls. Expression of Toll-like receptor 3 (TLR3) mRNA was increased in the lungs of Sftpc(-/-) mice relative to Sftpc(+/+) mice before and after RSV infection. Consistent with the increased TLR3 expression, BAL inflammatory cells were increased in the Sftpc(-/-) mice after exposure to a TLR3-specific ligand, poly(I:C). Preparations of purified SP-C and synthetic phospholipids blocked poly(I:C)-induced TLR3 signaling in vitro. SP-C deficiency increases the severity of RSV-induced pulmonary inflammation through regulation of TLR3 signaling.

Fig. 1.

Respiratory syncytial virus (RSV)-induced pulmonary pathology. Lungs from FVB/N Sftpc^+/+ and Sftpc^−/− mice infected with 4 × 10⁷ pfu RSV and from media-instilled control mice were prepared for histology and stained with hematoxylin and eosin. a: photomicrographs from day 5 post-RSV infection reveal mild perivascular and alveolar infiltration in Sftpc^+/+ mice (A and B) with increased infiltration in the lungs of Sftpc^−/− mice (C and D). Representative low-magnification images are shown in A and C (×20 magnification), and high-magnification images in B and D (×40 magnification). High-magnification images demonstrate the mixed cell infiltrates and pronounced interstitial changes in the lungs of Sftpc^−/− mice (D). Insets in B and D are high-magnification images that indicate the enlarged appearance of macrophages (arrows) and increased interstitial changes including thickened alveolar septum (arrowheads) in the lungs of infected Sftpc^−/− mice relative to Sftpc^+/+ mice. b: the morphology of lungs from untreated and mock-infected adult FVB/N Sftpc^+/+ (A and C) and Sftpc^−/− (B and D) mice appeared normal with no overt inflammation. Representative images of untreated mice are shown at the top; mock-infected mice are shown at the bottom (×20magnification).

Fig. 2.

RSV-induced goblet cell hypertrophy. Photomicrographs are of lungs sections immunostained with an antibody specific to the mucin granule protein Clca3 on day 5 post-RSV infection. Images of lungs from control mock-infected FVB/N Sftpc^+/+ mice (A) and FVB/N Sftpc^−/− mice (C) are Clca3 negative. Small numbers of Clca3-positive cells were detected in the airway epithelium of RSV-infected FVB/N Sftpc^+/+ mice (B). Clca3-positive cells were increased in the large airways of infected FVB/N Sftpc^−/− mice (D). RSV-induced Clca3 staining was even more extensive in the lungs of 129S6 Sftpc^−/− mice. Rare Clca3-positive cells were detected in the lungs of 129S6 Sftpc^+/+ mice following RSV infection (E), whereas Clca3 staining was abundant along the airways of 129S6 Sftpc^−/− mice (F) and extended to bronchoalveolar junctions. The inset in F is a high-magnification image of the Clca3-positive cells at the junction of terminal bronchiole and alveolar ducts. The arrow indicates the region of F enlarged in the inset. In A–D, ×20 magnification; in E and F, ×10 magnification; inset in F, ×40 magnification.

Fig. 3.

Increased total cell counts in bronchoalveolar lavage fluid (BALF) from FVB/N Sftpc^−/− mice. Cells were recovered by low-speed centrifugation of BALF, resuspended in 150 μl of PBS and an aliquot stained with trypan blue, and counted under light microscopy. RSV-infected Sftpc^−/− mice had greater cell counts on days 1–9. Data are presented as means ± SE. *P < 0.05 compared with Sftpc^+/+ mice, 5 mice/group.

Fig. 4.

Increased RSV in the lungs of FVB/N Sftpc^−/− mice. Viral titers were determined by quantitative plaque assays of lung homogenates from Sftpc^+/+ and Sftpc^−/− mice. Viral titers were increased in the lung homogenates from Sftpc^−/− mice compared with Sftpc^+/+ mice on days 3 and 5 postinfection and undetectable by day 7. Day 3 values were significant (*P < 0.05). Data are presented as means ± SE.

Fig. 5.

Viral gene expression is increased in the lungs of FVB/N Sftpc^−/− mice. Expression of RSV genes encoding F and G proteins was measured by RT-PCR of RNA isolated from the lungs of Sftpc^+/+, Sftpc^+/−, and Sftpc^−/− mice 3 days after mock infection (media) or RSV infection (5 mice/genotype). RSV F gene expression was increased in the lungs of heterozygote +/− mice and −/− mice. RSV G gene expression was present in +/+ lungs and increased in +/− and −/− lungs. Viral gene expression was not detected in the lungs of control (media) mice.

Fig. 6.

Pulmonary collectin levels in BALF after RSV infection. The concentrations of SP-A and SP-D in the BALF of Sftpc^+/+ and Sftpc^−/− mice were determined by Western blot analysis and densitometry. SP-A levels were similar between both genotypes of mice. There was a proportional increase in SP-A levels after RSV infection that was not statistically different between the Sftpc^+/+ and Sftpc^−/− mice. SP-D levels were identical in the BALF of media-instilled and RSV-infected Sftpc^+/+ and Sftpc^−/− mice. Data are presented as means ± SE (n = 3 mice/group).

Fig. 7.

Inflammation is sustained in the lungs of Sftpc^−/− mice. FVB/N Sftpc^+/+ and Sftpc^−/− mice were infected with RSV and maintained in a pathogen-free barrier facility, and the lungs were inflation-fixed and processed for histology 30 days after RSV infection. There were no indications of inflammation remaining in the lungs of Sftpc^+/+ mice (top). Perivascular and mild alveolar inflammation remained in the lungs of Sftpc^−/− mice (bottom). These results indicate that RSV-induced inflammation is unresolved in the SP-C-deficient lung relative to recovery in the normal lung. Photomicrographs are representative of 5 mice/time point.

Fig. 8.

TLR3 expression is increased in the lungs of 129S6 Sftpc^−/− mice. On a 129S6 background, the Sftpc^−/− mice develop progressive lung inflammation and an irregular interstitial lung disease with age. TLR3 gene expression was determined by RT-PCR of lung RNA from Sftpc^+/+, Sftpc^+/−, and Sftpc^−/− mice on day 3 following RSV infection (5 mice/lane). The relative levels of TLR3 expression was increased incrementally in the lungs of uninfected heterozygote +/− and −/− mice relative to Sftpc^+/+ mice (media). TLR3 expression was stimulated by RSV infection.

Fig. 9.

Double-stranded RNA (dsRNA) induced inflammation in the lungs of 129S6 Sftpc^+/+ and Sftpc^−/− mice. To determine if the inflammation induced in the lungs of Sftpc^−/− mice is mediated by TLR3 activity, Sftpc^+/+ and Sftpc^−/− mice were treated with poly(I:C) and evaluated 3 days postinstillation. Diffuse cell infiltrates were present in the lungs of Sftpc^+/+ mice (left), whereas increased cell infiltration with areas of consolidation were present in the lungs of Sftpc^−/− mice (right), 6 mice/treatment group.

Fig. 10.

SP-C inhibits TLR3 activity in vitro. Inhibition of dsRNA-induced TLR3 activity in HEK-293 cells was assessed using either a phospholipid preparation (lipid + dsRNA), the same phospholipid containing 5% bovine SP-C (lipid/SP-C + dsRNA), or SP-C containing bovine surfactant extract (Survanta + dsRNA). HEK-293 cells were transfected with a constitutive TLR3 signaling plasmid and NF-κB-dependant promoter-luciferase reporter plasmid. Cells were pretreated with the preparations 1 h before poly(I:C) stimulation of TLR3 activity. Cell lysates were prepared, and the luciferase activity was determined. Results are an average of 3 independent experiments. Data are presented as means ± SE. SP-C inhibition of poly(I:C) stimulated NF-κB activity, P = 0.02; Survanta inhibition of poly(I:C) stimulated NF-κB activity, P = 0.11.