Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome

Abstract

Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single-mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double-mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell function was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage-tracing and organoid-modeling studies demonstrated that HPS AT2 cells were primed to persist in a Keratin-8-positive reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.

Keywords: Cell biology; Fibrosis; Genetic diseases; Lysosomes; Pulmonology.

Figure 1. Loss of AT2 cells in HPS mice starting at 8 weeks of age.

(A) IF staining of whole-mount lung issue for pro-SPC and AGER in WT and HPS1/2 mice at 48 weeks of age. Arrows indicate regions of AT2 cell loss. (B) Schematic to evaluate AT2 cell loss in WT, HPS1, HPS2, and HPS1/2 mice over time. (C) Staining of paraffin-embedded lung tissue for proSP-C in WT, HPS1, HPS2, and HPS1/2 mice at 8 and 48 weeks of age. (D) Quantification of percentage of proSP-C⁺ cells as a percentage of total cells (by DAPI) in WT, HPS1, HPS2, and HPS1/2 mice at 4, 8, 24, and 48 weeks of age. (E) Schematic of AT2 cell loss in HPS mice and possible etiologies. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: adjusted * P < 0.05; ** P < 0.01, *** P < 0.001 after Benjamini-Hochberg correction for multiple comparisons. n = 4–6 per group per time point. Scale bars in A, 50 μm; C, 20 μm. Schematics created with BioRender.com.

Figure 2. Impaired regeneration of HPS AT2 cells after acute influenza injury.

(A) Schematic of influenza experiment. (B and C) Mean change in (B) weight and (C) oxygen saturation (SpO₂) in WT, HPS1, and HPS2 mice from 0 to 14 dpi. (D) IF staining of lung tissue for proSP-C and EdU in WT, HPS1, and HPS2 mice at 14 dpi in severely and mildly injured regions. Arrows indicate proliferating AT2 cells (EdU⁺proSP-C⁺). (E and F) Quantification of the percentage difference in proliferating AT2 cells (EdU⁺proSP-C⁺) in (E) severely injured and (F) mildly injured regions at 14 dpi in HPS1 and HPS2 mice relative to WT. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: adjusted * P < 0.05, ** P < 0.01, **** P < 0.0001 after Benjamini-Hochberg correction for multiple comparisons. n = 3–10 per group. All scale bars, 20 μm. Schematic created with BioRender.com.

Figure 3. Impaired proliferation of HPS AT2 cells ex vivo and in vivo.

(A) Schematic of lung organoid culture system. (B) Bright-field imaging and (C) IF staining of lung organoids for AGER and proSP-C generated with AT2 cells isolated from WT, HPS1, or HPS2 mice with WT fibroblasts at day 21 of culture. (D) Quantification of WT, HPS1, and HPS2 organoid CFE at day 21 of culture. (E) Quantification of WT, HPS1, and HPS2 organoid size from day 9 to day 21 of culture. (F) Schematic of keratinocyte growth factor (KGF) experiment. (G) IF staining of lung tissue for proSP-C and EdU in WT, HPS1, and HPS2 mice 48 hours after KGF administration. Arrows indicate proliferating AT2 cells (EdU⁺proSP-C⁺). (H) Quantification of the percentage of proliferating AT2 cells (EdU⁺proSP-C⁺) in WT, HPS1, and HPS2 mice treated with vehicle (PBS) versus KGF. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: adjusted * P < 0.05, ** P < 0.01, **** P < 0.0001 after Benjamini-Hochberg correction for multiple comparisons. For D and E, each point represents the average of at least 3 replicate wells from 1 mouse for a total of n = 3–6 mice per group. For H, n = 4–10 per group per treatment. Scale bars in B 1 mm; in C and G 20 μm. Schematics created with BioRender.com.

Figure 4. Lineage tracing of AT2 cells in HPS mice demonstrates aberrant differentiation at 8 weeks of age.

(A) Schematic of lineage labeling AT2 cells in WT, HPS1, HPS2, and HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ mice. (B) From images in Supplemental Figure 6A, quantification of EYFP⁺proSP-C^– cells as a percentage of EYFP⁺ lineage cells in WT, HPS1, HPS2, and HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ mice at 8 and 24 weeks of age. (C) IF staining of whole-mount lung tissue for EYFP, AGER, and LAMP3 in WT, HPS1, HPS2, and HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ mice at 8 weeks of age. Dashed line boxes represent squamous EYFP⁺AGER⁺LAMP3^– AT2-derived AT1 cells with AGER and EYFP and merged images shown below. (D) IF staining of HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ mice at 8 and 24 weeks of age, with arrows indicating cuboidal EYFP⁺AGER^–LAMP3^– cells with EYFP and LAMP3 images shown below. HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ 8-week images (C) and demonstrated again (D) capture simultaneous presence of squamous EYFP⁺AGER⁺LAMP3^– and cuboidal EYFP⁺AGER^–LAMP3^– cells. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: *** P < 0.001; **** P < 0.0001. n = 3–4 per group per time point. Scale bars in C and D, 20 μm (inset boxes equal scale to main image). Schematic created with BioRender.com.

Figure 5. Transcriptomic analysis suggests aberrant differentiation and p53-mediated senescence in HPS AT2 cells.

(A) Experimental schematic for bulk mRNA sequencing of HPS AT2 cells. (B) Principal component analysis (PCA) of AT2 cells from WT, HPS1, HPS2, and HPS1/2 mice at 8 weeks of age. (C) Differential expression analysis of AT2, transitional, and AT1 cell genes and p53 signaling pathway and cellular senescence genes in AT2 cells from WT, HPS1, HPS2, and HPS1/2 mice at 8 weeks of age. (D) Overall gene set score for cellular senescence. (E) Representative β-galactosidase staining of AT2 cells isolated from WT and HPS1/2 mice at 8 and 48 weeks of age. (F) Quantification of the percentage of β-galactosidase⁺ cells in WT, HPS1, HPS2, and HPS1/2 mice at 48 weeks of age. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: ** P < 0.01. n = 4–8 per group for bulk mRNA sequencing, n = 3 per group for β-galactosidase staining. Scale bars in E, 50 μm.

Figure 6. A p53-mediated Krt8⁺ reprogrammed transitional cell state in HPS mice.

(A) IF staining of paraffin-embedded lung tissue for EYFP, KRT8 or CLDN4, and LAMP3 in WT and HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ mice at 8 weeks of age. Dashed line boxes represent EYFP⁺ cells with inset boxes displaying EYFP, KRT8 or CLDN4, and LAMP3 staining. (B) IF staining of whole-mount lung tissue for EYFP, KRT19, and LAMP3 in WT, HPS1, HPS2, and HPS1/2 Sftpc^CreERT2/+ R26R^EYFP/+ mice at 8 weeks of age. White arrows indicate squamous EYFP⁺KRT19^–LAMP3^– AT2-derived AT1 cells, and yellow solid arrows indicate cuboidal EYFP⁺KRT19⁺ cells; dashed line boxes and insets display EYFP and KRT19 and merged images. (C) Schematic for alveolosphere culture system with feeder-free alveolar maintenance media (AMM) and alveolar differentiation media (ADM) and treatment with pifithrin-α (PFTα). (D) Bright-field imaging and (E) IF staining of alveolospheres for AGER and LAMP3 generated with AT2 cells from WT or HPS1/2 mice with and without treatment with PFTα at day 14 (D14) of culture. (F) Relative gene expression of selected reprogrammed transitional cell (Krt8, Cldn4) and AT1 cell (Hopx) genes in alveolospheres at day 0 (D0), day 7 (D7), and day 14 (D14) of culture with and without treatment with PFTα. Relative gene expression compared with WT at D0. Each point represents 4 replicate wells per time point for a total of n = 3–4 mice. DAPI stains nuclei (blue). All quantification data are represented as mean ± SEM. Statistics using 2-tailed unpaired Student’s t tests: * P < 0.05; ** P < 0.01. Scale bars in A, B, and E 20 μm (inset boxes equals to main image); D 1 mm. Schematic created with BioRender.com.

Figure 7. Senescence and a Krt8⁺ reprogrammed transitional cell state in a patient with HPS-1.

(A) IF staining of paraffin-embedded lung tissue from an age- and sex-matched control donor patient and HPS-1 patient for proSP-C and β-galactosidase. Arrows and dashed line boxes represent proSP-C⁺ cells with inset boxes displaying proSP-C and β-galactosidase staining with merged images. (B) IF staining of paraffin-embedded lung tissue from an age- and sex-matched control donor patient and HPS-1 patient for HTII-280, KRT8, and NKX2.1. DAPI stains nuclei (blue). All scale bars, 20 μm (inset boxes equal scale to main image).

Figure 8. Time course of AT2 cell dysfunction in HPS-PF.

Our current working hypothesis is that the HPS genetic mutations disrupt AT2 progenitor cell function early, with activation of the p53 pathway, impairing AT2 cell proliferation and driving aberrant AT2 cell differentiation. Over time, this results in accelerated AT2 cell loss with senescence and depletion of the stem cell pool and progressive fibrotic remodeling.