Animal care, genotyping, and handling
Laboratory mice were managed and handled according to EU and Spanish guidelines for the use and care of animals in research. All the conducted experiments were approved by the Bioethics Committee of the University of Salamanca (animal license number #409). All NRas and HRas knockout strains64,65 were maintained on pure C57Bl/6 background and kept on a 12 h light/dark cycle. Single heterozygous HRas+/− or NRas+/− mice, as well as double heterozygous (HRas+/−;NRas+/−) mice are phenotypically indistinguishable from wild-type animals. Thus, we routinely set out parental crosses between mouse strains as already stated in19 to more quickly and efficiently generate comparable sets of littermates of the four genotypes of interest for our studies with equal proportion (HRas+/−;NRas+/−, designated hereafter as Control; HRas−/−;NRas+/− designated as HRas-KO; HRas+/−;NRas−/− designated NRAS-KO; and HRas−/−;NRas−/− designated as DKO).
Genotyping was done by PCR analysis of genomic DNA isolated from mouse tails using specific primers for the wild-type (WT) or the null-mutant alleles of HRas or NRas, as appropriate as already described in19.
For dexamethasone (DEX) treatment of pregnant females, the beginning of gestation (E0.5) was timed via the detection of vaginal plugs, and pregnancy was later confirmed by weighting the females from day 10 post coitum (pc). DEX (SIGMA, D2915) or saline control (NaCl 0.9%) was injected subcutaneously (0.4 mg/kg)66,67 to pregnant females on days E17.5 and E18.5 of embryonic development, and survival of the newborn pups was monitored daily (weight, phenotype, activity, survival). Embryos collected at E18.5 received only one dose of dexamethasone at E17.5.
For N-acetylcysteine (NAC) treatment, pregnancy was timed at E0.5 after the detection of vaginal plugs. NAC (SIGMA, A7250) was administered in tap water (0.5%, pH 7.4) using the breeding bottle ad libitum68 throughout the pregnancy, starting on the E0.5 embryonic day. NAC solution was changed weekly and pregnant females, together with the resulting litters, were monitored daily (weight, phenotype, activity, survival).
Histology and Immunohistochemistry
Mouse lung tissues were fixed in 4% paraformaldehyde overnight at 4ºC for 3 days before dehydration and paraffin embedding. Three-micrometer sections were used for tissue staining with Hematoxylin-Eosin (H&E) and five-micrometer sections for periodic acid-Schiff (PAS) according to standard procedures.
Immunohistochemical procedures were performed using deparaffinized and rehydrated three-micrometer-thick sections, as previously described19. Antigen retrieval was routinely performed to facilitate antibody binding to antigen using citrate buffer 0.01 M pH 6.0 and heating in a microwave oven (3x3 min each, 250 W).
Detection of neutrophils in lung sections was carried out using an avidin-biotin-peroxidase procedure69. Sections were rinsed in PBS (3x10 min) and endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide for 15 min. Sections were sequentially incubated in (1) primary antibody Neutrophil elastase (NE) (1:400, Abcam, ab68672) in PBS, 0.1% Triton X-100 (Sigma, T8787),, 2% Bovine serum albumin (BSA, Sigma Aldrich, 9048-46-8) and 2% goat serum (Sigma, G9023), overnight at 4 ºC; (2) 1:250 biotinylated goat anti-rabbit IgG (Vector); and (3) 1:250 Vectastain Elite ABC reagent (Vector) in PBS for 1 h at room temperature (RT). The sections were rinsed in PBS (3 × 10 min) between each step. The reaction product was visualized by incubating the sections in 0.05% 3,3′-diaminobenzidine and 0.0033% hydrogen peroxide in PBS until the desired staining intensity was reached.
For immunofluorescence studies, sections were washed with PBS and blocked in PBS containing 0.1% Triton X-100, 5% BSA, and 2% goat serum. Primary antibodies were diluted in PBS, 0.1% Triton X-100, 2% BSA, and 2% goat serum, and incubated with the sections overnight at 4 ºC. Primary antibodies (dilution, supplier and catalogue number) used in this study included: β-Tubulin (β-Tub) (1:500, Sigma, T5293), Ceramide (1:100, Enzo, ALX-804-196), Cleaved-caspase-3 (CC3) (1:400, Cell Signaling, 9661), Ricinus communis agglutinin-I (RCA-I) (1:1000, Atom, FL-1081), Uteroglobin (Scgb1a1) (1:1000, Abcam, ab40873), prosurfactant protein-C (SftpC) (1:500, Merck Millipore, AB3786), Sex-determining region Y-box 9 (Sox9) (1:500, Cell Signaling, 82630S). After 3x PBS washes, sections were incubated with secondary antibodies (from Jackson ImmunoResearch; diluted 1:500) including, as appropriate, goat anti-mouse Alexa 488 or Cy3, goat anti-rabbit Alexa 488 or Cy3, and counterstained with nuclear DAPI (Sigma) for 1 h at RT, washed with PBS and mounted with ProLong Diamond anti-fading reagent (P36970, Life Technologies).
Images were acquired using a Leica TCS SP5 confocal microscope with the pinhole set to 1 Airy units and 40x1.25NA or 63x1.40NA immersion oil objectives. The proper laser lines, 405 nm, 488 nm, and 561 were employed to excite Hoechst 33342, Alexa 488/ FITC, and Cy3, respectively. Images were acquired sequentially, starting first with Hoechst 33342 and following with the Cy3 and Alexa 488 staining. Images were imported to ImageJ software (NIH, Bethesda, MD, USA) using the LOCI Bio-formats plug-in and Adobe Photoshop CS6 version 13.0.
Image analysis and quantifications
For SftpC+, SfptC+/RCA-I+, Sox9 + and NE + cell density analyses, images from E18.5 or P0 lung sections from the four genotypes (Control, HRas-KO, NRas-KO, and HRas/NRas-DKO), obtained as described above were selected for the analysis. Numbers of SftpC+, SftpC+/RCA-I+, Sox9 + or NE + cells were normalized to those of total nuclei, after counting them counted using the command “Cell Counter” of the ImageJ software. Cell density data are represented as a percentage of SftpC+, SftpC+/RCA-I+, Sox9+, or NE + cells.
For Ceramide and RCA-I quantification, digital images taken from equivalent alveolar or bronchiolar lung sections of control and KO animals were treated to balance the signal-to-noise ratio in such a way that the positive element (Ceramide or RCA-I) was clearly distinguishable from the background. The surface analyzed was then delimited using the original image as a reference, and both the average fluorescent intensity and the total number of nuclei were measured in the chosen area using ImageJ software.
For PAS + quantification, digital images were taken from equivalent lung sections of Control and KO animals. They were manually transformed into binary images in which only PAS-positive staining elements appeared as black pixels. Then, the surface analyzed was delimited, using the original image as reference, and average PAS staining was measured as the black/white pixel ratio in the chosen area using ImageJ software.
For alveolar area quantification, images taken from equivalent lung sections of Control and KO animals were manually transformed into binary images with ImageJ software where the alveolar spaces were recognized as positive elements (Black). The alveolar surface analyzed was then delimited using the original image as reference, and the area (µm2) of each alveolus was calculated using the ImageJ software.
For Ciliate and Clara cell length measurements, equivalent images of bronchiolar areas of Control and KO animals were obtained, and the length of each cell type was manually measured using ImageJ software.
Microarray hybridizations
Lungs were dissected from P0 neonate mice and RNA was extracted using Trizol following the manufacturer’s instructions. After the extraction, the RNA was further purified using RNAse Mini Kit columns (QIAGEN, 74104). RNA quantification and quality were checked by RNA capillary electrophoresis columns (Agilent Technologies, RNA 6000 Nanochips).
Chip microarray hybridizations and data generated with Affymetrix GeneChip Mouse Gene 2.0 ST Array (26,515 genes) were used in this study. All microarray hybridization data were deposited and are available at the NCBI Gene Expression Omnibus (GEO) database (GEO GSE186161 accession viewer). The RNAs were pre-amplified before microarray hybridization using the Gene Chip Expression 3'-Amplification Two-Cycle cDNA Synthesis kit (Affymetrix, Santa Clara, CA, USA; #900432), the Gene Chip Sample Cleanup Module (Affymetrix #900371) and the MEGAscript T7 High Yield Transcription Kit (Ambion, Austin, TX, USA; #1334), according to Affymetrix instruction manual #701025 rev. 5. The pre-amplified RNAs were then submitted to the Gene Chip microarray hybridization protocol (Affymetrix Expression Analysis Technical Manual, as previously described19,20. Using Bioconductor70 and R71 as computational tools, the limma algorithm72 was applied for background correction and normalization of fluorescent hybridization signals. The significance analysis of microarrays (limma) algorithm was used to identify probe sets displaying significant differential expression when comparing NAC-treated and untreated DKO samples. This method uses permutations to provide a robust statistical inference of the most significant genes and provides P-values adjusted to multiple testing using a false discovery rate (FDR)73. The Gene Codis 4 74 (Gene Annotation Co-occurrence Discovery) software package (https://genecodis.genyo.es/) was used for functional annotation analysis of differentially expressed gene sets to identify specific gene subsets sharing co-occurrent functional annotations linking them, with high statistical significance, to particular Gene Ontology (GO) Biological Process or Molecular Function categories and KEGG Signaling Pathways.
Reactive oxygen species analysis in lung tissue
Total hydrogen peroxide (H2O2), as well as total and mitochondrial superoxide (O2−) ROS were measured in total lung homogenates from newborn animals using 2',7'-dichlorofluorescein diacetate (DCFH-DA; hereinafter DCF, Molecular Probes, D-399), dihydroethidium (DHE, Molecular Probes, D11347), and MitoSOX™ (Molecular Probes, M36008), respectively.
Lungs were dissected, quickly minced using scissors, digested in 500 µl of 1 mg/ml Collagenase/Dispase® (Roche, 10269638001), and diluted in Hank’s Balanced Salt solution (HBSS) for 30 min at 37 ºC in gentle agitation. Digested lungs were then passed through a 100 µm cell strainer (Falcon, 352360) and further processed for staining. For DCF/DHE staining, samples were incubated with 5 µM of each probe in DMEM (Gibco, A14430.01) supplemented with 0.1% of fetal bovine serum (FBS) and 10 mM of glucose (Glu) for 30 min at 37 ºC in the dark with gentle agitation. For DCF/MitoSOX™, samples were incubated with 5 µM of MitoSOX™, in the same media, for 30 min at 37 ºC in the dark with gentle agitation, then 5 µM of DCF was added to each sample, followed by 30 min of incubation at 37 ºC in the dark with gentle agitation.
The reactions were quickly stopped by adding 1 ml of cold HBSS, followed by centrifugation of the samples at 13000 rpm at 4 ºC for 5 min. Supernatants were discarded and cell pellets were resuspended in 200 µl of DMEM supplemented with 0.1% FBS. Samples were analyzed using a BD Accuri™ C6 cytometer, with a minimum of 50.000 events analyzed per sample.
Mitochondrial respiration assays in lung tissue
Using Seahorse-based technology and the MitoStress Test (Agilent, 103015-100) we evaluated the mitochondrial respiration status, as well as function, using measuring the oxygen consumption rate (OCR) of the cells in real-time.
Lung lobes were dissected from newborn animals, rinsed in 1X PBS, and maintained o/n at 4 ºC in a 2 ml Eppendorf filled with DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin (P/S). Lung lobes were subsequently sliced into 1 mg pieces and inserted onto the Islet capture microplate (Agilent, 101122-100) following the manufacturer’s instructions. These were then incubated at 37 ºC without CO2 for 45 min in Seahorse XF DMEM medium, pH 7.4 (Agilent, 103575-100) supplemented with 10 mM Glu, 1 mM Pyruvate, and 2 mM L-Glutamine. Cartridge injection ports were filled with OL, FCCP, and ROT/AA so the final concentration in each well was 15, 16, and 3/12 µM respectively.
Microplates were assessed in a Seahorse XFe24 Extracellular Flux Analyzer system (Agilent, Santa Clara, CA), following MitoStress Test protocol with some alterations: 3 measures of basal OCR, 6 measures of OCR after OL injection, 3 measures after FCCP injection and 5 measures after ROT/AA injection. Data was analyzed using Wave Desktop 2.6 software.
Statistical Analysis
Power calculation analyses to establish the sample size (n = 2 SD2 (Zα/2 + Zβ)2 / d2) were performed, establishing a difference of at least 30%, a type I error of 5%, 80% power and standard deviation (SD) based on previous studies from our laboratory. With these parameters, we calculated a sample size of 3–7 mice for genotype, depending on the specific experiment, with experimental n = values being specified in each figure or figure legends. Animals were selected randomly and genotyped by the technician so that the investigators were blinded during the experiments and data acquisition. Data is expressed as mean ± standard error of the mean (S.E.M.). Normal distribution of the data was tested using the IBM SPSS Statistics 26 package (SPSS, Chicago, IL, USA) and the Kolmogorov-Smirnov test. One-way ANOVA followed by the Bonferroni post-hoc test correction was used for the comparison of parametric values. Survival analysis was performed by the Kaplan-Meier method and differences in survival between groups were tested using the Log-rank (Mantel-Cox) test (GraphPad Prism 8 Software, Inc.). Differences between groups were considered statistically significant when p < 0.05.