NOS2 is critical to the development of emphysema in Sftpd deficient mice but does not affect surfactant homeostasis

Abstract

Rationale: Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.

Objective: In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).

Methods: Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd(-/-)) and iNOS single knockout mice (NOS2(-/-)) as well as wild type (WT) littermates.

Measurements and results: DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd(-/-) demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.

Conclusion: iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.

Figure 1. Ablation of NOS2 reduces NO metabolite production in DiNOS mice.

BAL nitrates were measured by NOA as a marker of NOS activity. Values are mean ± S.E. (n = 6 to 15). Statistically significant differences between groups (p<0.05) are indicated as: * vs WT; ξ vs NOS2^−/−; # vs Sftpd^−/− mice.

Figure 2. Lung histology reveals parenchymal inflammation in Sftpd^−/− mice and reduced enlargement of distal airspaces in DiNOS.

(A) Representative micrographs of lung sections from WT (upper left), NOS2^−/− (upper right), Sftpd^−/− (lower left) and DiNOS (lower right) mice (toluidine blue staining). Normal lung architecture can be seen in WT and NOS2^−/− mice. In Sftpd^−/− the distal airspaces are enlarged and filled with foamy appearing alveolar macrophages. Enlargement of distal airspaces appears to be less pronounced in DiNOS compared to Sftpd^−/− mice. (B) Histological scoring of lung inflammation. Median inflammation scores were determined by blinded evaluation of stained sections from each genotype group as described in Online Supplement Methods. Data shown are Median, n = 7 animals per genotype.

Figure 3. Lungs of DiNOS and Sftpd^−/− mice exhibit AE2 hyperplasia and hypertrophy with increased numbers of lamellar bodies.

Representative electron micrographs of AE2 cells of WT (a), NOS2^−/− (b), Sftpd^−/− (c) and DiNOS (d) mice. The profiles of AE2 cells in Sftpd^−/− and DiNOS are bigger and seem to contain more lamellar bodies compared to WT and iNOS^−/− mice.

Figure 4. NOS2 ablation does not correct AE2 cell size and surfactant per lung in the absence of SP-D.

Hypertrophy of AE2 cells ( (typeII)) is present in Sftpd^−/− and DiNOS to a similar extent (A). The increased volume of lamellar bodies per lung V(lb,lung) (B) is unaffected by the additional ablation of the NOS2-gene in Sftpd deficient mice. Data shown are mean and individual data, n = 5–6 animals per genotype. Statistically significant differences between groups are shown in Table 3.

Figure 5. NOS2 ablation improves emphyatous phenotype resulting from the absence of SP-D.

The number of alveoli per lung N(alv, lung) (A) is increased whereas the number-weighted mean volume of alveoli (alv) (B) is decreased in the DiNOS mice compared to Sftpd^−/− mice, indicating an attenuation of the pulmonary emphysema due to the additional ablation of the iNOS-gene in Sftpd deficient mice. Data shown are mean and individual data, n = 5–6 animals per genotype. Statistically significant differences between groups are shown in Table 2.

Figure 6. NOS2 ablation corrects lung resistance and elastance changes seen in Sftpd^−/− mice.

Lung resistance (A) and elastance (B∫) spectra as a function of frequency, at a positive end-expiratory pressure (PEEP) of 3 cm H₂O, are shown. Each point represents the mean of 3–6 measurements ± S.E., lines are the empirical fit using the model outlined in the Online Supplement. Sftpd^−/− spectra are significantly different from WT as determined by χ² test (p<0.05).

Figure 7. DiNOS mice exhibit partial normalization of BAL cell counts.

Lungs were lavaged with 0.5-ml aliquots of sterile saline to a total of 5 ml. Recovered BALF samples were centrifuged (300 g for 10 min) and the cell pellet was gently resuspended in 1 ml of PBS (with Ca²⁺ and Mg²⁺). (A) Total cell count was determinated using a Z1 Counter particle counter (Beckman Coulter). (B) Aliquots of cells were spun on a Thermo Shandon Cytospin-3 at 750 rpm for 3 min and stained with standard Diff-Quik for manual determination of cell differentials. Cells were identified as macrophages, eosinophils, neutrophils, and lymphocytes by standard morphology. Data shown are mean ±S.E., n = 7 animals per genotype. Statistically significant differences between groups (p<0.05) are indicated as: * vs WT; ξvs NOS2^−/−; # vs Sftpd^−/− mice. (C) Representative Diff-Quik staining of cytospins from WT and Sftpd^−/−, DiNOS and NOS2^−/− mice.

Figure 8. NOS2 ablation alters the inflammatory phenotype of BAL in DiNOS mice.

RNA was extracted from BAL cells isolated from WT, Sftpd^−/−, NOS2^−/− and DiNOS mice. Gene markers were quantified by RT-qPCR as described. Ct values obtained were normalized to β-actin signals and further analyzed using the relative quantization (ΔΔCt) method. Data are expressed as fold change (means ± S.E.M, n = 5–8 in each group). Statistically significant differences between groups (p<0.05) are indicated as: * vs WT, ξ vs NOS2^−/−, # vs Sftpd^−/−.