Mitophagy promotes lung repair and regeneration by restoring epithelial metabolic fitness

Pei Wu; Jiawei Chen; Li Liu; Ping Wang; Tiantian Lu; Shengxi Shen; Yiyuan Cao; Yuandong Sui; Run Liu; Xiajuan Huan; Ning Ding; Ying Xi

doi:10.1038/s41467-026-71728-x

. 2026 Apr 14;17:5170. doi: 10.1038/s41467-026-71728-x

Mitophagy promotes lung repair and regeneration by restoring epithelial metabolic fitness

Pei Wu ¹, Jiawei Chen ¹, Li Liu ¹, Ping Wang ¹, Tiantian Lu ¹, Shengxi Shen ¹, Yiyuan Cao ¹, Yuandong Sui ¹, Run Liu ¹, Xiajuan Huan ¹, Ning Ding ², Ying Xi ^1,^✉

PMCID: PMC13249882 PMID: 41980941

Abstract

Alveolar Type II cells (AT2s) are the stem cells responsible for both lung homeostasis and regeneration. Mitochondrial dysfunction in AT2 cells has been implicated in both chronic and acute injury-induced alveolar diseases, including idiopathic pulmonary fibrosis (IPF) and viral pneumonia. However, the role of mitochondrial homeostasis in post-injury lung repair and regeneration remains elusive. Here we demonstrate that genetic depletion of Ubiquitin Specific Peptidase 30 (USP30), a negative regulator of mitophagy, boosts mitophagy and restores mitochondrial function in AT2 cells, leading to protection from injury-induced apoptosis and enhanced stem cell activity. Both global and AT2-specific Usp30 knockout (KO) promote alveolar regeneration, protecting the mice from bleomycin-induced lung fibrosis and influenza pneumonia. Moreover, pharmacological inhibition of USP30 effectively alleviates these conditions. Together, our findings reveal a previously underappreciated role for mitophagy in lung injury and repair and highlight USP30 inhibition as a promising therapeutic strategy for treating alveolar diseases.

Subject terms: Regeneration, Influenza virus, Respiratory distress syndrome

USP30, a negative regulator of mitophagy, is identified as a potential therapeutic target for lung fibrosis and viral pneumonia. Inhibition of USP30 protects alveolar epithelial cells from apoptosis and enhances alveolar regeneration.

Introduction

Mitochondria are vital energy factories and key regulators of cellular communication, lipid metabolism, calcium homeostasis, and apoptosis^1–10. Mitochondrial dysfunction, characterized by reduced ATP synthesis and the accumulation of mitochondrial reactive oxygen species (mtROS), has been implicated in numerous human diseases, including neurodegenerative diseases and respiratory conditions like IPF and viral pneumonia^11–17. Mitophagy, the process by which damaged mitochondria are removed and recycled, coordinates with mitochondrial dynamics and biogenesis to maintain mitochondrial function^18,19. Impaired mitophagy results in the accumulation of damaged mitochondria and contributes to the pathogenesis of various diseases.

Accumulating evidences suggest that mitochondrial dysfunction and abnormalities in mitophagy contribute to respiratory diseases. For example, IPF is a chronic and devastating lung disease caused by repeated injuries to the alveolar epithelium, leading to abnormal or failed alveolar repair^20–25. AT2 cells, which act as stem cells in the alveoli, from patients with IPF exhibit an accumulation of damaged mitochondria, impaired mitophagy and reduced expression of PTEN-induced kinase 1 (PINK1)^26,27. PINK1, along with Parkin, plays a crucial role in mitochondrial quality control. Pink1^-/- mice show enlarged mitochondria, defective mitochondrial function and increased susceptibility to lung fibrosis²⁶. Parkin deletion also exacerbates lung fibrosis in a mouse model²⁸. The absence of mitochondrial fusion proteins mitofusin1 (MFN1) and mitofusin2 (MFN2) in mouse AT2 cells leads to spontaneous lung fibrosis²⁹. On the other hand, viral pneumonia is a leading cause of morbidity and mortality worldwide, which may lead to acute respiratory distress syndrome (ARDS)^30,31. The severity of the infection is often associated with mitochondrial dysfunction. For example, oxidative phosphorylation (OXPHOS) impairment, modeled by Ndufs4 deficiency, can exacerbate influenza pathogenesis by reprogramming lung epithelial metabolism to enhance viral attachment³². Ripk2 knockout impairs mitophagy, enhances NLRP3 inflammasome activation, and increases mortality upon influenza virus infection³³. These findings underscore the link between these respiratory diseases and mitochondrial dysfunction and support that restoring mitochondrial homeostasis may confer therapeutic benefits on patients.

USP30 is a universally expressed deubiquitinase that is primarily localized to the outer mitochondrial membrane and antagonizes Parkin-mediated ubiquitination, thus functions as a key negative regulator of mitophagy^34,35. It preferentially removes ubiquitin chains connected via Lys6 and Lys11 linkages^35–37. Knockout or inhibition of USP30 rescues the defective mitophagy in parkin or pink1 mutant flies, functionally protects dopaminergic neurons in Parkinson’s disease mouse model and reduces oxidative stress in human neurons^34,38,39, validating USP30 as a potential therapeutic target for Parkinson’s disease. Recently, the knockout of Usp30 has also been shown to improve heart failure in mice caused by cardiomyocyte-specific knockout of carnitine palmitoyl transferase 2 (CPT2) by promoting mitophagy⁴⁰. However, it is unknown whether USP30 plays a role in lung diseases.

Here, we demonstrated mitochondrial dysfunction in both IPF lungs and influenza-induced pneumonia. Using both genetic and therapeutic models, we showed that either USP30 knockout or inhibition protects the mice from bleomycin-induced lung fibrosis and influenza-induced pneumonia. Mechanistically, USP30 depletion reduces the apoptosis of AT2 cells after injury and increases AT2 cell proliferation capacity, thus restricting alveolar injury and promoting alveolar regeneration, respectively. These data therefore support USP30 as a promising therapeutic target to treat injury induced pulmonary diseases such as IPF and influenza pneumonia by promoting lung repair and regeneration.

Results

Usp30 KO mice are protected from bleomycin-induced lung fibrosis

A re-analysis of published transcriptomic data of pulmonary fibrosis (PF) lungs⁴¹ (GSE110147) revealed “oxidative phosphorylation”, “aerobic respiration”, “ATP synthesis coupled electron transport” were the top Gene Ontology (GO) terms enriched in the downregulated genes (Fig. 1A and Fig. S1A), suggesting defective mitochondrial function in PF lungs. Gene Set Enrichment Analysis (GSEA) elucidated significant downregulation of oxidative phosphorylation and mitophagy in PF lungs (Fig. 1B, C). In align with the analysis, the expression levels of PINK1 and PRKN were negatively correlated with fibrillar collagen expression (Fig. S1B). Re-analyzing published single-cell RNA sequencing (scRNA-seq) data of IPF lungs⁴² (GSE135893) further revealed a significant downregulation of oxidative phosphorylation in the mesenchymal, epithelial, and immune cells (Fig. S1C, D). Mitophagy was significantly decreased in the epithelial cells, endothelial and immune cells of IPF lungs, with the most pronounced changes occurring in epithelial cells (Fig. 1D). These results collectively demonstrated that in patients with IPF, mitochondrial dysfunction is associated with impaired mitophagy, which correlates with the degree of fibrosis. Given mitochondrial dysfunction has been reported to contribute to fibrogenesis^26–29, we hypothesized that enhancing mitophagy may restore mitochondrial function and thereby inhibit lung fibrosis.

Fig. 1 — A GO enrichment of 1246 significantly downregulated genes (log₂FC ≤−1, p ≤ 0.05) in PF lungs (GSE110147), showing the top ten biological processes. B, C GSEA of GSE110147 demonstrating enrichment of oxidative phosphorylation pathway (B) and the positive regulation of mitophagy pathway (C) in control versus PF samples. D Single-cell RNA-seq analysis (GSE135893) depicting cell type-specific mitophagy scores across pulmonary populations. E Validation of knockout efficiency by qRT-PCR under homeostatic conditions (n = 3/group). F Experimental design for bleomycin-induced lung fibrosis model. G Body weight changes monitored for 18 days (saline: n = 3/group; bleo/WT: n = 18; bleo/KO: n = 20). H, I Images (H) and quantification (I) of Masson’s trichrome staining showing collagen deposition (blue) in lung sections (saline: n = 3/group; bleo/WT: n = 9; bleo/KO: n = 10). Scale bars: 1000 µm (overview); 200 µm (magnified). J Relative hydroxyproline content in bleomycin-injured lungs, normalized to saline controls (saline/WT: n = 6; saline/KO: n = 3; bleo/WT: n = 28; bleo/KO: n = 24). K qRT-PCR of fibrotic genes *Col1a1*, *Col1a2*, and *Fn1* on day 18 (saline: n = 3/group; bleo/WT: n = 9; bleo/KO: n = 10). L Images and quantification of ACTA2 (red) and DAPI (blue) immunofluorescence of lung sections on day 18 (saline: n = 3/group; bleo/WT: n = 9; bleo/KO: n = 10). Scale bars: 200 µm (overview); 50 µm (magnified). M Images and quantification of mito-Dendra2 (green), LC3B (red) and DAPI (blue) immunofluorescence in lung sections on day 18 (Saline/WT: n = 99; Saline/KO: n = 73; bleo: n = 135/group). Scale bars: 5 µm (overview); 1 µm (magnified). White arrows indicate mitochondria undergoing mitophagy. P values for GO enrichment (A) were calculated using a one-sided hypergeometric test followed by Benjamini–Hochberg correction. For GSEA (B, C), the normalized enrichment score (NES) and p values were determined using an empirical permutation test. Data in E, G, I–M represent mean ± SEM. Similar results were seen in at least three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t-test (E) and ordinary two-way ANOVA with multiple comparisons (G, I–M). Exact p values are provided in the respective panels. Source data are provided as a Source Data file. In all experiments, n represents biologically independent animals or samples. WT wild-type, KO knockout, PF pulmonary fibrosis, Bleo bleomycin.

To test our hypothesis, we generated a Usp30, a gene encoding a negative regulator of PINK1 and PARKIN-mediated mitophagy pathway³⁴, knockout (KO) mouse model by deleting exon 4 of the Usp30 gene. The knockout efficiency was confirmed by qRT-PCR (Fig. 1E). We further validated protein depletion using immunofluorescence with Tyramide Signal Amplification, which confirmed reduction of USP30 protein levels in the lung tissue (Fig. S1E). Usp30^−/− mice developed and bred normally without gross histological phenotypes, consistent with previously reported^38,43. The bleomycin model is a widely used lung fibrosis model. Upon bleomycin challenge, Usp30^−/− mice exhibited significantly less body weight loss compared to wild-type (WT) control mice (Fig. 1F, G). Masson’s trichrome staining revealed a substantial reduction in the fibrotic areas in Usp30^−/− mice (Fig. 1H, I). Usp30 KO significantly reduced hydroxyproline levels and fibrotic gene expression (Fig. 1J, K). Immunostaining of the myofibroblast marker ACTA2 confirmed a significant decrease in myofibroblast accumulation (Fig. 1L). These data collectively support that Usp30 KO alleviates bleomycin-induced lung fibrosis.

To examine whether Usp30 KO enhances mitophagy, we crossed Usp30 KO mice with PhAM^excised mice, which exhibit ubiquitous expression of mito-Dendra2 fluorescence localized specifically to mitochondria⁴⁴. By quantifying the proportion of mitochondria colocalizing with LC3B⁺ autophagic puncta, we were able to quantify the mitophagy levels in mouse lungs. Our results demonstrated that Usp30 KO increased the level of mitophagy under both homeostatic conditions and following bleomycin injury (Fig. 1M).

Usp30 deletion improves mitochondrial function and promotes alveolar regeneration

To explore the potential mechanisms by which Usp30 KO alleviates pulmonary fibrosis, we performed scRNA-seq of the bleomycin- injured lungs on day 18 post injury, analyzing both CD45⁺ immune cells and CD45⁻ non-immune cells (Fig. S1F). After rigorous quality control, we retained a total of 78,379 high-quality cells and identified 21 distinct cell types based on established marker genes (Fig. S1G, H)^42,45–48. Usp30 was universally expressed in all the cell types (Fig. S1H). Changes in cell composition between the WT and Usp30 KO groups were then compared. Consistent with decreased severity of fibrosis, Usp30 KO lungs exhibited a reduced proportion of “Proliferating” and “Peribronchial” fibroblast clusters, which were highly enriched for the pathological fibroblast marker Cthrc1, Postn and the myofibroblast marker Acta2 (Fig. S1I–K). The most pronounced change was observed in AT2 cells, which were significantly more abundant in the KO group compared to the WT group (Fig. S1I).

AT2 cells serve as alveolar stem cells, capable of self-renewal and differentiation into mature alveolar type 1 (AT1) cells, which are essential for gas exchange^49–51. AT2 cells are also responsible for maintaining alveolar surface tension⁵². Given the critical role of AT2 cells in maintaining alveolar integrity and their reliance on mitochondria for energy production and survival^49,53, we next assessed mitochondrial morphology, mitochondrial function, and apoptosis of USP30-deficient AT2 cells.

Previous studies have reported frequent enlarged and swollen mitochondria in AT2 cells from both IPF patients and Pink1^−/− mice, as shown by transmission electron microscopy (TEM) images²⁶. To assess mitochondrial architecture, we employed scanning electron microscopy (SEM) and 3D reconstruction. Under baseline conditions, the mitochondria of Usp30-KO AT2 cells displayed smaller, more condensed cross-sectional areas compared to WT controls (Fig. S2A, B). Following bleomycin injury, mitochondria in WT AT2 cells exhibited marked swelling and a globular morphology, while Usp30-KO AT2 cells showed significantly reduced mitochondrial size and a fragmented, short rod-like morphology. Quantitative analysis revealed that while injury induced a significant increase in mitochondrial counts, there was no significant difference in the number of mitochondria per cell between WT and KO groups (Fig. S2A). Therefore, we hypothesized that Usp30 KO may enhance mitophagy and mitochondrial biosynthesis in AT2 cells.

Next, we examined mitophagy, mitochondrial biogenesis and dynamics in the AT2 cells. The AT2 marker SFTPC was co-stained with LC3B in PhAM^excised mice, with or without Usp30 deletion. Usp30-KO AT2 cells exhibited significantly higher levels of mitophagy compared to WT controls both under homeostatic conditions and following bleomycin injury (Fig. S2C), along with elevated LC3B signal intensity (Fig. S2D). These data suggest that USP30 deletion enhances mitophagy levels in AT2 cells.

We also examined gene expression related to mitochondrial biogenesis and dynamics using scRNA-seq data. Usp30-KO AT2 cells displayed increased expression of Ppargc1a, the master regulator of mitochondrial biogenesis, as well as higher expression of mitochondrial genes that encode essential components of the mitochondrial respiratory chain (Fig. S2E). Furthermore, Usp30-KO AT2 cells maintained higher mitochondrial OXPHOS levels while exhibiting lower glycolysis levels and reduced mitochondrial stress (Fig. S2F), indicating a healthier metabolic profile.

To validate the transcriptome analysis, we then assessed mitochondrial function by measuring oxygen consumption rate (OCR) in cultured WT and Usp30^−/− AT2 cells. Usp30 KO significantly increased the maximum respiration rate, basal respiration rate, and ATP production capacity under both baseline and bleomycin-treated conditions (Fig. 2A, B), suggesting improved mitochondrial function. A higher maximum respiration rate often correlates with enhanced energy production capability, thereby improving cell viability and function^54,55. Bleomycin induces DNA damage, thus leading to apoptosis of the damaged epithelial cells^56,57. Usp30 deletion significantly inhibited bleomycin-induced apoptosis in both AT2 cultures and bleomycin-injured lungs (Fig. 2C–F). This result was further supported by the observation of more remaining epithelial cells in the bleomycin-injured Usp30^−/− lungs on day 8, when inflammation and injury peaked (Fig. S3A).

Fig. 2 — A Experiment design for (B, C) using WT and *Usp30*^−/− AT2 cells. B Oxygen consumption rate (OCR) measurement in cultured AT2s stimulated with 1 mg/mL bleomycin or vehicle for 8 h prior to assessment (n = 12/group). C Real-time analysis of cell apoptosis in cultured AT2 cells treated with vehicle or 1 mg/mL bleomycin (Vehicle WT: n = 4; Vehicle *Usp30*^−/−: n = 5; Bleo: n = 7/ group). D Experimental design for (E, F). E Apoptotic activity in lung tissues 18 days post bleomycin injury (saline: n = 3 mice/group; bleo: n = 9 mice/group). F Representative immunoblots and quantification of lung lysates from WT and KO mice on day 18. β-Actin served as loading control (saline: n = 1 mice/group; bleo: n = 5 mice/group). G Representative immunoblots and quantification of lung lysates from IPF patients and healthy donors. β-Actin served as loading control (Normal: n = 4 donors; IPF: n = 6 donors). H Experimental design for AT2 organoid cultures. I–N Representative images and quantification of AT2 organoids (I, J), proliferating cells (EdU, green; DAPI, blue) (K, L), and AT1 cells (RAGE, red; DAPI, blue) (M, N) (n = 8 droplets/group). Scale bars: 100 µm (I); 20 µm (K, M). O Experimental design for EdU analysis in the bleomycin model. P Percentage of proliferating AT2 cells (EdU⁺) measured by flow cytometry (saline: n = 3 mice/group; bleo: n = 11 mice/group). Q, R Representative images (Q) and quantification (R) of SFTPC (white) and RAGE (green) immunofluorescence in lung sections on day 18 (bleo/WT: n = 10 mice; bleo/KO: n = 9 mice). Scale bars: 200 μm (overview); 50 μm (magnified view). Data in (B, C, E–G, J, L, N, P, R) represent mean ± SEM. Similar results were seen in at least three independent experiments. Statistical significance was determined using ordinary two-way ANOVA with multiple comparisons (B, C, E, P) and a two-tailed unpaired Student’s t-test (F, G, J, L, N, R). Exact p values are provided in the respective panels. Source data are provided as a Source Data file. In all experiments, n represents biologically independent animals or samples. WT wild-type, KO knockout, Bleo bleomycin, IPF idiopathic pulmonary fibrosis.

Next, we explored the mechanism by which Usp30 deletion inhibits apoptosis. Among the numerous deubiquitination substrates of USP30, voltage-dependent anion channel 1 (VDAC1) has been reported as a key player of mitochondrial-dependent apoptosis^34,58. VDAC1 forms channels on the outer mitochondrial membrane through oligomerization, regulating the release of cytochrome c to the cytosol, and thereby leading to mitochondrial-dependent apoptosis^59,60. We found that VDAC1 overexpression was sufficient to increase bleomycin-induced apoptosis (Fig. S3B, C). In bleomycin-injured lungs, Usp30 KO decreased VDAC1 protein levels, leading to reduced apoptosis (Fig. 2F). To validate the clinical relevance of the USP30-VDAC1 axis identified in the mouse model, we analyzed lung tissue samples from IPF patients and healthy donors. Immunoblotting of whole lung lysates revealed significantly elevated levels of USP30 and its substrate, VDAC1, in IPF lungs compared to healthy controls (Fig. 2G), suggesting a conserved regulatory role of USP30 on VDAC1 protein levels in both mouse and human lung fibrosis and also suggesting a therapeutic potential of targeting USP30 for IPF treatment.

Epithelial injury induces apoptosis of the damaged epithelial cells, while the surviving AT2 cells proliferate and differentiate into AT1 cells to repair the damaged regions^61–64. We then asked if AT2 stem cell activity is impacted by USP30 deletion by using organoid cultures. Interestingly, Usp30^−/− AT2 organoids exhibited significantly higher colony formation efficiency (CFE) and increased proliferation and differentiation, resulting in a significantly greater number of proliferating AT2 cells and differentiated AT1 cells (Fig. 2H–N). Consistent with organoid culture results, Usp30^-/- mice displayed a significantly higher rate of AT2 proliferation following bleomycin injury (Fig. 2O, P), which leads to larger areas of AT2 and AT1 cells as well as a significant increase of KRT8⁺ intermediate cells (DATP/PATS/ADI)^65–68, suggesting improved alveolar regeneration (Fig. 2Q, R and Fig. S3D).

To further explore the molecular basis underlying increased AT2 proliferation, we assessed mitochondrial ROS levels, mitochondrial quantity, and changes in mitochondrial membrane potential both in vitro and in vivo. In cultured WT and Usp30-KO AT2 cells, Usp30 KO cells exhibited moderately elevated baseline mitochondrial ROS (mtROS) and cytosolic ROS (cROS) levels, along with a transient increase of mtROS following bleomycin injury, which then decreased, suggesting effective mitigation of injury-induced oxidative stress (Fig. S3E–H). There were no significant differences in mitochondrial mass and mitochondrial membrane potential between WT and Usp30-KO AT2 cells at baseline. However, following injury, mitochondrial mass gradually increased until 6 h and then declined at 8 h in WT cells, while KO cells exhibited an accelerated decline starting at 4 h, suggesting enhanced mitophagy. Consistent with this, KO cells had increased mitochondrial membrane potential at 6 h post injury and enhanced mitophagy flux as evidenced by P62 and LC3-II protein level changes (Fig. S3I).

In bleomycin-injured lungs, Usp30 KO did not affect cROS levels, mitochondrial quantity or mitochondrial membrane potential (Fig. S3J–N). Instead, Usp30 KO increased mtROS levels in AT2 cells on day 8, coinciding with the increase of AT2 proliferation on day 8, followed by a rapid decline of mtROS on day 14 during the later stage of repair (Fig. S3K). mtROS are considered byproducts of efficient oxidative phosphorylation⁶⁹, but it has been reported that moderate levels of ROS can promote basal cell proliferation⁷⁰. To test if ROS levels affect AT2 cell proliferation, we introduced hydrogen peroxide into AT2 organoid cultures to induce ROS accumulation (Fig. S3O). Lower concentrations of hydrogen peroxide promoted AT2 cell proliferation, while higher concentrations inhibited it (Fig. S3P, Q). These results suggest that Usp30 KO induced a transient increase of mtROS in AT2 cells, thereby facilitating the repair of the epithelial barrier.

In addition to AT2 cells, we explored the potential effects of Usp30 KO on other cell types, such as

macrophages and fibroblasts. Our results showed that Usp30 KO did not significantly affect macrophage polarization (Fig. S4A, B). Next, to determine if the reduction in fibrosis was due to direct effects on mesenchymal cells, we isolated primary lung fibroblasts from Usp30 KO and WT mice. These cells exhibited comparable proliferation rates, activation, myofibroblast differentiation, and apoptosis levels in vitro (Fig. S4C–H). We also examined transcriptional profiles of fibroblasts in vivo using the scRNA-seq dataset. Usp30 deletion did not significantly affect gene expression related to apoptosis, proliferation, or differentiation in any fibroblast subpopulation (Fig. S4I). These data collectively suggest that the reduced fibrotic burden in Usp30 KO mice is not due to cell-autonomous effects on fibroblasts.

In summary, our study demonstrated that Usp30 KO significantly improved mitochondrial function, inhibited AT2 apoptosis, possibly by decreasing VDAC1, and promoted AT2 stem cell activity, likely by increasing mitochondrial ROS levels, leading to alveolar regeneration and thus ameliorated lung fibrosis.

AT2-specific Usp30 deletion effectively mitigates bleomycin injury and ameliorates pulmonary fibrosis

Next, we asked whether AT2-specific Usp30 KO is sufficient to induce the protective effect on pulmonary fibrosis. To explore this, we established an AT2-specific Usp30 KO mouse model, Sftpc^CreERT2;R26^tdT;Usp30^flox/flox (hereafter Usp30^ΔSftpc). Four doses of tamoxifen injections decreased 50% of USP30 expression in AT2 cells (Fig. 3A, B). Immunofluorescence further validated ~50% deletion of protein specifically in lineage-labeled AT2 cells (Fig. 3C).

Fig. 3 — A Experimental design for the bleomycin-induced fibrosis model. B Validation of knockout efficiency by qRT-PCR under homeostatic conditions (n = 3 / group). C Representative immunofluorescence images and quantification of USP30 protein (white) within *Sftpc*-traced AT2 cells (tdTomato⁺) (n = 8 / group). Scale bars: 10 μm. D Body weight changes monitored for 18 days (saline: n = 3 / group; bleo: n = 8 / group). E Representative images and quantification of Masson’s trichrome staining showing collagen deposition (blue) in lung sections (saline, n = 3 / group; bleo: n = 8 / group). Scale bars: 1000 μm (overview); 200 μm (magnified view). F Relative hydroxyproline content in bleomycin-injured lungs, normalized to saline controls (saline/WT: n = 6 / group; bleo/WT: n = 23; bleo/cKO: n = 16). G qRT-PCR analysis of fibrotic genes *Col1a1*, *Col1a2* and *Fn1* on day 18 (saline: n = 3/group; bleomycin: n = 8 / group). H Representative images and quantification of ACTA2 staining (green) with DAPI counterstain (blue) in the lung sections on day 18 (saline: n = 3 / group; bleo, n = 8 / group). Scale bars: 200 µm (overview); 50 µm (magnified view). I Apoptotic activity in lung tissues 18 days post bleomycin injury (saline: n = 3/group; bleo, n = 8/group). J Percentage of proliferating AT2 cells measured by flow cytometry (saline: n = 3/group; bleo: n = 8/group). K, L Representative immunofluorescence images and quantification of *Sftpc*-lineage-traced cells expressing KRT8 (K, white) and RAGE (L, green) on day 14 (n = 7 / group). Scale bars: 100 μm. Data in (B–L) represent mean ± SEM. Similar results were seen in at least three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t-test (B, C, K, L) and ordinary two-way ANOVA followed by multiple comparisons test (D–J). Exact p values are provided in the respective panels. Source data are provided as a Source Data file. In all experiments, n represents biologically independent animals or samples.

Subsequently, the mice were challenged with bleomycin. Compared to the control group, Sftpc^CreERT2;R26^tdT;Usp30^wt/wt (hereafter WT^ΔSftpc), Usp30^ΔSftpc mice exhibited significantly less body weight loss (Fig. 3D) and less severe lung fibrosis as evidenced by Masson’s trichrome staining, quantification of fibrotic areas, hydroxyproline levels, fibrotic gene expression and ACTA2 immunofluorescence staining (Fig. 3E–H). The apoptosis levels in Usp30^ΔSftpc mice were significantly lower than those observed in the control group (Fig. 3I), while there were more proliferating AT2 cells and a higher percentage of Sftpc-lineage-traced KRT8⁺ intermediate cells and RAGE⁺ AT1 cells in Usp30^ΔSftpc mice (Fig. 3J–L), indicating less alveolar injury and better alveolar regeneration, respectively. These results demonstrated that AT2-specific Usp30 KO attenuates lung fibrosis to a similar extent as global Usp30 KO. Taken together, these results established that AT2-specific Usp30 KO is sufficient to mitigate lung fibrosis by inhibiting AT2 apoptosis and promoting AT2 proliferation.

To unravel the molecular mechanism underlying enhanced regenerative capacity, we performed transcriptomic profiling of primary AT2 cells isolated from Sftpc-CreERT2 mice both at baseline and post bleomycin injury. GSEA and cumulative distribution function (CDF) analysis of AT2 cells at baseline revealed enrichment of proton motive force-driven mitochondrial ATP synthesis, mitochondrial translation, respirasome assembly and increased mitochondrial gene expression in Usp30-KO AT2 cells (Fig. S5A, B), resembling the scRNA-seq and OCR results. Pathway analysis in Usp30-KO AT2 cells at baseline showed enrichment of Notch, Wnt, MAPK, and PI3K-AKT signaling, which are important for stem cell activity and cell proliferation (Fig. S5C). Following bleomycin injury, Usp30-KO AT2 cells downregulated immune responses and senescence markers (Cdkn1a, Cdkn2a, and Cdkn2b) while upregulating AT2 identity markers (Sftpc and Etv5) (Fig. S5D, E). Notably, multiple genes associated with Wnt/β-catenin signaling, FGF signaling, and Hippo-YAP signaling, which are critical for AT2 cell regeneration after injury^71–74, were upregulated, including Axin2, Fzd1/2, Ctnnb1, Fgfr2, and Yap1. Pathway analysis showed enrichment of PI3K-AKT and MAPK signaling (Fig. S5F). These findings suggest Usp30 KO in AT2 cells improved mitochondrial function and upregulated signaling pathways related to stem cell activity.

Usp30 deletion confers protection against influenza pneumonia

Given Usp30 KO protects AT2 cells from apoptosis and promotes AT2 cell proliferation, we reasoned that Usp30 deletion may also provide beneficial effects in respiratory viral infections. H1N1 influenza A virus (IAV) induces severe respiratory epithelial damage that may lead to acute respiratory distress syndrome and potentially progress to lung failure^75,76. To monitor the dynamic changes of mitochondria specifically in AT2 cells after IAV infection, we collected the mouse lungs on multiple time points post infection and examined mitochondrial morphology and gene expression (Fig. 4A). Scanning electron microscopy (SEM) images demonstrated that the mitochondria in AT2 cells, identified by the presence of their characteristic lamellar bodies (LBs), from IAV infected mouse lungs were enlarged and swollen as early as day 2 post infection, and the number of mitochondria enclosed by autophagic vesicles increased by day 11, indicative of undergoing mitophagy (Fig. 4B). The RNA-seq analysis of flow-sorted AT2 cells showed that mitochondrial gene expression was suppressed at various time points post infection (Fig. 4C), suggesting defective mitochondrial function.

Fig. 4 — A Experimental design for analyzing mitochondrial morphology and RNA expression in AT2 cells post-influenza. B Representative TEM images of AT2 cells showing mitochondrial morphology at the indicated time points post-influenza infection. Asterisks indicate autophagic vesicles; arrows denote damaged mitochondria (MT). LB lamellar body. Scale bars: 1 μm (overview); 300 nm (magnified view). Similar ultrastructural changes in mitochondria were consistently observed in at least three independent animals per time point. C GSEA analysis of mitochondrial gene expression in AT2 cells on day 11 post flu infection versus PBS controls (left). Heatmap of downregulated mitochondrial-encoded genes across time points (right). D Experimental design for influenza infection. E–H Body weight changes (E), blood oxygen saturation (F), BAL immune cell counts (G), and BAL protein concentrations (H) on day 14 post infection (PBS: n = 3 mice; influenza/WT: n = 16 mice; influenza/cKO: n = 19 mice). I Apoptotic activity in lung tissues 14 days post infection (PBS: n = 3 mice/group; influenza: n = 21 mice/group). J, K Representative images (J) and quantification (K) of SFTPC (white) and RAGE (green) co-staining in lung sections on day 14 (influenza/WT: n = 16 mice; influenza/cKO: n = 18 mice). Scale bar: 200 μm. L Relative *Sftpc* mRNA expression levels on day 14 (PBS: n = 3 mice/group; influenza/WT: n = 17 mice; influenza/cKO: n = 18 mice). M Experimental design and quantification of proliferating AT2 cells (EdU⁺) by flow cytometry (PBS: n = 3 mice/group; influenza: n = 13 mice/group). For GSEA (C), the NES and p values were determined using an empirical permutation test. Data in (E–I, K–M) represent mean ± SEM. Similar results were observed in at least three independent experiments. Statistical significance was determined using ordinary two-way ANOVA followed by multiple comparisons test (E–I, L, M) and a two-tailed unpaired Student’s t-test (K). Exact p values are provided in the respective panels. Source data are provided as a Source Data file. In all experiments, n represents biologically independent animals or samples. cKO conditional knockout, BAL bronchoalveolar lavage.

The role of USP30 in IAV-induced lung injury was first investigated using Usp30^−/− mice (Fig. S6A). All Usp30^−/− mice survived the influenza challenge, while half of the WT mice succumbed (Fig. S6B). Moreover, the lung function of Usp30^−/− mice, as measured by arterial oxygen saturation, declined less and recovered more quickly (Fig. S6C). Inflammatory cell counts and protein levels in the bronchoalveolar lavage (BAL) were comparable at early time points, however, significantly lower in the KO mice by day 14, when the tissue injury peaked (Fig. S6D, E), suggesting the initial injury was at similar levels, but there was less immune infiltration and tissue damage in the KO mice over time. Consistently, cell apoptosis levels were significantly lower in the KO mice by day 14 compared to the WT mice (Fig. S6F), suggesting Usp30 KO protects cells from apoptosis.

AT2 marker gene expression and alveolar epithelium-covered areas were significantly higher in the Usp30^−/− mice (Fig. S6G–I). EdU labeling revealed a significantly higher proportion of proliferating AT2 cells in the KO mice (Fig. S6J). We confirmed higher levels of mitophagy in the Usp30^−/⁻ mice after influenza infection (Fig. S6K). Consistent with the results in the bleomycin model, no significant changes in total ROS levels (Fig. S6L, M), mitochondrial quantity (Fig. S6N) or mitochondrial membrane potential (Fig. S6O) were observed. However, the levels of mitochondrial ROS exhibited more pronounced dynamic changes in the KO group (Fig. S6P). Specifically, Usp30^−/− mice exhibited higher mitochondrial ROS levels on day 14, followed by a decline during the later phase of repair on day 24, indicating a greater adaptability of the mitochondria.

Given the critical role of mitochondria in antiviral responses⁷⁷, we examined whether Usp30 deletion impacts virus control. No significant differences in viral load were observed between the WT and KO mouse lungs (Fig. S6Q, R). Additionally, viral infection in AT2 organoids did not reveal any notable differences in viral load (Fig. S6S, T). These findings indicate Usp30 deletion protects the mice from influenza infection by decreasing cell apoptosis and promoting AT2 proliferation, but not antiviral responses.

We then further validated the role of USP30 in the IAV model using AT2-specific Usp30 KO mice. Usp30^ΔSftpc mice exhibited faster recovery of body weight and lung function than the control group following infection (Fig. 4D–F). There were significantly less inflammatory cell counts and protein levels in the BAL as well as cell apoptosis levels in the cKO mice on day 14 compared with those in control mice (Fig. 4G–I), indicating less tissue damage. Furthermore, AT1 and AT2 cell-covered areas, which represent epithelial barrier integrity, were restored to a higher extent in the Usp30^ΔSftpc mice compared to the control group (Fig. 4J, K). Sftpc mRNA levels were also higher in the Usp30^ΔSftpc group (Fig. 4L). Usp30-null AT2 cells showed a significantly higher proliferation rate (Fig. 4M). Taken together, our results showed that Usp30 deletion has a conserved protective role across different lung injury models by limiting AT2 cell apoptosis and promoting AT2 cell proliferation.

To investigate the molecular basis, we performed transcriptomic analysis on flow-sorted AT2 cells on day 14 post infection. Usp30-KO AT2 cells exhibited a marked upregulation of cell cycle and mitotic pathways, along with a downregulation of immune response-related genes, such as chemokines and alarmins (Cxcl2, Ccl3, S100a8, Il1b), consistent with the attenuated lymphocyte activation and recruitment observed in GSEA (Fig. S7A, B). Pathway analysis also highlighted enrichment in PI3K-AKT and MAPK signaling (Fig. S7C). These transcriptomic findings validated the enhanced proliferation of AT2 cells and improved tissue regeneration observed.

Pharmacological inhibition of USP30 ameliorates lung fibrosis and influenza pneumonia

Our results of global and conditional knockout mouse models demonstrated that USP30 is a potential therapeutic target to alleviate both chronic and acute lung injury. We thus tested this notion by using MF094, a commercially available USP30 inhibitor that has been validated with highly selective inhibitory effect both in vitro and in vivo^78–80. We first confirmed the inhibitory effects of MF094 on USP30 enzyme activity and its impact on bleomycin-induced AT2 cell apoptosis (Fig. 5A). The results showed that MF094 inhibited USP30 enzyme activity with an IC50 of 69.60 nM (Fig. 5B) and exhibited a dose-dependent effect in suppressing bleomycin-induced apoptosis in mouse lung epithelial cell line MLE12 (Fig. 5C). We also examined the ubiquitination of a known USP30 substrate, phospho-Ser65-Ubiquitin (p-S65-Ub), which is phosphorylated at Ser65 by PINK1⁸¹. MF094 treatment resulted in a dose-dependent accumulation of p-S65-Ub following mitochondrial depolarization in MLE12 cells (Fig. S8A), confirming effective inhibition of USP30 deubiquitinase activity.

We then tested the effects of MF094 in vivo. MF094 was intraperitoneally injected daily starting on the third day after bleomycin injury until tissue harvest on day 14 (Fig. 5D). Mice treated with the USP30 inhibitor exhibited decreased lung fibrosis severity and less myofibroblast accumulation (Fig. 5E–I). The apoptotic levels were significantly lower in the inhibitor-treated group (Fig. 5J).

In the IAV model, MF094 was daily administered starting from the third day post infection till day 14 (Fig. 5K). The inhibitor-treated mice exhibited less body weight loss, faster recovery of body weight and better lung function (Fig. 5L, M). Inflammatory cell counts and protein levels in BAL were significantly lower in the inhibitor group (Fig. 5N, O). Sftpc expression as well as AT2 and AT1 covered areas were significantly higher in the inhibitor-treated group (Fig. 5P–R), while tissue apoptosis was significantly lower (Fig. 5S). Taken together, our studies using the two lung injury models support USP30 as a promising therapeutic target for lung injury-induced fibrosis and viral pneumonia.

Discussion

Respiratory diseases resulting from acute and chronic lung injuries represent significant challenges to human health, with limited therapeutic options currently available. For example, only three medications have been approved for IPF patients, and these medications only serve to slow down disease progression with notable side effects. On the other hand, treatment for respiratory pneumonia typically addresses viral infections but not specifically promotes lung healing. Here, our study demonstrated that the genetic knockout and pharmacological inhibition of USP30, a negative regulator of mitophagy, improves mitochondrial function in AT2 cells, protects AT2 cells from apoptosis and promotes the proliferation and differentiation of the surviving AT2 cells, thus ameliorating alveolar damage and enhancing regeneration. Mitochondrial dysfunction has been observed in various pulmonary diseases, including chronic conditions such as IPF and chronic obstructive pulmonary disease (COPD), as well as in infection-induced acute lung injury^{16,17,26,28,82–85}. Therefore, targeting aberrant mitophagy could represent a promising therapeutic strategy for tackling various injury induced alveolar diseases.

Extensive research has been conducted on USP30 in the context of human diseases such as Parkinson’s disease and chronic kidney disease. Yet the role of USP30 in respiratory diseases remains poorly understood. By employing both genetic models and a small molecular inhibitor, we show that USP30 deletion or inhibition effectively ameliorates pulmonary fibrosis and influenza pneumonia. Mechanistically, we showed that both global and AT2-specific Usp30 KO achieve similar protective effects, emphasizing the critical role of USP30 in AT2 cells. Moreover, our data support VDAC1, one of the key regulators of apoptosis, as a USP30 substrate. USP30 deletion decreases VDAC1 protein levels, thus reducing apoptosis. Notably, both USP30 and VDAC1 protein levels are elevated in IPF lungs compared with healthy donors. On the other hand, USP30 deletion promotes damaged mitochondria clearance, thus restoring mitochondrial respiration and transiently boosting mitochondrial ROS production in AT2 cells, which leads to increased AT2 proliferation and improved alveolar regeneration.

This transient mtROS increase is consistent with the concept of mitohormesis, where mild mitochondrial stress elicits adaptive cytoprotective responses that enhance cellular function and cause a long-term reduction of oxidative stress^86–88. Collectively, our findings demonstrate that USP30 deletion or inhibition promotes mitophagy, boosts mitochondrial biosynthesis, increases ATP synthesis, and transiently induces mtROS, which is required for regenerative proliferation.

Deletion of the mitochondrial electron transport chain complex I subunit Ndufs2 has been shown to activate the integrated stress response, leading to the accumulation of KRT8⁺ intermediate cells during postnatal alveolar development⁸⁹. The persistence of KRT8⁺ intermediate cells is associated with dysfunctional or maladaptive repair responses^65–68,90. However, USP30 deletion did not induce the formation of KRT8⁺ intermediate cells in uninjured mice, nor did it result in a persistent intermediate state after injury (Fig. S9). Although there were more KRT8⁺ intermediate cells in the USP30-KO mice following bleomycin injury, both KRT8⁺ cells and AT1 cells increased, suggesting that the cells were not stalled in an intermediate state. Instead, USP30 deletion enhanced AT2 cell proliferation, leading to a larger pool of AT2 cells, which subsequently differentiate into KRT8⁺ and AT1 cells. These findings indicate that USP30 deletion promotes both the proliferation and differentiation of AT2 cells, resulting in improved regeneration.

Mitochondrial dysfunction has been shown to increase apoptosis of AT2 cells, which ultimately promotes lung fibrosis as observed in the Pink1-²⁶, Sirt3-⁹¹, and Dio2-⁹² knockout mice and when ATF3 was overexpressed⁹³. However, defective mitophagy in non-AT2 cells, such as macrophages and fibroblasts, has also been linked to lung fibrosis^28,94–96. To determine which cell type mediates USP30 function in lung fibrosis, we explored the role of USP30 in AT2 cells, macrophages and fibroblasts and found that Usp30 KO protects the AT2 cells from apoptosis and promotes AT2 stem cell activity, but does not significantly affect fibroblast proliferation, activation, apoptosis, myofibroblast differentiation or macrophage polarization. Furthermore, AT2-specific Usp30 knockout animals exhibit comparable anti-fibrotic effects to Usp30 whole-body knockout animals in the bleomycin model, supporting an important role of AT2 cells in mediating USP30 function in lung fibrosis. The role of USP30 in bronchiolar stem/progenitor cells and whether there is cell type-specific function of USP30 in respiratory diseases warrants future investigations.

While they are critical for alveolar repair and regeneration, AT2 cells are vulnerable to various injuries^49,57. Repeated or severe epithelial injuries together with aging contributes to failed or abnormal repair of alveolar epithelium, leading to lung fibrosis^97–101. The severity and mortality rates of fibrotic lung diseases, as well as certain infectious lung diseases, increase with age¹⁶. Mitochondrial dysfunction is one of the prominent features of the aging process¹⁴. Our findings demonstrate that boosting mitophagy by targeting USP30 could provide a therapeutic avenue to enhance alveolar repair and regeneration and mitigate chronic and acute alveolar diseases, including IPF and influenza pneumonia, offering a great hope for regenerative medicine to treat age and injury-related diseases.

Methods

Mice

Usp30^−/− mice, generated by utilizing Cas9 technology to knock out a 64 bp sequence in exon 4 (gRNA1: 5’-GGCGTCCAAGACCTCGTCCT-3’, gRNA2: 5’-ATGGCAGATCTCGTCCTTTG-3’), resulting in premature termination of the encoded protein, and Usp30^fl/fl mice, created by inserting flox sites at both ends of exon 2 via Cas9 technology, allowing for deletion of exon 2 upon Cre expression, were purchased from GemPharmatech. Sftpc-CreERT2 mice¹⁰² were generously provided by Dr. Chuwen Lin at Sun Yat-sen University. Rosa26-tdTomato mice¹⁰³ were generously provided by Dr. Harold A. Chapman at the University of California, San Francisco. PhAM^excised mice⁴⁴ were generously provided by Dr. Yifeng Zhang at ShanghaiTech University and crossed with Usp30^−/− mice to examine mitophagy levels. All experiments were carried out using mice aged 12–20 weeks, with age and sex matched, and mice of both sexes were used in equal proportions. The mice used in this study were housed in the Animal Core Facility at ShanghaiTech University under pathogen-free conditions, maintained at an ambient temperature of 20–24 °C and 40–60% humidity, in a 12 h light/dark cycle room with water and food ad libitum. All mice used in this study were on a C57BL/6 background.

Bleomycin model of lung fibrosis

After anesthetizing the mice with isoflurane, 50 µL of bleomycin (1.6 mg/kg body weight, Nippon Kayaku) dissolved in saline was administered into the lungs via intratracheal intubation. Both control and experimental groups consisted of sex and weight-matched mice to minimize experimental variation. Following the model induction, the mouse body weight was monitored regularly, and the mouse lungs were collected on days 14–18.

Influenza infection

The mice were anesthetized using isoflurane to agonal breathing and intranasally administrated with 40 µL of H1N1 influenza virus PR8 (diluted in cold phosphate-buffered saline (PBS)). The viral dose administered was ~36 PFU. The H1N1 influenza virus was received as a generous gift from Dr. Haikun Wang at the Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences. Mice were weighed three times per week and euthanized at the indicated time points for tissue harvest.

Tamoxifen treatment

Tamoxifen (Sigma-Aldrich, T5648) was dissolved in corn oil at a concentration of 40 mg/mL. To knock out Usp30, Usp30^ΔSftpc mice and the control WT^ΔSftpc mice were given tamoxifen at a dose of 200 mg/kg via intraperitoneal injection every other day for a total of four injections. After a 4-week waiting period, knockout efficiency was validated, followed by experimental modeling.

Arterial blood oxygen saturation measurement

Arterial blood oxygen saturation was assessed using the MOUSEOX Pulse Oximeter system (STARR Life Sciences). Before measurements, the thighs of the mice were shaved. During measurement, the mice were anesthetized with isoflurane, and readings of oxygen saturation were taken using the thigh sensor mode. After obtaining the first successful reading, measurements were conducted for at least 2 min per mouse, and the stable, continuous readings were averaged as the final result for each mouse.

Lung cell isolation

The mouse lung cells were isolated based on an established protocol¹⁰⁴. After perfusion and lavage, the lungs were inflated with 1 mL of pre-cold sterile Dispase II (Gibco, 17105041, 15 U/mL, dissolved in HBSS and filtered through a 0.22 µm filter) via a catheter into the trachea, followed by 0.2 mL of warm 1% low melt agarose (BBI, A600015, dissolved in sterile ddH₂O). Individual lobes were separated, with large airways filled with agarose discarded, and the lobes were submerged in 1 mL of Dispase II in 50 mL tubes, rocked at room temperature for 45 min. The tissue was chopped using a 10 mL pipette until no visible clumps remained. Then 10 mL of DMEM (Invitrogen, 31053036) with 2% fetal bovine serum (FBS) (Ausbian, VS500T), 1% penicillin/streptomycin (Thermo, 15140122), and 50 U/mL DNase I (Roche, 10104159001) was added to the digested lobes, and pipetting was performed vigorously. The mixture was swirled at 37 °C for 15 min on a rotating platform, and the cell suspension was filtered sequentially through 100, 70, and 40 µm strainers. The filtered suspension was centrifuged for 3 minutes at 300×g to pellet the cells. If necessary, red blood cells were lysed by 1 mL of RBC lysis buffer (Beyotime, C3702) at room temperature for 1 min, followed by stopping the reaction with 5 mL of DMEM supplemented with 2% FBS and 1% penicillin/streptomycin.

Flow cytometry

After lung tissue dissociation, cells were resuspended in DMEM supplemented with 2% FBS and 1% penicillin/streptomycin at a density of 20 million/mL. For identifying lineage⁺ cells, cells were stained with Fixable Viability Dye eFluor^TM 780 (1:2000, Thermo Fisher Scientific, 65-0865-14), FITC-conjugated rat anti-mouse CD45 (1:1000, BD, 553080), BV711-conjugated rat anti-mouse CD31 (1:500; BioLegend, 102449), BV421-conjugated rat anti-mouse EpCAM (1:500; Biolegend, 118225), and Alexa Fluor 647-conjugated rat anti-mouse Integrin β4 (1:300; BioLegend, 123608). All cell sorting was performed on a BD FACSAria Fusion Cell Sorter. EpCAM⁺ cells were sorted as epithelial cells, EpCAM⁺ Integrin β4^- cells as AT2 cells, CD45⁺ cells as immune cells, CD31⁺ cells as endothelial cells, and EpCAM^- CD31^- CD45^- as fibroblasts.

To assess mitochondrial-related markers in epithelial cells, BUV395-conjugated rat anti-mouse CD45 (1:300; BD, 564279), BV711-conjugated rat anti-mouse CD31 (1:500, BioLegend, 102449), BV421-conjugated rat anti-mouse EpCAM (1:500; Biolegend, 118225), and Alexa Fluor 647-conjugated rat anti-mouse Integrin β4 (1:300; BioLegend, 123608) were used as lineage markers. Mitotracker green (200 nM; Invitrogen, M7514) was used to measure mitochondrial content, TMRM (100 nM; Invitrogen, T668) for mitochondrial membrane potential, H2DCFDA (10 μM; MCE, HY-D0940) for cellular ROS levels, and MitoSOX red mitochondrial superoxide indicator (5 μM; Yeasen, 40778ES50) for mitochondrial ROS levels. Flow cytometry analysis was performed using the BD LSRFortessa X-20 and BD FACSDiva software (version 8.0.3). Data analysis was performed using FlowJo software (version 10.4).

Bronchoalveolar lavage (BAL)

After exposing the trachea of the mouse, a 20-G catheter was inserted into the trachea for lavage. One milliliter of cold PBS was instilled into the lungs, followed by gentle aspiration, repeated three times. All bronchoalveolar lavage fluid was collected and centrifuged at 700×g. The supernatant was collected to measure total protein content using the BCA assay (Thermo, 23227). The cell pellet was lysed in red blood cell lysis buffer for 1 min, the reaction was stopped with PBS, and the cells were counted using a hemocytometer.

Hydroxyproline assay

For hydroxyproline detection, the manufacturer’s protocol for the hydroxyproline kit (Sigma-Aldrich, MAK008-1KT) was followed. Mouse lung tissues were snap frozen in liquid nitrogen and ground into a fine powder. The powdered tissue was then hydrolyzed with 1 mL of 6 M hydrochloric acid at 120 °C for 3 h. After cooling, the hydrolysate was neutralized with sodium hydroxide. Subsequently, 5 µL of the hydrolysate was transferred to the center of a well in a 96-well plate. The assay reagent, containing a chromogenic agent specific for hydroxyproline, was added to each well to facilitate a color change. Absorbance was measured at 550 nm using a spectrophotometer, and hydroxyproline concentrations were calculated based on a standard curve generated from known hydroxyproline concentrations.

EdU incorporation and analysis

To detect AT2 cell proliferation after bleomycin-induced injury, 100 µg of EdU (Thermo Fisher Scientific, A10044) was administered to mice via intraperitoneal injection on days 2, 4, and 6 after injury, with lung tissue collected on day 8. The Click-iT EdU Alexa Fluor 647 flow cytometry assay kit (ThermoFisher Scientific, C10424) were used to examine proliferating cells via FACS. For influenza-induced injury, proliferating AT2 cells were labeled using the same method on day 8, 10, and 12 after infection, with lung tissue collected on day 14 for analysis.

Caspase-3/7 activity assay

After grinding the snap-frozen mouse lung tissue into a powder, protein was extracted using RIPA buffer (50 mM Tris-HCl, PH 7.5, 150 mM NaCl, 1% deoxycholate, 0.1% SDS, and 1% Triton X-100) supplemented with Protease Inhibitor Cocktail (Roche, 11697498001), 1 mM phenylmethyl sulfonyl fluoride (PMSF), and Phosphatase Inhibitor Cocktail (Sigma-Aldrich, 524625-1SET). Protein concentration was quantified using the BCA assay (Thermo, 23227). An equal amount of protein was used to detect caspase-3/7 activity with the Caspase-Glo® 3/7 Assay (Promega, G8091). Briefly, 5 µg of total protein was diluted in PBS to a volume of 40 µL, then mixed with 40 µL of the reaction solution in a clear-bottom 96-well plate. The mixture was shaken at 300–500 rpm for 5 min and incubated at room temperature for 30 min. The solution was then transferred to a white opaque 96-well plate and measured using a Spark Multimode Microplate Reader (Tecan, Switzerland). Two replicates were set up for each sample, with the average of the two replicates as the final reading.

Masson’s trichrome staining

Trichrome stain was performed on the frozen lung sections according to the standard procedure of Masson Stain Kit (Yeasen, 60532ES74) with modifications. Briefly, OCT-embedded frozen sections were equilibrated to room temperature and rehydrated in distilled water for 1 min before sequentially stained with hematoxylin for 1 min, acidic fuchsin for 2 min, phosphomolybdic acid for 5 min, and aniline blue for 3 min, with three washes with washing solution between each staining step. After the final dehydration step in anhydrous ethanol, the sections were mounted for microscopic analysis.

For quantitative assessment of fibrosis, stained sections were analyzed using Fiji/ImageJ software. The color deconvolution plugin was first applied using the Masson Trichrome vector set to isolate the collagen-specific blue channel. The threshold function was then used to select and quantify collagen-positive areas, while total lung area was determined using the WEKA Trainable Segmentation plugin for 2D image analysis. The percentage of fibrotic areas was calculated as the percentage of collagen-positive areas relative to total lung areas.

AT2 organoid culture

Mouse AT2 cells were flow sorted from WT or Usp30^−/− mouse lungs, resuspended at 3000 cells per 40 μL droplet in a 1:1 mixture of AT2 Maintenance Medium (AMM)¹⁰⁵ (without CHIR99021 and IL-1β) and Matrigel® (Corning, 356231), plated in 24-well plates, and allowed to solidify at 37 °C for 30 min before adding 500 μL prewarmed AMM (without CHIR99021 and IL-1β). Medium was changed every 3 days. Samples were collected after 10 days of culture. For oxidative stress experiments, organoids were treated with H₂O₂ (Sinopharm, 10011208) at various concentrations of 1, 5, or 10 μM in fresh AMM (without CHIR99021 and IL-1β) throughout the entire duration of the experiment.

To analyze the colony formation efficiency, each droplet was imaged using a Nikon TI-E microscope in bright-field mode. Z-stack images were combined, and the number of organoids was counted using ImageJ. In ImageJ, the image contrast was enhanced using the “Subtract Background” function. A threshold was applied to distinguish colonies from the background. If colonies appeared clumped, the watershed function was used to separate them. Finally, colonies were counted using the “Analyze Particles” function, with appropriate size settings to exclude noise and adjustments to the circularity parameter. Only organoids with more than five nuclei were included.

Influenza infection of AT2 organoids

For viral infection, AT2 organoids were initially cultured at a density of 5000 cells in the complete AMM medium for 10 days. Then, the droplets were digested with Dispase II (15 U/mL; Gibco, 17105041) at 37 °C for 30–40 min to be released from Matrigel. One to two droplets from each group were further digested with TrypLE^TM (Thermo Fisher Scientific, 12604013) for 5–10 min to obtain single cells for cell count reference. The rest of the organoids were centrifuged, washed with PBS and then mechanically sheared using 1 mL pipette tips to expose the apical surface for viral infection. The virus was added at a multiplicity of infection (MOI) of 0.01 and incubated at 37 °C for 1 h. After infection, the organoids were washed three times with PBS, reseeded in 40 µL of a 1:1 Matrigel:Medium mixture in a 24-well plate at a density of 100,000 cells per well. Organoids were collected at various time points, washed three times with PBS, and snap-frozen in liquid nitrogen for RNA extraction to measure the viral load.

Oxygen consumption rate (OCR) measurement

Cells from AT2 organoids after 10-day culture were digested using Dispase II and TrypLE^TM as above mentioned and seeded for OCR measurement. Prior to cell plating, each well of the Seahorse XF96 plate (Agilent) was coated with Matrigel at a 1:20 dilution in PBS and incubated for 2 h at 37 °C. AT2 cells were then seeded at a density of 30,000 cells per well in the complete AMM medium and allowed to attach for 24 h in a humidified incubator at 37 °C with 5% CO₂. Eight hours prior to OCR measurement, cells were treated with 1 mg/mL bleomycin (Vokai Biotechnology, 9041-93-4) or vehicle control.

The Seahorse assay was performed according to the manufacturer’s protocol. The culture medium was replaced with Seahorse XF assay medium supplemented with appropriate substrates. Plates were equilibrated at 37 °C without CO₂ for 1 h before OCR measurements. The OCR was measured under basal conditions and after the sequential addition of inhibitors (1.5 μM oligomycin, 1 μM FCCP, and 0.5 μM each of rotenone/antimycin A) to assess mitochondrial function. Each measurement included three baseline readings followed by injections of oligomycin and FCCP to evaluate ATP production and maximal respiratory capacity, respectively. Data were analyzed using Seahorse Wave software, and OCR was expressed in pmol/min.

In vitro mitochondrial function and mitophagy flux assay

Cells from AT2 organoids were dissociated as described above and seeded in 12-well plates precoated with Matrigel (1:20 diluted) at a density of 250,000 cells per well. After 24 h, cells were treated with bleomycin (2 mg/mL; Vokai Biotechnology, 9041-93-4) or vehicle control. For flow cytometric analysis, cells were digested with 0.25% trypsin at the indicated time points. Mitochondrial parameters were assessed using Mitotracker green (200 nM; Invitrogen, M7514), TMRM (100 nM; Invitrogen, T668), H2DCFDA (10 μM; MCE, HY-D0940), and MitoSOX red (5 μM; Yeasen, 40778ES50). Data acquisition was performed on a BD LSRFortessa X-20. For mitophagy flux analysis, cells were treated with bleomycin (2 mg/mL) or vehicle for 4 h. Bafilomycin A1 (100 nM) was added 2 h prior to harvest, followed by protein extraction and Western blot analysis.

Cell line culture

MLE12 cells (ATCC, CRL-2110) were cultured in HITES medium (Dulbecco’s:Ham’s F12, 50:50 mix supplemented with 2% FBS (Ausbian, VS500T), 0.005 mg/mL insulin (Tocris, 3435/10), 0.01 mg/mL transferrin (Roche, 10652202001), 30 nM sodium selenite (Sigma-Aldrich, S9133), 10 nM hydrocortisone (MCE, HY-N0583), 10 nM β-estradiol (Sigma-Aldrich, E2758), and 2 mM L-glutamine (Solarbio, G0200) at 37 °C with 5% CO₂. HEK293T cells (ATCC, SNL-015) were cultured in DMEM (Invitrogen, 31053036) supplemented with 10% FBS (Ausbian, VS500T) at 37 °C with 5% CO₂. Both cell lines were authenticated by STR profiling by the supplier and were routinely tested to ensure they were free of mycoplasma contamination.

Plasmid

The full-length human VDAC1 coding sequence was amplified from human cDNA and cloned into the pcDNA3.1(+) vector to construct the pcDNA3.1-VDAC1 plasmid. All newly generated plasmids are available from the corresponding author upon request.

USP30 inhibitor

The MF094 inhibitor (MCE, HY-112438) was dissolved in dimethyl sulfoxide (DMSO) to create a stock solution of 125 mg/mL. Prior to administration, the stock solution was diluted tenfold with corn oil (Abmole, M9109). Daily intraperitoneal injections of the diluted solution were administered to mice at a dose of 30 mg/kg.

Fluorescence-based USP30 enzyme activity assay

Recombinant human USP30 (rhUSP30; R&D Systems, #E-582) was diluted to 25 nM in assay buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.1 mg/mL BSA, 0.05% Tween-20, and 1 mM DTT) and 5 µL was added to each well of black, clear-bottom 384-well plates (PE, #6008280). Two point five µL of MF094 was added to each well. After 30 min pre-incubation at room temperature, 2.5 µL of Ub-Rho110 substrate (R&D Systems, #U-555; final 50 nM) was added to initiate the reaction (final volume: 10 µL). The plate was incubated for 1–2 h at room temperature, with inhibitor-only and enzyme-only controls included. Fluorescence was measured at 485 nm excitation and 540 nm emission.

Cell-based apoptosis assessment

Cells from AT2 organoids were digested using Dispase II and TrypLE^TM as above mentioned and seeded for apoptosis assay at a density of 50,000 cells per well in a 96-well plate, which has been precoated with a 1:20 diluted Matrigel for 2 h. MLE12 cells were seeded at 20,000 cells/well in a 96-well plate and pretreated with MF094 for 2 h. HEK293T cells were seeded at 20,000 cells/well in a 96-well plate and transfected with 200 ng pcDNA3.1-VDAC1 plasmid using Lipofectamine 2000 (Invitrogen, 11668) 24 h before the following assay.

For apoptosis detection, the cells were treated with 1 mg/mL bleomycin (Vokai Biotechnology, 9041-93-4), incubated with IncuCyte® Caspase-3/7 Green (Sartorius, 4440) at 1:1000 dilution and monitored by IncuCyte® live cell system in real time. The ratio of cells undergoing apoptosis was calculated as the number of green fluorescent cells divided by the total cell areas.

Bone marrow-derived macrophage (BMDM) culture

Both WT and Usp30^−/− BMDMs were isolated and cultured as follows: mouse femurs and tibias were ground, and the bone marrow was flushed with sterile PBS, filtered through a 40 µm cell strainer and centrifuged. The cell pellet was resuspended in RPMI-1640 medium (Invitrogen, C11875500CP) supplemented with 10% FBS (Ausbian, VS500T), 1% penicillin/streptomycin (Thermo, 15140122), and 1% HEPES (Invitrogen, 15630080), plated at 8 million cells per 10 cm nontreated dish and incubated at 37 °C in a 5% CO₂ atmosphere. On day 4, 5 mL of fresh medium was added. On day 7, cells were digested with 2 mM EDTA for 10 min at 4 °C. The macrophages were collected and plated at 800,000 cells per well in a 12-well plate. For polarization, cells were stimulated with LPS (100 ng/mL; InvivoGen, tlrl-eklps) and IFN-γ (20 ng/mL; R&D, 485-MI-100) for M1 polarization, or IL-4 (20 ng/mL; PeproTech, 214-14) for M2 polarization. After 24 h, cells were collected for RNA extraction and qPCR analysis.

Lung fibroblast culture

Both WT and Usp30^−/− fibroblasts were isolated from mouse lungs by flow sort based on EpCAM^-CD31⁻ CD45⁻, as detailed in the flow cytometry section. The flow-sorted fibroblasts were cultured in DMEM (Invitrogen, 31053036) supplemented with 10% FBS (Ausbian, VS500T) and 1% penicillin/streptomycin (Thermo, 15140122) and used between passages 2 and 5. For fibroblast activation assays, WT and Usp30^−/− fibroblasts were seeded in a 12-well plate at a density of 50,000 cells per well for RNA extraction, or in eight-well chamber slides at a density of 200,000 cells per well for immunofluorescence. Cells were stimulated with 4 ng/mL of TGF-β1 (R&D, 240-B-010) for 48 h, followed by RNA extraction for qRT-PCR or fixation and staining. To evaluate fibroblast proliferation, WT and Usp30^−/− fibroblasts were seeded in a 96-well plate or in eight-well chamber slides at the density of 20,000 per well and cultured with or without 10% FBS (Invitrogen, 31053036) for 48 h followed by CellTiter-Glo assay (Promega, G7571) or EdU analysis. For EdU analysis, 10 µM EdU was added 4 h prior to harvest, followed by staining as described in the EdU incorporation section. For apoptosis assessment, fibroblasts were seeded in 96-well plates at a density of 20,000 cells per well. The following day, cells were changed to serum-free medium and cultured for 48 h, followed by caspase-3/7 activity determination using IncuCyte® Caspase-3/7 Green (Sartorius, 4440) as described in the cell-based apoptosis assessment section.

Scanning electron microscopy (SEM)

Freshly collected lung samples were fixed in 2.5% glutaraldehyde in PBS at room temperature for 2 h, followed by PBS wash and post-fixation in 1% osmium tetroxide in PBS for 1 h. After that, the samples were dehydrated through a graded series of ethanol (30, 50, 70, 90, and 100%) and embedded in epoxy resin. Ultrathin sections (70 nm) were cut using a Leica Ultracut UCT microtome and collected on copper grids. The sections were then stained with 2% uranyl acetate for 15 min and lead citrate for 5 min to enhance contrast. SEM imaging was performed using a GeminiSEM460 field-emission scanning electron microscope, with a minimum of 20 cells per sample examined to ensure representative imaging.

To quantify mitochondrial morphological parameters and perform 3D reconstruction, SEM images were analyzed using a customized computational pipeline. Mitochondrial segmentation was performed using Cellpose v4¹⁰⁶ with a CPSAM model that was pretrained on mitochondrial datasets and subsequently fine-tuned through multiple rounds of iterative training (≥10 rounds) using manually annotated SEM images from our samples until achieving a segmentation accuracy exceeding 90%. The resulting binary masks were analyzed in ImageJ (Fiji) using the Analyze Particles function to quantify mitochondrial counts and cross-sectional areas. For 3D reconstruction, serial ultrathin sections (70 nm thickness, n ≥ 100 consecutive sections per sample) were first aligned and registered in TrakEM2 (Fiji) to correct for cutting-induced distortions, followed by mitochondrial segmentation using the optimized Cellpose model. Aligned segmentation masks were subsequently imported into Amira software (v6.0, Thermo Fisher Scientific), where the following steps were performed sequentially: interactive thresholding, surface generation, surface smoothing, and surface visualization. This workflow enabled precise assessment of mitochondrial morphology and spatial structure.

Tissue preparation and immunofluorescence (IF)

The freshly dissected mouse left lungs were inflated with 600 μL fixation solution (4% PFA, 50% OCT) and fixed at room temperature for 1 h, followed by PBS wash, overnight dehydration in 15% sucrose and 50% OCT, and subsequently OCT embedding. The tissue block was sectioned into 6- μm slices using a Cryostat Microtome (Thermo Fisher Scientific). The sections were fixed with 4% PFA for 5 min, treated with 0.1% sodium borohydride (Sigma-Aldrich, 213462) in PBS for 10 min, blocked for 1 h in blocking buffer (PBS supplemented with 1% BSA, 5% horse serum, and 0.1% Triton X-100), and incubated with the primary antibody at 4 °C overnight. The next day, the sections were stained with a fluorescent secondary antibody for 1 h at room temperature, followed by DAPI staining for 15 min, and then mounted for imaging.

For tyramide signal amplification (TSA) staining, the tissue sections were processed using a TSA kit (ABclonal, RK50026P) with modifications to the standard immunofluorescence protocol described above. Following sodium borohydride treatment, endogenous peroxidase activity was quenched by incubation with Peroxidase Blocking Buffer for 15 min at room temperature in the dark. After three washes with PBS, blocking and primary antibody incubation were performed as previously described. The sections were then incubated with HRP-conjugated rabbit polymer secondary antibody (1:1000) for 1 h at room temperature. Immunofluorescence signals were developed by incubation with the fluorescent tyramide working solution for 10 min at room temperature. Nuclei were counterstained with DAPI for 15 min, and the sections were mounted for imaging.

The antibodies used in this study included rabbit anti-pro-SPC (1:3000; Millipore, AB3786), goat anti-pro-SPC (1:1000; Santa Cruz Biotechnology, sc-7706), mouse anti-pro-SPC (1:50; ServiceBio, GB154201), rat anti-RAGE (1:400; R&D, MAB1179-100), rabbit anti-phospho-Histone H3(Ser10) (1:500; Sigma-Aldrich, 06-570), rabbit anti-LC3B (1:100; Sigma-Aldrich, L7543), mouse anti-ACTA2-conjugated FITC (1:200; Sigma-Aldrich, F3777), rabbit anti-ACTA2 (1:500; Abcam, ab5694), rat anti-KRT8 (1:100; DSHB, MABT329M), and rabbit anti-USP30 (1:50; Thermo Fisher Scientific, PA5106762).

Quantification of immunofluorescence

For quantification of immunofluorescence staining of tissue sections, the entire lung lobes were imaged using the Nikon Ti2-E CSU-W1 Sora camera system (Nikon, Japan), except that Cy7 signals identifying mitochondrial autophagy in AT2 cells were acquired using the STELLARIS 8 Falcon FLIM Confocal microscope (Leica Microsystems, Germany). The Trainable Weka Segmentation (TWS) plugin in Fiji/ImageJ was employed to quantify the areas of ACTA2⁺, RAGE⁺, and their corresponding DAPI⁺ regions. SPC⁺ areas were manually outlined in ImageJ by selecting regions with homogeneous SPC distribution. For quantification of organoid cultures, SPC⁺, RAGE⁺, phospho-Histone H3⁺, and EdU⁺ cells were quantified using QuPath v0.5.1 with “Analyze → Cell Detection → Positive Cell Detection” module. For the quantification of mitophagy, QuPath v0.5.1 software and module were employed. Each mitochondrion was identified based on the mito-Dendra2 signal. The colocalization of LC3B with each mitochondrion was used to determine the mitochondrion undergoing mitophagy, and the mitophagy index was calculated as the ratio of LC3B⁺mito-Dendra2⁺ to total mito-Dendra2⁺ mitochondria.

Western blot

Snap-frozen mouse lungs were ground into powder on dry ice and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Roche, 11697498001), 1 mM phenylmethyl sulfonyl fluoride (PMSF) and Phosphatase Inhibitor Cocktail (Sigma-Aldrich, 524625-1SET). Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific, 23227), and equal amounts of protein lysates were loaded for SDS-PAGE. The primary antibodies employed were β-Actin (1:1000; Santa Cruz Biotechnology, sc-47778), VDAC1 (1:400; Santa Cruz Biotechnology, sc-390996), Cleaved Caspase-3 (1:1000; Cell Signaling Technology, 9661S), USP30 (1:500; Thermo Fisher Scientific, PA5106762), COL1A1 (1:1000; Cell Signaling Technology, 72026S), ACTA2 (1:200; Abcam, ab5694), LC3B (1:500; MBL M152-3), P62 (1:500; Santa Cruz, sc-48402), Phospho-Ser65-Ubiquitin (1:1000; Cell Signaling Technology, 62802S) and pro-SPC (1:400; Millipore, AB3786). The secondary antibodies used were goat anti-mouse conjugated HRP (1:5000; Thermo Fisher Scientific, G21040) and goat anti-rabbit conjugated HRP (1:5000; Thermo Fisher Scientific, G21234).

Measurement of phospho-Ser65-Ubiquitin levels

MLE12 cells were seeded in 12-well plates at a density of 200,000 cells per well. Cells were pretreated with MF094 at concentrations of 0, 1, 5, 10, and 20 µM for 2 h prior to the addition of 10 µM CCCP for 4 h. Cells were harvested, and the protein lysates were prepared for Western blot analysis.

RNA extraction and qPCR analysis

For RNA extraction, the Eastep™ Super Total RNA Extraction Kit (Promega, LS1040) was used for tissue samples, while the HiPure Total RNA Nano Kit (Magen, R4125) was employed for cell samples. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Vazyme, R222-01). Subsequent qPCR analysis was conducted with SYBR qPCR Master Mix (Vazyme, Q711-03). Relative gene expression levels were determined using the ΔΔCT method and normalized to HPRT. The primers utilized in this study are listed in Supplementary Table 1.

RNA-seq analysis

AT2 cells (CD45^- EPCAM⁺ CD31^- Integrin β4^-) were flow-sorted from the lungs of influenza-infected mice at 0, 2, 6, and 11 days post infection. Sftpc-traced AT2 cells (tdTomato⁺) were flow-sorted from tamoxifen-treated WT^ΔSftpc and Usp30^ΔSftpc mice at day 14 after saline or bleomycin administration, or at day 14 after influenza infection. Following RNA extraction, library preparation was performed using the TruSeq RNA sample preparation kit (Illumina, RS-122-2102) according to the manufacturer’s protocol. Sequencing was conducted on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Raw sequencing data were processed using bcl2fastq (Illumina) to convert base call files to FASTQ format. Quality control was performed using FastQC (V0.12.1) to assess the FASTQ files¹⁰⁷. Reads were aligned to the Mouse Genome Assembly GRCm38 using HISAT2 (V2.2.1)¹⁰⁸. The resulting alignment files were processed to generate gene expression counts using featureCounts (V1.22.2)¹⁰⁹. For the time-course samples, differential expression analysis was conducted using edgeR (V3.40.2)¹¹⁰. For Sftpc-traced AT2 cell datasets (saline, bleomycin, and influenza groups), differential expression analysis was performed using DESeq2 (V1.38.3)¹¹¹. Gene set enrichment analysis (GSEA)¹¹² and KEGG pathway analysis were performed using clusterProfiler¹¹³ (V4.1.2). For saline-treated Sftpc-traced AT2 cells, the log₂ fold changes of mitochondrial genes were compared to those of background genes (non-mitochondrial genes). Cumulative distribution function (CDF) curves were generated using the ggplot2 package (V3.4.4) in R, and the statistical significance of the distribution shift was assessed using the Kolmogorov–Smirnov (KS) test.

For re-analysis of published microarray data (GSE110147)⁴¹, raw CEL files were processed using the R packages affy¹¹⁴ (V1.76.0) and limma¹¹⁵ (V3.54.0), with differentially expressed genes defined as |log₂FC|≥1 and p value ≤0.05. GSEA and correlation analyses were performed using clusterProfiler¹¹³ (V4.1.2) and cor.test function, respectively.

Single-cell RNA-seq analysis

CD45⁺ and CD45⁻ cells were flow-sorted from bleomycin-treated WT and USP30^–/− mice (n = 4 mice/group) and processed using the Chromium Single-Cell Platform (10X Genomics, Pleasanton, CA). Libraries were prepared according to the manufacturer’s protocol and sequenced on an Illumina NovaSeq 6000 system. The CD45⁺ and CD45^- library yielded 79,493 cells with an average sequencing depth of 22,802 reads per cell. Raw sequencing data were processed using Cell Ranger v8.0.1 (10X Genomics) for initial quality control. Subsequent analyses were performed using the Seurat package¹¹⁶ (v4.3.0) in R. We applied stringent quality filters, excluding cells with fewer than 200 detected genes or >25% mitochondrial gene content. After quality control, 78,379 high-quality cells were retained for downstream analysis.

Data normalization and feature selection were performed using SCTransform. Dimensionality reduction was achieved through principal component analysis (PCA), with the top 20 principal components serving as input for UMAP visualization. Cell clustering was performed using the FindClusters function (resolution = 0.8), and cell type annotation was based on canonical marker gene expression patterns. To evaluate specific cellular phenotypes and metabolic states, pathway activity scores were computed using the AddModuleScore function in Seurat with default parameters. For fibroblasts, functional states were assessed using gene sets for proliferation (Mki67, Pcna, Top2a, Ccna2, Cdk1, Ccnb1), apoptosis (Bax, Casp3, Casp8, Fas, Bid, Bcl2l11), and differentiation (Acta2, Col1a1, Fn1, Postn, Cthrc1, Spp1). For AT2 cells, metabolic profiles were characterized using gene sets for glycolysis (Pkm, Ldha, Gapdh, Hk2, Eno1, Pgk1, Pfkp), mitochondrial stress (Hspd1, Hspa9, Clpp, Lonp1, Yme1l1), nuclear-encoded OXPHOS (Ndufa9, Ndufb8, Sdhb, Uqcrc2, Atp5a1, Cox5a, Cox4i1), and mitochondria-encoded OXPHOS (mt-Nd1, mt-Nd2, mt-Co1, mt-Co2, mt-Atp6, mt-Cytb).

The published scRNA-seq data of IPF lungs (GSE135893)⁴² were reanalyzed using the Seurat¹¹⁶ (V4.3.0) package in R (V4.2.2). Differential gene expression analysis between IPF and control samples was performed using the FindMarkers function with default parameters, followed by gene set enrichment analysis (GSEA)¹¹² to identify significantly altered pathways. To assess mitophagy and oxidative phosphorylation activity scores, we retrieved the corresponding gene sets from the Gene Ontology database (GO:0000423 for mitophagy and GO:0006119 for oxidative phosphorylation). Cellular pathway activity scores were calculated using Seurat’s AddModuleScore function¹¹⁶ (V4.3.0) with default parameters, enabling quantitative comparison across cell populations.

Human sample collection

Human lung tissues were obtained from Tongji University Affiliated Shanghai Pulmonary Hospital. Specifically, normal lung tissues were obtained from macroscopically normal tissues adjacent to pulmonary nodules during surgical resection, and IPF lung tissues were obtained from the explanted lungs of IPF patients undergoing lung transplantation. Participants were not compensated. To protect participant anonymity, exact ages have been replaced with age ranges in our data presentation. Following the SAGER guidelines, participant sex (three females and seven males) was determined based on self-reported medical records. Due to the limited sample size used exclusively for immunoblotting, sex-based analysis was not performed. All human tissue samples were completely destroyed during the protein extraction process for immunoblotting analysis, and no samples were conserved for further analysis. Detailed de-identified characteristics of the human tissues are listed in Supplementary Table 2.

Statistics and reproducibility

Custom scripts in R v4.2.2 were utilized for RNA-seq and scRNA-seq data analysis and plotting. All other statistical calculations were analyzed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA). Statistical tests used, and the sample size is indicated in the corresponding figure legends. The exact p values are shown in figures or figure legends. A p value of <0.05 was considered statistically significant. All data were generated from three or more independent experiments with at least three biological replicates per genotype and condition. Investigators were blinded to group allocation during the quantitative assessment of imaging analysis and lung function monitoring. For biochemical assays and computational sequencing analysis, blinding was not applicable as group identity is required for downstream comparative pipelines. No statistical methods were used to predetermine sample sizes. Sample sizes were empirically chosen based on established standards in pulmonary research, which provide sufficient statistical power to detect biological differences in injury models.

Ethical compliance

This research complies with all relevant ethical regulations regarding animal research and human tissue usage. All animal studies were approved by the Animal Ethics Committee of ShanghaiTech University (protocol: 20210928001) and were performed in compliance with ethical guidelines and the approved protocols. The human study protocol for the use of human lung tissues was approved by the Human Ethics Committee of Shanghai Pulmonary Hospital (protocol: K21-282), and written informed consent was obtained from all participants prior to inclusion.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information^{(10.4MB, pdf)}

Reporting Summary^{(3.2MB, pdf)}

Transparent Peer Review file^{(9.8MB, pdf)}

Source data

Source Data^{(7MB, zip)}

Acknowledgements

This work was supported by the National Key R&D Program of China (No. 2024YFA1108500 to Y.X.), the Major Project of Guangzhou National Laboratory (No. GZNL2023A01003 to Y.X.), the National Natural Science Foundation of China (No. 82570002 to Y.X.), and the Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University (to Y.X.). We thank Rui Wang for critical technical contributions on 3D SEM for preparing tissue sections, collecting samples, and acquiring SEM data with Zeiss GeminiSEM 460, also thank Rui Wang and Dr. Xiaoming Li for image alignment, Cellpose-SAM training, and mitochondrial 3D reconstruction via Python, napari and Amira. We thank Dr. Xiucheng Liu at Tongji University Affiliated Shanghai Pulmonary Hospital for providing the human lung tissue samples. We thank Professors Harold A. Chapman, Chuwen Lin, Yifeng Zhang, and Haikun Wang for providing mouse strains and influenza virus. We also thank the Molecular Imaging Core Facility, Molecular and Cell Biology Core Facility, and Animal Core Facility at the School of Life Science and Technology, ShanghaiTech University and Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences for providing technical support. Additionally, we thank the Biomedical Big Data Platform at the Institute of Immunochemistry, ShanghaiTech University, for their assistance with single-cell sequencing.

Author contributions

P. Wu conceived the project, designed and performed experiments, analyzed data, and wrote the manuscript. J.C., L.L., X.H. and P. Wang conducted experiments and contributed to data acquisition. T.L. participated in experimental design and execution. S.S. performed influenza time-course RNA-sequencing experiments. Y.C., Y.S. and R.L. managed mouse breeding and colony maintenance. N.D. provided intellectual input and revised the manuscript. Y.X. conceived and supervised the project, analyzed the data, and wrote the manuscript.

Peer review

Peer review information

Nature Communications thanks Bin Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The single-cell RNA-seq and bulk RNA-seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession codes GSE295598, GSE317252, GSE317253, and GSE301256. The published transcriptomic datasets used in this study are available in the GEO database under accession codes GSE110147 and GSE135893. The processed count matrices and metadata generated in this study have been deposited alongside the raw sequencing data in the NCBI GEO database. All numerical data underlying the statistical plots and uncropped scans of all immunoblots are provided in the “Source Data” file. There are no restrictions on data access. Source data are provided with this paper.

Code availability

All bioinformatics analyses were performed using standard, publicly available software packages, and functions in R, as comprehensively detailed in the Methods section. The customized R scripts utilized to process the scRNA-seq and bulk RNA-seq data, execute the analysis pipelines, and generate the figures in this study have been deposited in the Zenodo public repository and are available under the 10.5281/zenodo.18831801.

Competing interests

N. Ding is an employee of BeOne Medicines. The remaining authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-71728-x.

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PERMALINK

Mitophagy promotes lung repair and regeneration by restoring epithelial metabolic fitness

Pei Wu

Jiawei Chen

Li Liu

Ping Wang

Tiantian Lu

Shengxi Shen

Yiyuan Cao

Yuandong Sui

Run Liu

Xiajuan Huan

Ning Ding

Ying Xi

Abstract

Introduction

Results

Usp30 KO mice are protected from bleomycin-induced lung fibrosis

Fig. 1. Usp30 KO mice are protected against bleomycin-induced lung fibrosis.

Usp30 deletion improves mitochondrial function and promotes alveolar regeneration

Fig. 2. Usp30 deletion improves mitochondrial function, inhibits AT2 apoptosis and promotes AT2 proliferation.

AT2-specific Usp30 deletion effectively mitigates bleomycin injury and ameliorates pulmonary fibrosis

Fig. 3. AT2-specific Usp30 deletion effectively mitigates bleomycin-induced pulmonary fibrosis.

Usp30 deletion confers protection against influenza pneumonia

Fig. 4. AT2-specific Usp30 deletion effectively mitigates influenza pneumonia.

Pharmacological inhibition of USP30 ameliorates lung fibrosis and influenza pneumonia

Fig. 5. USP30 inhibition ameliorates lung fibrosis and influenza pneumonia.

Discussion

Methods

Mice

Bleomycin model of lung fibrosis

Influenza infection

Tamoxifen treatment

Arterial blood oxygen saturation measurement

Lung cell isolation

Flow cytometry

Bronchoalveolar lavage (BAL)

Hydroxyproline assay

EdU incorporation and analysis

Caspase-3/7 activity assay

Masson’s trichrome staining

AT2 organoid culture

Influenza infection of AT2 organoids

Oxygen consumption rate (OCR) measurement

In vitro mitochondrial function and mitophagy flux assay

Cell line culture

Plasmid

USP30 inhibitor

Fluorescence-based USP30 enzyme activity assay

Cell-based apoptosis assessment

Bone marrow-derived macrophage (BMDM) culture

Lung fibroblast culture

Scanning electron microscopy (SEM)

Tissue preparation and immunofluorescence (IF)

Quantification of immunofluorescence

Western blot

Measurement of phospho-Ser65-Ubiquitin levels

RNA extraction and qPCR analysis

RNA-seq analysis

Single-cell RNA-seq analysis

Human sample collection

Statistics and reproducibility

Ethical compliance

Reporting summary

Supplementary information

Source data

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Code availability

Competing interests

Footnotes

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

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