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

<p>The number of alveoli per lung N(alv, lung) (<b>A</b>) is increased whereas the number-weighted mean volume of alveoli (alv) (<b>B</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085722#pone-0085722-t002" target="_blank">Table 2</a>.</p

Ablation of NOS2 reduces NO metabolite production in DiNOS mice.

<p>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: * <i>vs</i> WT; ξ <i>vs</i> NOS2^−/−; # <i>vs</i> Sftpd^−/− mice.</p

Surfactant homeostasis is not corrected by additional NOS2 genetic ablation.

<p>Phospholipid content of BAL as well as small and large aggregate fractions was determined by the method of Bartlett. Data shown are mean ± S.E.. Statistically significant differences between groups (p<0.05) are indicated as:</p>*<p><i>vs.</i> WT,</p>ξ<p><i>vs.</i> NOS2^−/−,</p>#<p><i>vs.</i> Sftpd^−/− mice.</p

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

<p>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: * <i>vs</i> WT, ξ <i>vs</i> NOS2^−/−, # <i>vs</i> Sftpd^−/−.</p

Stereological data: AE2 cells and intracellular surfactant.

<p>Values are given as mean ± S.E. of n = 5–6 mice per genotype. Abbreviations: V = volume, V_V = volume fraction,  = number-weighted mean volume,  = volume-weighted mean volume, lb = lamellar body, type II = AE2. Statistically significant differences between groups (p<0.05) are indicated as:</p>*<p><i>vs.</i> WT,</p>ξ<p><i>vs.</i> NOS2^−/−,</p>#<p><i>vs.</i> Sftpd^−/−.</p

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

<p>Representative electron micrographs of AE2 cells of WT (<b>a</b>), NOS2^−/− (<b>b</b>), Sftpd^−/− (<b>c</b>) and DiNOS (<b>d</b>) 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.</p

Stereological data of lung architecture.

<p>Values are given as mean ± S.E. of n = 5–6 mice per genotype. Abbreviations: V = volume, S = surface area, S_V = surface area density, N = number, N_V = numerical density,  = number-weighted mean volume,  = volume-weighted mean volume, par = parenchyma, alvepi = alveolar epithelium, alv = alveoli. Statistically significant differences between groups (p<0.05) are indicated as:</p>*<p><i>vs.</i> WT,</p>ξ<p><i>vs.</i> NOS2^−/−,</p>#<p><i>vs.</i> Sftpd^−/−.</p

DiNOS mice exhibit partial normalization of BAL cell counts.

<p>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⁺). (<b>A</b>) Total cell count was determinated using a Z1 Counter particle counter (Beckman Coulter). (<b>B</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: * <i>vs</i> WT; ξ<i>vs</i> NOS2^−/−; # <i>vs</i> Sftpd^−/− mice. (<b>C</b>) Representative Diff-Quik staining of cytospins from WT and Sftpd^−/−, DiNOS and NOS2^−/− mice.</p