Surfactant protein C mutation links postnatal type 2 cell dysfunction to adult disease

Abstract

Mutations in the gene SFTPC, encoding surfactant protein C (SP-C), are associated with interstitial lung disease in children and adults. To assess the natural history of disease, we knocked in a familial, disease-associated SFTPC mutation, L188Q (L184Q [LQ] in mice), into the mouse Sftpc locus. Translation of the mutant proprotein, proSP-CLQ, exceeded that of proSP-CWT in neonatal alveolar type 2 epithelial cells (AT2 cells) and was associated with transient activation of oxidative stress and apoptosis, leading to impaired expansion of AT2 cells during postnatal alveolarization. Differentiation of AT2 to AT1 cells was also inhibited in ex vivo organoid culture of AT2 cells isolated from LQ mice; importantly, treatment with antioxidant promoted alveolar differentiation. Upon completion of alveolarization, SftpcLQ expression was downregulated, leading to resolution of chronic stress responses; however, the failure to restore AT2 cell numbers resulted in a permanent loss of AT2 cells that was linked to decreased regenerative capacity in the adult lung. Collectively, these data support the hypothesis that susceptibility to disease in adult LQ mice is established during postnatal lung development, and they provide a potential explanation for the delayed onset of disease in patients with familial pulmonary fibrosis.

Keywords: Cell Biology; Cell stress; Protein misfolding; Pulmonary surfactants; Pulmonology.

Figure 1. Augmented lung injury and parenchymal fibrosis in adult LQ/LQ mice.

(A) Schematic of repetitive bleomycin challenge. (B) Representative 10× H&E-stained, 20× trichrome-stained, and 10× second harmonic images of left lung sections. H&E image for LQ/LQ mouse shows injured area with inflammation (arrow) including lymphocytic aggregate (arrowhead) and proximalization of distal airspaces (boxed region), juxtaposed to normal lung parenchyma. Note parenchymal collagen in trichrome (in blue) and second harmonic images (in red). Scale bars: 50 μm (H&E, trichrome) and 500 μm (second harmonic image). (C) Representative 10× tile scans (black and white image) and maximum intensity projection of LQ/LQ lung sections stained with proSP-C, ABCA3, and proximal epithelial cell transcription factor SOX2. Scale bar: 50 μm. (D) Histological score for area of left lung injured in response to bleomycin challenge on day 71 of the study. Area of injury was normalized to total area of the lung lobe. (E) Area of second harmonic signal (representing collagen) scored from images of 5 μm–thick paraffin sections obtained from bleomycin-challenged mice on day 71 of the study. Area of collagen signal was normalized to area of the lung parenchyma imaged with auto fluorescence of the section. For D and E, *P < 0.05, **P < 0.01 by unpaired 2-tailed t test. (F) Hydroxyproline concentration obtained from the right middle lung lobe. (G) Lung compliance measured by FlexiVent. For F and G, *P < 0.05, **P < 0.01, ****P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparison test.

Figure 2. AT2 cell numbers are decreased in adult LQ/LQ mice.

(A) Representative maximum intensity projections from confocal Z-stacks of lung sections obtained from 10- to 12-week-old mice stained with proSP-C, ABCA3, and distal epithelial cell transcription factor NKX2.1. Insets show individual channels for AT2 cell markers. Arrows point to NKX2.1⁺ABCA3⁺proSP-C^– AT2 cell. Scale bars: 50 μm. (B) Frequency distribution for AT2 cell markers, proSP-C, and ABCA3 in WT and LQ/LQ lung sections. All WT AT2 cells were proSP-C⁺ ABCA3⁺ at all time points. (C) Morphometric analyses of AT2 cells from 10- to 12-week-old mice. Each data point represents an individual mouse. ****P < 0.0001 by unpaired 2-tailed t test. (D) Morphometric analyses of AT2 cells in lung sections obtained during postnatal alveolarization. Dashed and dotted lines represent curve fits for WT (exponential) and LQ/LQ (linear) data points, respectively. **P < 0.01, ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple comparison test. DAPI counts remained similar between genotypes (Supplemental Figure 4A).

Figure 3. Accumulation of proSP-C in P4 LQ/LQ AT2 cells.

(A) Western blot analyses of 20 μg of AT2 cell lysates separated by SDS-PAGE. Gel was stained after transfer with Coomassie-based instant blue to assess protein loading. (B) Normalization of proSP-C levels in A to total protein using Bio-Rad ImageLab software. (C) Relative Sftpc mRNA levels in isolated AT2 cells obtained by quantitative PCR of 20 ng of cDNA. Data were normalized to Ppia expression. Cycle threshold for Ppia was constant in all genotypes and across developmental time points (Supplemental Figure 5D). RQ, relative quantitation. For B and C, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparison test. (D) Lung slices (1 mm) from P3 mice were labeled with [³⁵S] methionine/cysteine for 30 minutes, followed by immunoprecipitation of 4.6 × 10⁶ counts with an antibody directed against the mature SP-C peptide (detects both SP-C proprotein and mature peptide). Lung homogenates were separated by SDS-PAGE, followed by phosphor imaging to detect newly synthesized proSP-C. Top and bottom panels were exposed for 2 and 7 days, respectively. Red box in the bottom portion shows absence of the mature peptide in LQ/LQ lung homogenates. Arrowheads in A and D point to SP-C proprotein. Unedited blots are available online with this article.

Figure 4. Activation of oxidative stress in P4 LQ/LQ AT2 cells.

(A) UMAP embedding shows AT2 cell clusters (n, WT = 415 and LQ/LQ = 540) obtained from scRNAseq of P4 lungs. (B) Toppfun analyses show pathways and biological processes characterizing the P4 LQ/LQ AT2 cell population. Groups are ordered by descending P values (x axis, –log₁₀ of P value). (C) Freshly isolated P4 AT2 cells were incubated with DCFDA for measurement of ROS levels. Cells without DCFDA were used as a control to measure background fluorescence. RFU, relative fluorescence unit; TBHP, tert-butyl hydrogen peroxide (inducer of oxidative stress). (D) Glutathione (GSH) concentrations were measured in freshly isolated, deproteinized P4 AT2 cells. AT2 cell lysates were incubated with 2-vinylpyridine for measurement of oxidized glutathione disulfide (GSSG). For C and D, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparison test.

Figure 5. Oxidative stress is associated with proapoptotic signaling and impaired differentiation.

(A) Relative Bax mRNA levels in isolated AT2 cells obtained by quantitative PCR of 25 ng of cDNA. Data were normalized to Ppia expression. (B) Western blotting for proapoptotic protein, BAX. In total, 30 μg of AT2 cell lysates was separated by SDS-PAGE. (C) Densitometry for BAX levels in B. Data were normalized to Actin. (D) Confocal images of organoids generated from P4 WT and LQ/LQ AT2 cells. Organoid cultures were treated with or without the antioxidant, butylated hydroxyanisole (BHA). HOPX, AT1 cell marker; CCSP, club cell (proximal airway epithelial cell) marker; α-SMA: α-smooth muscle actin; proSP-C, AT2 cell marker; ECAD, pan-epithelial cell marker; AGER, AT1 cell marker. Scale bars: 100 μm. (E–H) Morphometric analyses of HOPX⁺ (E), AGER⁺ (F), proSP-C⁺ (G), and CCSP⁺ (H) organoids (circles, WT; squares, LQ/LQ). For A, C, and E–H, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparison test. Unedited blots are available online with this article.

Figure 6. Chronic stress promotes adaptation in P21 LQ/LQ AT2 cells.

(A) GO analyses of proteomics data show overrepresented processes in P4 LQ/LQ AT2 cells compared with P4 WT AT2 cells. Biological processes are ordered by descending P values (x axis, –log₁₀ P value). (B) Venn diagram representing the overlap between genes characterizing the P21 LQ/LQ and P4 WT AT2 cell populations compared with P4 LQ/LQ AT2 cell population. (C) Toppfun analyses show pathways characterizing the P21 LQ/LQ AT2 cells compared with P4 LQ/LQ AT2 cells. (D) Toppfun analyses show pathways characterizing the common genes (710 genes) from B. For C and D, pathways are ordered by descending P values (x axis, –log₁₀ P value).

Figure 7. Resolution of stress responses in P21 LQ/LQ AT2 cells.

(A) BiP protein levels normalized to total protein in isolated AT2 cells (blot shown in Supplemental Figure 13A). (B) Relative expression of spliced Xbp1 (sXbp1) mRNA obtained by quantitative PCR of 20 ng of AT2 cell cDNA. Data were normalized to Ppia expression (shown in Supplemental Figure 5D). (C) Ratio of peIF2α to total eIF2α levels in isolated AT2 cells (blot shown in Supplemental Figure 13B). (D) GADD34 protein levels normalized to total protein in isolated AT2 cells (blot shown in Supplemental Figure 13C). (E and F) Levels of proteasome subunits RPT5 (E) and α 1-7 (F) normalized to total protein in isolated AT2 cells (blot shown in Supplemental Figure 13D). For E and F, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparison test.