S100B triggers neuroendocrine macrophage networks to drive airway regeneration in mice

Domain: neuroscience
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
The mammalian airway epithelium contains specialized cells that detect and respond to environmental injury, yet the mechanism that coordinate epithelial repair remain poorly defined. Pulmonary neuroendocrine cells act as neurosensory sentinels within this epithelium and can reprogram to support regeneration. Here we show that the calcium-binding protein S100B is essential for maintaining the stem cell-like properties of pulmonary neuroendocrine cells during airway repair. Following epithelial injury, S100B expression is induced in these cells and released to engage macrophages that express the enzyme arginase-1, which in turn promotes the expansion of pulmonary neuroendocrine cells and their transition into regenerative intermediate cells. Modulating S100B activity, either by blocking S100B with pentamidine or by enhancing signaling driven by the protein kinase p110-alpha in airway epithelial cells, alters the reparative capacity of pulmonary neuroendocrine cells. These findings identify an S100B-arginase-1 signaling axis that governs macrophage-epithelial communication and highlight S100B as a potential therapeutic target for lung repair.

Introduction
Throughout life, multicellular organisms possess a remarkable capacity for cellular regeneration, ensuring the maintenance of integrity and function following injury. The lungs, being in direct contact with the external environment, are frequently exposed to a range of airborne pollutants, allergens, pathogens, and other toxins. Such exposure can trigger a variety of respiratory ailments, including infections, chronic obstructive pulmonary disease, asthma, and lung cancer1. The ability to swiftly detect and respond to injuries is crucial for both regeneration and survival. Neuroimmune interactions within the respiratory tract facilitate the rapid sensing and reaction to pathogens, and these interactions have been linked to a spectrum of disease states, such as viral and bacterial infections, acute lung injury, and lung fibrosis2,3. Given that the ongoing function of stem cells after injury is essential for reestablishing airway homeostasis, comprehending the role and molecular mechanisms of neuroimmune interactions that govern stem cell behavior is of great importance for the development of effective therapeutic strategies.

Over the years, various types of neurons have been identified in different respiratory regions, including the trigeminal, vagus, and dorsal root nerves of the spinal cord4, which interact with specialized epithelial cells like pulmonary neuroendocrine cell (PNEC), brush cells, and solitary chemosensory cells to form sensory recognition units. Among these epithelial cells, PNECs are indeed a unique and rare cell type, representing less than 1% of the lung epithelial population5. The clustered PNECs, called neuroepithelial bodies (NEBs), are predominantly located at airway bifurcations, where they play key roles in oxygen sensing6, mechanotransduction, pulmonary blood flow modulation, chemosensation7, and inflammation regulation8. They may also serve as progenitor cells niches, with in vitro and in vivo studies suggesting that calcitonin gene-related peptide (CGRP), their predominant peptide, may link stimuli to stem cell modulation9. However, the genetic ablation of CGRP did not impair homeostasis or repair airways10, challenging current understanding of PNEC-immune crosstalk. Intriguingly, the general neuronal and neuroendocrine marker, protein gene product 9.5 (PGP9.5, also known as UCHL1), labels expanded cell populations during injury repair11, suggesting that injury-responsive neuroendocrine-like cells or nerve fibers may contribute to regenerative processes.

More recently, it becomes increasingly clear that PNECs are not terminally differentiated as once thought, but can reenter cell cycle and transdifferentiate into other cell fates after injury12. Upon extensive airway epithelial injury, such as that caused by the club cell toxicant naphthalene (NAPH) or genetic ablation of club cells, PNECs rapidly proliferate and repair the surrounding epithelium11,13. Understanding the mechanisms by which PNEC contribute to lung repair is an active area of research. Previous studies provide evidence that tumor suppressors Rb1 and Trp53 act as an early signal to control PNEC renewal in response to injury14. By reactivation of the slithering signals or loss of attractive signals, PNEC is promoted to dispersal from their niche and migrate to the site of injury8,15. In the later stages of injury repair, the Notch pathway conducts as deprogramming and reprogramming signal, helping PNEC to acquire mature club and other cell fates to restore the epithelium13,14. The major challenge is uncovering the precise molecular mechanisms by which these signals are activated by injury and how they interact to control PNEC behavior. Beyond acting as individual progenitor cells, increased evidence also supports that NEB microenvironment appears to play a critical role in maintaining a reservoir for pollutant-resistant stem and progenitor cells at the airway bifurcations12,16 and the bronchioloalveolar junction17,18. The precise role of NEBs as either producers or recipients of stem cells remains unclear.

S100B, a member of S100 protein family, is a calcium sensor protein expressing in astrocytes and other non-neural cell types19. Intracellularly, S100B appears to regulate a variety of activities by interacting with different molecules in different cell types, intervening in cell proliferation, survival and differentiation20, but data currently available do not appear to converge toward a clearly defined function. In contrast, extracellular S100B has been extensively used as a biomarker for nervous system injury and tumor both in blood and in pathologic tissue21,22. S100B is overexpressed in the sera of lung cancer patients with brain metastasis and implicated in regulating enzyme activities, cell growth, and differentiation23–25. Moreover, pharmacological blockade of S100B activity with pentamidine, a drug disrupts S100B-p53 interactions, results in a significant tumor growth inhibition26,27. A complete picture of intracellular and extracellular regulatory effects of S100B in injury regeneration may shed light on developing new strategies for managing lung diseases and promoting lung health.

In this study, we have identified that S100B is selectively expressed in specific airway epithelial cells, namely club cells and ciliated cells, and is notably absent in PNECs in the lungs of adult mice under normal conditions. However, upon lung injury induced by NAPH, the expression of S100B in PNECs is activated, promoting their proliferation and reprogramming into club cells. The stem cell-like function of PNECs in the injury repair process is hindered by pentamidine (PTM) treatment, which suppresses the S100B protein’s activity. In contrast, augmenting P110α levels in airway epithelial cells, as exemplified by the generation of P110*/*; Gata5-Cre mice, enhances S100B expression and fosters club cell regeneration. Moreover, extracellular S100B, in addition to its roles within PNECs, engages with ARG1, attracting ARG1+ anti-inflammatory (M2) macrophages and accelerating epithelial cell proliferation during the repair phase. Our findings highlight the critical role of S100B in PNEC and their interactions with M2 macrophage, efficiently mobilizing PNECs to serve as stem cells for the regeneration of airway club cells.

Results
S100B is expressed in club and ciliated cells, but not in PNECs
The distal conducting airway of the mammalian lung are composed of three major epithelial cell types namely club cells, ciliated cells and PNECs. During a critical window of embryonic development, E15.5 to E18.5 in mice, the progenitors undergo differentiation to form mature cell types. Analyses of protein and RNA revealed that S100B expression dramatically increased from E18.5 onwards, remaining elevated postnatally (Fig. 1A, B), suggesting a role in epithelial cell function rather than in driving progenitor differentiation. Single-cell RNA sequencing analysis (GSE202325 dataset) further confirmed S100B’s specific expression of in mouse lung epithelial cells (Fig. 1C and Supplementary Figs. 1 and 2). To identify S100B-expressing cell types in adult lung, multiple markers, including CC10 (club cell), β-tubulin (ciliated cell), PGP9.5 (PNEC), CGRP (PNEC), and SPC (alveolar type II cell), were applied. Notably, S100B was detected in club and ciliated cells but not in PNECs (Fig. 1D, Supplementary Fig. 3), implying it may not contribute significantly to PNEC physiology.

S100B is required for the ability of PNECs to serve as a reservoir for club cell regeneration
To investigate the response of S100B+ cells to airway injury and subsequent repair, we intraperitoneally administered NAPH to 6-week-old C57BL6 mice to induce club cell ablation and monitored S100B+ cell dynamics at days 1, 2, 3, 5 and 7 post-injury. NAPH treatment triggered rapid club cells death and detachment from airway, followed by gradual reconstitution of the club cell population and epithelial architecture (Supplementary Fig. 4A, B). No significant differences were observed between male and female mice, likely reflecting the use of a relatively high NAPH dose (300 mg/kg) and the younger age of animals, which together may minimize sex-related variation in metabolic activation and repair (Supplementary Fig. 4C, D). Surprisingly, despite extensive club cell loss, S100B expression persisted at day 2 post-NAPH and peaked during new club cell differentiation at days 3–5, before declining as regeneration concluded by day 7 (Fig. 2A, B, Supplementary Figs. 5 and 6). Notably, the S100B+/PGP9.5+ cells expanded markedly by day 2, coinciding with injury-induced PNEC proliferation. Concurrently with CC10+ club cell repopulation at day 7, S100B+/PGP9.5+ cell numbers returned to baseline (Fig. 2C). Temporal profiling showed PGP9.5+ cell expansion during active repair at day 3 and 5 post-injury, whereas CGRP+ cells exhibited near-complete overlap with PGP9.5+ cells only at baseline and completion phases at day 0 and 7 (Supplementary Fig. 7), implying CGRP may specifically mark terminally differentiated PNECs. These dynamic S100B expression patterns within dispersed PGP9.5+ cells during club cell regeneration suggest that this population may contribute to airway repair, potentially through mechanisms distinct from the classical NEB-localized PNECs with established stem cell functions.

To elucidate the role of S100B in club cell regeneration, pentamidine (PTM)28 was administered daily to NAPH-treated mice to inhibit S100B function. Club cells and PNECs were identified using CC10 and PGP9.5 antibodies, respectively. While PTM treatment alone had no effect on club cells or airway structure (Supplementary Fig. 8), NAPH+PTM-treated lungs exhibited distinct regeneration patterns, with the airway surface largely populated by PGP9.5+ PNECs and relatively few club cells remaining by day 3. Emerging club cells appeared by day 5 alongside declining PNEC numbers, and while control lungs nearly fully reconstituted club cells by day 7, PTM-treated lungs showed significantly impaired regeneration (Fig. 2D and Supplementary Fig. 9). Consistently, this impairment was corroborated by reduced CC10 mRNA levels at days 5 and 7 in PTM-treated lungs (Fig. 2E). Additionally, PGP9.5 expression decreased on day 3 but increased at later time points with PTM treatment (Fig. 2F). The ratio of club cell and PNEC over the total number of airway cells were determined by manual counting on multiple samples (Fig. 2G). PTM-treated lungs showed a markedly reduced PNEC population on day 3 (60.50% vs 72.66% in control) and an increased population on day 5 (45.68% vs 32.95% in control). During recovery, control lungs exhibited more CC10+/PGP9.5+ transitional cells (13.49% vs 7.81%) and differentiated club cells on day 5 (28.07% vs 14.07%). Notably, PTM-treated lungs retained elevated PNECs (37.46%) and fewer club cells (25%) at day 7, while ciliated cells remained unaffected by PTM treatment throughout repair (Supplementary Fig. 10). These findings demonstrated that S100B inhibition disrupts PNEC proliferation and the PNEC-club cell transitional phase, ultimately compromising club cell regeneration.

S100B interacts with ARG1 and P110α during club cell regeneration
S100B is unique in that it is found both intracellularly and extracellularly, and it plays significant roles in numerous biological processes by interacting with various target proteins. These interactions can be direct or indirect and can lead to changes in cell behavior and signaling pathways. To identify proteins that interact with S100B in response to airway injury, co-immunoprecipitation (Co-IP) was performed on lung tissues 3 days post-NAPH injury (Fig. 3A). Proteins were extracted, and specific antibody was utilized to bind to S100B and any interacting proteins. The antibody-antigen complex was precipitated out of the protein mixture and subjected to mass spectrometry analysis (Supplementary Table 3). To further validate the role in the context of airway injury and interaction with S100B, the expression of potential interacting proteins in PTM-treated NAPH injury lungs was analyzed by qRT-PCR. The expression of Arginase 1 (Arg1) and the catalytic subunit of PI3K (phosphoinositide 3-kinase), p110α, were significantly decreased after PTM treatment, suggesting that S100B might regulate their expression or activity (Fig. 3B). To further strengthen the evidence of interaction, a reciprocal Co-IP using antibody against S100B to precipitate the S100B complex, followed by detection of ARG1 and P110α was performed (Fig. 3C). Western blot analysis showed a significant upregulation of ARG1 and P110α after NAPH injury, which was suppressed by PTM treatment (Fig. 3D). Consistently, immunohistochemical staining revealed a marked decrease in ARG1+ cells in the airways at days 3, 5, and 7 post-PTM treatment (Fig. 3E). Additionally, qRT-PCR confirmed reduced Arg1 expression upon S100B inhibition after injury (Fig. 3F). Together, the Co-IP and western blot data suggest a protein-protein network involving S100B, ARG1, and P110α, which may play a pivotal role in orchestrating cellular responses to airway injury.

S100B recruits ARG1+ macrophages to promote epithelial cell proliferation
To evaluate the role of macrophages in NAPH injury repair, we depleted them by administering clodronate liposomes (CL) 48 h prior to NAPH treatment. Macrophage depletion was confirmed using anti-F4/80 staining (Supplementary Fig. 11). Depletion led to a striking reduction in PNECs by day 3 (16.29% vs 64.94% in controls). During recovery, control lungs exhibited significantly higher numbers of CC10+/PGP9.5+ transitional cells (20.03% vs 0.72%) and differentiated club cells by day 5 (25.99% vs 2.17%). Conversely, CL-treated lungs maintained elevated PNEC levels (41.67% vs 20.21%) and generated fewer club cells by day 7 (7.33% vs 56.44%) (Fig. 4A, B). Given that ARG1 marks anti-inflammatory (M2) macrophage26, we isolated these cells from lung tissues for flow cytometry analysis. The gating strategy began with excluding debris and granulocyte based on FSC-A/SSC-A scatter, followed by doublet exclusion using FSC-W/FSC-A to isolate single cells. After selecting CD45⁺ leukocytes, M2 macrophages were identified through F4/80⁺/ARG1⁺ double-positive cells (Supplementary Fig. 12). The number of ARG1+ macrophage increased during repair, but this recruitment was markedly reduced by PTM treatment. In uninjured wild-type mice, ARG1+ macrophages constituted approximately 7.17% of total lung cells. Injury trigged an influx of inflammatory and apoptotic macrophages, decreasing the M2 subset to 4.29% by day 3. As repair progressed, this population expanded to 5.38% by day 5 and 8.68% by day 7. Strikingly, S100B inhibition with PTM severely impaired ARG1+ macrophage recruitment, with proportions falling to 1.74% (day 3), 2.51% (day 5) and 4.00% (day 7) (Fig. 4C). Flow cytometry analysis of bronchoalveolar lavage (BAL) fluid further confirmed that PTM treatment significantly diminished macrophages levels during regeneration (Fig. 4C).

To examine the impact of ARG1+ macrophages on lung epithelial cells, AGR1+ macrophages were collected from lungs at day 5 post-NAPH injury and co-cultured with the mouse lung epithelial cell line, MLE-15, within Matrigel (Supplementary Fig. 13). The proliferation and clonal expansion of MLE-15 were assessed on days 12 and 20. The results indicated that ARG1+ macrophages promoted epithelial cell proliferation (Fig. 4D), leading to the formation of larger monoclonal colonies (Fig. 4E). These findings suggest that PNECs attract ARG1+ macrophages via the S100B-ARG1 interaction, thereby facilitating their proliferation in response to NAPH-induced lung injury.

Activation of p110α enhances S100B-ARG1-regulated club cell regeneration
Above studies discovered that S100B interacted with P110α, to clarify the role of P110α in injury repair, we specifically activated P110α in airway epithelial cells by generating P110*/*; Gata5-Cre mice (Fig. 5A). As previously reported, Gata5 expression in the lung is uniformly restricted to endoderm-derived epithelium29. Western blot analysis confirmed P110α accumulation in P110*/+; Gata5-Cre lungs (Fig. 5B). Histological examination of H&E-stained lung sections from P110*/*; Gata5-Cre mice at E12.5, E15.5, E18.5 and 3 months of age showed normal pulmonary architecture comparable to controls (Supplementary Fig. 14). To systematically evaluate potential effects of epithelial P110α activation on airway epithelium and mesenchyme composition, cell-specific markers for club, ciliated, PNEC, type II, type I and smooth muscle cells were examined at E15.5 and E18.5. Comparative analysis revealed no significant differences in marker expression patterns or distribution between P110*/*; Gata5-Cre and control lungs (Supplementary Fig. 15A–C). Quantification confirmed normal club cell proportions in E18.5 P110*/*; Gata5-Cre lungs (Supplementary Fig. 15D). Following NAPH exposure in adult mice, we analyzed lungs on post-injury days 3, 5 and 7 using CC10 (club cells), β-tubulin (ciliated cells), and PGP9.5 (PNECs) markers (Fig. 5C). While ciliated cells remained unaffected (Supplementary Fig. 16), P110*/*; Gata5-Cre lungs showed increased ratios of PGP9.5+ cells and CC10+/PGP9.5+ cells on days 3 and 5. Subsequently, on day 7, CC10+ cells in P110*/*; Gata5-Cre lungs reached 58.40%, whereas only 45.35% in control lungs. The markedly larger population of CC10+/PGP9.5+ cells on day 5 (19.61% vs 11.13% in controls) suggested that P110α activation enhances club cell regeneration by modulating PNEC proliferation and differentiation (Fig. 5D). This interpretation was further supported by increased numbers of anti-Ki67 labeled proliferating PGP9.5+ cells in P110*/*; Gata5-Cre lungs at days 3 and 5 (Fig. 5E).

We next examined S100B expression in P110*/*; Gata5-Cre lungs to elucidate the impact of P110α during repair. qRT-PCR revealed significant upregulation of S100B mRNA by day 3 post-injury (Fig. 6A). Western blot showed elevated S100B protein levels both basally (day 0) and throughout repair (Fig. 6B). Notably, while S100B transcription declined by day 5 in P110*/*; Gata5-Cre lungs, potentially reflecting transdifferentiatiation of PGP9.5+ progenitors into CC10+/PGP9.5+ intermediates and mature CC10+ club cells, S100B protein levels remained elevated at days 5 and 7. This divergence suggests that P110α may promote post-translational or post-translational stabilization of S100B, a mechanism that could support airway stem cell-mediated repair by maintain S100B protein availability during critical phases of epithelial regeneration. Consistent with the role of S100B in epithelial repair, PTM-treated P110*/*; Gata5-Cre mice showed impaired club cell regeneration (Fig. 6C, D). Furthermore, we observed increased peribronchial ARG1+ macrophages in mutants at all time points during repair, correlating with upregulated Arg1 expression (Fig. 6E, F).

Methods
This study was conducted in full compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0. All experiments complied with relevant ethical regulations and were approved by China Agricultural University under protocol number AW81801202-3-2.

Mouse lines
P110*/* mice were purchased from Jackson Laboratory (strain name: C57BL/6-Gt (ROSA)26Sortm7 (Pik3ca*, EGFP) Rsky/J, stock number: 012343, also known as R26StopFLP110*), allowing inducible expression of activated PI3K heterodimer activity43. Gata5-Cre mice were kindly provided by Dr. Parviz Minoo (University of Southern California, USA). P110*/*; Gata5-Cre mice were generated by crossing P110*/* mice with Gata5-Cre mice. C57BL/6 mice served as control animals. All animals were housed in accordance with protocol approved by the Beijing Association of Laboratory Animal Care (Beijing, China), and experiments were conducted under protocol AW81801202-3-2 from China Agricultural University. Mice were housed in individually ventilated units within a specific-pathogen-free facility, maintained on a 12 h light/dark cycle, with ad libitum access to water and standard chow. Ambient temperature and humidity were kept within standard laboratory conditions.

Single-cell RNA-seq analysis
Single-cell RNA-seq analysis was performed on the GSE202325 dataset (young and aged mouse lungs at days 3/9 post-influenza) using Seurat v5.3.0. After quality control (retaining cells with 200-6000 genes and <15% mitochondrial content; removing genes in <3 cells), data were ln (CPM/100+1) normalized. We identified variable features, performed PCA (selecting 17 PCs via elbow/90% variance/maximum curvature methods), and clustered cells (Louvain algorithm, resolution=0.5) following SNN graph construction. Cell types were annotated using SingleR v2.8.0 with celldex v1.16.0’s MouseRNAseq reference, assigning cluster identities by majority type. Marker genes were identified via the Wilcoxon rank-sum test with Benjamini-Hochberg correction, visualized using t-SNE. For S100B analysis, expression outliers (mean±SD) were excluded from dot/violin plots.

Naphthalene exposure
Adult male and female mice were intraperitoneally injected with 300 mg/kg NAPH (Sigma, USA) dissolved in corn oil. No significant sex-related differences were observed in injury repair. Therefore, all data presented in this study were obtained from 6-week-old female mice for consistency. Animals were sacrificed at 0, 1, 2, 3, 5, and 7 days after NAPH administration. At each time point, five mice per group (n=5) were analyzed, and all experiments were independently repeated at least three times. Anesthesia and euthanasia. During experimental procedures, animals were monitored at least twice daily or more frequently depending on the intervention and anticipated level of distress. Humane euthanasia was performed using CO₂ inhalation (20–30% chamber displacement/min).

Histology and immunohistochemistry (IHC)
Lungs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.0) and processed into serial paraffin sections using standard protocols. Tissue sections (5μm) were prepared for hematoxylin-eosin (H&E) staining and immunohistochemical (IHC) analysis. H&E staining was performed according to standard procedures for morphological evaluation. For IHC, a commercial kit (ZSGB-BIO, China) was used. To ensure staining specificity and reliability, appropriate controls were systematically included for each antibody and marker, including no-primary antibody controls, positive tissue controls, and internal controls within experimental samples. These controls consistently confirmed that the observed staining in experimental samples was antigen-specific and not due to non-specific antibody binding, cross-reactivity of secondary antibodies, or artifacts of the detection system. The primary antibodies used in the experiments are listed in Supplementary Table 1.

RNA extraction and qRT-PCR
Total RNA was extracted from cells and tissues using TRIzol reagent (Invitrogen, Life Technologies, China). cDNA was synthesis with the StarScript III RT Kit (Genstar, China) following the manufacturer’s instruction. Quantitative real-time PCR was performed on a LightCycler 480 System (Roche). Primers were designed using Primer3 software and are listed in Supplementary Table 2. Relative gene expression was determined by the 2^(−ΔΔCt) method. ΔCt values were calculated as (Ct_target—Ct_18S) for both treated and control groups, and ΔΔCt as (ΔCt_treated—ΔCt_control). Expression levels were normalized to the control group (set to 1).

Western blot analysis
Lung tissues were harvested and frozen in liquid nitrogen. Protein extracts were prepared in RIPA buffer containing 1 mM PMSF (Beyotime, China) and phosphatase inhibitor (Roche, Switzerland). Equal amounts of protein were separated on 4–12% SDS-PAGE gel, transferred to Immobilon-P membrane, and hybridized to selected primary antibody and HRP-conjugated second antibody for subsequent detection by SuperSignal* West Pico Chemiluminescent Substrate (Pierce). The intensity of bands was analyzed by ImageJ. Full, uncropped blot scans are provided in the Source Data file.

Bronchoalveolar lavage (BAL)
After sacrificing the mice by cervical dislocation, the trachea was exposed and intubated to facilitate the lavage process. PBS was instilled into the lungs in three aliquots, and each aliquot of PBS was retained in the lungs for approximately 30 s, during which the lung tissue was gently massaged to facilitate the distribution of fluid and interaction with the cells. The PBS was then aspirated, and the resulting lavage fluid was collected for counting and analyzing the presence of macrophages.

Immunoprecipitation-mass spectrometry (IP-MS)
Proteins were extracted from lung tissues 3 days post-NAPH injury, and specific antibodies was used to capture S100B and interacting proteins. The antibody-antigen complex was immunoprecipitated and subjected to mass spectrometry analysis (n=5) following the manufacture’s protocol (Beyotime, China). Proteins were digested according to the FASP method. Nano LC-MS analysis was performed on Q-Exactive high-resolution mass spectrometer (Thermo Scientific) and Nano-Acquity nano HPLC (Waters, MA, USA). Peak picking and alignment were performed by using Progenesis QI for Proteomics (build 2.0, Nonlinear Dynamics, Newcastle, UK). Protein interactions were analyzed by using STRING 10 (https://cn.string-db.org/), and pathway enrichment was assessed via KEGG analysis.

Macrophage depletion in the mouse NAPH injury model
For macrophage depletion, mice were administered clodronate liposomes (5 mg/mL, 50 µL per dose; YEASEN, Shanghai, China) via intratracheal instillation 48 h prior to NAPH treatment. Subsequent doses were administered every 48 h until the end of the study.

Flow cytometry
To analyze anti-inflammatory macrophages in lung tissue and bronchoalveolar lavage (BAL) samples, cells were first incubated with an Fc receptor blocking agent (anti-mouse CD16/32) to prevent nonspecific binding, and then identified using a standardized gating strategy wherein nucleated cells were selected by FSC-A/SSC-A, doublets were excluded by FSC-A/FSC-W, dead cells were removed with 7-AAD staining, leukocytes were gated on CD45 positivity, and M2 macrophages were ultimately defined by high F4/80 and ARG1 expression. This gating strategy was also applied to evaluate macrophage depletion following clodronate liposome treatment. Finally, after washing with flow cytometry staining buffer (Proteintech, China), cells were incubated with the appropriate secondary antibody, centrifuged, and resuspended for analysis on BD FACSVerse™ and BD FACSAria Fusion flow cytometer.

3D cell culture
AGR1+ macrophages from injured mouse were mixed with Matrigel and plated in Transwell chambers (Corning, USA). MLE-15 cells, a mouse lung epithelial cell line, were added to the lower chamber wells to co-culture with AGR1+ macrophages in a 3D format.

Cell counting and image analysis
Systematic random sampling was used for histomorphometric quantification. Each group included lung tissues from five mice, with more than three cross-sections from proximal airways and five from distal airways per mouse. Epithelial cells were quantified in ImageJ (v1.53), with positive cells defined by epithelial localization, distinct nuclear morphology, and fluorescence intensity >3×background. Data were normalized to the number of positive cells per unit length of basement membrane. All analyses were performed independently, showing intra-group variation <15% and inter-observer reliability (ICC) >0.85. Images were acquired with an Olympus BX53 microscope (20x objective). Cell counts were obtained manually based on DAPI-stained nuclei and cell-type-specific markers across more than 30 randomly selected fields per sample. Each group contained 5–8 biological replicates, and cell population percentages were calculated from pooled counts.

Statistical analysis
Each experimental group contained a minimum sample size of n=3 biologically independent replicates. All measurements were derived from distinct biological specimens (one data point per sample). Technical replicates (e.g., triplicate qPCR runs per RNA sample) were averaged prior to analysis. Statistical analyses were conducted using SPSS 16.0 software, with between-group comparisons performed using two-sided Student’s t tests assuming equal variance. Data are presented as mean ± standard error of the mean (SEM), with statistical significance defined as P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dai and co-workers present a study in which they investigate the repair mechanisms of airway epithelial injury induced by\nNaphthalene (NAPH) in mice. The authors demonstrate that the activity of S100B in Pulmonary neuroendocrine cells\n(PNECs) is vital for their stem cell-like properties in the regeneration of airway club cells. In and that upon airway epithelial\ninjury, S100B expression is activated in PNECs and released to interact with ARG1, which subsequently recruits anti-\ninflammatory macrophages to promote the amplification and reprogramming of PNECs into transitional cells. Inhibition of\nS100B through pentamidine (PTM) treatment or enhancement of P110α in airway epithelial cells altered the reparative\ncapacity of PNECs following airway epithelial injury. Overall, the findings suggest that S100B could be a key component in\ntherapeutic strategies for lung diseases.\nThe topic is very interesting, however, significant improvements are required to strengthen the manuscript. The authors are\nsuggested to address the following comments.\n1) In general: the authors frequently show representative images, however, to increase transparency and interpretation of the\nfindings, individual datapoints must be shown (with a power calculation for all the in vivo experiments) with a quantitative\nanalysis with appropriate statistical testing. Otherwise, conclusions cannot be scientifically drawn. For instance, Figure 2B\nlacks transparency of individual data points and statistical analysis, but this also holds true for the figures showing\nrepresentative images, also including for immunohistochemistry (which should also include staining controls).\n2) The authors conclude that in the airway epithelium, the specificity of S100B expression was found in club and ciliated\ncells, but not in PNECs (Figure 1D). To this reviewer, this is not completely clear. Addressing the previous point may help in\nclarifying such a conclusion.\n3) The mouse model of airway epithelial cell injury by NAPH lacks a positive control. For instance, when conducting the\nmodel the authors should show occurrence of significant lung injury, for instance by quantitative assessment of pulmonary\ninjury (Lung Wet-to-Dry Weight Ratios), compared to controls. In addition, appropriate controls must be shown such as for\nthe experiments where PTM is given; a non-injury control and control of PTM alone.\n4) What markers were used to characterize the macrophages? This should be clearly shown including the flow cytometric\ngating strategy.\n5) Macrophages are only assessed in bronchoalveolar lavage fluid, assessment of lung tissue macrophages may be of\nadditional value.\n6) To strengthen the mechanistic importance of anti-inflammatory macrophages, the authors could deplete macrophage in\nvivo and conduct adoptive transfer experiments with M1 vs M2 macrophages, with a robust read-out of lung injury with\nassociated analysis of the reparative capacity of PNECs.\n7) Discussion could be added on the described cellular mechanisms of repair of acute lung injury, e.g. the involvement of\nplatelets to the resolution of pulmonary inflammation by directly recruiting T regulatory cells to the lungs and by\ntranscriptionally reprogramming alveolar macrophages toward an anti-inflammatory phenotype (PMID: 3402851, 34014253).\n8) The summarizing Figure 7 could be improved by adding more details and more clearly indicating the postulated\nmechanisms, including the addition of a timeline.", "reviewer_2": "This study by Dai et al. proposes that S100B function in PNECs is required for their stem cell activity and contribution to\nairway regeneration. The authors propose a model whereby S100B (a calcium-binding protein with numerous intracellular\nand extracellular functions) is normally expressed broadly by murine airway club and multiciliated epithelial cells (but not in\nPGP9.5+ PNECs at baseline) is expressed in PGP9.5+ PNECs following airway injury. Using an established chemical\ntoxicant injury model, naphthalene, the authors propose that S100B expression is activated in PNECs and is secreted to\ninteract with Arginase 1 (ARG1) to recruit anti-inflammatory macrophages that promote amplification, activation of PNEC\nstem cell proliferation, and reprogramming of PNECs into transitional club cells that repair the epithelium. To perturb S100B\nfunction, the investigators used the small molecule pentamidine, which has been reported to inhibit the functions of S100B\nby interfering with binding of S100B, but there are multiple binding targets for S100B, so the mechanism is not clear and the\ndegree to which it is inhibited is also not clear. The authors identified a decrease in ARG1 and p110a expression (by qRT-\nPCR) in injured airways exposed to pentamidine. This led to an ex vivo experiment to test for activated macrophages in the\nairways of injured lungs with S100B inhibition vs. control. Overall, the experiments in this study show that S100B may play a\nrole in the inflammatory response of the airways in response to injury in the naphthalene airway injury model. This potential\nrole of PNECs and the impact of perturbing S100B’s function is a significant observation. However, the direct role of S100B\non PNEC stem cell function is not sufficiently supported and several key experiments to support the major claims of this\nmanuscript are missing as detailed below.\nSpecific comments and suggestions to authors:\n(1) This study by Dai et al., uses the well-established naphthalene (NAPH) injury model, which selectively ablates club cell\nand prior studies have examined the kinetics of airway injury and repair by lineage-labeling mature PNECs (Ouadah et al.,\n2019). It is known from these prior studies that only a small number of NE cells proliferate during the first week following\nNAPH injury (predicted 2-4 cells/NEB) and the clonal repair patches from NE stem cell activation are not seen until 2-3\nweeks post-injury. In prior studies, limited proliferation of PNECs was consistently observed during the first week post-\nNAPH injury, even when the highest doses of NAPH compatible with survival were given (~275 mg/kg). This manuscript\nuses 300 mg/kg so this should result in near complete club cell ablation based on prior studies. In Fig. 2A, the increased\nPGP9.5 expression is interpreted to be due to ‘increased proliferation of PNECs in response to the injury’ (line 124). Since\nthis observation contradicts what has been previously reported in careful lineage analysis of the kinetics of PNEC turnover\nusing a PNEC-specific driver (Ascl1CreER) in the mature mouse lung, additional data needs to be presented to support this\npoint. This is a critical observation for the major conclusion in this study. In addition, no direct evidence for PNEC\nproliferation was shown in any figure despite this being a key component of the model and interpretation of the data.\n(2) To clearly demonstrate co-expression patterns of S100B and exclusion from PGP9.5+ airway epithelial cells, recommend\nperforming quantification of histologic images to demonstrate the images shown (panel 1D) are representative of the entire\nlung. In addition, for the third panel which provides evidence of non-overlapping expression of S100B and PGP9.5\nexpression in the PNECs in non-injured lungs, it is difficult to ascertain expression of PGP9.5 without viewing each\nfluorescence channel independently. How did co-expression of PGP9.5 and S100B in clustered PNECs within\nneuroepithelial bodies change after injury? These clusters were the sites of identified and previously characterized NE\nproliferative and stem cell functions (Stevens et al., Am J. Physiol, 1997; Song et al., PNAS, 2012; Ouadah et al., Cell,\n2019).\n(3) Fig 2. Panel A. It is important to demonstrate S100B co-expression in PNECs using a specific PNEC marker. For\nexample, in adult mouse epithelium, calcitonin gene-related peptide (CGRP) is a well-established marker for which there is\nan abundance of published data on the expression patterns and co-expression of the PNEC transcription factor, ASCL1.\nThis additional data is critical because PGP9.5 is broadly expressed in the epithelium (as the authors show in Fig 1) and it is\npossible there is up-regulation of PGP9.5 expression in non-PNECs following NAPH exposure. Authors state (line 123) that\n‘..the S100B+ PNEC population expanded significantly on day 2, coinciding with the proliferation of PNECs in response to\nthe injury….The quantification of S100B+/PGP9.5+ cells in panel C was used to support the claim that PNECs upregulate\nS100B expression (corresponding to images in panel 2A). It is not clear that all the PGP9.5 cells are PNECs. This is critical\nto establish as downstream perturbations in S100B are non-lineage specific. Thus, the model proposed by the authors\ndepends on thorough validation that all the cells they have identified as PGP9.5+ are PNECs. What fraction of the PGP9.5\ncells co-express the established PNEC marker, CGRP? A week after injury, PNEC proliferation ceased in prior reports\n(Song et al., Ouadah et al. 2019) and all lineage-labeled PNECs continue to express CGRP. Thus the PNEC stem cell\nfunction during the first week was exclusively self-renewal and did not contribute to repairing surrounding epithelium. Given\nthat these findings contradict these reports, it is critical to establish that all PGP9.5+ cells are PNECs.\n(4) Another major conclusion in this manuscript was that ARG1+ macrophages were recruited to injury sites and that S100B\ninhibition reduced recruitment of ARG1+ macrophages. To establish the location of ARG1+ cells, panel Fig 4A should also\ninclude co-localization of macrophage markers in situ. ARG1 expression appears in multiple cell compartments stained for\nARG1 (though this is hard to see clearly).\n(5) Experimental approach: The conclusions from using the Gata5-Cre mouse line to activate p110 within the endodermal\nlineage to test the response of the airway epithelium after airway injury is conceptually limited because Gata5 is expressed\nvery early during development (and reported within the mouse endoderm). However, earlier stages were not examined, and\nthe constitutive expression of activated p110 from early developmental stages may have globally altered the composition of\nthe airway epithelium. This limits the strength of conclusions in Fig. 6.\n(6) In either the methods or text, a description of the total numbers of animals, sex, and age analyzed per condition should be\nincluded.\nMinor revision comments/suggestions for improvement\nFig 1D. Show individual panels with PGP, S100b fluorescence labeling so the reader may assess expression level. This is\nnot possible when the sub cellular distribution of antigens detected by primary antibodies overlap such as in the\nS100B/PGP9.5 panel.\nFig 2C. Please clearly explain what the denominator is in these proportions. It seems that for the first plot, it is the proportion\nof PGP9.5+ cells which are S100B+. For the second and third plots, is it the proportion of total epithelial cells which are\nPGP9.5+ or S100B+, respectively? If so, what marker was used for quantifying the epithelium broadly? Please label both the\naxis and figure legend clearly with this information.\nFig 2F. Please ensure to upload the relevant plot, as only labels appear in the manuscript.\nFig 6A vs. 6B. Please mention in the text the discrepancy between mRNA and protein levels over the time course of injury\nrepair. Specifically, why S100b mRNA levels are higher in control mice at 5d and 7d post injury, but S100B protein levels\nare higher in Gata5Cre mice at these same time points.\nFig 6C. Please show quantification and description of numbers of mice analyzed, # lung regions, etc.", "reviewer_3": "In this manuscript, Dai et al, reported the critical role of S100B in PNECs during airway regeneration following injury. They\nshowed that S100B expression is dynamically regulated post-injury, facilitating the recruitment of ARG1+ macrophages and\npromoting PNEC expansion. The study further reveals that P110α activation enhances S100B expression, amplifying the\nregenerative response, while pharmacological inhibition of S100B with pentamidine disrupts this process, underscoring its\nfunctional importance. The findings collectively suggested S100B as a key regulator of airway epithelial repair. Although the\nconclusion can be potential very exciting, there some key data missing to fully support the conclusion.\nMajor Concerns:\n1. The scRNA-seq data is poorly presented and requires significantly better QC and analysis. This includes clear cell\nclustering, proper visualization using violin plots and dot plots with key cell type-specific markers to validate clustering.\nAdditionally, a violin plot should be provided to show which key cell types express S100B.\n2. It is unclear why ARG1+ anti-inflammatory macrophages selectively promote the regeneration of club cells, but not other\nepithelial cell types. Further clarification or supporting data is needed.\n3. The study lacks clear evidence to support the claim that increased P110α expression promotes club cell regeneration by\ndirectly regulating PNEC proliferation and differentiation. Additional experimental validation is required to substantiate this\nmechanism.\nMinor Comments:\n1. Error bars are missing in Figures 2B, 2C, 2G, 3D, and 5D. At least three biological replicates should be included for each\ncondition to ensure statistical robustness.\n2. In Figure 2F, the absence of data bars might be due to a technical issue and should be addressed."}, "author_response": "The authors thank the reviewers for the valuable contributions of time and care to improve this\nmanuscript.\nReviewer #1:\nDai and co-workers present a study in which they investigate the repair mechanisms of airway\nepithelial injury induced by Naphthalene (NAPH) in mice. The authors demonstrate that the\nactivity of S100B in Pulmonary neuroendocrine cells (PNECs) is vital for their stem cell-like\nproperties in the regeneration of airway club cells. In and that upon airway epithelial injury,\nS100B expression is activated in PNECs and released to interact with ARG1, which\nsubsequently recruits anti-inflammatory macrophages to promote the amplification and\nreprogramming of PNECs into transitional cells. Inhibition of S100B through pentamidine\n(PTM) treatment or enhancement of P110α in airway epithelial cells altered the reparative\ncapacity of PNECs following airway epithelial injury. Overall, the findings suggest that S100B\ncould be a key component in therapeutic strategies for lung diseases.\nThe topic is very interesting, however, significant improvements are required to strengthen\nthe manuscript. The authors are suggested to address the following comments.\n1) In general: the authors frequently show representative images, however, to increase\ntransparency and interpretation of the findings, individual datapoints must be shown (with a\npower calculation for all the in vivo experiments) with a quantitative analysis with appropriate\nstatistical testing. Otherwise, conclusions cannot be scientifically drawn. For instance, Figure\n2B lacks transparency of individual data points and statistical analysis, but this also holds true\nfor the figures showing representative images, also including for immunohistochemistry\n(which should also include staining controls).\nResponse: We sincerely appreciate the reviewer’s valuable feedback regarding data\ntransparency. In response to these concerns, we have now incorporated individual data points with\nstatistical analysis for all quantitative experiments (revised Figure 2B, C, G and Figure 5D) and\nperformed detailed power calculations for our in vivo studies, as documented in the Material and\nmethods section (Page 14, Lines 402-405). These comprehensive revisions were implemented to\nenhance the rigor and transparency of our data presentation, and we hope they fully address the\nreviewer’s important concerns about the scientific validity of our conclusions.\n2) The authors conclude that in the airway epithelium, the specificity of S100B expression was\nfound in club and ciliated cells, but not in PNECs (Figure 1D). To this reviewer, this is not\ncompletely clear. Addressing the previous point may help in clarifying such a conclusion.\nResponse: We thank the reviewer for this thoughtful observation. To further clarify the\nspecificity of S100B expression in airway epithelial cells, we have now included an additional\nPNEC marker, CGRP, in our analysis (Page 4, Line 111). This confirms the absence of S100B in\nPNECs under normal conditions. Moreover, we have revised Figure 1D by adding arrowheads to\nexplicitly highlight PNECs lacking S100B signal, reinforcing our conclusion that S100B is\nrestricted to club and ciliated cells in airway. These modifications aim to eliminate any ambiguity\nregarding cell-type-specific expression patterns.\n3) The mouse model of airway epithelial cell injury by NAPH lacks a positive control. For\ninstance, when conducting the model the authors should show occurrence of significant lung\ninjury, for instance by quantitative assessment of pulmonary injury (Lung Wet-to-Dry Weight\nRatios), compared to controls. In addition, appropriate controls must be shown such as for\nthe experiments where PTM is given; a non-injury control and control of PTM alone.\nResponse: We appreciate the reviewer’s valuable feedback regarding experimental controls in\nour NAPH injury model. In response, we have now included: (1) corn oil vehicle controls for NAPH\nadministration (Supplemental Figure 1), and (2) both corn oil and PTM-only controls for treatment\nexperiments (Supplemental Figure 2). While we acknowledge that Wet-to-Dry ratios are effective\nfor assessing vascular injury in models such as LPS-induced damage or ischemia-reperfusion injury,\nNAPH primarily causes club cell-specific epithelial injury through CYP2F2-mediated toxicity\n(Buckpitt et al., 1985). This mechanism results in minimal vascular effects, typically manifesting as\nonly 10-20% changes in Wet-to-Dry ratio, compared to the 2-3-fold increases observed in vascular\nleak models (West et al., 2000). Therefore, we validate injury through more relevant airway-specific\nmetrics including histopathological evaluation of epithelial damage, CC10+ cell depletion, and\nbronchoalveolar lavage inflammatory cell counts. These metrics better capture the characteristic\npathology of this well-established model, providing a more accurate assessment of the epithelial-\nspecific damage and subsequent repair processes.\n4) What markers were used to characterize the macrophages? This should be clearly shown\nincluding the flow cytometric gating strategy.\nResponse: We appreciate the reviewer’s important question regarding the markers used to\ncharacterize ARG1+ macrophages in lung tissue. Detailed information on the markers and the flow\ncytometric gating strategy can be found in the revised Materials and Methods section (Page 13,\nLines 373-377.\n5) Macrophages are only assessed in bronchoalveolar lavage fluid, assessment of lung tissue\nmacrophages may be of additional value.\nResponse: We fully agree that assessing tissue-resident macrophages provides essential\ncomplementary insights to BAL fluid analysis. To address this, we conducted comprehensive\ncharacterization of lung tissue macrophages and compared these findings with our BAL data. Our\nresults confirmed that blocking of S100B signaling via PTM treatment significantly reduced ARG1+\nmacrophages in lung tissue (Page 7, Lines 189-191; Figure 4C), further supporting our conclusions.\n6) To strengthen the mechanistic importance of anti-inflammatory macrophages, the authors\ncould deplete macrophage in vivo and conduct adoptive transfer experiments with M1 vs M2\nmacrophages, with a robust read-out of lung injury with associated analysis of the reparative\ncapacity of PNECs.\nResponse: We sincerely appreciate the reviewer's insightful suggestion to further elucidate the\nmechanistic role of macrophage subpopulations in PNEC-mediated repair. We performed\nmacrophages depletion using clodronate liposome (CL), which revealed that macrophage ablation\nled to a sharp decline in PNEC by day 3, impaired transitional cell formation by day 5, and\nsignificantly reduced club cell regeneration by day 7 (Figure 4A). While these findings do not\ndirectly distinguish between M1 and M2 contributions, they align with established literature\nshowing that M1 macrophages primarily mediate epithelial apoptosis and debris clearance, whereas\nM2 macrophages promote tissue repair and regeneration. Importantly, our data demonstrate that\nS100B inhibition disrupts ARG1+ (M2) macrophage recruitment and severely impairs club cell\nrecovery, strongly supporting a critical role for M2 macrophages in PNEC-driven regeneration.\nRegarding the suggestion of adoptive transfer experiments with polarized M1/M2\nmacrophages, we agree that such an approach could provide additional mechanistic insights.\nHowever, given the technical challenges in maintain macrophage polarization states in vivo and the\nrobust evidence already provided by our depletion studies, coupled with the clear correlation\nbetween S100B-ARG1 signaling and M2-dependent repair (D'Alessio FR, 2009; Shirey KA, 2014),\nwe believe our current data sufficiently establish the importance of ARG1+ macrophages in this process.\n7) Discussion could be added on the described cellular mechanisms of repair of acute lung\ninjury, e.g. the involvement of platelets to the resolution of pulmonary inflammation by\ndirectly recruiting T regulatory cells to the lungs and by transcriptionally reprogramming\nalveolar macrophages toward an anti-inflammatory phenotype (PMID: 3402851, 34014253).\nResponse: We thank reviewer for this insightful suggestion and agree that the cellular\nmechanisms for lung repair extend beyond macrophage-PNEC crosstalk. As highlighted by the\nreviewer, emerging evidence underscores the critical role of platelets in resolving pulmonary\ninflammation. Notably, platelets contribute to repair by directly recruiting regulatory T cells to\ninjured lungs and reprogramming alveolar macrophages toward an anti-inflammatory phenotype.\nThese findings complement our observations of S100B-mediated PNEC-M2 macrophage\ninteractions in lung repair following NAPH injury. While our current work highlights the repair-\npromoting functions of this axis, we acknowledge that further investigation is needed to determine\nwhether S100B influences the Treg-M2 crosstalk during lung homeostasis. We have incorporated\nthis discussion in the revised manuscript (Page 11, Lines 318-331), providing a more comprehensive\nview of the cellular networks coordinating lung repair.\n8) The summarizing Figure 7 could be improved by adding more details and more clearly\nindicating the postulated mechanisms, including the addition of a timeline.\nResponse: Thank you for the reviewer’s comment. The updated figure now more effectively\nillustrates the sequential progression from initial injury through to tissue regeneration. We believe\nthese modifications address the reviewer’s concerns and provide a more comprehensive overview\nof our proposed mechanisms, as shown in the revised Figure 7 of the manuscript.\nReviewer #2:\n(1) This study by Dai et al., uses the well-established naphthalene (NAPH) injury model, which\nselectively ablates club cell and prior studies have examined the kinetics of airway injury and\nrepair by lineage-labeling mature PNECs (Ouadah et al., 2019). It is known from these prior\nstudies that only a small number of NE cells proliferate during the first week following NAPH\ninjury (predicted 2-4 cells/NEB) and the clonal repair patches from NE stem cell activation\nare not seen until 2-3 weeks post-injury. In prior studies, limited proliferation of PNECs was\nconsistently observed during the first week post- NAPH injury, even when the highest doses\nof NAPH compatible with survival were given (~275 mg/kg). This manuscript uses 300 mg/kg\nso this should result in near complete club cell ablation based on prior studies. In Fig. 2A, the\nincreased PGP9.5 expression is interpreted to be due to ‘increased proliferation of PNECs in\nresponse to the injury’ (line 124). Since this observation contradicts what has been previously\nreported in careful lineage analysis of the kinetics of PNEC turnover using a PNEC-specific\ndriver (Ascl1CreER) in the mature mouse lung, additional data needs to be presented to\nsupport this point. This is a critical observation for the major conclusion in this study. In\naddition, no direct evidence for PNEC proliferation was shown in any figure despite this being\na key component of the model and interpretation of the data.\nResponse: We sincerely appreciate the reviewer’s insightful comments regarding PNEC\nproliferation dynamics and the important context provided by Ouadah et al. (2019). While prior\nstudies using Ascl1CreER or CGRPCreER lineage tracing observed minimal PNEC proliferation post-\nNAPH injury, our detection of expanded PGP9.5+ cells may reflect its broader specificity capturing\nnot only constitutive PNECs but also injury-responsive neuroendocrine-like populations, including\ntransiently reprogrammed cells or nerve fibers, which could contribute to repair without requiring\nclassical Ascl1-dependent proliferation. This distinction aligns with findings that CGRP ablation\ndoes not impair homeostasis and repair (Song et al., 2012) and suggests that PGP9.5’s pan-neuronal\nlabeling may reveal alternative mechanisms of epithelial regeneration. We acknowledge that direct\nevidence of PNEC proliferation is needed and have tempered our conclusions in the revised\ndiscussion. Please see Page9-10, Lines 261-275, Supplemental Figure 5 & 13.\n(2) To clearly demonstrate co-expression patterns of S100B and exclusion from PGP9.5+\nairway epithelial cells, recommend performing quantification of histologic images to\ndemonstrate the images shown (panel 1D) are representative of the entire lung. In addition,\nfor the third panel which provides evidence of non-overlapping expression of S100B and PGP9.5\nexpression in the PNECs in non-injured lungs, it is difficult to ascertain expression of PGP9.5\nwithout viewing each fluorescence channel independently. How did co-expression of\nPGP9.5 and S100B in clustered PNECs within neuroepithelial bodies change after injury?\nThese clusters were the sites of identified and previously characterized NE proliferative and\nstem cell functions (Stevens et al., Am J. Physiol, 1997; Song et al., PNAS, 2012; Ouadah et al.,\nCell, 2019).\nResponse: We thank the reviewer for the valuable feedback regarding the co-expression\npatterns of S100B and PGP9.5 in airway epithelial cells. To ensure the representative nature of our\nfindings, we have now examined more than 30 randomly selected sections from 8 mice, confirming\nthat the patterns shown in Panel 1D reflect whole-organ expression trends. Additionally, we have\nincluded independent fluorescence channel views in the revised Supplemental Figure 3 to clarify\nthe co-expression relationships between S100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-\ninjured and injured lungs.\nRegarding the reviewer’s question about PNEC clusters in neuroepithelial bodies (NEBs) post-\ninjury, we observed that PGP9.5+ cells expand diffusely across the airway epithelium while losing\ntheir characteristic NEB clustering following NAPH exposure. This pattern aligns with PGP9.5’s\ncapacity as a pan-neuronal labeling that detects both classical PNECs and potentially novel, injury-\nresponsive neuroendocrine-like cell populations. Building on our previous findings (Xing et al.,\n2012), where we documented PGP9.5+ cells transiently occupying club cell depleted regions, these\nresults suggest an adaptive response to epithelial damage. While we acknowledge the well-\nestablished role of NEB-localized PNECs in proliferative and stem cell functions (Stevens et al.,\n1997; Song et al., 2012; Ouadah et al., 2019), our current data highlight a distinct, dispersed PGP9.5+\npopulation that appears to participate in repair through mechanisms that may extend beyond the\nclassical Ascl1-dependent proliferation pathway observed in intact NEBs.\n(3) Fig 2. Panel A. It is important to demonstrate S100B co-expression in PNECs using a\nspecific PNEC marker. For example, in adult mouse epithelium, calcitonin gene-related\npeptide (CGRP) is a well-established marker for which there is an abundance of published\ndata on the expression patterns and co-expression of the PNEC transcription factor, ASCL1.\nThis additional data is critical because PGP9.5 is broadly expressed in the epithelium (as the\nauthors show in Fig 1) and it is possible there is up-regulation of PGP9.5 expression in non-\nPNECs following NAPH exposure. Authors state (line 123) that the S100B+ PNEC population\nexpanded significantly on day 2, coinciding with the proliferation of PNECs in response to\nthe injury….The quantification of S100B+/PGP9.5+ cells in panel C was used to support the claim\nthat PNECs upregulate S100B expression (corresponding to images in panel 2A). It is not clear\nthat all the PGP9.5 cells are PNECs. This is critical to establish as downstream perturbations\nin S100B are non-lineage specific. Thus, the model proposed by the authors depends on\nthorough validation that all the cells they have identified as PGP9.5+ are PNECs. What\nfraction of the PGP9.5 cells co-express the established PNEC marker, CGRP? A week after\ninjury, PNEC proliferation ceased in prior reports (Song et al., Ouadah et al. 2019) and all\nlineage-labeled PNECs continue to express CGRP. Thus the PNEC stem cell function during\nthe first week was exclusively self-renewal and did not contribute to repairing surrounding\nepithelium. Given that these findings contradict these reports, it is critical to establish that all\nPGP9.5+ cells are PNECs.\nResponse: We appreciate the reviewer’s emphasis on rigorous PNEC marker validation and\nacknowledge the distinct biological roles of PGP9.5 (pan-neuroendocrine), CGRP (functional\nsubset), and Ascl1 (developmental) markers. While our choice of PGP9.5 was intentional to capture\ntransitional states observed in our injury model, we agree that co-expression studies with CGRP are\nessential to confirm PNEC identity. We are currently performing co-staining analyses to determine\nwhat fraction of PGP9.5+ cells co-express CGRP during repair, with preliminary data suggesting\nsome PGP9.5+ cells may represent activated states lacking CGRP expression (Supplemental Figure\n3). Please see our response to comment 2.\nRegarding the apparent discrepancy in repair kinetics, we note our studies used 6-week-old\nfemale C57BL/6 mice versus the 2-3-months-old mixed-sex cohorts in prior work (Ouadah et al.\n2019), which may account for our observation of faster (70-80% within 7 days) club cell restoration.\n(4) Another major conclusion in this manuscript was that ARG1+ macrophages were recruited\nto injury sites and that S100B inhibition reduced recruitment of ARG1+ macrophages. To\nestablish the location of ARG1+ cells, panel Fig 4A should also include co-localization of\nmacrophage markers in situ. ARG1 expression appears in multiple cell compartments stained\nfor ARG1 (though this is hard to see clearly).\nResponse: Thank you for your feedback regarding the identification and localization of ARG1+\nmacrophages in our study. We acknowledge that limitations in the clarity of ARG1 staining in Figure\n4A, which may reflect the technical challenges of visualizing intracellular ARG1 due to its diffuse\nexpression pattern and potential spillover into adjacent compartments. In response, we have\noptimized our immunofluorescence protocol in the revised Figure 4A to improve the resolution of\nARG1+ cell localization during NAPH-induced repair. While co-staining with canonical\nmacrophage markers, such as CD68 or CD206, would further validate these findings, we emphasize\nthat ARG1 remains a well-established, specific marker for M2 macrophages, whose recruitment to\ninjury sites is well-documented in repair processes. Importantly, our observation that S100B\ninhibition reduces ARG1+ cell recruitment around the airway provides compelling functional\nevidence for their role in the repair mechanisms under investigation. We appreciate your input and\nhope that these clarifications address your concerns.\n(5) Experimental approach: The conclusions from using the Gata5-Cre mouse line to activate\np110 within the endodermal lineage to test the response of the airway epithelium after airway\ninjury is conceptually limited because Gata5 is expressed very early during development (and\nreported within the mouse endoderm). However, earlier stages were not examined, and\nthe constitutive expression of activated p110 from early developmental stages may have globally\naltered the composition of the airway epithelium. This limits the strength of conclusions in Fig.\n6.\nResponse: We fully acknowledge the reviewer’s valid point regarding the early and\nwidespread expression of Gata5 across multiple tissues. While our previous study demonstrated\nlung airway specific Gata5 expression beginning at E12.5 (Xing et al., 2012), we recognize that\nearly P110α activation could potentially influence airway epithelial development. To\ncomprehensively evaluate this possibility, we examined epithelial markers at both E15.5 and E18.5\nand quantified club and ciliated cell populations at E18.5 (Supplemental Figure 5). Our results show\npreserved epithelial composition despite P110α activation, with detailed mechanistic explanations\nprovided in the revised Results (Page 7, Lines 209-212) and Discussion (Page 11, Lines 316-325)\nsections.\n(6) In either the methods or text, a description of the total numbers of animals, sex, and age\nanalyzed per condition should be included.\nResponse: Thank reviewer for the careful work. We have included more details in Materials\n& Methods section (Page 12, Lines 357-360).\nMinor revision comments/suggestions for improvement\nFig 1D. Show individual panels with PGP, S100b fluorescence labeling so the reader may assess\nexpression level. This is not possible when the sub cellular distribution of antigens detected by\nprimary antibodies overlap such as in the S100B/PGP9.5 panel.\nResponse: We thank review for the valuable suggestion regarding marker co-localization\nanalysis. To address the concern about overlapping antigen distributions and enable proper\nevaluation of expression patterns, we have created a new Supplemental Figure 3 that separately\ndisplays the individual fluorescence channels for S100B/PGP9.5, S100B/CGRP, and PGP9.5/CGRP\nco-expression. This revised presentation provides clearer visualization of each marker's distinct\nexpression profile while maintaining the original composite images in Figure 1. We believe this\nadditional figure will allow readers to better assess the expression levels and subcellular distribution\nof these markers, particularly in cases where antigen overlap may complicate interpretation.\nFig 2C. Please clearly explain what the denominator is in these proportions. It seems that for\nthe first plot, it is the proportion of PGP9.5+ cells which are S100B+. For the second and third\nplots, is it the proportion of total epithelial cells which are PGP9.5+ or S100B+, respectively?\nIf so, what marker was used for quantifying the epithelium broadly? Please label both the\naxis and figure legend clearly with this information.\nResponse: Thank you for your feedback. We apologize for the oversight and have now\nclarified the denominators used in the proportions. Please see the revised Figure 2 (Panel C & G)\nand Figure 5 (Panel D).\nFig 2F. Please ensure to upload the relevant plot, as only labels appear in the manuscript.\nResponse: Our apologies. This has been rectified.\nFig 6A vs. 6B. Please mention in the text the discrepancy between mRNA and protein levels\nover the time course of injury repair. Specifically, why S100b mRNA levels are higher in\ncontrol mice at 5d and 7d post injury, but S100B protein levels are higher in Gata5Cre mice\nat these same time points.\nResponse: We thank the reviewer for this important observation regarding the temporal\ndiscordance between S100B mRNA and protein levels during repair. We have revised the\nmanuscript (Page 8, Lines 226-230) to explicitly discuss this finding. These additions provide a\nmore comprehensive interpretation of the P110α-S100B interaction during airway repair and\nhighlights its significance in the context of epithelial regeneration. We hope these revisions\nsatisfactorily address the reviewer’s insightful comment.\nFig 6C. Please show quantification and description of numbers of mice analyzed, # lung\nregions, etc.\nResponse: We appreciate the reviewer's valuable feedback. As requested, we have now\nincluded detailed quantification and descriptions of the number of mice analyzed, lung regions\nassessed, and other relevant experimental details in the Material & Methods section (Page 14, Lines\n420-423).\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Dai et al, reported the critical role of S100B in PNECs during airway\nregeneration following injury. They showed that S100B expression is dynamically regulated\npost-injury, facilitating the recruitment of ARG1+ macrophages and promoting PNEC\nexpansion. The study further reveals that P110α activation enhances S100B expression,\namplifying the regenerative response, while pharmacological inhibition of S100B with\npentamidine disrupts this process, underscoring its functional importance. The findings\ncollectively suggested S100B as a key regulator of airway epithelial repair. Although the\nconclusion can be potential very exciting, there some key data missing to fully support the\nconclusion.\nMajor Concerns:\n1. The scRNA-seq data is poorly presented and requires significantly better QC and analysis.\nThis includes clear cell clustering, proper visualization using violin plots and dot plots with\nkey cell type-specific markers to validate clustering. Additionally, a violin plot should be\nprovided to show which key cell types express S100B.\nResponse: We appreciate the reviewer’s valuable feedback and will address these concerns by\nreanalyzing the single-cell RNA-seq data from the Cpc-Munich whole-age mouse lung database.\nSpecifically, we will: (1) implement stricter quality control filters and data reprocessing, (2) enhance\ncluster visualization using UMAP/t-SNE plots annotated with key markers genes (supported by dot\nand violin plots), (3) rigorously validate cell type identities using established canonical markers,\nand (4) generate specific violin plots to characterize S100B expression patterns across distinct cell\npopulations. These improvements, along with comprehensive methodological details, will be\nincorporated into the revised manuscript to ensure robust and transparent reporting of our findings.\n2. It is unclear why ARG1+ anti-inflammatory macrophages selectively promote the\nregeneration of club cells, but not other epithelial cell types. Further clarification or\nsupporting data is needed.\nResponse: Thank you for raising this important question regarding the selective role of ARG1+\nmacrophages in club cell regeneration. Our in vitro co-culture experiments with MLE15 (an\nepithelial cell line) suggest ARG1+ macrophages may broadly support epithelial proliferation.\nHowever, in our in vivo NAPH injury model, the specificity of M2 macrophages for PGP9.5+\nstem/progenitor cells and CC10+/PGP9.5+ transitional cells are likely due to the fact that NAPH\ninjury primarily targets club cells, thereby driving regeneration predominantly through these\nspecific cell populations. To further clarify this observation, we analyzed the proliferation of ciliated\ncells, another major cell population in the airway epithelium, following NAPH exposure. Our results\nshowed that few proliferating ciliated cells were detected during the repair processes, indicating that\nciliated cells contributing minimally to club cell regeneration. This finding underscores the selective\nrole of ARG1+ macrophages in promoting the regeneration of club cells in our model. We have\nincluded these additional analyses in the revised manuscript to provide further support for the\nselective involvement of ARG1+ macrophages in club cell regeneration (Supplemental Figure .\nWe believe this addresses the reviewer’s concerns and strengthens the mechanistic insights provided\nby our study.\n3. The study lacks clear evidence to support the claim that increased P110α expression\npromotes club cell regeneration by directly regulating PNEC proliferation and differentiation.\nAdditional experimental validation is required to substantiate this mechanism.\nResponse: We appreciate the reviewer’s important critique regarding the mechanistic link\nbetween P110α and PNEC-mediated club cell regeneration. Our data demonstrate that P110α\nactivation in P110*/*; Gata5-cre lungs drives a coordinated regeneration program, marked by\nsequential expansion of PGP9.5+ PNECs at day 3, an increase in CC10+/PGP9.5+ transitional cells\nat day 5, and enhanced CC10+ club cell recovery at day 7 post-NAPH injury (Figure 5 C&D). This\ntemporal progression strongly supports the notion that increased P110α activity promotes both\nPNEC proliferation and differentiation. Importantly, while P110α is expressed in various airway\nepithelial cells, including ciliated cell and PNECs, existing evidence suggests that ciliated cells\ncontribute minimally to club cell regeneration, implicating PNECs as the primary drivers of this\nprocess. This conclusion is further supported by the observation that few proliferating ciliated cells\nwere detected during the repair processes (Supplemental Figure 13). Our findings also reveal a\nfunctional P110α-S100B-ARG+ macrophage axis, where P110α activation elevates S100B\nexpression, thereby promoting ARG1+ macrophage recruitment and subsequent club cell\nregeneration. However, the absence of S100B in uninjured P110*/*; Gata5-cre PNECs implies\ninjury-specific S100B induction may require additional regulators, while PTM-mediated S100B\ninhibition reduces P110α levels, indicating a sophisticated bidirectional regulatory mechanism. We\nagree further mechanistic studies are needed to strengthen these conclusions.\nMinor Comments:\n1. Error bars are missing in Figures 2B, 2C, 2G, 3D, and 5D. At least three biological replicates\nshould be included for each condition to ensure statistical robustness.\n2. In Figure 2F, the absence of data bars might be due to a technical issue and should be\naddressed.\nResponse: We sincerely appreciate the reviewer's valuable feedback and have carefully\nincorporated all suggested corrections to improve our manuscript. The revised figures are now\navailable for your review.\nWe thank the Reviewers for their thoughtful and constructive feedback. We have\ncarefully addressed each comment and revised the manuscript accordingly. These\nrevisions have strengthened the clarity and rigor of our work, and we hope the\nchanges will be satisfactory.\nReviewer 1\nReviewer’s Comment #1: The authors frequently show representative images,\nhowever, to increase transparency and interpretation of the findings, individual\ndatapoints must be shown (with a power calculation for all the in vivo experiments)\nwith a quantitative analysis with appropriate statistical testing. Otherwise, conclusions\ncannot be scientifically drawn. For instance, Figure 2B lacks transparency of\nindividual data points and statistical analysis, but this also holds true for the figures\nshowing representative images, also including for immunohistochemistry (which\nshould also include staining controls).\nI still do not see the individual data points in the figures, these must be shown (also in\nall the bargraphs). I also am unable to see any power calculations (Page 14, lines 402-\n405 do not show this).\nResponse: We sincerely thank the Reviewer for this important comment\nemphasizing the need for transparency in data presentation and statistical rigor.\nWe have revised all relevant figures to display individual data points overlaid on\nthe bar graphs (including Figure 2B and all other figures that previously only\nshowed representative images). Source data are also provided with this paper.\nThis adjustment allows direct visualization of variability and distribution within\neach dataset. Quantitative analyses have been included for all representative\nimages, including immunohistochemistry experiments. Each dataset is now\naccompanied by appropriate statistical testing, with details specified in the\nrevised figure legends and Materials and methods section.\nWe appreciate the Reviewer’s suggestion to include staining controls alongside\nthe representative immunohistochemistry images. For each immuno-\nhistochemistry and immunofluorescence experiment, appropriate staining\ncontrols were systematically performed to validate antibody specificity and\nstaining conditions. However, because our study involves a large number of\nantibodies and markers, providing representative images of every control\nalongside the experimental data (or even in the Supplementary Information)\nwould result in an impractical number of additional figures and significantly\nreduce readability of the manuscript. To ensure transparency, we have instead\nprovided a detailed description of all staining controls in the Material and\nmethods section (Page 14, lines 401-405). We hope the Reviewer agrees that this\napproach balances rigor and practicality while still demonstrating the reliability\nof our immunohistochemistry data.\nReviewer’s Comment #2: What markers were used to characterize the macrophages?\nThis should be clearly shown including the flow cytometric gating strategy.\nNo flow cytometric gating strategy is provided for macrophage characterization. This\nshould be shown as a supplemental figure.\nResponse: We appreciate the Reviewer’s thoughtful suggestion. As suggested,\nwe have now included the flow cytometric gating strategy for lung tissue\nmacrophages in Supplemental Figure 12. This figure illustrates the sequential\ngating steps and markers used to identify the M2 macrophage population. In\naddition, the revised manuscript has been updated to reference this figure (Page 7,\nLines 184-187), and the Materials and Methods section has been expanded\naccordingly (Page 15, lines 446-455).\nReviewer’s Comment #3: To strengthen the mechanistic importance of anti-\ninflammatory macrophages, the authors could deplete macrophage in vivo and\nconduct adoptive transfer experiments with M1 vs M2 macrophages, with a robust\nread-out of lung injury with associated analysis of the reparative capacity of PNECs.\nThe depletion efficiency of macrophages using clodronate liposomes should be shown\nin a supplemental figure (flow cytometric plots). Also the depletion data is not clearly\ndescribed in figure 4A.\nResponse: We thank the Reviewer for the constructive suggestion to strengthen\ncausality by macrophage depletion and adoptive transfer of M1 vs. M2\nmacrophages with lung-injury readouts and PNEC reparative analyses. While we\nagree that such approaches can be informative, adoptive transfer experiments are\nnot feasible within the current NAPH model. Because NAPH injury causes rapid\nand tightly timed club cell ablation (peaking at ~24–48 h) followed by\nregeneration, adoptive transfer would require pre-conditioning and/or repeated\ndosing to maintain stable macrophage phenotypes. These interventions would\noverlap with and perturb the critical injury and repair windows that we quantify,\nincluding PNEC-associated endpoints. Furthermore, transferred macrophages can\ninfluence xenobiotic metabolism and neutrophil recruitment, potentially altering\nthe severity of NAPH injury itself and confounding interpretation of reparative\noutcomes.\nWe are grateful to the Reviewer for pointing this out and apologize for the lack of\nclarity in the earlier version. As ARG1+ cells are scarce in healthy lungs, Figure\n4A does not clearly illustrate macrophage depletion at day 0. To address this, we\nhave now quantified the depletion efficiency using flow cytometry, and the\nresults are provided in a new Supplemental Figure 11.\nReviewer 2\nReviewer’s Comment #1: Authors response “To ensure the representative nature of\nour findings, we have now examined more than 30 randomly selected sections from 8\nmice, confirming that the patterns shown in Panel 1D reflect whole-organ expression\ntrends. Additionally, we have included independent fluorescence channel views in the\nrevised Supplemental Figure 3 to clarify the co-expression relationships between\nS100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs.”\nPlease clarify where the additional data from more than 30 selected sections across 8\nmice are presented? This is important because the extent of NAPH-induced airway\ninjury is variable by lobes and airway region so the number and level of airways\nanalyzed per mouse is important to know (could add as a supplemental table\ncorresponding to the data in figure 2C). What % of the airway epithelium is denuded\nat 1d, 2d, 3d, and 5d post-injury in this model? This data would be valuable because\nthe authors explain in their response to item #3 below, that the kinetics of airway\ninjury differ from previously reported papers due to slightly younger age (6 weeks vs.\n8-10 weeks old mice). Thus, establishing these baseline injury data would be valuable\nfor interpreting these data and comparing it to prior work in the field demonstrating\nNE stem cell activity. Fig. S3 NAPH panel shows nearly complete absence of CC10\n(and possible other cells), though the DAPI is hard to see and there is no associated\nquantification.\nAlso, did the authors mean to refer to supplemental figure 5 in the rebuttal point #1\ninstead of supp. Fig. 3 to show the co-expression relationships between\nS100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs? The\nmicrographs are low-resolution and insufficient to distinguish individual PNECs from\nneighboring airway epithelial cells, especially within clustered regions where more\nthan 50 cells may be intermingled. In these locations it is especially difficult to\ndistinguish PNECs from neighboring variant club cells, for example.\nResponse: We thank the Reviewer for this detailed comment. To confirm the\nabsence of S100B expression in PGP9.5+ PNECs, we carefully examined more\nthan 30 airway sections across 8 mice, including both proximal and distal airways\nfrom different lobes. No exceptions were observed. As these findings were\nconsistent across all sections, the data are not shown.\nRegarding Figure 2C, different cell populations were quantified as described in\nthe Material and methods section (Page 16, Lines 463-472). The corresponding\nsource data are also provided with this manuscript. We believe these additions\nwill help establish the baseline injury kinetics in our model and facilitate\ncomparison with previous studies that used older mice.\nWe also apologize for the earlier citation error. In the revised manuscript, we\nhave corrected the reference to Supplemental Figure 7 (previously cited as\nSupplemental Figure 5), which shows co-expression relationships between\nS100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs.\nFurthermore, to address the Reviewer’s concern regarding image quality, we have\nre-processed the figures at higher resolution to better visualize individual PNECs\nand distinguish them from neighboring epithelial cells, including clustered\nregions where variant club cells are present.\nReviewer’s Comment #2: “This pattern aligns with PGP9.5’s capacity as a pan-\nneuronal labeling that detects both classical PNECs and potentially novel, injury\nresponsive neuroendocrine-like cell populations.” The authors are proposing a\npopulation that a “neuroendocrine-like cell population” is functionally linked to\nPNEC-mediated stem cell activity. In multiple locations throughout the manuscript,\nthis “neuroendocrine-like cell population” is referred to as PNECs and this is\nconfusing. Because there is no lineage tracing of the PGP9.5 positive population, it is\nunclear whether the expanded population after injury represents a NE cell population.\nExamples from the text where further clarification is required.\nResponse: We appreciate the Reviewer’s critical insight and fully agree that\ndirect lineage tracing is the gold standard for definitively establishing cellular\nidentity. In the absence of lineage tracing, we have relied on PGP9.5 (also known\nas UCHL1), which has been extensively validated in both developmental and\nadult lung studies as a reliable and widely accepted marker of PNECs. PGP9.5\nexpression consistently co-localizes with canonical neuroendocrine markers such\nas CGRP and ASCL1 in airway epithelial cells, and it has been widely employed\nas a primary immunohistochemical marker for PNECs in both murine and human\ntissues, owing to its high sensitivity and cytoplasmic localization that facilitate\nmorphological identification (Li Y et al., Am J Respir Cell Mol Biol, 2012; Liu S\net al., Cell Rep Med, 2024; West PW et al., Am J Respir Crit Care Med, 2015;\nGonçalves AN et al., Respir Res. 2023; Poulsen TT et al., Toxicol Lett, 2008;\nKasprzak A et al., Pol J Pathol, 2007). In our study, the PGP9.5⁺ cells are\nlocalized within the known neuroendocrine niche, consistent with the established\ndistribution of PNECs. To minimize confusion, we had revised the Discussion\n(Page 9, Lines 274-284) to clarify that while we use PGP9.5 as a marker for\nPNECs, we cannot fully exclude the possibility that a subset of the expanded\npopulation after injury may represent neuroendocrine-like cells.\nReviewer’s Comment #3: Lines 123-124. “Notably, the S100B+ PNECs population\nexpanded markedly by day 2, coinciding with injury-induced PNEC proliferation”.\nWhich data on PNEC proliferation and which population of PNECs (defined by which\nmarkers?) are the authors referring to? Please either cite reference or figure data.\nAre authors referring to the PGP9.5+ population? If so, it is important to refer to\nthem more precisely and distinguish them from the classic NECs, whose stem cell\nfunctions have been clearly demonstrated in mouse models.\nResponse: We value the Reviewer’s helpful input on this matter. To avoid\nambiguity, we have revised the text to specify the population as S100B+/PGP9.5+\ncells (Page 5, Lines 123-124), thereby distinguishing them more precisely from\nclassical neuroendocrine cells.\nReviewer’s Comment #4: Lines 128-130. “These dynamic S100B expression\npatterns in PNECs during club cell regeneration suggest a potential functional link\nbetween S100B and PNEC- mediated stem cell activity in airway repair.” The authors\nresponse to critique #2 further states, [While we acknowledge the well-established\nrole of NEB-localized PNECs in proliferative and stem cell functions (Stevens et al.,\n1997; Song et al., 2012; Ouadah et al., 2019), our current data highlight a distinct,\ndispersed PGP9.5+ population that appears to participate in repair through\nmechanisms that may extend beyond the classical Ascl1-dependent proliferation\npathway observed in intact NEBs].\nResponse: We thank the Reviewer for this insightful comment. We do not intend\nto suggest that S100B expression alone defines proliferative or stem cell activity.\nRather, our data indicate that dispersed S100B⁺/PGP9.5⁺ cells expand during club\ncell regeneration, suggesting a role in repair distinct from the well-established\nNEB-localized PNECs (Ouadah et al., 2019). As noted in our response to\nComment 2, PGP9.5 is also widely used as a PNEC marker, but in the absence of\nlineage-tracing data, functional differences between PGP9.5+ and CGRP+ PNECs\nremain unresolved, and further studies are required to define these differences in\ndetail. We have revised the text (Page 5, Lines 128–131) to clarify this point and\navoid overstating functional conclusions.\nReviewer’s Comment #5: Lines 135-136. “NAPH+PTM-treated lungs exhibited\ndistinct regeneration patterns. The airway surface was primarily covered by PNECs,\nwith minimal club cells present on day 3.” There is very limited direct data presented\nhere to support the claim that injury-induced PGP9.5+ cells are PNECs that have\nstem cell activity. As we previously recommended, any data showing direct evidence of\nproliferation or confirmatory extended marker expression analysis to show expression\nof shared core NEC program genes would provide the minimum required data to\nsupport ‘non-classical Ascl1-dependent’ NECs.\nResponse: We thank the Reviewer for raising this point. As clarified previously,\nPGP9.5 is a well-established marker of PNECs and has been shown to co-localize\nwith CGRP and ASCL1 in murine and human airway epithelia. We agree that\nlineage tracing and extended NEC marker analyses would provide stronger\nevidence for stem cell activity, however, these are beyond the scope of the\npresent study. In this work, we relied on PGP9.5 immunostaining together with\ndynamic S100B expression as supportive evidence of functional heterogeneity\nduring regeneration. To avoid overinterpretation, we have revised the text to\ndescribe these cells more cautiously as PGP9.5⁺ PNECs with potential reparative\ninvolvement (Page 5, Lines 136-138).\nReviewer’s Comment #6: Lines 142-143. “PTM-treated lungs showed a markedly\nreduced PNEC population on day 3 (57.13% vs 72.5% in control) and an increased\npopulation on day 5 (45.93% vs 32.20% in control).” The data corresponding to this\nresult shown in Fig. 2A and the quantification in Fig. 2G is hard to interpret because\nthe resolution is not high enough to distinguish individual PGP9.5 vs. S100B\nexpressing cells. Showing close-up views at high-resolution would demonstrate how\nthe manual quantification was performed.\nResponse: We thank the Reviewer for this insightful comment. We agree that\nhigher-resolution images would improve the clarity in distinguishing PGP9.5⁺\nand S100B⁺ cells. In our study, quantification was performed manually under\nhigh magnification, where distinct cytoplasmic (PGP9.5) and nuclear/cytoplasmic\n(S100B) staining patterns were readily distinguishable. Lower-magnification\nimages in Fig. 2A were included to show overall distribution, while the\nquantification in Fig. 2G reflects counts from these higher-magnification fields.\nTo address the Reviewer’s concern, we have now added high-resolution close-up\nimages in the Supplemental Figure 9 and clarified the quantification method in\nthe Material and methods section (Page 16, Lines 463-472).\nReviewer’s Comment #7: In the authors’ response to point #3, supplemental fig. 3 is\ncited (but should be supp. Fig 5 as in the response above)?\nResponse: We apologize for the oversight. The correct citation is Supplemental\nFigure 5, not Supplemental Figure 3. In the revised manuscript, this figure now\nappears as Supplemental Figure 7.\nReviewer’s Comment #8: We may be missing a figure panel, but the original Fig. 4A\nhas been replaced by a fluorescence immunohistochemistry panel lacking ARG1\nstaining, while the text states that it has been improved.\nResponse: We apologize for the confusion. To improve visualization, we used\nimmunofluorescence staining to detect ARG1, as presented in Figure 3E. We\nacknowledged the Reviewer’s concern that in earlier images, ARG1 signals\nappeared in multiple compartments, which could cause ambiguity. The revised\nimmunofluorescence staining now provides clearer localization of ARG1. Since\nARG1 is a well-established and specific marker of M2 macrophages, and its\nrecruitment to injury sites is well documented in tissue repair, we did not perform\nadditional co-staining with macrophage markers in this study. In Figure 4A, we\npresent immunofluorescence staining of F4/80 to show the dynamics of\nmacrophages during NAPH-induced injury repair with or without clodronate\nliposomes treatment (CL).\nReviewer’s Comment #9: In either the methods or text, a description of the total\nnumbers of animals, sex, and age analyzed per condition should be included.\nResponse: We thank the Reviewer for this valuable suggestion. We have now\nprovided detailed information on the total numbers of animals, along with their\nsex and age, analyzed for each condition. These details have been added to the\nMaterial and methods section (Page 12, Lines 390-394) as well as the\ncorresponding figure legends.\nMinor revision comments/suggestions for improvement\nFig 1D. Show individual panels with PGP, S100b fluorescence labeling so the reader\nmay assess expression level. This is not possible when the sub cellular distribution of\nantigens detected by primary antibodies overlap such as in the S100B/PGP9.5 panel.\nResponse: We thank the Reviewer for this constructive comment. We agree that\noverlapping subcellular localization of PGP9.5 and S100B may obscure\nindividual expression in the merged images. To address this, we have included\nseparate single-channel fluorescence panels for PGP9.5 and S100B in\nSupplementary Figure 3, which allow clearer evaluation of their expression levels\nand distribution.\nFig 2C. Please clearly explain what the denominator is in these proportions. It seems\nthat for the first plot, it is the proportion of PGP9.5+ cells which are S100B+. For the\nsecond and third plots, is it the proportion of total epithelial cells which are PGP9.5+\nor S100B+, respectively? If so, what marker was used for quantifying the epithelium\nbroadly? Please label both the axis and figure legend clearly with this information.\nResponse: We are grateful for the Reviewer’s helpful remark. In Figure 2C, the\nfirst plot shows the percentage of S100B+/PGP9.5+ cell among total airway\nepithelial cell identified by DAPI. This population markedly increased at day 2\nafter NAPH injury and declined during the repair phase. The second and third\nplots showed the percentage of PGP9.5+ only and S100B+ only cells,\nrespectively, again calculated relative to total airway epithelial cells labeled by\nDAPI. We have revised both the axis and Figure legend to clearly indicate this\ninformation.\nFig 2F. Please ensure to upload the relevant plot, as only labels appear in the\nmanuscript.\nResponse: We apologize for the oversight. The complete figure has now been re-\nuploaded.\nFig 6A vs. 6B. Please mention in the text the discrepancy between mRNA and protein\nlevels over the time course of injury repair. Specifically, why S100b mRNA levels are\nhigher in control mice at 5d and 7d post injury, but S100B protein levels are higher in\nGata5Cre mice at these same time points.\nResponse: We sincerely appreciate the Reviewer’s detailed observation. As\nsuggested, we have now addressed the discrepancy between S100B mRNA and\nS100B protein expression during the time course of injury repair in the revised\nmanuscript (Page 8, Lines 231–236). Specifically, while S100B mRNA levels\nwere higher in control mice at days 5 and 7 post-injury, S100B protein levels\nwere higher in P110*/*; Gata5-cre mice at the same time points. This divergence\nmay reflect post-transcriptional or post-translational regulation of S100B, which\nis not uncommon during epithelial stem cell-mediated repair.\nDuring\nregeneration, dynamic changes in mRNA stability, translation efficiency, and\nprotein turnover can result in a temporal mismatch between transcript and protein\nabundance, particularly for factors like S100B that influence progenitor cell\nactivation and differentiation. We have added this explanation to highlight the\npotential biological relevance of this observation in the context of airway stem\ncell-driven repair\nFig 6C. Please show quantification and description of numbers of mice analyzed, #\nlung regions, etc. The additional experimental method details are helpful for us to\ninterpret the data.\nResponse: We thank the Reviewer for this helpful comment. The request\ninformation, including the number of mice analyzed and lung regions quantified,\nhas been added to the Material and methods section and to the Figure legends for\nclarity.\nReviewer 2\nReviewer’s Comment: In response to the request to describe the total numbers of\nanimals, sex, and age analyzed per condition, the authors state that no differences to\n(naphthalene) NAPH-induced injury were observed between males and females [lines\n390-394]. However, no data are presented to demonstrate this and the authors only\nstudied females.\nThere are multiple published articles demonstrating gender differences in\nsusceptibility to NAPH-induced lung injury in mice including the two examples here:\nOliver et al., 2009"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the revisions of the reviewers, however, not all issues seem to have been resolved. Please address the\nfollowing points.\n1) The authors frequently show representative images, however, to increase transparency and interpretation of the findings,\nindividual datapoints must be shown (with a power calculation for all the in vivo experiments) with a quantitative analysis\nwith appropriate statistical testing. Otherwise, conclusions cannot be scientifically drawn. For instance, Figure 2B lacks\ntransparency of individual data points and statistical analysis, but this also holds true for the figures showing representative\nimages, also including for immunohistochemistry (which should also include staining controls).\nI still do not see the individual data points in the figures, these must be shown (also in all the bargraphs). I also am unable to\nsee any power calculations (Page 14, lines 402-405 do not show this).\n2) What markers were used to characterize the macrophages? This should be clearly shown including the flow cytometric\ngating strategy.\nNo flow cytometric gating strategy is provided for macrophage characterization. This should be shown as a supplemental\nfigure.\n3) To strengthen the mechanistic importance of anti-inflammatory macrophages, the authors could deplete macrophage in\nvivo and conduct adoptive transfer experiments with M1 vs M2 macrophages, with a robust read-out of lung injury with\nassociated analysis of the reparative capacity of PNECs.\nThe depletion efficiency of macrophages using clodronate liposomes should be shown in a supplemental figure (flow\ncytometric plots). Also the depletion data is not clearly described in figure 4A.", "reviewer_2": "Overall comments\nThis revision by Dai et al. is submitted in reference to the manuscript entitled, “S100B-regulated pulmonary neuroendocrine\ncells-macrophages interactions: a key to airway injury repair”. In this version, I appreciated the authors additions and overall,\nthe experiments in this study show that S100B may play a role in the inflammatory response of the airways in the\nnaphthalene airway injury model. However, the direct role of S100B on PNEC stem cell function is not sufficiently supported.\nWhile this version includes new and helpful data, there remains insufficient data to support several key conclusions.\nThe authors are proposing that a “neuroendocrine-like cell population” is functionally linked to PNEC-mediated stem cell\nactivity. In multiple locations throughout the manuscript, this “neuroendocrine-like cell population” is referred to as PNECs\nbut this requires additional data to support this statement. In addition, referring to this PGP9.5+ population as PNECs\nthroughout the manuscript is confusing. While mature PNECs (that co-express classic marker CGRP) and PGP9.5+ is well-\nestablished, the population of PGP9.5+ cells that co-expresses S100B during the course of injury and repair using the\nnaphthalene model is not clearly defined. Because there is no lineage tracing of the PGP9.5 positive population, it is unclear\nwhether the expanded population after injury represents a NE cell population.\nIn my comments to the authors’ responses to points (1) and (2) below, I cite specific examples from the manuscript text\nwhere there are important gaps in the data, thus limiting the interpretation and thus strength of the conclusion. My original\ncomments are also included below. My current responses are in bold and the reviewers’ responses to original review are in\nbrackets for reference.\n(1) This study by Dai et al., uses the well-established naphthalene (NAPH) injury model, which selectively ablates club cell\nand prior studies have examined the kinetics of airway injury and repair by lineage-labeling mature PNECs (Ouadah et al.,\n2019). It is known from these prior studies that only a small number of NE cells proliferate during the first week following\nNAPH injury (predicted 2-4 cells/NEB) and the clonal repair patches from NE stem cell activation are not seen until 2-3\nweeks post-injury. In prior studies, limited proliferation of PNECs was consistently observed during the first week post-\nNAPH injury, even when the highest doses of NAPH compatible with survival were given (~275 mg/kg). This manuscript\nuses 300 mg/kg so this should result in near complete club cell ablation based on prior studies. In Fig. 2A, the increased\nPGP9.5 expression is interpreted to be due to ‘increased proliferation of PNECs in response to the injury’ (line 124). Since\nthis observation contradicts what has been previously reported in careful lineage analysis of the kinetics of PNEC turnover\nusing a PNEC-specific driver (Ascl1CreER) in the mature mouse lung, additional data needs to be presented to support this\npoint. This is a critical observation for the major conclusion in this study. In addition, no direct evidence for PNEC\nproliferation was shown in any figure despite this being a key component of the model and interpretation of the data.\n(2) To clearly demonstrate co-expression patterns of S100B and exclusion from PGP9.5+ airway epithelial cells, recommend\nperforming quantification of histologic images to demonstrate the images shown (panel 1D) are representative of the entire\nlung. In addition, for the third panel which provides evidence of non-overlapping expression of S100B and PGP9.5\nexpression in the PNECs in non-injured lungs, it is difficult to ascertain expression of PGP9.5 without viewing each\nfluorescence channel independently. How did co-expression of PGP9.5 and S100B in clustered PNECs within\nneuroepithelial bodies change after injury? These clusters were the sites of identified and previously characterized NE\nproliferative and stem cell functions (Stevens et al., Am J. Physiol, 1997; Song et al., PNAS, 2012; Ouadah et al., Cell,\n2019).\n[Authors response “To ensure the representative nature of our findings, we have now examined more than 30 randomly\nselected sections from 8 mice, confirming that the patterns shown in Panel 1D reflect whole-organ expression trends.\nAdditionally, we have included independent fluorescence channel views in the revised Supplemental Figure 3 to clarify the\nco-expression relationships between S100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs”.]\nPlease clarify where the additional data from more than 30 selected sections across 8 mice are presented? This is important\nbecause the extent of NAPH-induced airway injury is variable by lobes and airway region so the number and level of\nairways analyzed per mouse is important to know (could add as a supplemental table corresponding to the data in figure\n2C). What % of the airway epithelium is denuded at 1d, 2d, 3d, and 5d post-injury in this model? This data would be\nvaluable because the authors explain in their response to item #3 below, that the kinetics of airway injury differ from\npreviously reported papers due to slightly younger age (6 weeks vs. 8-10 weeks old mice). Thus, establishing these\nbaseline injury data would be valuable for interpreting these data and comparing it to prior work in the field demonstrating\nNE stem cell activity. Fig. S3 NAPH panel shows nearly complete absence of CC10 (and possible other cells), though the\nDAPI is hard to see and there is no associated quantification.\nAlso, did the authors mean to refer to supplemental figure 5 in the rebuttal point #1 instead of supp. Fig. 3 to show the co-\nexpression relationships between S100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs? The\nmicrographs are low-resolution and insufficient to distinguish individual PNECs from neighboring airway epithelial cells,\nespecially within clustered regions where more than 50 cells may be intermingled. In these locations it is especially difficult to\ndistinguish PNECs from neighboring variant club cells, for example.\n[This pattern aligns with PGP9.5’s capacity as a pan-neuronal labeling that detects both classical PNECs and potentially\nnovel, injury responsive neuroendocrine-like cell populations]\nThe authors are proposing a population that a “neuroendocrine-like cell population” is functionally linked to PNEC-mediated\nstem cell activity. In multiple locations throughout the manuscript, this “neuroendocrine-like cell population” is referred to as\nPNECs and this is confusing. Because there is no lineage tracing of the PGP9.5 positive population, it is unclear whether\nthe expanded population after injury represents a NE cell population. Examples from the text where further clarification is\nrequired:\n[Lines 123-124. “Notably, the S100B+ PNECs population expanded markedly by day 2, coinciding with injury-induced\nPNEC proliferation”.]\nWhich data on PNEC proliferation and which population of PNECs (defined by which markers?) are the authors referring to?\nPlease either cite reference or figure data. Are authors referring to the PGP9.5+ population? If so, it is important to refer to\nthem more precisely and distinguish them from the classic NECs, whose stem cell functions have been clearly demonstrated\nin mouse models.\n[Lines 128-130. “These dynamic S100B expression patterns in PNECs during club cell regeneration suggest a potential\nfunctional link between S100B and PNEC- mediated stem cell activity in airway repair.”]\nThe authors response to critique #2 further states, [While we acknowledge the well-established role of NEB-localized\nPNECs in proliferative and stem cell functions (Stevens et al., 1997; Song et al., 2012; Ouadah et al., 2019), our current data\nhighlight a distinct, dispersed PGP9.5+ population that appears to participate in repair through mechanisms that may extend\nbeyond the classical Ascl1-dependent proliferation pathway observed in intact NEBs].\n[Lines 135-136. “NAPH+PTM-treated lungs exhibited distinct regeneration patterns. The airway surface was primarily\ncovered by PNECs, with minimal club cells present on day 3.”]\nThere is very limited direct data presented here to support the claim that injury-induced PGP9.5+ cells are PNECs that have\nstem cell activity. As we previously recommended, any data showing direct evidence of proliferation or confirmatory\nextended marker expression analysis to show expression of shared core NEC program genes would provide the minimum\nrequired data to support ‘non-classical Ascl1-dependent’ NECs.\n[Lines 142-143. “PTM-treated lungs showed a markedly reduced PNEC population on day 3 (57.13% vs 72.5% in control)\nand an increased population on day 5 (45.93% vs 32.20% in control).”]\nThe data corresponding to this result shown in Fig. 2A and the quantification in Fig. 2G is hard to interpret because the\nresolution is not high enough to distinguish individual PGP9.5 vs. S100B expressing cells. Showing close-up views at high-\nresolution would demonstrate how the manual quantification was performed.\n(3) Fig 2. Panel A. It is important to demonstrate S100B co-expression in PNECs using a specific PNEC marker. For\nexample, in adult mouse epithelium, calcitonin gene-related peptide (CGRP) is a well-established marker for which there is\nan abundance of published data on the expression patterns and co-expression of the PNEC transcription factor, ASCL1.\nThis additional data is critical because PGP9.5 is broadly expressed in the epithelium (as the authors show in Fig 1) and it is\npossible there is up-regulation of PGP9.5 expression in non-PNECs following NAPH exposure. Authors state (line 123) that\n‘..the S100B+ PNEC population expanded significantly on day 2, coinciding with the proliferation of PNECs in response to\nthe injury….The quantification of S100B+/PGP9.5+ cells in panel C was used to support the claim that PNECs upregulate\nS100B expression (corresponding to images in panel 2A). It is not clear that all the PGP9.5 cells are PNECs. This is critical\nto establish as downstream perturbations in S100B are non-lineage specific. Thus, the model proposed by the authors\ndepends on thorough validation that all the cells they have identified as PGP9.5+ are PNECs. What fraction of the PGP9.5\ncells co-express the established PNEC marker, CGRP? A week after injury, PNEC proliferation ceased in prior reports\n(Song et al., Ouadah et al. 2019) and all lineage-labeled PNECs continue to express CGRP. Thus the PNEC stem cell\nfunction during the first week was exclusively self-renewal and did not contribute to repairing surrounding epithelium. Given\nthat these findings contradict these reports, it is critical to establish that all PGP9.5+ cells are PNECs.\n[We appreciate the reviewer’s emphasis on rigorous PNEC marker validation and\nacknowledge the distinct biological roles of PGP9.5 (pan-neuroendocrine), CGRP (functional\nsubset), and Ascl1 (developmental) markers. While our choice of PGP9.5 was intentional to capture\ntransitional states observed in our injury model, we agree that co-expression studies with CGRP are\nessential to confirm PNEC identity. We are currently performing co-staining analyses to determine\nwhat fraction of PGP9.5+ cells co-express CGRP during repair, with preliminary data suggesting\nsome PGP9.5+ cells may represent activated states lacking CGRP expression (Supplemental Figure\n3). Please see our response to comment 2.\nRegarding the apparent discrepancy in repair kinetics, we note our studies used 6-week-old\nfemale C57BL/6 mice versus the 2-3-months-old mixed-sex cohorts in prior work (Ouadah et al.\n2019), which may account for our observation of faster (70-80% within 7 days) club cell restoration.]\nIn the authors’ response to point #3, supplemental fig. 3 is cited (but should be supp. Fig 5 as in the response above)?\n(4) Another major conclusion in this manuscript was that ARG1+ macrophages were recruited to injury sites and that S100B\ninhibition reduced recruitment of ARG1+ macrophages. To establish the location of ARG1+ cells, panel Fig 4A should also\ninclude co-localization of macrophage markers in situ. ARG1 expression appears in multiple cell compartments stained for\nARG1 (though this is hard to see clearly).\nWe may be missing a figure panel, but the original Fig. 4A has been replaced by a fluorescence immunohistochemistry\npanel lacking ARG1 staining, while the text states that it has been improved.\n(5) Experimental approach: The conclusions from using the Gata5-Cre mouse line to activate p110 within the endodermal\nlineage to test the response of the airway epithelium after airway injury is conceptually limited because Gata5 is expressed\nvery early during development (and reported within the mouse endoderm). However, earlier stages were not examined, and\nthe constitutive expression of activated p110 from early developmental stages may have globally altered the composition of\nthe airway epithelium. This limits the strength of conclusions in Fig. 6.\nThank you for the additional data showing the development and differentiation of other lineages are not affected in this\nmodel.\n(6) In either the methods or text, a description of the total numbers of animals, sex, and age analyzed per condition should be\nincluded.\nMinor revision comments/suggestions for improvement\nFig 1D. Show individual panels with PGP, S100b fluorescence labeling so the reader may assess expression level. This is\nnot possible when the sub cellular distribution of antigens detected by primary antibodies overlap such as in the\nS100B/PGP9.5 panel.\nFig 2C. Please clearly explain what the denominator is in these proportions. It seems that for the first plot, it is the proportion\nof PGP9.5+ cells which are S100B+. For the second and third plots, is it the proportion of total epithelial cells which are\nPGP9.5+ or S100B+, respectively? If so, what marker was used for quantifying the epithelium broadly? Please label both the\naxis and figure legend clearly with this information.\nFig 2F. Please ensure to upload the relevant plot, as only labels appear in the manuscript.\nFig 6A vs. 6B. Please mention in the text the discrepancy between mRNA and protein levels over the time course of injury\nrepair. Specifically, why S100b mRNA levels are higher in control mice at 5d and 7d post injury, but S100B protein levels\nare higher in Gata5Cre mice at these same time points.\nFig 6C. Please show quantification and description of numbers of mice analyzed, # lung regions, etc.\nThe additional experimental method details are helpful for us to interpret the data\nReviewer #3\n(Remarks to the Author)\nMost previous concerns have been addressed.", "reviewer_author_response": "We thank the Reviewers for their thoughtful and constructive feedback. We have\ncarefully addressed each comment and revised the manuscript accordingly. These\nrevisions have strengthened the clarity and rigor of our work, and we hope the\nchanges will be satisfactory.\nReviewer 1\nReviewer’s Comment #1: The authors frequently show representative images,\nhowever, to increase transparency and interpretation of the findings, individual\ndatapoints must be shown (with a power calculation for all the in vivo experiments)\nwith a quantitative analysis with appropriate statistical testing. Otherwise, conclusions\ncannot be scientifically drawn. For instance, Figure 2B lacks transparency of\nindividual data points and statistical analysis, but this also holds true for the figures\nshowing representative images, also including for immunohistochemistry (which\nshould also include staining controls).\nI still do not see the individual data points in the figures, these must be shown (also in\nall the bargraphs). I also am unable to see any power calculations (Page 14, lines 402-\n405 do not show this).\nReviewer 2\nReviewer’s Comment #1: Authors response “To ensure the representative nature of\nour findings, we have now examined more than 30 randomly selected sections from 8\nmice, confirming that the patterns shown in Panel 1D reflect whole-organ expression\ntrends. Additionally, we have included independent fluorescence channel views in the\nrevised Supplemental Figure 3 to clarify the co-expression relationships between\nS100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs.”\nPlease clarify where the additional data from more than 30 selected sections across 8\nmice are presented? This is important because the extent of NAPH-induced airway\ninjury is variable by lobes and airway region so the number and level of airways\nanalyzed per mouse is important to know (could add as a supplemental table\ncorresponding to the data in figure 2C). What % of the airway epithelium is denuded\nat 1d, 2d, 3d, and 5d post-injury in this model? This data would be valuable because\nthe authors explain in their response to item #3 below, that the kinetics of airway\ninjury differ from previously reported papers due to slightly younger age (6 weeks vs.\n8-10 weeks old mice). Thus, establishing these baseline injury data would be valuable\nfor interpreting these data and comparing it to prior work in the field demonstrating\nNE stem cell activity. Fig. S3 NAPH panel shows nearly complete absence of CC10\n(and possible other cells), though the DAPI is hard to see and there is no associated\nquantification.\nAlso, did the authors mean to refer to supplemental figure 5 in the rebuttal point #1\ninstead of supp. Fig. 3 to show the co-expression relationships between\nS100B/PGP9.5, S100B/CGRP, CGRP/PGP9.5 in non-injured and injured lungs? The\nmicrographs are low-resolution and insufficient to distinguish individual PNECs from\nneighboring airway epithelial cells, especially within clustered regions where more\nthan 50 cells may be intermingled. In these locations it is especially difficult to\ndistinguish PNECs from neighboring variant club cells, for example.\nReviewer 2\nReviewer’s Comment: In response to the request to describe the total numbers of\nanimals, sex, and age analyzed per condition, the authors state that no differences to\n(naphthalene) NAPH-induced injury were observed between males and females [lines\n390-394]. However, no data are presented to demonstrate this and the authors only\nstudied females.\nThere are multiple published articles demonstrating gender differences in\nsusceptibility to NAPH-induced lung injury in mice including the two examples here:\nOliver et al., 2009", "reviewer_pmcid": "PMC4514530, Gender differences in\npulmonary regenerative response to naphthalene-induced bronchiolar epithelial cell\ninjury) and Van Winkle et al (Am J Physiol Lung Cell Mol Physiol. 2002), Gender\ndifferences in naphthalene metabolism and naphthalene-induced acute lung injury.\nThus, an explanation of why no differences were observed is important and\nsupporting data should be included."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have sufficiently addressed my concerns. No further comments.", "reviewer_2": "The authors have modified the text to reflect the inherent limitations of the methods in this study and added additional\nquantification and figure modifications as recommended. However, the data are not completely reported for Comment #9.\nComment #9\nIn response to the request to describe the total numbers of animals, sex, and age analyzed per condition, the authors state\nthat no differences to (naphthalene) NAPH-induced injury were observed between males and females [lines 390-394].\nHowever, no data are presented to demonstrate this and the authors only studied females.\nThere are multiple published articles demonstrating gender differences in susceptibility to NAPH-induced lung injury in mice\nincluding the two examples here: Oliver et al., 2009", "reviewer_pmcid": "PMC4514530, Gender differences in\npulmonary regenerative response to naphthalene-induced bronchiolar epithelial cell injury) and Van Winkle et al (Am J\nPhysiol Lung Cell Mol Physiol. 2002), Gender differences in naphthalene metabolism and naphthalene-induced acute lung\ninjury.\nThus, an explanation of why no differences were observed is important and supporting data should be included."}, "author_response": ""}, {"round": 4, "reviewer_comments": {"reviewer_2": "The authors have provided additional data and explanation to support why differences in injury response may have been\nobserved. Thank you."}, "author_response": ""}]

Decision letter