Conditional BCL-2 Expression in Fibroblasts Promotes Persistent Pulmonary Fibrosis which is Reversible by Therapeutic BCL-2 Inhibition

Domain: chemistry
Published: 2026-02-28
Editorial outcome: major_revision

Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease that develops in response to chronic epithelial injury. Unlike injury-induced homeostatic lung repair during which fibroblasts undergo apoptosis and clearance, the lungs of IPF patients continue to accumulate apoptosis-resistant, pro-fibrotic, extracellular matrix-producing fibroblasts. Here, we show that prevention of PDGFRα+ fibroblast apoptosis by conditional BCL-2 expression leads to the emergence and persistence of senescent, pro-fibrotic fibroblasts along with enduring, pathologic fibrotic lung remodeling. Additionally, spatial transcriptomic studies of human IPF lungs confirmed the presence of senescent, BCL-2 expressing α-smooth muscle actin+ myofibroblasts in fibrotic regions. Of translational significance, selective BCL-2 inhibition with ABT-199 in fibrotic mice re-engaged the apoptotic pathway in fibroblasts, reduced senescence, and promoted fibrosis resolution and lung regeneration. Our findings suggest that sustained BCL-2 expression in fibroblasts prevents homeostatic lung repair, drives persistent fibrosis and is a therapeutically relevant target to reverse persistent pulmonary fibrosis.

Introduction
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease in which progressive scarring of the alveolar-capillary units leads to eventual respiratory failure 1. With a median survival of ~3-5 years following diagnosis and limited availability of effective therapies, the prognosis for IPF patients is bleak 2,3. IPF is thought to develop following injury to the alveolar and terminal bronchiolar epithelium followed by an aberrant repair process which leads to the abundant accumulation of pro-fibrotic fibroblasts and excessive deposition of collagen and other fibrotic matrix components 1,4. Unlike normal homeostatic repair 5 where fibroblasts undergo apoptosis and clearance, fibroblasts develop resistance to apoptosis induction, gain a senescent phenotype and survive in fibroblastic foci in IPF patients 6-8. Understanding the fundamental mechanisms that lead to fibroblast resistance to apoptosis an d the subsequent acquisition of a senescent phenotype could thereby provide insights into targeted therapeutic opportunities to reverse this key pathogenic event.

Previous studies have shown that both down-regulation of the cell surface death receptor, Fas, 9-11 and expression of the anti-apoptotic proteins PTPN13, XIAP and BCL-2 contribute to apoptosis resistance in cultured human fibrotic lung fibroblasts 12-18. Among these, BCL-2 has been shown to block apoptosis by partnering with pro-apoptotic mitochondrial activators and pore formers preventing them from activating the mitochondrial apoptotic pathway 19,20. Increased levels of BCL-2 have been found in lung fibroblasts from IPF patients and in mouse models of pulmonary fibrosis 12,21,22. However, the mechanism by which fibroblast resistance to apoptosis and senescence contribute to persistent fibrotic lung disease remains largely unknown. To address this knowledge gap, we generated transgenic mice in which human BCL-2 can be conditionally expressed in fibroblasts. Herein, we demonstrate that BCL-2 expression in fibroblasts prevents lung repair and regeneration in bleomycin-instilled mice and leads to fibroblast persistence, acquisition of a senescent phenotype, augmentation of pro-fibrotic gene expression, abnormal lung remodeling and persistent pulmonary fibrosis. Of significance to the treatment of patients with IPF, our pre-clinical studies in mice show that BCL-2 inhibition with the FDA-approved BCL-2 BH3-mimetic drug ABT-199 (VenetoclaxTM) reversed fibroblast senescence, re-engaged mitochondrial apoptosis pathways, promoted fibrosis resolution and improved lung architecture and function.

Results
Conditional BCL-2 expression in fibroblasts inhibits apoptosis and promotes their persistence in bleomycin-injured lungs.

We engineered mice in which human BCL- 2 and the lineage tracer mCerulean3 cyan fluorescent protein (CFP) can be conditionally expressed through Cre-mediated recombination (Fig. 1a,b,c). We refer to this strain as CFP/BCL-2stopfl/fl. We bred PDGFRα-CreERT2;CFP/BCL-2stopfl/+ mice, which are referred to as PDGFRα-CFP/BCL-2+ mice following tamoxifen treatment.

Lung fibroblasts from PDGFRα-CFP/BCL-2+ mice exhibit robust expression of predominantly mitochondrial BCL-2 and cytoplasmic CFP (Figs. 1b,c,d) and resisted apoptosis induction by both the mitochondrial and death receptor pathways (Fig. 1e,f). PDGFRα-CFP/BCL-2+ mice exhibit colocalization of BCL-2 and CFP in PDGFRα+ and PDGFRα+/β+ fibroblasts located in alveolar walls and septae (Fig. 1g and Suppl. Fig. 1). CFP was not detected in PDGFRβ+ (i.e. PDGFRα-) pericytes, pro-SPC alveolar type II epithelial cells (AEC2), CCSP+ club cells, CD45+ alveolar macrophages or CD31+ endothelial cells, and had minimal expression in αSMA+ parabronchial or arterial smooth muscle (Fig. 1g and Suppl. Fig. 1). Using the flow cytometry strategy shown in Fig 1h, CFP was abundantly detected in PDGFRα+ and PDGFRα+/β+ fibroblasts (Fig. 1i), but not in endothelial cells, epithelial cells, hematopoietic cells or PDGFRβ+ pericytes (Figs. 1i,j). CFP was not expressed in the absence of tamoxifen treatment (Figs. 1i,j). Thus, BCL-2 is specifically expressed in lung fibroblasts and blocks fibroblast apoptosis in PDGFRα-CFP/BCL-2+ mice.

Lung delivery of bleomycin in mice leads to acute epithelial injury followed by peak fibroblast accumulation and fibrosis at 2-3 weeks. The peak of fibrosis coincides with a wave of fibroblast apoptosis followed by fibrosis resolution and lung regeneration by 6-8 weeks 23,24. To investigate the consequences of BCL-2 expression on fibroblast apoptosis in vivo, PDGFRα-CFP/BCL-2fl/+ mice were instilled with bleomycin or saline, treated with tamoxifen or corn oil (Fig. 2a) and apoptotic lung fibroblasts were identified by staining for PDGFRα, PDGFRβ and TUNEL at 3 weeks. Figs. 2b,c show that TUNEL+ fibroblasts were detected in bleomycin-instilled PDGFRα-CFP/BCL-2- mice, but were significantly reduced (p<0.01) in the fibrotic lungs of PDGFRα-CFP/BCL-2+ mice. TUNEL+ staining was negligible in the lungs of saline-instilled PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- mice.

PDGFRα+ fibroblasts and PDGFRβ+ pericytes have been implicated in the development of pulmonary fibrosis 25-28. To determine if BCL-2 expression led to increased lung mesenchymal cell numbers, we quantified PDGFRα+ fibroblasts, PDGFRα+/β+ fibroblasts and PDGFRβ+ pericytes in bleomycin- and saline-instilled PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- mice for up to 12 weeks (Fig. 3a). Figs. 3b,c,d and Suppl. Fig. 2 show that PDGFRα+ fibroblasts, PDGFRα+/β+ fibroblasts and PDGFRβ+ pericytes were increased (p<0.001) to similar extents in bleomycin-instilled mice of both genotypes at 3 weeks. However, while the numbers of PDGFRα+ fibroblasts, PDGFRα+/β+ fibroblasts and PDGFRβ+ pericytes returned to baseline by 6 weeks in PDGFRα-CFP/BCL-2- mice, they remained significantly elevated for up to 12 weeks in PDGFRα-CFP/BCL-2+ mice (Figs. 3b,c,d). No differences in fibroblast or pericyte accumulation were observed when comparing PDGFRα-CFP/BCL-2- and wild type C57Bl/6 mice (Suppl. Fig. 3a,b). The frequency of CFP expression in PDGFRα+ and PDGFRα+/β+ lung fibroblasts was >95% in saline and bleomycin-instilled PDGFRα-CFP/BCL-2+ mice and <10% in PDGFRβ+ pericytes (Figs. 3e,f,g). The observed deep penetrance of CFP in both PDGFRα+ and PDGFRα+/β+ fibroblasts suggests that these subsets were derived from PDGFRα+ cells.

To determine the spatial localization of PDGFRα+ and PDGFRβ+ cells within fibrotic areas, we co-immunostained lung sections for PDGFRα, PDGFRβ and CFP. Additional sections were stained for human BCL-2. An expansion of both PDGFRα+ and PDGFRα+/β+ fibroblasts was observed to be coincidental with CFP staining. PDGFRβ+ (i.e. CFP-) pericytes were also noted within fibrotic areas in bleomycin-instilled PDGFRα-CFP/BCL-2+ mice at 3 weeks and persisted for up to 12 weeks (Fig. 3h, Suppl. Fig. 4). BCL-2 staining was also observed in fibrotic PDGFRα-CFP/BCL-2+ mice. As expected, neither CFP nor human BCL-2 were detected in the lungs of PDGFRα-CFP/BCL-2- mice and the expansion of PDGFRα+ fibroblasts and PDGFRβ+ pericytes was only seen at 3 weeks post bleomycin (Fig. 3i). By 6 weeks, PDGFRα+ fibroblasts and PDGFRβ+ pericytes were detected in resolving fibrotic areas and by 9-12 weeks were once again observed in the alveolar walls of bleomycin-instilled PDGFRα-CFP/BCL-2- mice, similar to naïve mice (Fig. 3i).

Conditional BCL-2 expression in fibroblasts leads to persistent pulmonary fibrosis and fibrotic remodeling.

We next determined if the BCL-2 driven fibroblast persistence in bleomycin-instilled PDGFRα-CFP/BCL-2+ mice prevented lung repair and regeneration leading to sustained fibrosis and architectural remodeling. Assessment of lung hydroxyproline levels (Fig. 4a) revealed that bleomycin-instilled PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- mice developed similar levels of fibrosis at 3 weeks (p=0.185). However, while fibrosis had largely resolved in PDGFRα-CFP/BCL-2- mice at 6 weeks, it persisted in PDGFRα-CFP/BCL-2+ mice for up to 12 weeks (p<0.01) (Fig. 4a). Histologic examination of Masson’s trichrome stained sections (Fig. 4b) from bleomycin-instilled PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- mice indicated that they developed similar airway distortion and alveolar collapse with patchy fibrosis, septal thickening and increased inflammation at 3 weeks. However, whereas lung regeneration was evident in PDGFRα-CFP/BCL-2- mice at 6 weeks, PDGFRα-CFP/BCL-2+ mice exhibited non-resolving lung fibrosis, sustained pathologic airway distortion and architectural collapse, and areas of focal bronchiolization and cysts (Fig. 4b).

The development of lung fibrosis is thought to involve initial epithelial injury which in turn drives fibroblast accumulation and pro-fibrotic fibroblast activation. The generation of PDGFRα-CFP/BCL-2+ mice provided an opportunity to determine if prolonging the lifespan of pro-fibrotic fibroblasts through conditional BCL-2 expression promotes aberrant epithelial cell accumulation, cyst formation and parenchymal bronchiolarization 29. We observed that the areas of remodeled lung in PDGFRα-CFP/BCL-2+ mice exhibited a significant accumulation of CCSP+KRT8+ cells at 3 weeks, which persisted up to 12 weeks (Fig. 4c,e upper panels). Additionally, staining for pro-SPC revealed a significant increase in SPC+ AEC2 cells throughout the fibrotic areas also persisting for up to 12 weeks in contrast to PDGFRα-CFP/BCL2- mice, where AEC2 cells were localized to normal alveoli (Fig. 4d,f). Bleomycin-instilled PDGFRα-CFP/BCL2- mice also showed limited numbers of KRT8+ cells in fibrotic areas and were absent during lung regeneration between 6 and 12 weeks (Fig. 4c,e lower panels). Naïve and saline-instilled PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- mice showed no histological abnormalities (Suppl. Fig. 5a,b), while bleomycin-instilled PDGFRα-CFP/BCL-2- and control C57Bl/6 mice show identical patterns of fibrosis development and resolution (Fig. 4b lower panels and Suppl. Fig. 5c).

To validate the findings in PDGFRα-CFP/BCL-2+ mice, we bred Col1a1-CreERT2;CFP/BCL-2stopfl/+ mice to allow tamoxifen-inducible conditional expression of BCL-2 in fibroblasts 24. Using the bleomycin and tamoxifen application timeline shown in Fig. 3a, bleomycin-instilled Col1a1-CFP/BCL-2+ mice were found to phenocopy PDGFRα-CFP/BCL-2+ mice with persistently elevated numbers of PDGFRα+ fibroblasts and PDGFRβ+ pericytes, sustained elevations in lung hydroxyproline and histologically evident fibrosis with focal areas of bronchiolization and cysts for up to 12 weeks (Suppl. Fig. 6). Col1a1-CFP/BCL-2- mice displayed similar fibrosis development at 3 weeks followed by lung regeneration at 6 weeks (Suppl. Figs 5c and 6). Thus, using two Cre-drivers, conditional expression of BCL-2 in fibroblasts: (i) prevented normal, homeostatic fibroblast apoptosis following bleomycin-induced lung injury, (ii) led to the prolonged increases in PDGFRα+ fibroblasts and PDGFRβ+ pericytes, (iii) impeded lung repair and regeneration, and (iv) resulted in persistent pulmonary fibrosis along with aberrant epithelial cell differentiation and architectural remodeling.

Apoptosis resistant BCL-2 expressing fibroblasts exhibit sustained pro-fibrotic and senescence-related transcriptomic programming.

In view of the findings presented above, we hypothesized that BCL-2 might exert additional pressure on fibroblast pro-fibrotic programming, beyond simply blocking apoptosis. To test this hypothesis, we isolated lung fibroblasts from naïve PDGFRα-CFP/BCL-2+ mice and bleomycin-instilled PDGFRα-CFP/BCL-2- and PDGFRα-CFP/BCL-2+ mice at 3 and 6 weeks and conducted bulk RNA-sequencing and analysis (Fig. 5). Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) at 3 weeks showed similar enrichment in proliferation regulation genes in both PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- mice compared to naïve mice (Suppl. Table 1). Other pathways that were enriched in both mouse strains at 3 weeks included ECM structure, collagen fibril organization, collagen-containing ECM, and ECM organization, together with their associated genes (e.g. Col1a1, Col1a2, Col5a1, Col8a1, Col12a1, Eln, Fn1, Postn and Cthrc1) (Fig. 5a,b,c and Suppl Table 4). These findings suggest overlap in the pro-fibrotic programming patterns of lung fibroblasts at 3 weeks, though this was enhanced in PDGFRα-CFP/BCL-2+ mice. However, a notable difference between the two genotypes was that fibroblasts from PDGFRα-CFP/BCL-2+ mice showed significant enrichment in biological processes associated with positive regulation of cellular senescence (including transcripts encoding Cdkn2a (p16Ink4a), Glb1 (senescence-associated β-galactosidase (SA-β-gal)) and p53 signaling (Tp53) although there was no significant difference in Cdkn1a (p21Waf1) expression between PDGFRα-CFP/BCL-2+ and PDGFRα-CFP/BCL-2- fibroblasts at 3 weeks. There was also an increased signature for the negative regulation of apoptosis, compared to fibrotic fibroblasts from PDGFRα-CFP/BCL-2- mice (Fig. 5a,d,e,f, and Suppl. Table 1).

Fibroblasts isolated from the fibrotic lungs of PDGFRα-CFP/BCL-2+ mice at 6 weeks showed continued enrichment in pro-fibrotic terms together with genes associated with positive regulation of cellular senescence and p53 signaling (Fig. 5g). By contrast, fibroblasts from the regenerating lungs of bleomycin-instilled PDGFRα-CFP/BCL-2- mice at 6 weeks lacked enrichment in pro-fibrotic biological processes (Suppl. Fig. 7), but exhibited enhanced enrichment in pathways associated with ECM disassembly, wound repair (Wnt and Hippo signaling), cell migration, wound healing, epidermal cell differentiation and cell population proliferation compared to fibroblasts from PDGFRα-CFP/BCL-2+ mice (Fig. 5g,h,I,k,l and Suppl. Table 2, 3 and 4). Additionally, PDGFRα-CFP/BCL-2- fibroblasts, but not PDGFRα-CFP/BCL-2+ fibroblasts, were enriched for terms associated with the positive regulation of apoptotic signaling at 6 weeks compared to 3 weeks (Suppl. Table 2), consistent with the notion that these cells are capable of undergoing apoptosis in the absence of BCL-2 expression (Fig. 5j). Thus, whereas PDGFRα-CFP/BCL-2- fibroblasts develop a transcriptomic program consistent with a beneficial role in lung repair and regeneration, conditional expression of BCL-2 in PDGFRα-CFP/BCL-2+ mice promoted lung fibroblast transcriptomic programs associated with fibrosis and senescence but had no enrichment in pathways associated with positive regulation of apoptosis signaling.

Further analysis of the cellular senescence GO terms identified enrichment of a senescence signature in PDGFRα-CFP/BCL-2+ fibroblasts compared to PDGFRα-CFP/BCL-2- and naïve fibroblasts at 6 weeks (Fig. 6a). The composite senescence score (using 111 senescence associated genes 30) was increased in PDGFRα-CFP/BCL-2+ fibroblasts compared to PDGFRα-CFP/BCL-2- fibroblasts (p=0.06)(Fig. 6b). In view of this enrichment, we generated a network correlation plot between BCL-2 and the senescence genes. We identified a strong (|r|≥0.5) to very strong (|r|≥0.75) Pearson correlation between BCL-2 and 54 senescence genes (p≤0.05) expressed in PDGFRα-CFP/BCL-2+ fibroblasts (Fig. 6c, Suppl. Table 5). We next investigated the location and abundance of senescent fibroblasts at 6 weeks by immunostaining for the markers p16INK, p21WAF and SA-β-gal, along with human BCL-2, PDGFRα and PDGFRβ (Fig. 6d-k). Expression of BCL-2, p16INK, p21WAF and SA-β-Gal in PDGFRα+ cells were all significantly increased (p<0.001) in the fibrotic areas of PDGFRα-CFP/BCL-2+ mice but were minimally detected in PDGFRα-CFP/BCL-2- mice (Fig. 6d-k). Furthermore, all three senescence markers co-localized with BCL-2 (Fig. 6h,i,j,k).

To determine the clinical relevance of our findings in PDGFRα-CFP/BCL-2+ mice, we performed spatial transcriptomics on IPF and non-diseased lung tissues (Fig. 6l,m). Myofibroblasts (yellow = cell boundaries, cluster defined by COL1A2, ACTA2, IGFBP5,COL6A1, COL6A3, and IGFBP3) were present within fibrotic foci with co-localized expression of BCL2 (red = transcripts) (Fig. 6n). Areas with a comprehensive senescence score (based on 10 genes associated with cell senescence; ATF3, C11orf96, CDKN1A, DUSP1, FOS, GADD45B, IL6, JUN, JUNB, ZFP36) were spatially-associated with BCL-2+ myofibroblasts (Fig. 6o). BCL-2+ myofibroblasts had enriched expression of profibrotic and senescence-associated genes compared to BCL-2- myofibroblasts (Fig. 6p) and were significantly increased in IPF lungs compared to control lungs (Fig. 6q). We immunostained and quantified human non-diseased and IPF lung tissue for 1) BCL-2 and αSMA+ myofibroblasts (Fig. 6r,s), which are the predominant apoptosis-resistant fibroblast subset in persistent human fibrotic lung diseases 31,32, and 2) senescence associated proteins (Fig. 6t,u,v). Minimal staining of BCL-2, αSMA, p21WAF, p16INK, or SA-β-Gal was detected in non-diseased lung tissue (Fig. 6s,t,u,v). However, abundant p21WAF, p16INK, SA-β-Gal and BCL-2 staining was observed in α-SMA+ fibroblasts in lung tissues from IPF patients (Fig. 6s,t,u,v). We confirmed this protein data by querying two publicly available scRNA-seq datasets, namely the IPF Cell Atlas (GEO:GSE135893 33) and the Integrated Human Lung Cell Atlas 34. We observed increased expression of senescence-associated genes CDKN1A, CDKN2A, GLB1 in fibrosis-associated mesenchymal cells, including αSMA+ myofibroblasts, in ILD lung tissues compared to non-diseased control subjects (Suppl. Fig. 8). Notably, BCL2 was expressed in all fibrotic mesenchymal cell populations, but at low levels in these end-stage IPF samples (Suppl. Fig. 8). Thus, conditional expression of BCL-2 in mouse lung fibroblasts resulted in the development of pro-fibrotic and senescent fibroblast phenotypes in the persistently fibrotic lungs of bleomycin-injured mice, which was also observed in BCL-2-expressing αSMA+ myofibroblasts in the lungs of IPF patients, reinforcing the translational significance of the mouse studies.

Linkage between apoptosis resistance and senescence in pro-fibrotic lung fibroblasts

We next investigated potential relationships between fibroblast persistence, fibrosis and the acquisition of a sustained senescence phenotype in 2 additional models of persistent lung fibrosis (i.e. 24 weeks after repetitive instillation of bleomycin 35 and 6 weeks after a single bleomycin instillation in Col1a1-CreERT2;Fas-/- mice 11). Bulk sequencing of fibroblasts isolated from both models exhibited a similar senescence-associated gene enrichment signature to PDGFRα-CFP/BCL-2+ fibroblasts (Fig. 6a and Fig. 7a), including increased expression of senescence-associated genes Cdkn1a, Cdkn2a, the secretory-associated senescence phenotype (SASP) genes, Il6, IL1a, and Serpine1, downstream IL-6 signaling (Il6st) and control of cell growth (mTor, Pten) (Fig. 7a). Fibroblasts isolated after repetitive bleomycin also exhibited a significant increase in senescence score (p<0.01) (Fig. 7b) and a 55% positive (p<0.05) Bcl-2:senescence gene Pearson correlation in the network plots (Fig. 7c, Suppl. Table 6). Examination of lung fibroblasts from Col1a1-CreERT2;Fas-/- mice also revealed an increase in senescence score trending towards significance (p=0.08) (Fig. 7b) and a Bcl-2:senescence gene Pearson correlation of 29% (p<0.05) (Fig. 7c, Suppl. Table 7). Fibroblasts from all 3 models exhibited a significant positive correlation between Bcl-2 and SASP associated genes (Fgf7, PDGFα, Il-6, MMPs, TIMPs and Nox4) and a significant negative correlation between Bcl-2 and cell cycle (Ccnb1, Ccnd1) and cytokeratin genes (Cdk1, Cdk2b) (Fig. 6a, 7a and Suppl. Fig. 9).

To determine the location and abundance of senescent fibroblasts from these additional models of persistent fibrosis, lung sections were stained for p21WAF, p16INK, SA-β-gal and BCL-2 together with PDGFRα and PDGFRβ (Figs. 7d,e,f,g,h). There was significant expression of BCL-2, p21WAF, p16INK and SA-β-Gal in PDGFRα+ and PDGFRβ+ fibrotic areas in the lungs of mice repetitively-instilled with bleomycin compared to saline instilled mice (p<0.001, Fig. 7d and Suppl. Fig. 10). Col1a1-CreERT2;Fas-/- mice also showed increased, though less robust, expression of senescence markers p21WAF (p<0.05), p16INK (p=0.057), and SA-β-gal (p=0.0814) (Figs. 7d,e,f,g,h). Taken together, these data suggest that BCL-2 augments fibroblast senescence in the persistently fibrotic lungs of PDGFRα-CFP/BCL-2+ mice, in C57Bl/6 mice after repetitively-instilled bleomycin and in fibroblasts in which apoptosis has been inhibited by Fas deletion.

Therapeutic inhibition of BCL-2 rescues mice with persistent pulmonary fibrosis and promotes fibrosis resolution.

ABT-199 (VenetoclaxTM) is an orally available FDA-approved BCL-2 BH3-mimetic that inhibits the interaction between BCL-2 and BH3-only activators and sensitizers 36. To determine if ABT-199 reduces lung fibroblast persistence and resolves the persistent fibrosis in bleomycin-instilled PDGFRα-CFP/BCL-2+ mice, ABT-199 or vehicle was administered by oral gavage for 4 weeks, beginning 5 weeks after bleomycin instillation (Fig. 8a). Following treatment with ABT-199, the numbers of PDGFRα+ lung fibroblasts were significantly reduced (p<0.001) compared to vehicle-treated mice (Fig. 8b). The numbers of PDGFRβ+ pericytes (Fig. 8c) were unaffected by treatment with ABT-199, possibly reflecting the acquisition of apoptosis resistance through an ABT-199 insensitive pathway. CD45+ leukocytes and EpCAM+ epithelial cells were also significantly reduced after treatment with ABT-199 (Suppl. Fig. 11). Analysis of lung hydroxyproline levels (Fig. 8d), micro-CT quantification of non-aerated lung volume (Fig. 8e,f,h) and measurements of arterial oxygen saturation (Fig. 8g) showed that treatment with ABT-199 led to a significant reduction in established fibrosis and improved arterial oxygenation (p<0.01, p<05 and p<0.001 respectively). Lung histology analysis showed that treatment with ABT-199 also led to a loss of bronchiolization in the lung parenchyma and partially restored alveolar architecture (Fig. 8i), whereas vehicle treated mice had sustained pathologic airway distortion, alveolar collapse and bronchiolization (Fig. 8i). To determine if treatment with ABT-199 reduced the senescent phenotype of the remaining PDGFRα+ fibroblasts and PDGFRβ+ pericytes, we stained lung sections for p16INK, p21WAF and SA-β-gal. Following treatment with ABT-199, fibroblast expression of p16INK and SA-β-gal were significantly reduced (p<0.001), with no change in p21WAF expression (p=0.3896) compared to vehicle control (Fig. 8j,k). Taken together, these pre-clinical data suggest that pharmacologic targeting of BCL-2 leads to the loss of senescent, apoptosis-resistant pro-fibrotic PDGFRα+ fibroblasts, resolved persistent fibrosis and promoted the restoration of lung architecture with improved gas exchange.

Methods
Study Approvals.
All animal studies were approved by the National Jewish Health Institutional Animal Care and Use Committee (AS2741). Euthanasia was conducted in accordance with AVMA guidelines. All human lung tissue samples were de-identified and determined by the National Jewish Health and University of Colorado Anschutz Medical Campus Institutional Review Boards (IRBs) to constitute non–human subjects research; therefore, informed consent and additional IRB approval for this study were not required. Tissue procurement followed approved IRSB protocols at both institutions. Healthy lungs were obtained through Donor Alliance, Inc. (Denver, CO). IPF lungs were obtained as explants during lung transplantation.

Mice
Generation of CFP/BCL-2stopfl/fl mice
Mice capable of conditionally expressing human BCL-2 were generated by synthesizing a targeting vector in which a loxP-flanked transcriptional stop cassette was inserted downstream of a synthetic CAG enhancer. This was followed by the coding sequence of human BCL-2 fused to a P2A protease cleavage site 84 followed by the mCerulean Blue (CFP) 85,86 to allow lineage tracing of BCL-2 expressing cells. B6N JM8.A3 embryonic stem cells 87 were electroporated with the ROSA26-STOPfl BCL-2-2A-CFP targeting construct (Suppl. Fig. 12a), placed under antibiotic selection and the resulting clones were picked and expanded. gDNA was prepared for each clone and used for qPCR analysis by loss of allele (LOA)88. Putative positive clones were then subject to in vitro transfection of with a Cre expression plasmid, which led to the expected recombination-mediated excision of the floxed-stop-cassette and expression of eCFP in multiple clones. Blastocyst injection was then performed using B6J blastocysts and the functionally verified ES cell clones (untransfected). Chimeric mice of genotype Rosa26R-stopfl/fl-CFP/BCL-2 (abbreviation: CFP/BCL-2stopfl/fl) were generated and breeding with B6J wildtype mice established germline transmission, which was confirmed by genotypic analysis (Suppl. Fig. 12b). CFP/BCL-2stopfl/+ mice were crossed with mice expressing tamoxifen-inducible Cre recombinase driven by the PDGFRα (Jax strain: 18280 - B6N.Cg-Tg(Pdgfra-cre/ERT)467Dbe/J) and Col1a1 (Jax strain:016241 - B6.Cg-Tg (Col1a1-cre/ERT2)1Crm/J) (The Jackson Laboratory, Bar Harbor, ME) promoters. Recombination was induced by intraperitoneal injection of tamoxifen (0.25 mg/g body weight in corn oil, Sigma Aldrich, St. Louis, MO) or corn oil as a control starting at day 4 after bleomycin. Mice received 5 injections between day 4 and 21. Mice receiving corn oil or tamoxifen are referred to as PDGFRα-CFP/BCL-2- or PDGFRα-CFP/BCL-2+ mice, respectively. All mice were maintained in a specific pathogen-free facility under 12-hour light/dark cycles with ad libitum access to autoclaved water and standard chow (Diet 2919, Inotiv, West Lafayette, IN).

Primary Fibroblasts
Primary mouse lung fibroblasts were sorted from healthy male PDGFRα-CFP/BCL-2- and PDGFRα-CFP/BCL-2+ lungs on a FACSAria Fusion (BD Biosciences, San Jose, CA), as previously described 9,89. The cells were maintained in DMEM containing 10% (v/v) FBS and used between passages 3-8. Intrinsic CFP expression was confirmed by fluorescence microscopy (Fig. 1b). Fibroblast mitochondria were stained with Mitotracker Red CMXRos (M7512, Invitrogen/ThermoFisher, Waltham, MA) while actin filaments were stained with phalloidin (AF488, #A12379, Invitrogen/ThermoFisher). Stained cells were imaged on a Zeiss LSM 700 Confocal microscope with image acquisition using ZenBlack (Zeiss, White Plains, NY). Detection of BCL-2 (1:1000 anti-rabbit, Abcam #59348, Waltham, MA), GFP (AvesLab #GFP-1020, chicken, 1:1000, Davis, CA), β-actin (Millipore #MAB150, mouse, 1:1000, St. Louis, MO), GAPDH (Abcam #8245, mouse, 1:1000 ) and Tom70 (Cell Signaling #65675, rabbit, 1:1000, Danvers, MA) was done by Western blot analysis in the mitochondrial and cytosolic fractions, as described 90. Fibroblasts were sensitized with TNF-α (10 ng/mL) and IFN-γ (50 U/mL) for 48 hours prior to stimulation with agonistic anti-Fas antibody (Jo2, 250 ng/mL BD Pharmingen, San Diego, CA) for 4 hr or were incubated with 1 µM staurosporine (Sigma Aldrich) for 4 hours. Active caspase-3/7 was measured using a Caspase-Glo kit (Promega, Madison, WI) as previously described 15,79.

Assessment of fibrotic lung disease
Pulmonary fibrosis was initiated by the intratracheal instillation of 50 µL of bleomycin (1.5U/kg, Amneal Biosciences, Bridgewater, NJ dissolved in sterile saline) to anesthetized male and female mice 8-10 weeks of age 91. Fibrosis was assessed by lung measurements of collagen in the upper right lobe (hydroxyproline assay) 11,91 and histology was evaluated by Masson’s trichrome staining of sections from the left lung 91. Images were taken on an upright Olympus BX51 or by Aperio Scanning (Leica Biosystems, Buffalo Grove, IL). Where specifically indicated C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME) were used as controls. For therapeutic intervention with ABT-199, mice were dosed with ABT-199 (100 mg/kg) by daily oral gavage for 28 days or vehicle (60% phosal 50 propylene glycol (PG), 30% polyethylene glycol 400, 10% ethanol), starting 5 weeks after fibrosis was established 81.

Flow cytometry
Single cell suspensions were obtained from perfused lungs by enzymatic dispersion with collagenase (1mg/ml, Sigma Aldrich, C2139), elastase (4U/ml, Worthington, LS002274 Lakewood, NJ) and dispase (5U/ml, Worthington LS02104) incubation (30 min, 37oC) followed by a 0.25% trypsin-EDTA (ThermoFisher, 2520056) + DNaseI (20 µl/10ml, Worthington LS006331) digest (20 min, 37oC) 11. Cells were stained with fluorescently-tagged monoclonal antibodies against CD45 (17-0451-82), CD326/EpCAM (17-5791-80), CD31 (541814, BD Biosciences, San Jose, CA), PDGFRα/CD140a (25-1401-82) and PDGFRβ/CD140b (ThermoFisher, Waltham, MA) at a 1:200 dilution. Cell analysis data were acquired with the LSRFortessa (BD Biosciences) and analyzed with FlowJo software (V10, Tree Star, Ashland, OR). Lineage (CD326/EpCAM, CD31, CD45) negative cells (Lin-) were gated, (Suppl Fig 13) 11, and assessed for PDGFRα and PDGFRβ staining. Lin- fibroblasts were found to be comprised of PDGFRα+, PDGFRα+/β+ and PDGFRβ+ cells.

Immunofluorescent staining
Formalin fixed, paraffin embedded sections, were deparafinized and rehydrated followed by antigen retrieval using citrate buffer. Non-specific binding was reduced by incubation in 10% mouse serum and mouse IgG serum from the M.O.M immunodetection blocking kit (#BMK-2202, Vector Laboratories, Burlingame, CA). Primary antibodies for BCL-2 (BioRad #MCA1550, mouse, 1:100 Hercules, CA), Bcl-2 (Cell Signaling Technology # 3498, rabbit, 1:500), PDGFRα (Cell Signaling, #3174S rabbit, 1:100), PDGFRβ (R&D Systems #AF1032 goat, 1:100, Minneapolis, MN), GFP (which identifies CFP; AvesLab #GFP-1020, chicken, 1:200), αSMA (Sigma Aldrich #2547, mouse, 1:500), p16INK (Abcam #ab211542 rabbit, 1:100), p21WAF (Abcam #ab188224 rabbit, 1:250), β-galactosidase (Cell Signaling #27198 rabbit, 1:200), CCSP (Seven Hills Bioreagents #WRAB-3950 rabbit, 1:200, Cincinnati, OH), pro-SPC (Millipore #AB3786 rabbit, 1:500), Krt8 (DSHB #AB531826 rat, 1:100), CD45 (Invitrogen, 17-0451-82 rat, 1:100), CD31 (Invitrogen, #14-0311-82 rat, 1:100) were incubated overnight at 4oC followed by incubation with fluorescently tagged goat anti mouse-A647 (A21236), donkey anti-rabbit-A555 (A31572), goat anti rat-A555 (A21434), and donkey anti goat-A647 (A21447) secondary antibodies at 1:100 dilution (Invitrogen, Carlsbad, CA). TUNEL staining to detect apoptotic cells was completed prior to antibody staining per manufacturer’s instructions (Promega, Madison, WI). TUNEL positive cells were counted and averaged from 10 images from each animal as described 24. Images were captured in fibrotic areas using PDGFRα and PDGFRβ positive staining as an indicator of fibroblast accumlation. TUNEL positive cells located in the airway epithelium were excluded from the analysis. Quantitative cell counts from CCSP, KRT8, BCL-2, p16INK, p21WAF, and β-Gal images were compiled using steriology grids and averaged from 10 images from each animal. Frozen 4% PFA fixed OCT (Fisher Scientific, Hampton, NH) sections were mounted with Fluoroshield Mounting Media containing DAPI (Vector Laboratories). Images were acquired on a Zeiss Axioplan 2 epi-fluorescence microscope and analyzed with Axiovision software (Zeiss, Jena, Germany).

Bulk RNA sequencing
Prior to cell sorting, single cell suspensions were enriched for lineage negative cells by incubating with CD45 (130-052-301), CD31 (130-097-418) and CD326 (130-105-958) MicroBeads and purified off of LS columns per manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany/Auburn, CA). At least 300,000 live (DAPI-) lineage negative cells were collected per sample on the FACSAria Fusion (BD Biosciences). Purified cells were pelleted, lysed, and RNA was prepared, as described 11. Libraries were sequenced as barcoded-pooled samples and processed for next-generation sequencing (NovaSeq 6000 - Illumina platform).

Bulk RNA sequencing analysis
To improve downstream mapping quality, raw sequencing reads were trimmed using skewer with parameters (end-quality=15, mean-quality=25 and min=30). Trimmed reads were aligned to the mouse reference genome GRCm38 using Hisat2. Gene quantification was performed with htseq-count using GRCm38 Ensembl v84 GTF. Differential expression analysis and gene correlation between groups was conducted with the R (3.5.1) package DESeq2 (1.22.2). FDR less than or equal to 0.05 (|r|≥0.5) and a Pearson correlation equal to or greater than 0.7 (|r|≥0.75) were used as thresholds of significance. Pathway enrichment analysis was conducted with Enricher to identify Gene Ontology:Biological Processes (GO:BP) significantly genes by pair-wise comparison 92,93. The Col1a1-CreERT2;Fas-/- mice data set has been deposited in the National Center for Biotechnology Information/Gene Expression Omnibus under accession number GSE161648 11, the CFP/BCL-2+ mice is under accession number GSE307983 and the spatial transcriptomics data is under accession number GSE282639.

Spatial transcriptomic analysis
We performed Nanostring CosMx Digital Spatial Profiler to obtain subcellular resolution spatial transcriptomic data on 15 IPF and 13 unaffected controls using methods reported in the “Supplemental Methods” and by Blumhagen et al. 94. For this manuscript, we focused on BCL-2 expression in myofibroblasts and cell senescence. Following cell annotation, we applied the spatial correlation method InSituCor 95 to identify modules of gene co-expression in which we focused on a module that comprised 10 genes associated with cell senescence (ATF3, C11orf96, CDKN1A, DUSP1, FOS, GADD45B, IL6, JUN, JUNB, ZFP36). In addition to spatial clustering, we compared expression between BCL-2+ myofibroblasts and BCL-2- myofibroblasts using a linear mixed model on VST transformed counts adjusting for disease and including a random effect for subject.

Statistical Overview
Data are presented as the mean ± SEM. Data were analyzed using GraphPad Prism software Version 10 (GraphPad, San Diego, CA). Differences between conditions at specific time points were examined using Student’s unpaired 2-tailed test with Welch’s correction with p<0.05 considered to be significant. Box-and-whiskers plots show median, minimum and maximum values. Violin plots show median, upper and lower quartiles. Specific details about the replicates used where n = individual animal replicates are available in the figure legends. All assays and measurements were taken from distinct samples. Immunostained sections were cut from distinct individual animals.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript shows that BCL2 overexpression in Pdgfra+ fibroblasts inhibits fibroblast apoptosis, promotes senescence, and causes prolonged fibrosis in the bleomycin model. The concept aligns well with the literature showing the importance of myofibroblast clearance in wound healing and fibrosis resolution and provides additional support for the important role that death of activated fibroblasts plays in fibrosis resolution. However, the effects of artificial overexpression of BCL2 do not address the question of whether BCL2 overexpression is relevant to persistent fibrosis under physiological conditions. The authors try to make the case that upregulation of BCL2 in myofibroblasts in the lungs of patients with Idiopathic Pulmonary Fibrosis supports a functional role for BCL2 in the persistent fibrosis that characterizes that disease. However, the data included to support that contention are not convincing. Although the violin plots in supplemental figure 7 do appear do show no expression of BLC2 in myofibroblasts from normal lungs and some expression (albeit at quite low level) in myofibroblasts from IPF lungs, BCl2 expression appears to be higher in the cells labelled fibroblasts and PLIN2+ fibroblasts from normal lungs. This finding is consistent with data from other publicly available sources, such as the IPF Cell Atlas, where COL1A1-high fibroblasts express lower levels of BCL2 than other fibroblasts. In contrast, the violin plots showing expression of CDNK2A and CDNK1A, used as markers of senescence, show much more dramatic upregulation of these markers in myofibroblasts from IPF lungs. These findings suggest that, although forced overexpression of BCL2 in fibroblasts can induce expression of these senescence genes, BCL2 overexpression may not explain the upregulation of these genes in IPF.\nThe authors also try to demonstrate potential clinical relevance of their findings by showing that treatment with a drug that inhibits BCL2, ABT-199, has beneficial effects in their model. While it is interesting that ABT-199 improved the persistent fibrosis and functional endpoints in mice that over-expressed BCL2 and were treated with bleomycin, this result would be expected and could only reflect reversal of the effects of pharmacologic overexpression. It would be more impactful to show that ABT-199 was effective in improving fibrosis in mice with persistent fibrosis in a model with endogenous BCL2, such as repeated dose bleomycin, since that might be more analogous to the low levels of BCL2 expression in fibroblasts from patients with IPF.\nThe data presented in Figure 3 show that over-expression of BCL2 leads to increased expression of many genes associated with ECM production and organization in cells derived from PDGFRa expressing cells at 3 weeks after bleomycin treatment, but the authors don’t address the mechanism by which this occurs. It is unclear if this result is due to a direct effect of BCL2 on signaling pathways involved in ECM gene expression (for example TGF  signaling) or simply due to the selective persistent survival of activated fibroblasts at this time point.\nSpecific Comments\nFigure 1 – The images showing expression of CFP and staining for BCL2 and nuclei are hard to understand. It looks like every nucleus overlaps with CFP and BCL2 expression in Panel 1b and every DAPI stain nucleus overlaps with staining for BLC2 in Panel g, whereas expression of CFP and BCL2 should be only seen in the fraction of cells that are PDGFRa+ fibroblasts. It is also not clear from the images shown how the authors determined that BCL2 expression was localized to mitochondria.\nFigure 2 – Previous work has suggested that PDGFRb is induced by bleomycin in subsets of fibroblasts and PDGFRa expression is reduced. It is thus not clear how the authors determined that PDGFRb+ PDGFRa- CFP+ cells were pericytes rather than fibroblasts that had upregulated PDGFRb and downregulated PDGFRa. In Panel 2h, the authors used an anti-human BCL2 antibody produced in mouse and used an anti-mouse IgG secondary antibody. The authors did not describe how they avoided the binding of the secondary antibody to endogenous antibodies. This may be why BCL2 appears to be diffusely expressed in all cells in Figure 2h, including epithelial and endothelial cells. From panel 2I it is not clear that BCL2+ cells were confined to alveolar walls at 6, 9 and 12 weeks after bleomycin as described in the text. It looks like there was persistence of these cells throughout the lungs at substantially higher levels than seen at baseline.\nFigure 3 – It would be useful to quantify changes in Krt8, SPC. CCSP, etc.\nFigure 4 -Although the authors highlight differences between changes in senescence genes and P53 regulated genes between bcl2+ and bcl2- mice, differences in these gene sets looked similar to differences in ECM organization genes, with increases by bleomycin in both groups but quantitatively larger differences with bcl2 over-expression.\nAlthough the authors don’t show the heatmaps, they claim that at 6 weeks changes in ECM organization were no longer apparent in bcl2 – mice. However, the dot plots suggest that this category was similar in bcl2+ and bcl2- mice. For apoptotic signaling, ECM disassembly and Wnt signaling for some reasons the authors chose to show the data differently, ranking genes from highest to lowest for the bcl2- fibroblasts which makes it hard to compare these differences to those in panels B, D and E. Although the selected genes shown for qPCR suggest that for these processes overexpression of bcl2 partially reversed the effects of bleomycin the heatmaps seem to show a more complicated pattern, at least for regulation of wnt signaling. For all of these data it is not certain which effects were cell autonomous, and which might be explained by cross talk between bcl2+ and bcl2-fibroblasts and other adjacent cells.\nFigure 5 – Again listing the genes in expression order for one cell type makes it difficult to evaluate differences. Nonethless, it looks like some senescence genes were higher at 6 weeks in bcl2+ animals and some senescence genes were higher in fibroblasts from bcl2- animals. Furthermore, it appears that many senescence genes were actually expressed at higher levels in naïve animals, which probably explains why the difference in the 111 gene senescence score was not statistically significantly different between fibroblasts from bcl2+ and bcl2- animals. The tissue staining panels don’t all seem to line up as staining the same features (were these from adjacent slides?) and it is very difficult to tell if there was really co-localization of p16, p21 and B-gal in bcl2+ fibroblasts. It does appear that the bcl2 antibody staining was not sensitive enough to pick up endogenous bcl2 in any cells. In Figure 5q, pro-fibrotic genes in BCL2+ and BCL2- myofibroblasts are shown. However, it might be more informative to first define myofibroblasts and normal fibroblasts based on transcriptomics and then compare BCL2 expression between these two populations.\nFor the human tissues it is surprising that the normal lung has essentially no BCL2 signal, but there seems to be diffuse signal in the IPF lung with a pattern very different from that seen for p21 or SA-b-gal. This is a surprising result, since the violin plot in supplemental figure 7 seems to show only low-level expression of BCL2 in lung myofibroblasts in IPF, but higher expression in fibroblasts and PLIN2+ fibroblasts in normal lungs. In the stained human sections it is also not clear which p21, b-gal or BCL2+ cells are fibroblasts.\nI may be misreading the spatial transcriptomics panel, but it appears like the region at the bottom left that has the most cells annotated as myofibroblasts and the most BCL2 signal in the most leftward panel has essentially no senescence signature in the rightward panel. It also appears that most of the BCL2 expression is in different cells (or regions) than the myofibroblast signature.\nFigure 6 – It is somewhat surprising that the authors don’t include bcl2 itself in the heatmaps in panel A for the two other persistent fibrosis models. It would be reassuring if bcl2 was upregulated in fibroblasts in at least one of these models. This is especially true given the apparent dramatic upregulation of bcl2 protein in the examples shown if 6C-F. It would also be good to quantify bcl2+ fibroblasts/field as was done for the other endpoints. Finally, p21 should be expressed in many cell types, not only fibroblasts. It is surprising that the staining is completely negative in saline treated mice in panel 6F.\nFig s1 – clearly shows that many lung fibroblasts express PDGFRb, including a substantial number labelled as PDGFRa+/PDGFRb+. It is still not clear how immunostaining allowed the authors to determine if PDGFRb+ cells were fibroblasts or pericytes.\nS2 – The authors should show CFP flow to confirm that none of the PDGFRb+/PDGFRa low cells were derived from PDGFRa+ cells.\nS7 – Although there is definitely higher expression of p16 and p21 in IPF myofibroblast, expression of GLB1 and Bcl2 actually seems to be higher in the other fibroblast populations in both normal and IPF lungs. These data support previous reports of senescent myofibroblasts in IPF but do not strongly support a role for overexpression of bcl2 in this process.\nS8 – There doesn’t appear to be much overlap in expression of most of the included senescence genes among the 3 non-resolving models.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors present a manuscript investigating the role of BCL-2 expression in fibrotic lung remodeling. Primarily leveraging a mouse model expressing human BLC-2 in PDGFRA fibroblasts, the authors demonstrate the impact of BLC-2 expression on inhibition of apoptosis, fibroblast accumulation, and the emergence of pro-senescent phenotypes after bleomycin injury. The authors recapitulate some of their key findings in human tissue and demonstrate the ability of an FDA approved BCL-2 inhibitor (ABT-199) to reverse some of the effects seen in their model. Overall this paper will be of broad interest to the lung fibrosis community. However, I have detailed a number of concerns that should be addressed before publication of this work.\nMajor:\n1). There appears to be some discrepancy between figure 1g and 1h/i. Looking at the visual co-localization of CFP and PDGFRB/aSMA and to a lesser degree SPC and CCSP there appears to be many cells that are positive for both markers. This is particularly true for PDGFRB where nearly every + cells in the pop out window of 1g appears to be dual positive. This is at odds with the flow data from dissociated tissue. I’m not sure how to interpret these results but the apparently pervasive CFP shown in panel g co-localizing with many different cell types causes concern for the specificity of the lineage tracing.\n2) Similarly, in Fig. 2 h there appears to be expansive BLC-2 expression but PDGFRB, PDGFRA and BLC-2 are not shown co-localized in the same tissue section. Based on my concerns in the prior comment I’m not convinced that BCL-2 expression is not seen at some level in PDGFRB+ cells.\n3) In the Col1a1-CFP/BCL-2+ model the authors claim it phenocopies the PDGFRA model, but the accumulation and persistence of PDGFRB+ pericytes appears to be more muted (SFig. 5)\n4) It’s unclear what SFig 6 is supposed to be demonstrating. In a neither PC1 or PC2 split samples by BCL2+/-. Indeed PC1 seems to separate two groups of 6 week mice. Are these corresponding to some sort of technical batch? Similarly in b PC1 and PC3 are shown but it’s unclear why this plot only shows 6 week and naive mice or why PC3 was shown instead of PC2.\n5) The analyses and associated panels in Fig. 4 are not sufficiently described. In the methods the authors state they used DESeq2 to identify DE and use an FDR cutoff of 0.05. However it is unclear how the genes presented in Fig. 4 b/d/e/h/j/k were chosen. What were the comparisons being made for each plot? Is this naive vs BCL2+? BCL2- vs BCL2+? Are these genes that were significant for any pairwise comparison? The authors should clearly state their grouping and how they arrived at these results. They should also present a supplementary table with a full list of DE genes (with associated LFC and FDR) for every contrast. Furthermore, it is unclear how the reported contrasts in Fig. 4 c/f/i/l are derived. Are these the p-values from pairwise DE comparisons performed with DESeq2? In the figure legend it states “Brown-Forsythe and Welch's ANOVA with Dunnett correction for multiple comparisons. n=4 mice/group. *p<0.05, **p<0.01.” It is not statistically appropriate to perform a second association test on a subset of associations that were identified in a genome-wide test. The adjusted p-value from the DE analysis originally performed should be reported.\n6) On page 9 the text suggests that Cdkn1a and Cdk5r1 are upregulated in BCL2+ mice compared to BCL2- mice 3 weeks post bleomycin. However Cdkn1a is actually higher in BCL2- mice at this timepoint and I do not see Cdk5r1 reported anywhere.\n7) The color scales on Fig. 4g are a bit difficult to interpret. There are two enrichment with much higher values skewing the color scale making it very difficult to see the range between 1 and 4 where most of the values lie. Additionally, all of the heatmaps have a range of -1 to 1 expect for d/e.\n8) Figure 5a has no consistency in capitalization.\n9) Insufficient details are provided to interpret spatial transcriptomics results. The authors state in the methods: “We performed Nanostring CosMx Digital Spatial Profiler to obtain subcellular resolution spatial transcriptomic data on 15 IPF and 13 unaffected controls. The full methods and results will be available in a comprehensive manuscript (manuscript in preparation)”. This is inconsistent with Nature Communications data policies and makes all of these analyses uninterpretable. The authors provide no details around how these data were processed or how cell typing was performed. The authors must provide a full accounting of methods including the release of the data – in accordance with Nature Communications data release policy.\n10) In Fig. 5o BCL-2 and myofibroblasts appear to be poorly co-localized. It is unclear exactly what is being shown in this plot as both the red and yellow appear to be of similar “size”. Are the red points individually detected transcripts and the yellow points cell boundaries? The authors should show the breakdown of BLC expression across cell types in their dataset (similar to the results presented in SFig. 7). Similarly, their Senescence score appears poorly correlated with myofibroblasts. They should show how this senescence module is distributed across cell types and across disease.\n11) In Fig. 5q the authors should state how many BCL2+ myofibroblasts and BCL2- myofibroblasts were in the dataset. Are these only from disease individuals? How many are from controls vs disease? The authors also do not provide enough details in the methods to understand the analysis. Did the regression analysis treat each individual cell as an independent observation with a random effect for each individual? Or were these pseudobulked with a random effect? How were the data processed before this analysis and what was the significance cutoff?\n12) The color scales on Fig. 5q are misleading. Having the two heatmaps together with wildly different scales leads the reader to incorrect conclusions. It appears many of these senescence genes (including CDKN1A) are actually downregulated in BCL2+ myofibroblasts. This is in contrast the the point the authors are making and conflicts with the results from SFig. 7.\n13) Additionally I suggest the results from SFig. 7 should be revalidated in a larger cohort. There are very few cells from individuals without PF in either Myofibroblasts or PLIN2+ Fibroblasts. Thus it is difficult to be confident in the robustness of this analysis. Using one of the newer and larger datasets should improve these estimates of expression (e.g. PMC10287567 or PMC11018522).\n14) At the end of page 11 the authors describe correlations between BCL2 and senescence associated genes: “…Bcl-2:senescence gene correlation of 29% (p<0.05) (Fig. 6a,b). Fibroblasts from all 3 models had significant positive correlation between Bcl-2 and SASP associated genes (Fgf7, PDGFa , Il-6, MMPs, TIMPs and Nox4) and a significant negative correlation between Bcl-2 and cell cycle (Ccnb1, Ccnd1) and cytokeratin genes (Cdk1, Cdk2b).” However a direct correlation between BCL2 and these genes is never presented. 6a/b certainly don’t present a results with a gene correlation of 29%? The authors should directly demonstrate the relationship between BLC2 expression and these genes if they want to make this claim. Additionally, there is no consistency in capitalization on Fig. 6a.\n15) Did the authors quantify additional cell type changes associated with BCL-2 inhibition? Did epithelial cell populations remodel appropriately as well? Could the authors postulate a bit more about why pericyte numbers are not decreasing and where they are localized? They postulate a bit on the role of signaling between PDGFRA+ and PDGFRB+ cells in the discussion, but this doesn’t explain why pericytes persist when PDGFRA cells are depleted.\nMinor:\n1). On page 5 I believe PDGFRa-CFP/BCL-2fl/+ should be PDGFRa-CFP/BCL-2+\n2) There is no Fig. 5n and Fig. 5n-q is referenced twice in the manuscript in places where I believe it is supposed to reference Fig. 5 r-u\nIn accordance with Nature's public access policy, the authors should make all data generated as a part of this study publicly available on GEO (including both bulk RNA sequencing data and spatial transcriptomics data) and provide reviewer access tokens before resubmission. The authors should also make all code used for genomic analysis available.", "reviewer_4": "This is a well-conceived and well-conducted study by Redente, Riches, et al that provide new insights into parenchymal remodeling that accompanies persistent/progressive pulmonary fibrosis using both mouse and human models. Most notably, their studies show that BCL-2 dependent apoptosis-resistant fibroblasts are both necessary (loss-of-function) and sufficient (gain-of-function) to induce a non-resolving, persistent fibrotic response to lung injury. The discovery of an additional phenotype of epithelial-driven airway remodeling with bronchiolization and cyst formation in the fibroblast-specific, BCL-2 overexpressing mice provides new insights into pathogenesis of these morphologic lesions that accompany IPF and introduces a valuable tool (animal model) for the field.\nOverall, I have minimal concerns that primarily relate to data interpretation. Cellular senescence is generally thought to induce an anti-apoptotic phenotype that is, at least partly, driven by BCL-2. The new finding (inverse relationship) that BCL-2 overexpression can induce the generation of a senescent phenotype is intriguing and may indicate the acquisition of DNA damage responses in “long-lived” (apoptosis-resistant) fibroblasts; the authors provide potential other explanations, e.g. diminished autophagy. There is an opportunity to address this more mechanistically with ex-vivo studies of cultured fibroblasts (although not essential).\nThe translational studies with the FDA-approved BCL-2 are convincing in the BCL-2 overexpressing mice, but has this been shown to be effective in the repetitive bleomycin model or in an aging model (PMID: 24718857)? These studies may be outside the scope of the current studies but perhaps worthy of discussion and/or future studies."}, "author_response": "Point-by-point response to the reviewer’s questions\nWe have “cut” the unedited reviewer’s questions (italicized) from the original critique and “pasted” them into our responses below. Newly generated data and revised figures are also incorporated into our responses. Each figure is only displayed once after its first reference.\nReviewer 1\nSince Reviewer 1 raised issues in both the “General comments” and “Specific Comments” sections, we have addressed these separately and have assigned sequential numbers to allow cross-referencing.\nGeneral comments:\nPlease note: In this section, we have added callouts (a-d) to help define the reviewer’s questions and our responses below.\n1. a)The authors try to make the case that upregulation of BCL2 in myofibroblasts in the lungs of patients with Idiopathic Pulmonary Fibrosis supports a functional role for BCL2 in the persistent fibrosis that characterizes that disease. However, the data included to support that contention are not convincing. b) Although the violin plots in supplemental figure 7 do appear do show no expression of BLC2 in myofibroblasts from normal lungs and some expression (albeit at quite low level) in myofibroblasts from IPF lungs, BCl2 expression appears to be higher in the cells labelled fibroblasts and PLIN2+ fibroblasts from normal lungs. This finding is consistent with data from other publicly available sources, such as the IPF Cell Atlas, where COL1A1-high fibroblasts express lower levels of BCL2 than other fibroblasts. In contrast, the violin plots showing expression of CDNK2A and CDNK1A, used as markers of senescence, show much more dramatic upregulation of these markers in myofibroblasts from IPF lungs. These findings suggest that, although forced overexpression of BCL2 in fibroblasts can induce expression of these senescence genes, BCL2 overexpression may not explain the upregulation of these genes in IPF. c) The authors also try to demonstrate potential clinical relevance of their findings by showing that treatment with a drug that inhibits BCL2, ABT-199, has beneficial effects in their model. While it is interesting that ABT-199 improved the persistent fibrosis and functional endpoints in mice that over-expressed BCL2 and were treated with bleomycin, this result would be expected and could only reflect reversal of the effects of pharmacologic overexpression. It would be more impactful to show that ABT-199 was effective in improving fibrosis in mice with persistent fibrosis in a model with endogenous BCL2, such as repeated dose bleomycin, since that might be more analogous to the low levels of BCL2 expression in fibroblasts from patients with IPF. d) The data presented in Figure 3 show that over-expression of BCL2 leads to increased expression of many genes associated with ECM production and organization in cells derived from PDGFRa expressing cells at 3 weeks after bleomycin treatment, but the authors don’t address the mechanism by which this occurs. It is unclear if this result is due to a direct effect of BCL2 on signaling pathways involved in ECM gene expression (for example TGFb signaling) or simply due to the selective persistent survival of activated fibroblasts at this time point.\nResponse:\n1a: We feel confident that our studies in mice convincingly support the conclusion that human BCL-2 expression in fibroblasts functionally leads to persistent fibrosis. Regarding the studies in human tissue, we have carefully reviewed the manuscript and do not feel that we overstated the findings in IPF. Given the inherent limitations of observational studies in end-stage IPF lung tissues, our findings solely, but comprehensively, show that the prevalence of pro-fibrotic, senescent fibroblasts expressing BCL-2 was increased in IPF lung tissues. We have included new analyses and data to bolster the human special transcriptomics studies as requested by the reviewers (and as noted below).\n1b: We thank the reviewers for this thoughtful observation which we address in part here as well as later in “Specific Comment” #15.\nRegarding the first point, we have now included additional data analysis from the Integrated Human Lung Cell Atlas (PMC10287567 and PMC1170654) looking at (i) multiple mesenchymal cell subsets (fibroblasts, myofibroblasts and pericytes, Suppl. Fig 6c,d), (ii) the original data from the IPF cell atlas which identified three fibroblast subsets (GEO: GSE135893) (Suppl. Fig. 6a,b) and (iii) our primary data on myofibroblasts presented in Fig. 6l-v. Looking across these data sets, as well as within lung fibroblasts isolated from our three murine models of persistent fibrosis, we consistently observe low expression of BCL2/Bcl2 mRNA. However, this does not directly correlate with the increase in protein expression we show by immunostaining, reinforcing the importance of not simply relying on transcript expression data.\nSupplemental Figure 6c,d\nSecond, and we thank the reviewers for bringing up this insightful comment, we have revised the “Discussion” to propose that BCL2 expression itself, while not directly initiating fibroblast senescence, enables or licenses senescence development by preventing fibroblast apoptosis. As noted on page 17 we speculate that BCL-2 induced resistance to apoptosis and senescence are sequential events leading to persistent fibrosis. We address this point further in our response to “Specific Comment” #15.\n1c: Reviewer 3 also raised a similar point. Briefly, in currently submitted studies, we show that mice that have been repetitively instilled with bleomycin and treated with ABT-199 beginning at 10 weeks show reduced fibrosis progression. We have not specifically studied the effects of ABT-199 in aged mice, but feel that such studies are beyond the scope of this manuscript.\n1d: The reviewers raise an interesting question. In previous published studies (PMID: 33290280) we did not observe a similar enhancement of the pro-fibrotic signature in Fas-deficient fibroblasts isolated from bleomycin-instilled mice at 3 weeks. These mice also accumulate apoptosis-resistant fibroblasts and develop persistent fibrosis. These findings perhaps suggest (as the reviewers speculate) that conditional BCL-2 expression may impact signaling pathways involved in ECM gene expression. Regrettably, we feel experimentally addressing this is beyond the scope of the present manuscript.\nSpecific Comments.\n2. Figure 1 – The images showing expression of CFP and staining for BCL2 and nuclei are hard to understand. It looks like every nucleus overlaps with CFP and BCL2 expression in Panel 1b and every DAPI stain nucleus overlaps with staining for BLC2 in Panel g, whereas expression of CFP and BCL2 should be only seen in the fraction of cells that are PDGFRa+ fibroblasts. It is also not clear from the images shown how the authors determined that BCL2 expression was localized to mitochondria.\nResponse: Agreed. Due to the spectral overlap between CFP and DAPI we re-performed the staining and replaced Fig. 1g with new images omitting DAPI. Based on our flow cytometry data (Fig. 1i, j and Fig. 3e, g) >90% of the PDGFRa+ fibroblasts co-express CFP and, by inference, BCL-2.\nFigure 1g, i, j\nFigure 3e,g\nTo address the comment about demonstrating mitochondrial localization, we performed new co-immunostaining experiments in cultured primary PDGFRa-CFP/BCL-2+ and PDGFRa-CFP/BCL-2- fibroblasts. Cells were stained vitally with Mitotracker red and, after fixation, with anti-human BCL-2 (revised Fig. 1b). In confirmation of previous studies, the new data show that the majority of BCL-2 is co-localized with mitochondria (revised Fig. 1b), validating the data that was shown in Fig. 1d.\nFigure 1b\n3. Figure 2 – Previous work has suggested that PDGFRb is induced by bleomycin in subsets of fibroblasts and PDGFRa expression is reduced. It is thus not clear how the authors determined that PDGFRb+ PDGFRa- CFP+ cells were pericytes rather than fibroblasts that had upregulated PDGFRb and downregulated PDGFRa.\nResponse: This is a good point and we agree that the PDGFRa levels are reduced on fibroblasts from fibrotic lungs and we apologize for not clarifying how we addressed this in our initial submission. Basically, our flow cytometry gating strategy utilized a broad PDGFRa gate to accommodate these changes in PDGFRa expression. To inform the reader, we have combined the original Suppl. Figs. 2a,b and inserted it as Fig. 1h in the revised Fig.1. This gate contains the PDGFRa+ only subset and the PDGFRa+/PDGFRb+ double positive subset. We also see a distinct PDGFRb+ only (i.e. PDGFRa-) subset identified as pericytes. Both the PDGFRa+ and PDGFRa+/PDGFRb+ subsets are CFP+ indicating tamoxifen-induced recombination and labeling (Figs. 1g,i,j and Figs. 3e,g). The PDGFRb+ (PDGFRa-) pericyte population remains CFP- (Figs. 1g,i,j and Fig. 3h,i). To reinforce the identification of these subsets, Suppl. Fig. 1 shows our previously published single cell RNA-seq data in fibroblasts demonstrating distinct PDGFRa+ fibroblast and PDGFRb+ pericyte clusters in naïve mice (Suppl. Fig. 1b,c). In fibrotic lungs, pro-fibrotic fibroblasts express both PDGFRa and PDGFRb but the PDGFRb+ pericyte population remains distinct Suppl. Fig. 1d,e. Taken together, these data indicated that fibroblasts that gain PDGFRb (becoming double positive) during fibrosis (and are CFP+) are a distinct subset from the PDGFRb+ pericytes which remain CFP-.\nFigure 1h\nFigure 3h,i\nSupplemental Figure 1\n4. In Panel 2h, the authors used an anti-human BCL2 antibody produced in mouse and used an anti-mouse IgG secondary antibody. The authors did not describe how they avoided the binding of the secondary antibody to endogenous antibodies. This may be why BCL2 appears to be diffusely expressed in all cells in Figure 2h, including epithelial and endothelial cells.\nResponse: This is also a good point and we apologize for not fully detailing our immunostaining method. To mitigate the reviewer’s concern, we utilized the M.O.M® (mouse on mouse) immunodetection kit from Vector laboratories which is designed to reduce endogenous mouse Ig staining when using mouse primary antibodies on mouse tissue sections. We have revised the methods which now incorporate this procedure (page 21).\nTo address the concern that human BCL-2 is diffusely expressed in all cells, we provide new co-immunostaining for CFP, PDGFRa and PDGFRb. This is now presented in revised Figs. 3h,i.\n5. From panel 2I it is not clear that BCL2+ cells were confined to alveolar walls at 6, 9 and 12 weeks after bleomycin as described in the text. It looks like there was persistence of these cells throughout the lungs at substantially higher levels than seen at baseline.\nResponse: Thank you for bringing this to our attention. We have revised the “Results” section (page 6) to more accurately describe the fibroblast locations in the PDGFRa-CFP/BCL-2- mice at 6-12 weeks post bleomycin instillation. At 6 weeks, there were still minor patches of fibrosis that we have highlighted in our image that contain PDGFRa+ fibroblasts and PDGFRb+ pericytes. The PDGFRa+ fibroblasts are located within the alveolar walls by 9 and 12 weeks after fibrosis resolution has occurred. As a note, this is now revised Fig. 3h,i.\n6. Figure 3 – It would be useful to quantify changes in Krt8, SPC. CCSP, etc.\nResponse: Thank you for this helpful suggestion. We have now added the suggested analysis to a revised Fig. 4c,d.\nFigure 4c,d\n7. Figure 4 -Although the authors highlight differences between changes in senescence genes and P53 regulated genes between bcl2+ and bcl2- mice, differences in these gene sets looked similar to differences in ECM organization genes, with increases by bleomycin in both groups but quantitatively larger differences with bcl2 over-expression.\nResponse: Agreed. We hypothesized that the conditional expression of BCL2 drives the persistence of the PDGFRa+ and PDGFRa+/b+ fibroblasts in the lungs after bleomycin resulting in an enhanced fibrotic and eventual senescent phenotype. This overall gene signature is not substantially different from that observed at 3 weeks, but it is enriched when BCL-2 is conditionally expressed in fibroblasts. This is discussed on page 16. As a note, this is now referred to Fig. 5.\n8. Although the authors don’t show the heatmaps, they claim that at 6 weeks changes in ECM organization were no longer apparent in bcl2 – mice. However, the dot plots suggest that this category was similar in bcl2+ and bcl2- mice.\nResponse: To address this comment, we have now included two additional heat maps for the ECM organization pathway genes (GO:0030198) as Suppl Fig. 5a,b. Suppl Fig. 5a is a comparison between 6-week PDGFRa-CFP/BCL-2+ and PDGFRa-CFP/BCL-2-fibroblasts, while Suppl Fig. 5b is a comparison between naïve, PDGFRa-CFP/BCL-2+ and PDGFRa-CFP/BCL-2- fibroblasts at 3 and 6-weeks post bleomycin instillation. Both of these analyses support our statement that the genes in the ECM organizational GO BP pathway were no longer significantly enriched in PDGFRa-CFP/BCL-2+ fibroblasts at 6 weeks.\nSupplemental Figure 5a,b\n9. For apoptotic signaling, ECM disassembly and Wnt signaling for some reasons the authors chose to show the data differently, ranking genes from highest to lowest for the bcl2- fibroblasts which makes it hard to compare these differences to those in panels B, D and E.\nResponse: For the heat maps shown in Figs. 5b,d,e (3 week time point), we organized the data with highest gene expression rank occurring in the PDGFRa-CFP/BCL-2+ fibroblasts so it was easier for the reader to observe the increased enrichment in those pathways compared to the PDGFRa-CFP/BCL-2- and naïve fibroblasts. The data presented in Figs. 5h,j,k heat maps represent the 6-week time point and are not meant to be directly compared with the 3-week data (Figs. 5b,d,e). As pointed out by the reviewer, these heat maps are organized with the highest gene expression rank occurring in the PDGFRa-CFP/BCL-2- fibroblasts so that it is easier for the reader to observe the reduced enrichment for resolution associated pathways in the PDGFRa-CFP/BCL-2+ fibroblasts corresponding to their persistent fibrotic phenotype. Accordingly, we have ensured that the text in the “Results” section (page 8-9) does not imply a direct comparison between the data shown in Figs. 5b,d,e with that in Figs. 5h,j,k.\n10. Although the selected genes shown for qPCR suggest that for these processes overexpression of bcl2 partially reversed the effects of bleomycin the heatmaps seem to show a more complicated pattern, at least for regulation of wnt signaling. For all of these data it is not certain which effects were cell autonomous, and which might be explained by cross talk between bcl2+ and bcl2-fibroblasts and other adjacent cells.\nResponse: We apologize for the confusion. We have now revised the text in the Fig. 5 legend to clearly articulate that the data presented in Figs. 5c,f,i,l are the normalized TPM gene counts. We agree with the reviewer that as expected, a heat map with many genes derived from a GO:BP pathway analysis would be reflective of a more complex gene enrichment pattern. We believe that studies to determine cross talk between Bcl2+ and Bcl2- fibroblasts and other adjacent cells would require new spatial transcriptomics experiments which we consider to be beyond the scope of this manuscript but could be very important to pursue in future studies.\n11. Figure 5 – Again listing the genes in expression order for one cell type makes it difficult to evaluate differences. Nonethless, it looks like some senescence genes were higher at 6 weeks in bcl2+ animals and some senescence genes were higher in fibroblasts from bcl2- animals. Furthermore, it appears that many senescence genes were actually expressed at higher levels in naive animals, which probably explains why the difference in the 111 gene senescence score was not statistically significantly different between fibroblasts from bcl2+ and bcl2- animals.\nResponse: Given that senescence is a complex cellular process involving the dynamic differential regulation of a large number of genes in multiple cell types during fibrosis development, resolution and persistence, we felt that a heat map was the best way to show the overall pattern of gene changes. A transient senescent phenotype has been documented after bleomycin-induced fibrosis (PMID:31010465, PMID:35934931) and while our data do not quite reach statistical significance (Fig. 6b, p=0.06), the overall combined data show a GO:BP gene enrichment (Fig. 6a), an increased senescence score (Fig. 6b, p=0.60), a significant Pearson correlation between BCL2 and senescence genes, and increased quantitation of immunostaining of key senescence proteins (Fig. 6d-k). Taken together, these data reveal a previously unknown increased senescence signature in PDGFRa-CFP/BCL-2+ fibroblasts during persistent fibrosis.\n12. The tissue staining panels don’t all seem to line up as staining the same features (were these from adjacent slides?) and it is very difficult to tell if there was really co-localization of p16, p21 and B-gal in bcl2+ fibroblasts.\nResponse: The immunostaining for PDGFRa, PDGFRb, p16, p21 and b-gal was done on serial sections. To address the reviewers concern about difficulty in seeing co-localization, we have now added white arrow heads to help identify common features across the images in revised Figs. 6g, i-k.\nRevised Figure 6g,i-k\n13. It does appear that the bcl2 antibody staining was not sensitive enough to pick up endogenous bcl2 in any cells.\nResponse: The anti-BCL-2 antibody used in Fig. 6h is human specific and does not detect endogenous mouse Bcl-2. We used this antibody to enable us to identify human BCL-2 that was driven by Cre-mediated recombination. This information can be found in the Fig. 6 legend and the “Results” section (page 10).\n14. In Figure 5q, pro-fibrotic genes in BCL2+ and BCL2- myofibroblasts are shown. However, it might be more informative to first define myofibroblasts and normal fibroblasts based on transcriptomics and then compare BCL2 expression between these two populations.\nResponse: We appreciate the reviewer’s suggestion. The top 6 genes that were used to define this myofibroblast cluster have now been included in the “Results” (page 10). The average BCL2 expression between the myofibroblasts and fibroblasts in this data set was not statistically different. Thus, we have focused our spatial data analysis in Fig. 6n-r on the pathogenic myofibroblasts.\nFigure 6r\n15. For the human tissues it is surprising that the normal lung has essentially no BCL2 signal, but there seems to be diffuse signal in the IPF lung with a pattern very different from that seen for p21 or SA-b-gal. This is a surprising result, since the violin plot in supplemental figure 7 seems to show only low-level expression of BCL2 in lung myofibroblasts in IPF, but higher expression in fibroblasts and PLIN2+ fibroblasts in normal lungs. In the stained human sections it is also not clear which p21, b-gal or BCL2+ cells are fibroblasts.\nResponse: Under homeostatic conditions, endogenous BCL2 protein expression is low and difficult to detect by western blotting without mitochondrial isolation. Following fibrosis development in our persistent fibrosis models and in IPF lungs, we have previously published that multiple other cell populations, in addition to fibroblasts, increase their expression of BCL-2, including airway epithelial cells and macrophages (PMID: 36752201). Because multiple cell populations are engaging anti-apoptotic pathways and gaining senescent phenotypes in both humans and mice, we would also expect staining in these cells.\n16. I may be misreading the spatial transcriptomics panel, but it appears like the region at the bottom left that has the most cells annotated as myofibroblasts and the most BCL2 signal in the most leftward panel has essentially no senescence signature in the rightward panel. It also appears that most of the BCL2 expression is in different cells (or regions) than the myofibroblast signature.\nResponse: Thank you for this comment. We do not believe that you are misreading the spatial transcriptomics panel. We agree that there is a region at bottom left (revised Fig. 6 n,o) with annotated BCL-2- myofibroblasts that have a low senescence signature. We have modified Fig. 6n,o to include call out boxes that highlight the area with co-localized BCL-2+ myofibroblasts. We have also added text to the “Discussion” (page 17) to highlight that the IPF samples we are studying are from end-stage fibrotic lung.\nFigure 6 n,o\nAdditionally, we added text to the “Results” (page 10) to indicate that the ‘senescence module’ is comprised of 10 senescence-associated genes (ATF3, C11orf96, CDKN1A, DUSP1, FOS, GADD45B, IL6, JUN, JUNB, ZFP36) which is not reflective of an all-inclusive senescence signature. We confirmed the data with immunostaining of lung sections for representative senescence-associated proteins. And while these are highly senescent, the BCL-2 correlation may be less than what is seen in our murine model where we are hypothesizing that an increase in BCL-2 expression followed by a gaining of anti-apoptotic phenotype allows a permissive license/environment for the fibroblasts to become senescent. What we are measuring at this time point is the end result of this and perhaps not the journey. This is a limitation of our study and in order to address this, we would either need to examine lungs during an earlier time in fibrosis development which we feel is beyond the scope of this manuscript. We have addressed this in the “Discussion” (pages 16 and 17).\n17. Figure 6 – It is somewhat surprising that the authors don’t include bcl2 itself in the heatmaps in panel A for the two other persistent fibrosis models. It would be reassuring if bcl2 was upregulated in fibroblasts in at least one of these models. This is especially true given the apparent dramatic upregulation of bcl2 protein in the examples shown if 6C-F. It would also be good to quantify bcl2+ fibroblasts/field as was done for the other endpoints. Finally, p21 should be expressed in many cell types, not only fibroblasts. It is surprising that the staining is completely negative in saline treated mice in panel 6F.\nResponse: We did not originally include Bcl2 in the senescence heat map panels because it is not considered to be a senescence gene. Indeed, Bcl2 is not listed in the panel of 111 genes used to define senescence. However, to address this point, we have now included Bcl2 expression data as an independent new Suppl Fig. 7 for both the repetitive bleomycin model and the Fas deletion model to support that there is increased transcript expression that corresponds to the increased protein expression shown by immunostaining in Fig. 7d. As suggested, we have also now included Bcl-2 quantitation in Fig. 7d and a more representative p21 image for the naïve/saline lung has been included in Fig. 7f.\nSupplemental Figure 7\nFigure 7d and f\nWe would also like to thank the reviewer for bringing up Bcl-2 in this context. It allowed us to clarify part of our overall message in the “Discussion” (page 16). In this revision, we propose that increased BCL-2 inhibits fibroblast apoptosis, driving a persistent fibrosis phenotype. Thus, we propose that BCL-2 licenses (or enables) fibroblast to gain a stable senescent phenotype but does not directly promote senescence.\n18. Fig s1 – clearly shows that many lung fibroblasts express PDGFRb, including a substantial number labelled as PDGFRa+/PDGFRb+. It is still not clear how immunostaining allowed the authors to determine if PDGFRb+ cells were fibroblasts or pericytes.\nResponse: We have updated Figs. 1 and 2 and Suppl Fig. 1, and the \"Results” (pages 4-6) to clarify how we have defined our PDGFRa+, PDGFRa+/b+ and PDGFRb+ populations. Only the PDGFRb+ PDGFRa- cells are identified as pericytes (by flow cytometry and immunostaining). If cells were PDGFRa+ and CFP+ (and were also PDGFRb+ or CFP-) they were considered to be fibroblasts. We did not rely on our immunostaining alone to identify cells as fibroblast or pericytes. Only cells with PDGFRb staining and no CFP or PDGFRa were identified as pericytes. All quantitation of these populations was done by flow cytometry where we could more accurately separate out these two markers and also assess CFP+ at the same time. Additionally, to address this concern, co-immunostaining for CFP, PDGFRa and PDGFRb was performed on the mouse tissue and is now presented in a revised Fig. 3h,i.\n19. S2 – The authors should show CFP flow to confirm that none of the PDGFRb+/PDGFRa low cells were derived from PDGFRa+ cells.\nResponse: These data were presented in Fig. 2i in the original submission and are now Fig. 3g of the revised manuscript.\n20. S7 – Although there is definitely higher expression of p16 and p21 in IPF myofibroblast, expression of GLB1 and Bcl2 actually seems to be higher in the other fibroblast populations in both normal and IPF lungs. These data support previous reports of senescent myofibroblasts in IPF but do not strongly support a role for overexpression of bcl2 in this process.\nResponse: We agree that there is not equally increased expression of the senescence genes CDKN2A, CDKN1A, GLB1, nor of BCL2 across the different fibroblast subsets in IPF/ILD. When we examine our data set (Fig. 6n), and two publicly available data sets (PMC10287567 and PMC1170654/GSE135893) we see that BCL2 expression is very low. Based on the reviewer’s comments, we hypothesize that increased BCL-2 protein is sufficient to protect the fibroblast population from undergoing apoptosis, thereby “licensing” them to become senescent. This has been added to the “Discussion” (page 17).\n21. S8 – There doesn’t appear to be much overlap in expression of most of the included senescence genes among the 3 non-resolving models.\nResponse: We agree. Senescence is a complex cell state that can be reached through the differential regulation of a multitude of gene pattern leading to the lack of a precise definition or gene list within the literature. All three of the models we presented: 1) develop persistent fibrosis, 2) have an apoptotic resistant, pro-fibrotic fibroblast population that gains a senescent phenotype, and 3) have varying expression of Bcl2. However, the models arrive at these three endpoints through different mechanisms. Therefore, we expect there to be differences in the overall gene expression pattern, even if the phenotype of senescence is phenocopied across the models. We have added the underlined text above to the legend of Suppl Fig. 8 to expand on this point.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We appreciate the reviewer’s thorough, thoughtful and constructive review of our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors present a manuscript investigating the role of BCL-2 expression in fibrotic lung remodeling. Primarily leveraging a mouse model expressing human BLC-2 in PDGFRA fibroblasts, the authors demonstrate the impact of BLC-2 expression on inhibition of apoptosis, fibroblast accumulation, and the emergence of pro-senescent phenotypes after bleomycin injury. The authors recapitulate some of their key findings in human tissue and demonstrate the ability of an FDA approved BCL-2 inhibitor (ABT-199) to reverse some of the effects seen in their model. Overall this paper will be of broad interest to the lung fibrosis community. However, I have detailed a number of concerns that should be addressed before publication of this work.\nResponse: We appreciate the Reviewer’s comment about the broad interest of our manuscript to the lung fibrosis community. We address the Reviewer’s specific concerns below.\nMajor:\n1. There appears to be some discrepancy between figure 1g and 1h/i. Looking at the visual co-localization of CFP and PDGFRB/aSMA and to a lesser degree SPC and CCSP there appears to be many cells that are positive for both markers. This is particularly true for PDGFRB where nearly every + cells in the pop out window of 1g appears to be dual positive. This is at odds with the flow data from dissociated tissue. I’m not sure how to interpret these results but the apparently pervasive CFP shown in panel g co-localizing with many different cell types causes concern for the specificity of the lineage tracing.\nResponse: Thank you for spotting this issue, which was also raised by Reviewers 1 and 2. Due to the spectral overlap between CFP and DAPI we re-generated the data and replaced Fig. 1g with new images omitting DAPI. Based on our flow cytometry data (revised Fig. 1i,j and Fig. 3e,g) >90% of the PDGFRa+ fibroblasts co-express CFP and, by inference, BCL-2.\n2. Similarly, in Fig. 2 h there appears to be expansive BLC-2 expression but PDGFRB, PDGFRA and BLC-2 are not shown co-localized in the same tissue section. Based on my concerns in the prior comment I’m not convinced that BCL-2 expression is not seen at some level in PDGFRB+ cells.\nResponse: Many thanks for raising this question. Our flow cytometry data show that there is a fibroblast subset that expresses both PDGFRa and PDGFRb (PDGFRa+/PRGFRb+) that also expresses BCL-2 and CFP. The PDGFRb+ (PDGFRa-) subset is CFP negative (Fig.1i,j and Fig. 3e,g). To address this concern, we have conducted new experiments in which we co-stained with anti-CFP, PDGFRa and PDGFRb antibodies. These new data are shown in revised Figs. 3h,i.\nWe hope that these new data convince the reviewer that BCL-2 expression is limited to PDGFRa+ and PDGFRa+/PDGFRb+ fibroblasts, but not PDGFRb+ (PDGFRa-) pericytes.\n3. In the Col1a1-CFP/BCL-2+ model the authors claim it phenocopies the PDGFRA model, but the accumulation and persistence of PDGFRB+ pericytes appears to be more muted (SFig. 5)\nResponse: Thank you for this comment. Looking at the data at the 9-12 week time points for both strains (i.e. PDGFRa-CFP/BCL-2+ mice and Col1a1-CFP/BCL-2+), we can see that the PDGFRb+ pericyte numbers are approximately 1x106 for both strains (revised Fig. 3d and Suppl. Fig. 4d). It is only at the 6-week time point that there was a lower number of pericytes in the Col1a1-CFP/BCL-2+ mice. Thus, we have decided to keep our broad statement on page 8 that “Col1a1-CFP/BCL-2+ mice phenocopied PDGFRa-CFP/BCL-2+ mice with persistently elevated numbers of PDGFRa+ fibroblasts, and PDGFRb+ pericytes …” as we feel this represents the data presented.\nFigure 3d (teal lines) and Supplemental Figure 4d (red lines)\n4. It’s unclear what SFig 6 is supposed to be demonstrating. In a neither PC1 or PC2 split samples by BCL2+/-. Indeed PC1 seems to separate two groups of 6 week mice. Are these corresponding to some sort of technical batch? Similarly in b PC1 and PC3 are shown but it’s unclear why this plot only shows 6 week and naive mice or why PC3 was shown instead of PC2.\nResponse: We agree that these data are not helpful and so we have removed this Supplemental Figure.\n5. The analyses and associated panels in Fig. 4 are not sufficiently described. In the methods the authors state they used DESeq2 to identify DE and use an FDR cutoff of 0.05. However it is unclear how the genes presented in Fig. 4 b/d/e/h/j/k were chosen. What were the comparisons being made for each plot? Is this naive vs BCL2+? BCL2- vs BCL2+? Are these genes that were significant for any pairwise comparison? The authors should clearly state their grouping and how they arrived at these results. They should also present a supplementary table with a full list of DE genes (with associated LFC and FDR) for every contrast. Furthermore, it is unclear how the reported contrasts in Fig. 4 c/f/i/l are derived. Are these the p-values from pairwise DE comparisons performed with DESeq2? In the figure legend it states “Brown-Forsythe and Welch's ANOVA with Dunnett correction for multiple comparisons. n=4 mice/group. *p<0.05, **p<0.01.” It is not statistically appropriate to perform a second association test on a subset of associations that were identified in a genome-wide test. The adjusted p-value from the DE analysis originally performed should be reported.\nResponse: We thank the Reviewer for carefully reviewing these data. The genes presented in the heat maps shown in what is now Figs. 5b,d,e,h,j,k were genes identified as significantly different in each of the GO Pathway Enrichments by pair-wise comparison. We have now provided the appropriate analysis details for the heat maps shown in the “Methods” (page 23) and in Fig. 5 Figure Legend. As suggested, these DE genes and associated statistics are now available as Suppl. Table IV. The data presented in Figs. 5c,f,i,l are normalized TPM values. p-values were calculated from the TPM data, not from DE comparisons.\n6. On page 9 the text suggests that Cdkn1a and Cdk5r1 are upregulated in BCL2+ mice compared to BCL2- mice 3 weeks post bleomycin. However Cdkn1a is actually higher in BCL2- mice at this timepoint and I do not see Cdk5r1 reported anywhere.\nResponse: We apologize for this error and thank the Reviewer for catching it. We have modified the text to better reflect the minimal changes in Cdkn1a (page 9) between the BCL2+ and BCL2- fibroblasts at 3 weeks post bleomycin instillation. Also, we inadvertently listed Cdk5r1 (p35) in the “Results” which we have corrected to Tp53 (i.e. p53 not p35).\n7. The color scales on Fig. 4g are a bit difficult to interpret. There are two enrichment with much higher values skewing the color scale making it very difficult to see the range. Additionally, all of the heatmaps have a range of -1 to 1 expect for d/e.\nResponse: Thank you for this excellent suggestion. The color rage for both Figs. 5a,g has been increased to aid in interpretation of the pathway enrichment terms. Thank you for spotting this labeling error for the heatmap range for d/e. It has now been corrected.\nFigure 5 a,g\n8. Figure 5a has no consistency in capitalization.\nResponse: We apologize for this oversight which has now been corrected.\n9. Insufficient details are provided to interpret spatial transcriptomics results. The authors state in the methods: “We performed Nanostring CosMx Digital Spatial Profiler to obtain subcellular resolution spatial transcriptomic data on 15 IPF and 13 unaffected controls. The full methods and results will be available in a comprehensive manuscript (manuscript in preparation)”. This is inconsistent with Nature Communications data policies and makes all of these analyses uninterpretable. The authors provide no details around how these data were processed or how cell typing was performed. The authors must provide a full accounting of methods including the release of the data – in accordance with Nature Communications data release policy.\nResponse: We apologize that our methods were lacking during the initial submission. Full and detailed methods for the spatial transcriptomic analysis of the human samples are now provided in the Supplemental Methods. In addition, a new publication by our collaborators describing these methods (recently accepted for publication) is now cited and referenced.\n10. In Fig. 5o BCL-2 and myofibroblasts appear to be poorly co-localized. It is unclear exactly what is being shown in this plot as both the red and yellow appear to be of similar “size”. Are the red points individually detected transcripts and the yellow points cell boundaries? The authors should show the breakdown of BLC expression across cell types in their dataset (similar to the results presented in SFig. 7). Similarly, their Senescence score appears poorly correlated with myofibroblasts. They should show how this senescence module is distributed across cell types and across disease.\nResponse: As discussed above, detailed methods for the spatial transcriptomics experiments are now provided in the “Supplemental Methods”. For Fig. 6n, the red dots are representative of individually detected transcripts and the yellow points reflect cell boundaries identified by cell segmentation. This is described in the legend for Fig. 6 and in the results (page 10). We agree that the senescence score in the tissue is reflective of the entire section of end stage IPF lung tissue which has a senescent phenotype and that this is not restricted to only the BCL2+ myofibroblasts.\nRegarding the reviewer’s suggestion to analyze and show the breakdown of cell type expression of BCL2, we too are curious about this. However, we don’t think that this would strengthen manuscript and might in fact dilute the message of what is already a very large and data rich manuscript. For these reasons we have focused specifically on quantifying BCL2+ and BCL2- myofibroblasts.\n11. In Fig. 5q the authors should state how many BCL2+ myofibroblasts and BCL2- myofibroblasts were in the dataset. Are these only from disease individuals? How many are from controls vs disease? The authors also do not provide enough details in the methods to understand the analysis. Did the regression analysis treat each individual cell as an independent observation with a random effect for each individual? Or were these pseudobulked with a random effect? How were the data processed before this analysis and what was the significance cutoff?\nResponse: Thank you for this suggestion. We have now added the quantitative data about the number of BCL2+ myofibroblasts from control and IPF lungs in our spatial data set in revised Fig. 6q and in the “Results” (page 11). The complete analysis methods are now included within the “Supplemental Methods”. The regression analysis treated each individual cell as an independent observation with a random effect for each individual. It was not analyzed as pseudobulk with a random effect. Data was processed with a significance cutoff of FDR = 0.05.\nFigure 6q\n12. The color scales on Fig. 5q are misleading. Having the two heatmaps together with wildly different scales leads the reader to incorrect conclusions. It appears many of these senescence genes (including CDKN1A) are actually downregulated in BCL2+ myofibroblasts. This is in contrast the the point the authors are making and conflicts with the results from SFig. 7.\nResponse: We appreciate this comment and have changed the color scales between the two heat maps in revised Fig. 6p.\nRevised Figure 6p\n13. Additionally I suggest the results from SFig. 7 should be revalidated in a larger cohort. There are very few cells from individuals without PF in either Myofibroblasts or PLIN2+ Fibroblasts. Thus it is difficult to be confident in the robustness of this analysis. Using one of the newer and larger datasets should improve these estimates of expression (e.g. PMC10287567 or PMC11018522).\nResponse: Thank you for this suggestion which was also brought up by Reviewers 1/2 (“General Comments” #2). We have now included new analysis from the larger Integrated Human Lung Cell Atlas cohort (PMC10287567) in Suppl. Fig. 6c,d.\n14. At the end of page 11 the authors describe correlations between BCL2 and senescence associated genes: “…Bcl- 2:senescence gene correlation of 29% (p<0.05) (Fig. 6a,b). Fibroblasts from all 3 models had significant positive correlation between Bcl-2 and SASP associated genes (Fgf7, PDGFa , Il-6, MMPs, TIMPs and Nox4) and a significant negative correlation between Bcl-2 and cell cycle (Ccnb1, Ccnd1) and cytokeratin genes (Cdk1, Cdk2b).” However a direct correlation between BCL2 and these genes is never presented. 6a/b certainly don’t present a results with a gene correlation of 29%? The authors should directly demonstrate the relationship between BLC2 expression and these genes if they want to make this claim. Additionally, there is no consistency in capitalization on Fig. 6a.\nResponse: The genes and the Pearson Correlations score with Bcl2 are now included as Suppl. Table IV,V and VI. This was originally included in the “Methods” section (page 22) to indicate that we used a Pearson Correlation between Bcl2 and the 111 senescence gene list to determine percent gene correlation, but has been added to the “Results” section (pages 10 and 12) and the Figure legends for Fig. 6 and 7. We have also fixed the capitalization consistency in Fig. 7a and thank the reviewer for spotting these inconsistencies.\n15. Did the authors quantify additional cell type changes associated with BCL-2 inhibition? Did epithelial cell populations remodel appropriately as well? Could the authors postulate a bit more about why pericyte numbers are not decreasing and where they are localized? They postulate a bit on the role of signaling between PDGFRA+ and PDGFRB+ cells in the discussion, but this doesn’t explain why pericytes persist when PDGFRA cells are depleted.\nResponse: A good question and “yes”. We have now included flow cytometry quantitation of CD31+, CD45+ and EpCAM+ cells after treatment with ABT-199 which is now presented in Suppl Fig. 9.\nAs for the question concerning the persistence of pericytes after ABT-199 treatment, we too have been intrigued by this observation. As present, we can only speculate that they are surviving through an ABT-199 insensitive, BCL-2-independent mechanism, possibly involving other anti-apoptotic proteins. We have added text in the “Results” (page 13) to bolster the “Discussion” (pages 15 and 18). However, we feel that further experimental investigation is beyond the scope of the manuscript.\nMinor Comments:\n1. On page 5 I believe PDGFRa-CFP/BCL-2fl/+ should be PDGFRa-CFP/BCL-2+\nResponse: We use the term PDGFRa-CFP/BCL-2+ to refer to the mice after tamoxifen treatment. In these experiments, the mice are first instilled with bleomycin and then injected with tamoxifen between days 4-21. We therefore believe that PDGFR-a-CFP/BCL-2fl/+ is the correct nomenclature to describe the strain prior tamoxifen administration.\n2. There is no Fig. 5n and Fig. 5n-q is referenced twice in the manuscript in places where I believe it is supposed to reference Fig. 5 r-u\nResponse: Thank you for finding these inconsistencies in the figures and text. They have now been corrected. As a note, Fig. 5 has been renumbered and is now Fig. 6.\n3. In accordance with Nature's public access policy, the authors should make all data generated as a part of this study publicly available on GEO (including both bulk RNA sequencing data and spatial transcriptomics data) and provide reviewer access tokens before resubmission. The authors should also make all code used for genomic analysis available.\nResponse: Our data have now been accepted at GEO and the accession numbers are provided within the manuscript for both the bulk RNA sequencing and the spatial transcriptomics data. To review GEO accession GSE307983: ypgdkcuujrstjyb (Reviewer Token) and GSE282639 (PMID: 41091081).\nReviewer #4 (Remarks to the Author):\nThis is a well-conceived and well-conducted study by Redente, Riches, et al that provide new insights into parenchymal remodeling that accompanies persistent/progressive pulmonary fibrosis using both mouse and human models. Most notably, their studies show that BCL-2 dependent apoptosis-resistant fibroblasts are both necessary (loss-of-function) and sufficient (gain-of-function) to induce a non-resolving, persistent fibrotic response to lung injury. The discovery of an additional phenotype of epithelial-driven airway remodeling with bronchiolization and cyst formation in the fibroblast-specific, BCL-2 overexpressing mice provides new insights into pathogenesis of these morphologic lesions that accompany IPF and introduces a valuable tool (animal model) for the field.\nResponse: We appreciate the reviewer’s positive and constructive review of our manuscript.\n1. Overall, I have minimal concerns that primarily relate to data interpretation. Cellular senescence is generally thought to induce an anti-apoptotic phenotype that is, at least partly, driven by BCL-2. The new finding (inverse relationship) that BCL-2 overexpression can induce the generation of a senescent phenotype is intriguing and may indicate the acquisition of DNA damage responses in “long-lived” (apoptosis-resistant) fibroblasts; the authors provide potential other explanations, e.g. diminished autophagy. There is an opportunity to address this more mechanistically with ex-vivo studies of cultured fibroblasts (although not essential).\nResponse: Since the reviewer feels that these are not essential, we have not addressed these points experimentally for this revision. However, we thank the reviewers for these insightful and thoughtful ideas that we might pursue in future studies.\n2. The translational studies with the FDA-approved BCL-2 are convincing in the BCL-2 overexpressing mice, but has this been shown to be effective in the repetitive bleomycin model or in an aging model (PMID: 24718857)? These studies may be outside the scope of the current studies but perhaps worthy of discussion and/or future studies.\nResponse: As discussed above in response to Reviewer 1, we agree that it is important to study the therapeutic effects of ABT-199 in a model of with endogenous Bcl-2 expression that also incorporates aging. We currently have a manuscript in revision at the American Journal of Respiratory Cell and Molecular Biology that examines the effects of ABT-199 as well as an anti-fibrotic in combination in the repetitive dose bleomycin model. The manuscript was submitted shortly after submission of our manuscript to NCOMM. Briefly, these studies show that therapeutic treatment with ABT-199 reverses most fibrotic disease outcomes in mice repetitively instilled with ABT-199, including a reduction in non-aerated lung volume by microCT, increased oxygen saturation by pulse oximetry, improvement in histology by quantitative scoring of Masson’s Trichrome stained lungs and a reduction in fibroblast numbers. However, these studies did not fully recapitulate the robust lung regeneration seen by therapeutically targeting human BCL-2 in our novel mouse strain. This observation reinforces the importance of the findings presented herein and highlight how this new conditional mouse strain will benefit the BCL-2 research community by allowing human BCL-2 to be therapeutically targeted in mice.\nFinally, we agree that studying this further in aged mice addresses an important and relevant question, we respectfully feel this this is beyond the scope of the present study."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have effectively addressed all the comments raised in my initial review.\nMinor comments\nIt is notable that increased fibroblast BCL2 expression in mice leads to a persistent increase in pro-SPC immunostaining in mice, whereas in human IPF several lines of evidence suggest that there is a decrease rather than an increase in the number of AEC2s This difference should at least be mentioned and discussed. It would also be reassuring if the authors showed increased AEC2 numbers by an orthogonal method, for example by flow cytometry. In the absence of such data, the authors might consider discussing the possibility that increased pro-SPC, and increased numbers of pericytes and CCSP+/Krt8+ cells per high power field in sections could be in part explained by the decreased aeration (airspace collapse) seen in fibrotic lungs rather than a true increase in total numbers of these cells in the lungs.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors addressed many of my comments but I still have two significant concerns.\n1) Staining fidelity:\n- CFP stains in Fig. 1G. My original concerns remains. Nearly every cell appears to be positive for CFP with broad co-localziation of CFP and PDGFRB, SPC, CD45, and CD31. The only that appears to not co-localize with CFP is CCSP. Given this comment was raised by all three reviewers in the original comments I'm concerned that the new staining does not show a more restricted pattern.\n- The staining in 3H is not well matched with results from 3A-G. Namely there appears to be pervasive co-localization of CPF with PDGFRB+ PDGFRA- cells. There are also about an order of magnitude fewer PDGFRA/PDGFRB+ cells (according to 3C) than PDGFRA+/PDGFRB- cells (3B). However nearly all the PDGFRA signal co-localized with PDGFRB signal in 3H and 3I\n2) The statistics associated Fig. 5C, F, I, and L are not appropriate. A whole transcriptome DE analysis was carried out. Pulling out a smaller subset of genes and testing these with a separate linear regression and not accounting for multiple testing is effectively p-hacking. The authors should report only genes that were significant in the pairwise comparisons from the whole transcriptome DE analysis or adjust the p-values from the ANOVA to account for multiple testing.", "reviewer_4": "The authors have satisfactorily addressed my concerns."}, "author_response": "Point-by-point response to the reviewer’s questions second revision\nTo assist the reviewers, we have “cut” the unedited reviewer’s questions (italicized) from the original critique and “pasted” them into our responses below. Newly generated data and revised figures are also incorporated into our responses.\nReviewer #1:\n1. The authors have effectively addressed all the comments raised in my initial review.\nResponse: We thank the reviewer for this positive comment.\nMinor comments\n1. It is notable that increased fibroblast BCL2 expression in mice leads to a persistent increase in pro-SPC immunostaining in mice, whereas in human IPF several lines of evidence suggest that there is a decrease rather than an increase in the number of AEC2s This difference should at least be mentioned and discussed. It would also be reassuring if the authors showed increased AEC2 numbers by an orthogonal method, for example by flow cytometry. In the absence of such data, the authors might consider discussing the possibility that increased pro-SPC, and increased numbers of pericytes and CCSP+/Krt8+ cells per high power field in sections could be in part explained by the decreased aeration (airspace collapse) seen in fibrotic lungs rather than a true increase in total numbers of these cells in the lungs.\nResponse: We appreciate this insightful observation and thank the reviewer for the suggested revision, which we have incorporated on page 16 of the “Discussion”. Unfortunately, as the reviewer suspects, we did not perform epithelial cell specific digests or staining for AEC2s in our flow cytometry experiments.\nReviewer #2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We appreciate Reviewer 2’s participation in the review process. Together with Reviewer 1’s comments, we firmly believe that their combined input has markedly strengthened the manuscript.\nReviewer #3:\nThe authors addressed many of my comments but I still have two significant concerns.\n1) Staining fidelity:\n1\n- CFP stains in Fig. 1G. My original concerns remains. Nearly every cell appears to be positive for CFP with broad co-localziation of CFP and PDGFRB, SPC, CD45, and CD31. The only that appears to not co-localize with CFP is CCSP. Given this comment was raised by all three reviewers in the original comments I'm concerned that the new staining does not show a more restricted pattern.\nResponse: By way of perspective, we investigated lineage localization of CFP by 2 independent methods, namely multi-parameter flow cytometry and co-immunofluorescent staining. To address the last comment, we wish to note that the reviewers who raised similar concerns in the original submission were satisfied with our revisions and the new data presented in the revised Figure 1g.\nNevertheless, to further address Reviewer 3’s point of view, we have created a new Supplemental Fig. 1 which shows the individual staining channels for endogenous CFP (teal) and co-immunostaining for markers of specific cell populations (red or yellow) in naïve lung. The new figure shows colocalization of CFP in the expected populations, i.e. PDGFRa+/PDGFBb- and PDGFRa+/PDGFRb+ cells with minimal to no co-localization of CFP in pro-SPC+ AEC2 cells (which are closely adjacent to PDGFRa+ “niche support” alveolar fibroblasts and may explain the reviewer’s point of view), CCSP+ airway epithelial cells, aSMA+ smooth muscle cells, CD45+ immune cells and CD31+ endothelial cells (which are also closely adjacent to PDGFRa+ cells in alveolar walls). As expected, and as shown in Supplemental Fig. 1a, we observed precise co-localization of CFP with human BCL2. Finally, and as noted above, the data from our flow cytometry experiments, wherein we stained dispersed lung cells with antibodies directed against PDGFRa, PDGFRb, CD31, EpCAM and CD45, are in agreement with the co-immunofluorescent staining data.\n- The staining in 3H is not well matched with results from 3A-G. Namely there appears to be pervasive co-localization of CPF with PDGFRB+ PDGFRA- cells. There are also about an order of magnitude fewer PDGFRA/PDGFRB+ cells (according to 3C) than PDGFRA+/PDGFRB- cells (3B). However nearly all the PDGFRA signal co-localized with PDGFRB signal in 3H and 3I\nResponse: Regrettably, we have to respectfully disagree with the view expressed by Reviewer 3 that “the staining data in Fig. 3h is not well matched with the data in Figs. 3a-g. Namely, that there appears to be pervasive co-localization of CFP with PDGFRb+/ PDGFRa- cells”. To help clarify our view, please see the red boxes, highlighted in the Figure below, and please note that the “green-only” cells are CFP-/PDGFRb+/PDGFRa-. This corresponds to our flow cytometry data presented in Fig. 3g which shows <10% CFP expression in PDGFRb+/PDGFRa- cells.\nFigure 3h top panel\n2) The statistics associated Fig. 5C, F, I, and L are not appropriate. A whole transcriptome DE analysis was carried out. Pulling out a smaller subset of genes and testing these with a separate linear regression and not accounting for multiple testing is effectively p-hacking. The authors should report only genes that were significant in the pairwise comparisons from the whole transcriptome DE analysis or adjust the p-values from the ANOVA to account for multiple testing.\nResponse: We thank the reviewer for clarifying the original concern and apologize for misinterpreting the reviewer’s question. We have revised Fig. 5 which now shows expression on a gene level (TPM) in box plots with p-values adjusted for multiple comparisons from the original DE analysis. This is specified in the legend of the revised Fig. 5. Additionally, we have included a new Supplemental Table IV that contains the DE analysis and pAdj values for these genes.\nBy brief way of explanation, we originally selected the genes shown in the original Figs. 5 c, f, i and l because these were key genes associated with the presented GO-BP pathways and heat maps in Fig. 5b, d, e, and j. We bring this up because we wish to push back on the reviewer’s comment that we presented “p-hacked” data. However, we acknowledge and appreciate the reviewer’s point that the ANOVA statistics did not account for multiple testing.\nBox-and-whisker plots are normalized TPM values with pAdj from DE analysis *p<0.05, **p<0.01, ***p<0.01.\nSupplemental Table IV: Differential Gene Expression Analysis for genes shown by normalized gene expression level (TPM) in Figure 5.\nReviewer #4\nThe authors have satisfactorily addressed my concerns.\nResponse: We thank Reviewer 4 for his/her second positive review.\n5"}, {"round": 3, "reviewer_comments": {"reviewer_3": "1). I appreciate the authors inclusion of Sup. Fig 1. This did indeed help separate the signal. I will note that the intensities in the dual stained figures are sometimes different – most notable in the CFP/SPC – leading to the appearance of increased co-localization. In the example below co-localization appears more prevalent in the first panel then the second and third.\n2) It’s possible that this is a figure quality issue, but I’ve highlighted regions that look to be co-localizing CFP and PDGFR-B but not PDGFR-A from Fig. 3H. Again, it’s possible this is just the CFP showing up with a “green” tint on the background. Perhaps showing individual channels in a supplementary figure would help calrify this.\n3) The authors have addressed my concerns on this point. I also want to apologize for how my review was worded in the prior round I want to be clear, both to the editors and authors, that I do not believe the authors were intentionally trying to “p-hack”. I was trying to clarify the point from my initial review about why I was concerned about the approach. Again, the authors have correctly addressed this point and appropriately pushed back on the tone of my initial review."}, "author_response": "Point-by-point response to the reviewer’s questions\nReviewer 3.\n1). I appreciate the reviewers careful review of Suppl. Fig. 1. We think the comment may have stemmed from the position of the arrow in the left box (cyan only) which was obscuring the cell. We apologize for this and have moved the arrow to a more accurate position in Suppl. Fig. 1d.\n2) We thant the reviewer for this suggestion. A new Suppl. Fig. 4 has been provided which shows the individual staining channels for Figs. 3h and 3i.\n3) Thank you for these comments and we appreciate the careful review of the data."}]

Decision letter