Roles of lung-recruited monocytes and pulmonary Vascular Endothelial Growth Factor (VEGF) in resolving Ventilator-Induced Lung Injury (VILI)

Chin-Kuo Lin; Tzu-Hsiung Huang; Cheng-Ta Yang; Chung-Sheng Shi

doi:10.1371/journal.pone.0248959

. 2021 Mar 19;16(3):e0248959. doi: 10.1371/journal.pone.0248959

Roles of lung-recruited monocytes and pulmonary Vascular Endothelial Growth Factor (VEGF) in resolving Ventilator-Induced Lung Injury (VILI)

Chin-Kuo Lin ^1,², Tzu-Hsiung Huang ³, Cheng-Ta Yang ^4,⁵, Chung-Sheng Shi ^2,^6,^*

Editor: Heinz Fehrenbach⁷

PMCID: PMC7978382 PMID: 33740009

Abstract

Monocytes and vascular endothelial growth factor (VEGF) have profound effects on tissue injury and repair. In ventilator-induced lung injury (VILI), monocytes, the majority of which are Ly6C^+high, and VEGF are known to initiate lung injury. However, their roles in post-VILI lung repair remain unclear. In this study, we used a two-hit mouse model of VILI to identify the phenotypes of monocytes recruited to the lungs during the resolution of VILI and investigated the contributions of monocytes and VEGF to lung repair. We found that the lung-recruited monocytes were predominantly Ly6C^+low from day 1 after the insult. Meanwhile, contrary to inflammatory cytokines, pulmonary VEGF decreased upon VILI but subsequently increased significantly on days 7 and 14 after the injury. There was a strong positive correlation between VEGF expression and proliferation of alveolar epithelial cells in lung sections. The expression pattern of VEGF mRNA in lung-recruited monocytes was similar to that of pulmonary VEGF proteins, and the depletion of monocytes significantly suppressed the increase of pulmonary VEGF proteins on days 7 and 14 after VILI. In conclusion, during recovery from VILI, the temporal expression patterns of pulmonary growth factors are different from those of inflammatory cytokines, and the restoration of pulmonary VEGF by monocytes, which are mostly Ly6C^+low, is associated with pulmonary epithelial proliferation. Lung-recruited monocytes and pulmonary VEGF may play crucial roles in post-VILI lung repair.

Introduction

Mechanical ventilation is essential for the life support of patients with respiratory failure. Nevertheless, it has potential drawbacks that often induce lung damage and lead to ventilator-induced lung injury (VILI). Earlier studies have explored the pathophysiological mechanisms of VILI occurrence and identified inflammatory mediators and cytokines that participate in the initiating insult [1–3]. However, few studies have focused on the temporal expression patterns of inflammatory cytokine and growth factor expression during resolving from VILI and elucidated their associations with subsequent lung repair. Many growth factors are involved in the process of epithelial repair of injured lungs [4]. These are chiefly members of the epithelial growth factor and fibroblast growth factor families. However, it is uncertain whether vascular endothelial growth factor (VEGF) contributes to tissue repair. As one of the crucial angiogenic growth factors, VEGF promotes not only the proliferation of vascular endothelial cells but also maintains survival and regulates the proliferation of alveolar epithelial cells in injured lungs [1, 5–7]. In hyperoxia-induced acute lung injury (ALI), treatment with VEGF enhances healing of lung structure during late recovery [8]. Nevertheless, with respect to VILI, the contribution of pulmonary VEGF to the repair of injured lungs has not been investigated.

Circulating monocytes are derived from the mononuclear phagocytic cells population in the bone marrow. Monocytes, the heterogeneous cells that perform various physiological activities, can be divided into two subsets [9]. In mice, monocytes are classified according to the differential expression of surface markers, including CCR2, CD62L, and CX₃CR1 [10]. Mouse monocytes that express CCR2 are known as inflammatory monocytes, while those not expressing CCR2 are termed resident monocytes. Gr1, as well as Ly6C, which is part of the Gr1 epitope, are additional markers of CCR2⁺ monocytes [10, 11]. The different physiological activities of the monocytes in the vasculature are closely linked to the Gr1 expression (Ly6C) [12–14]. Monocytes play dual roles in tissue injury and repair of the heart and liver [15, 16]. Ly6C^+high monocytes participate in injury progression, whereas Ly6C^+low monocytes help tissue healing [15, 16]. In VILI, high-stretch mechanical ventilation promotes pulmonary margination of activated Gr1^high monocytes and contributes to the lung injury progression [17]. Our earlier study showed that the monocytes recruited to the injured lungs are primarily Ly6C^+high and can produce VEGF to regulate vascular permeability during the first couple of hours following VILI [18]. However, the phenotypes of lung-recruited monocytes during recovery from VILI have not been clarified.

In this study, we explore the post-VILI expression patterns of inflammatory cytokines and growth factors, and identify the phenotypes of monocytes recruited to the lungs during recovery from VILI to elucidate how monocytes recruited to lungs and pulmonary VEGF benefit epithelial proliferation.

Materials and methods

Animals and two-hit model of VILI

All experimental protocols using animals were performed following the Guide to the Care and Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital, Chiayi, Taiwan (Approval number: 2012102301 (September 21, 2013)). All animal experiments were performed in the Animal Center of Chang Gung Memorial Hospital at Chiayi, and mice were housed following IACUC, national, and international animal welfare protocols. The center introduced the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) certification system in 2013 and had obtained AAALAC certification in July 2014 (No: TTQ08384). To optimize environmental conditions and reduce stress, animals were housed in a room maintained with low noises and vibrations and under constant temperature (20–25°C), humidity (40–60%) and a 12‐h light/dark cycle; cage occupancy was limited to five mice per cage, where animals were provided with enrichment toys and given free access to food and water. Male C57BL/6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). A two-hit VILI model was used, according to a previously established protocol [18, 19]. The model consisted of the first hit of a subclinical dose of lipopolysaccharides (LPS) to prime circulating inflammatory cells and the second hit of a high tidal volume (HTV) mechanical ventilation to generate VILI. Briefly, mice were anesthetized using intraperitoneal Zoletil 50 (80 mg/kg; Tiletamine-Zolazepam, Virbac, Carros CEDEX, France) and received a single intravenous tail vein injection of 20 ng LPS (O111B4; Sigma-Aldrich, St Louis, MO, USA) immediately before mechanical ventilation [17]. The mice were intubated by inserting a sterile 20-gauge intravenous cannula through the vocal cords into the trachea and ventilated with a SAR-830 series small animal ventilator with multi-animal setups (CWE Inc., Ardmore, PA, USA) in room air and under general anesthesia with regular intraperitoneal injections of Zoletil 50 (10 mg/kg/h) for 4 h. The ventilator was set at a HTV of 40 mL/kg to produce high-stretch mechanical ventilation with an inspiratory time of 0.3 s, a respiratory rate of 80 breaths/min, and end-expiratory pressure (PEEP) of 0 cm H₂O. After ventilation, the day 0 mice were sacrificed immediately after administering an overdose of anesthetic (Zoletil 50, 50 mg/kg), while the others were extubated to allow resumption of spontaneous breathing and recovery. The extubated mice received post-treatment care following the IACUC guidelines. We checked the mice every day and measured their body weight twice per week. A full‐time veterinarian took care of animal health and well-being until the mice were sacrificed with an overdose of anesthetic on days 1, 3, 7, or 14 after VILI. Mouse lungs were dissected and prepared for further experiments. The numbers of animals used in the experiments and the numbers of animals that died during mechanical ventilation or recovery are detailed in S1 Fig. The number of animals used per group was as follows: normal control (n = 6), day 0 (n = 6 ), day 1 (n = 6), day 3 (n = 6), day 7 (n = 12, six animals were used for studying the depletion of pulmonary monocytes), and day 14 (n = 12, six animals were used for studying the depletion of pulmonary monocytes).

Histopathology and immunohistochemistry analysis

For histopathology, mouse lungs were manually inflated and fixed by injecting 10% buffered formalin and embedded in paraffin. Tissues were then sliced (4 μm) and stained with hematoxylin and eosin (H&E). The number of inflammatory cells in the alveoli was determined by counting 100 randomly sampled alveoli under a light microscope at a magnification of 400×. For immunohistochemistry (IHC) analysis, formalin-fixed, paraffin-embedded tissue sections were incubated overnight at 4°C with anti-VEGF antibodies (VEGF A-20: sc-152, 1:40; Santa Cruz Biotechnology, USA) or anti-Ki67 antibodies (ab15580, 1:100; Abcam, USA) using DAKO antibody dilution buffer (DAKO). The tissue sections were then treated with tetramethylrhodamine isothiocyanate-conjugated (Jackson ImmunoResearch Laboratories) or horseradish peroxidase (HRP)-conjugated secondary antibodies (DAKO) in the same buffer for 1 h at room temperature. In the tissue sections treated with HRP-conjugated antibodies, 3, 3’ diaminobenzidine (DAB) (DAKO) was added as the chromogen; hematoxylin or 4’,6-diamidino-2-phenylindole (DAPI) (DAKO) was used to counterstain the nuclei. To measure the correlation between pulmonary VEGF expression and epithelial cell proliferation, the number of Ki67-positive cells was counted under a light microscope (Olympus BX51) at a magnification of 400× in 20 consecutive alveoli, and the staining intensities of positive VEGF secretions were analyzed using Pax-It quantitative image analysis software (Paxcam). Images were standardized for light intensity. For H&E analysis, the number of animals used per group was as follows: normal control (n = 4), day 0 (n = 3), day 1 (n = 3), day 3 (n = 3), day 7 (n = 5), and day 14 (n = 3). For IHC analysis, the number of animals used was n = 6 for each group.

Preparation of lung single cells and flow cytometric analysis

Lungs were dissected, rinsed in a petri dish containing PBS (pH 7.2) on ice, and cut into pieces of approximately 1–2 mm². The tissues were milled by mechanical disruption and incubated in tubes with a digestion solution containing Dispase II and DNase I (Sigma) in a water bath at 37°C for 45 min. The digested lung tissues were filtered through a 100-μm cell strainer and then forced through a 40-μm filter. The cell suspension was treated with 10× RBC lysis buffer (BioLegend) to remove RBCs and suspended in 1 mL staining buffer. Before flow cytometry, the cells in suspension were counted using a hemocytometer. Cells (10⁵−10⁶) in 100 μL were dispensed into each tube. Cells were stained with rat anti-mouse CD11b FITC (eBio 11‐0112), rat anti-mouse F4/80 PE (eBio 12‐4801), and rat anti-mouse Ly6C APC (eBio 17-5932-82) antibodies. The specimens were incubated at 4°C for 40 min in the dark. The stained cells were analyzed using a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA) and the acquired data were analyzed using FlowJo software (Treestar, Ashland, OR, USA). A FACSCanto instrument and FACSDiva software (Becton Dickinson, San Jose, CA) were used for flow cytometric analysis, and the acquired data were analyzed with FlowJo software (Treestar, Ashland, OR, USA). For flow cytometric analysis, the number of animals used per group was as follows: normal control (n = 4), day 0 (n = 4), day 1 (n = 4), day 3 (n = 4), day 7 (n = 5), and day 14 (n = 5).

Quantitation of pulmonary growth factors and cytokines

The concentrations of VEGF, transforming growth factor-beta (TGF-β), interleukin 6 (IL-6), interleukin 1-beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) expressed by lung cells were measured by performing ELISA using the following commercial kits: mouse VEGF Quantikine ELISA Kit, cat# MMV00; mouse/rat/porcine/canine TGF-β Quantikine ELISA Kit, cat# MB100B; mouse IL-1β/IL-1F2 DuoSet ELISA, cat# DY401-05; and mouse TNF-α DuoSet ELISA, cat# DY410-05 (R&D Systems, Minneapolis, MN, USA) and mouse IL-6 ELISA Ready-SET-Go, cat#: 88–7064 (eBioscience, San Diego, CA, USA). ELISA plates were read on a multimode plate reader (BioTek, Winooski, VT, USA). For ELISA, the number of animals used per group was as follows: normal control (n = 4), day 0 (n = 4), day 1 (n = 4), day 3 (n = 4), day 7 (n = 6), and day 14 (n = 6).

Isolation of lung-recruited monocytes

For PCR experiments, lung-recruited monocytes were obtained by negative selection and isolated using an EasySep Mouse Monocyte Isolation Kit (Stemcell Tech) and EasySep magnet following the manufacturer’s instructions. Briefly, lung cells were suspended and prepared at a density of 1 × 10⁸ cells/mL in PBS/5% FBS. Cells were incubated in a mixture of 50 μL/mL normal rat serum and 50 μL/mL EasySep Mouse Monocyte Enrichment Cocktail at 4°C for 15 min. Cells were then washed with RoboSep Buffer and centrifuged at 300 ×g for 10 min at 4°C. The cell pellet was resuspended at the same density and incubated under the same conditions as described above. Subsequently, cells were mixed with EasySep D Magnetic Particles at 150 μL/mL cells and incubated at 4°C for 10 min. The cell suspension volume was adjusted to 2.5 mL with EasySep Buffer without rat serum. Finally, the centrifuge tube was placed into an EasySep Magnet for 5 min.

Subsequently, the supernatant, in which cells were enriched, was transferred to a new tube, centrifuged at 300 × g for 10 minutes, and resuspended at 1×10⁸ cells/mL in the recommended medium. The cell suspension was mixed and incubated with species-specific FcR blocking antibody for mouse cells at 10 μL/mL and anti-macrophage antibody [BM8] at a final concentration of 0.3–3.0 μg/mL at room temperature for 15 min. EasySep PE Selection Cocktail was added to a concentration of 100 μL/mL cells and incubated at room temperature for 15 min. EasySep magnetic nanoparticles were added to a concentration of 50 μL/mL cells and incubated at room temperature for 10 min. The cell suspension was adjusted to 2.5 mL by adding the recommended medium. The tube was placed into the magnet and set aside for 5 min. The desired fraction was collected into a new 5 mL polystyrene tube contained monocytes.

RT-PCR analysis of VEGF mRNA

Total RNA was extracted from the isolated monocytes using TRIzol reagent (Invitrogen, Life Technologies) following the manufacturer’s protocol. Five nanograms of total RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen, Life Technologies) and following the manufacturer’s protocol. Real-time PCR was performed to determine VEGF mRNA expression levels, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal control, on a BioRad iQ iCycler Detection System (BioRad Laboratories, Ltd) using SYBR green fluorophore. The primer sequences used in this experiment were as follows:

VEGF forward: 5’-ATCATGCGGATCAAACCTCACCA-3’

VEGF reverse: 5’-TCACCGCCTTGGCTTGTCACAT-3’

GAPDH forward 5’-CTCACTGGCATGGCCTTCCGTGT-3’

GAPDH reverse: 5’-GATGTCATCATATTTGGCAGGT-3’.

Reactions were performed in a total volume of 25 μL, including 12.5 μL 2× QuantiFast SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μL of each primer (10 mM), and 0.5 μL of reverse-transcribed cDNA. PCR was performed for 10 min at 95°C to activate the enzyme, denaturation for 10 s at 95°C, annealing for 15 s at 60°C, and extension for 10 s at 72°C for 50 cycles. Relative quantification was conducted using the ΔΔCt method. Results were normalized to the amplified housekeeping gene GAPDH. For RT-PCR analysis, the number of animals used per group was as follows: normal control (n = 4, day 0 (n = 4), day 1 (n = 4), day 3 (n = 4), day 7 (n = 5), and day 14 (n = 5).

Depletion of pulmonary monocytes

Monocytes were depleted using liposomal clodronates, Clophosome-A (FormuMax Scientific). Some of the mice that were sacrificed on day 7 were injected on day 3 with a single 200 μl dose of Clophosome-A in the tail vein after administering anesthesia with intraperitoneal Zoletil 50 (80 mg/kg); some of the mice that were sacrificed on day 14 received the same dose of Clophosome-A on day 3 and were reinjected with 100 μL dose after the anesthesia on days 7 and 11. Mouse lungs were isolated for VEGF ELISA, as described above.

Statistical analysis

Statistical evaluations were performed using PASW Statistics version 22. Data were expressed as means ± SD. The normality of the data was examined by Shapiro-Wilk test. For comparisons between two groups, data were analyzed by unpaired Mann–Whitney U test. For comparisons between multiple groups, one-way ANOVA with Bonferroni’s or Dunnett’s post hoc test or Kruskal-Wallis test with corrections by Bonferroni for multiple testing was used as appropriate. Spearmen’s correlation was used to evaluate the relationship between the IHC intensity of VEGF and the number of Ki67-positive cells in the alveolar epithelium of lung sections. Statistical significance was defined as p <0.05. Detailed illustrations of the statistical analysis are provided in the S1 Text.

Results

Abundance of inflammatory cells in lung tissues following VILI

C57BL/6 mice that had received HTV ventilation after LPS treatment were used to determine how VILI influenced the recruitment and abundance of inflammatory cells in lung tissues. H&E staining revealed that immediately after VILI (day 0), the number of inflammatory cells in the injured lung tissues increased and was significantly higher than that in the lung tissues of the normal control group (45 ± 3 vs. 6 ± 1 cells per 100 alveoli, respectively; p < 0.001, Fig 1). The number of inflammatory cells peaked on day 1 after VILI (53 ± 1 cells per 100 alveoli; p < 0.001 compared to the normal control group, and p < 0.01, compared to day 0 and day 3 groups, Fig 1). The number of inflammatory cells gradually decreased from day 3 to day 14, but remained higher than that of the normal control group (39 ± 4 to 14 ± 2 cells per 100 alveoli, p < 0.01, Fig 1).

Fig 1 — C57BL/6 mice received 20 ng LPS intraventricularly and HTV ventilation to produce a two-hit VILI. The mice were sacrificed immediately after VILI (day 0) or on days 1, 3, 7, or 14 after VILI. (A) H&E staining of the lung sections of mice sacrificed at different time points (original magnification: 200×). The closed arrow indicates lung-recruited inflammatory cells, including neutrophils and monocytes. (B) Quantification and comparison of the inflammatory cells in lung sections from individual mice sacrificed at different time points. Scale bar = 200 μm. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test. Data are expressed as means ± SD; n = 3–5 per group, * p< 0.01 vs. normal control (C) and ^#p < 0.01 vs. day 0 and day 3.

Phenotype of monocytes recruited to the lungs during the recovery phase following VILI

Flow cytometry was conducted to identify leukocytes recruited to the lungs and determine the phenotypes of monocytes during recovery after VILI. Lung-recruited leukocytes, which express CD11b, were selected from the lung single-cell suspension (Fig 2A). Monocytes in this leukocyte population were further selected based on the expression of F4/80. Finally, the monocytes were gated and divided into two populations based on the expression intensity of Ly6C. We found that the number of CD11b⁺ leukocytes recruited to the injured lungs increased following VILI and reached a peak level that was significantly higher than that of the normal control group, on day 1 after VILI (4.5 × 10⁶ ± 6.3 × 10⁵ vs. 2.8 × 10⁴ ± 1.7 × 10⁴ leukocytes per lung; adjusted p = 0.005, Fig 2B and 2C). However, the number of CD11b⁺ leukocytes declined and returned to the baseline after day 3 (9.7 × 10⁴ ± 4 × 10⁴ leukocytes per lung; adjusted p = 1, compared to the normal control group, Fig 2B and 2C). During recovery, from day 1 to day 14 after VILI, most monocytes recruited to the lungs were Ly6C^+low (Fig 2B and 2D).

Fig 2 — (A) Gating strategy for differentiating CD11b⁺ leukocytes and Ly6C⁺ monocyte subsets. CD11b⁺ population (G1) was gated for estimation of lung-recruited leukocytes (neutrophils and monocytes). In the G1 population, the F4/80 positive population, which represented monocytes, was gated further and divided into the Ly6C^+high (G2) and the Ly6C^+low (G3) population. (B) The time course of lung-recruited leukocytes and the Ly6C phenotypic change of lung-recruited monocytes immediately after VILI (day 0) or on days 1, 3, 7, and 14 after VILI. (C) Quantification and comparison of lung-recruited CD11b⁺ leukocytes of individual mice sacrificed at different time points. Statistical analysis was performed using the Kruskal-Wallis test with Bonferroni correction for multiple tests. Data are expressed as means ± SD; n = 4–5 per group, *p < 0.05, vs. normal control (C). (D) The percentages of pulmonary Ly6C^+high and Ly6C^+low monocytes of individual mice sacrificed at different time points. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test. Data are expressed as means ± SD; n = 4–5 per group, *p < 0.001 and ^#p = 0.002, vs. day 0.

Time courses of pulmonary growth factor and inflammatory cytokine expressions during recovery from VILI

The expression of growth factors and inflammatory cytokines in lung tissue lysates following VILI were determined by ELISA. Following VILI, the concentration of interleukin 6 (IL-6) increased from the baseline of 586.2 ± 189.6 pg/g to 15817.5 ± 703.5 pg/g (p = 0.006; adjusted p = 0.125, Fig 3A). However, the level decreased by 55% to 7170.8 ± 151.3 pg/g on day 3 after VILI (Fig 3A) and returned to the baseline on day 7 and 14 (1414.9 ± 900.9 and 399.5 ± 158 pg/g, respectively; adjusted p = 1 for both comparisons against the normal control group, Fig 3A). Interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) also showed similar expression patterns as IL-6. IL-1β concentration increased from 1485.4 ± 988 to 8861.8 ± 517.9 pg/g (adjusted p = 0.008, Fig 3B) and TNF-α concentration increased from 21293.8 ± 2792.6 to 52728.1 ± 4400.4 pg/g (p < 0.001, Fig 3C) on day 1 after VILI but gradually decreased from day 1 on and reached baseline levels on days 7 and 14 (Fig 3B and 3C).

Fig 3 — ELISA for inflammatory cytokines and growth factors in the lung tissue, (A) interleukin-6 (IL-6), (B) interleukin-1 beta (IL-1β), (C) tumor necrosis factor-alpha (TNF-α), (D) tumor growth factor-beta (TGF-β), and (E) vascular endothelial growth factor (VEGF). Mice were sacrificed immediately after VILI (day 0) or on days 1, 3, 7, or 14 after VILI. Statistical analysis was performed using parametric (one-way ANOVA with Bonferroni’s or Dunnett’s post hoc test) or nonparametric (Kruskal-Wallis test with Bonferroni correction) tests. Data are expressed as means ± SD; n = 4–6 per group, p < 0.05 for * vs. normal control (C), ⁺ vs. day 0, ^# vs. day 1 and ** vs. day 14.

Unlike the cytokines, the growth factors tested, tumor growth factor-beta (TGF-β) and VEGF, exhibited a different temporal pattern of expression. TGF-β concentration did not change significantly immediately after VILI at day 0 and remained unchanged until day 1 post-VILI (Fig 3D). VGEF concentration showed a non-significant decrease from the baseline (8190.2 ± 505.6 to 5276.6 ± 95.2 pg/g, p = 0.169 and adjusted p = 1, Fig 3E) on day 1 after VILI. On day 3, the concentrations of both growth factors increased compared to day 1 and continued to increase progressively until day 14. On day 14, TGF-β concentration increased to 11542.3 ± 117.2 pg/g (adjusted p = 0.009, compared to the normal control group, Fig 3D) and VEGF concentration reached 13755 ± 138.5 pg/g (p = 0.005; adjusted p = 0.099, Fig 3E) compared to the normal control group. Both concentrations were significantly higher than those at day 0 and day 1 (Fig 3D and 3E).

Correlation between pulmonary VEGF and alveolar epithelial repair

To assess the role of pulmonary VEGF in post-VILI alveolar epithelial repair, we performed IHC staining to estimate the expression of VEGF and the number of Ki67-positive cells in the alveolar epithelium of lung sections and evaluated the correlation between each measure (Fig 4A and 4B). Consistent with the ELISA results, the relative staining intensity of VEGF decreased by 69% from the baseline (252.8 ± 25.9 to 77.3 ± 7.3) on day 1 after VILI, although this difference did not reach statistical significance (p = 0.005 and adjusted p = 0.075, Fig 4C). The levels rebounded on day 3, and the relative intensity of VEGF staining increased to a level significantly higher on days 7 and 14 than on day 1 (991.8 ± 119.9 and 2165.8 ± 245.5; adjusted p = 0.001 and <0.001, respectively, Fig 4C). Similar to the expression pattern of VEGF, the number of Ki67-positive cells decreased by 44% from the baseline (87 ± 5 cells per 100 alveoli to 48 ± 5 cells per 100 alveoli) on day 1 after VILI, however, this decrease was not statistically significant (p = 0.018 and adjusted p = 0.276, Fig 4D); levels of Ki67-positive cells were higher on day 3 (84 ± 5 cells per 100 alveoli) and reached significantly higher levels on days 7 and 14 than observed on day 1 (106 ± 4 and 216 ± 36 cells per 100 alveoli; adjusted p = 0.003 and < 0.001, respectively, Fig 4D). There was a strong correlation between the IHC relative staining intensities for VEGF and the number of Ki67-positive cells in the alveolar epithelium (Spearmen’s correlation coefficient rho = 0.93, p < 0.001, Fig 4E).

Fig 4 — C57BL/6 mice received 20 ng LPS intraventricularly and HTV ventilation to produce a two-hit VILI. The mice were sacrificed immediately after VILI (day 0) or on day 1, 3, 7, or 14 after VILI. (A) Immunocytochemistry (IHC) for VEGF in mouse lung sections. (B) IHC for Ki67-positive cells of the alveolar epithelium in mouse lung sections. The closed arrow indicates Ki67-positive cells in the alveolar epithelium. (C-D) Histometric analysis of the time courses of VEGF expression and the number of Ki67-positive cells in the lung alveolar epithelium. Scale bar = 200 μm. Statistical analysis was performed using the Kruskal-Wallis test with Bonferroni correction. Data are expressed as means ± SD; n = 6 per group, p < 0.05 ⁺ vs. day 0 and ^# vs. day 1; normal control (C). (E) Correlation between the VEGF relative staining intensity and the number of Ki67-positive cells per 100 alveoli (Spearmen’s correlation coefficient rho = 0.93, p < 0.001).

Contribution of monocytes to pulmonary VEGF production during recovery from VILI

In the normal lung, pulmonary VEGF is mainly derived from alveolar epithelial cells [20]. However, large amounts of alveolar epithelial cells are destroyed during VILI, which reduces their contribution to pulmonary VEGF restoration during the recovery phase. To elucidate the role of lung-recruited monocytes in post-VILI restoration of pulmonary VEGF, we analyzed VEGF mRNA expression in lung-recruited monocytes following VILI, calculated the relative change in mRNA, and compared to the untreated control. We found that VEGF mRNA expression decreased immediately following VILI (0.19 ± 0.05 fold as compared to normal control at day 0; p = 0.009, Fig 5) and further decreased by 96% relative to the normal control on day 1 after VILI (0.04 ± 0.02 fold; p = 0.008, Fig 5). However, levels gradually returned to the baseline after day 7 (0.68 ± 0.22 fold; p = 0.32, Fig 5). The temporal expression pattern was consistent with that of pulmonary VEGF protein, as determined by ELISA. To ascertain whether monocytes are a cellular source of pulmonary VEGF during recovery from VILI, we depleted monocytes from mice using liposomal clodronates from day 3 after VILI, when the pulmonary VEGF began to restore. The depletion significantly suppressed the pulmonary VEGF restoration, which declined by 24% on day 7 (12556 ± 567.5 vs. 9544.4 ± 1069.9 pg/g; p = 0.002, Fig 6) and 47% on day 14 (13755 ± 138.5 vs. 7301.9 ± 1602.8 pg/g; p = 0.002, Fig 6).

Fig 5 — mRNA expression was presented as the relative change versus untreated control. Statistical analysis was performed using ANOVA. Data are expressed as means ± SD; n = 4–5 per group, *p < 0.05, vs. normal control (C).

Fig 6 — Mice treated with clodronate for depleting pulmonary monocytes underwent the same protocol as the two-hit VILI. The mice sacrificed on day 7 after VILI were injected with a single 200 uL dose of Clophosome-A intravenously on day 3 after VILI. The mice sacrificed on day 14 after VILI received the same treatment of Clophosome-A on day 3 and reinjection of 100 uL on days 7 and 11 after VILI. VEGF protein concentration in lung tissue was determined by ELISA. Data are expressed as mean ± SD; n = 6 per group, *p = 0.002, compared between without and with clodronate treatment groups using Mann-Whitney U Test.

Discussion

We used the two-hit model of lung injury to investigate the roles of VEGF and lung-recruited monocytes in post-VILI lung repair. The main findings of this study are as follows. First, during recovery from VILI, a majority of lung-recruited monocytes are Ly6C^+low. Second, the expression of growth factors, such as VEGF and TGF-β, in injured lungs increases progressively during recovery from VILI, while inflammation fades, as determined from the decreasing levels of IL-6, TNF-α, and IL-1β. Moreover, VEGF expression in lung tissue positively correlated with the expression of Ki67, a cell proliferation marker, in alveolar epithelial cells. Third, monocytes are a cellular source of VEGF and the depletion of monocytes suppresses the post-VILI restoration of pulmonary VEGF.

In patients on ventilator support, bacterial infection, as one of the activators, can activate circulating inflammatory cells. In the current study, a two-hit mouse model was used to mimic the clinical scenario, a subclinical dose of LPS was used to prime circulating inflammatory cells but not damage the alveolar epithelial barrier, and a setting of HTV resulting in VILI [17, 21, 22]. There remains a substantial difference between live bacterial infection and LPS treatment in terms of cytokine expression profiles [23, 24]. The overexpression of inflammatory cytokines in mice exposed to either LPS or peritoneal contamination and infection recovers after 72 h. However, cecal ligation and puncture treatment induce comparatively fewer effects with a more protracted inflammation course. Considering the experimental complexity and difficulty, the LPS model is more suitable and proper for investigating acute inflammation and subsequent tissue repair, as in our study.

This study found that Ly6C^+low monocytes are recruited to the lungs during recovery from VILI. The roles of monocytes in inflammatory tissue vary depending on their surface Gr1 (Ly6C) expression. The Gr1⁺ (Ly6C⁺) subset of monocytes recruited to inflamed tissues participates in inflammatory responses [10, 25]. In contrast, the Gr1^- (Ly6C^-) subset of monocytes, which surveys the vasculature, is involved in tissue repair [13, 14]. Nahrendorf et al. [15] discovered the biphasic responses and dual roles of monocytes in myocardial injury, in which Ly6C^+high monocytes, which dominate during the early stage after myocardial infarction, exhibit inflammatory functions, and Ly6C^+low monocytes, which become more abundant during the healing phase, attenuate inflammation. In ALI, pulmonary macrophages are present as diverse populations that mediate acute lung inflammation and resolution separately [26]. Pulmonary macrophages are derived from circulating monocytes [27]. Circulating monocytes primed by endotoxemia and recruited to the lungs can also predispose the lungs toward acute injury [21, 25]. In VILI, mechanical stress of HTV ventilation contributes to injury by upregulating intrapulmonary cytokines expression [28]. The lung-recruited monocytes, which mainly express Gr1^high, can be further activated during HTV ventilation and promote the progression of VILI [17, 29]. As in myocardial injury, we found that the biphasic monocyte response also exists in mouse VILI, where Ly6C^+high monocytes dominate during the initiation of VILI, and Ly6C^+low monocytes become more abundant during recovery from VILI. These findings suggest that Ly6C^+low monocytes may play a crucial role in resolving VILI.

During recovery from the VILI insult, the temporal expression of VEGF and TGF-β follows an opposite trend to that of inflammatory cytokines. The concentration of VEGF and TGF-β in the injured lungs increased progressively and was significantly higher than the baseline and those in the first 24 h after injury. To the best of our knowledge, most of the previous studies focused on the expression and contribution of inflammatory cytokines during the early inflammatory phase of lung injury. Few of the previous studies addressed the subsequent temporal expression pattern and explored the roles of inflammatory cytokines and growth factors during lung healing. However, it is of paramount importance to understand the temporal expression pattern of inflammatory cytokines and growth factors during the recovery phase after lung injury, which helps in elucidating the mechanism underlying lung repair. TNF-α, IL-1β, and IL-6, the proinflammatory cytokines, are known to be involved in VILI [30]. In the early phase of VILI, Wilson et al. [17] found that TNF-α expression was rapidly upregulated in bronchoalveolar lavage fluid (BALF) after 2 h of HTV. Our previous study based on a two-hit VILI mouse model also showed that the elevated IL-6 and TNF-α concentrations in BALF and serum peaked at 4–6 h after HTV ventilation [18]. In acid-induced ALI, Patel et al. [31] found that the TNF-α and IL-6 in BALF peaked between days 1 and 3, followed by a return to normal levels over days 5 to 10. All the above studies explored the time course of inflammatory cytokines in BALF but not in injured lung tissue. To understand the expression of inflammatory cytokines and growth factors in injured lungs, we investigated the temporal expression patterns of both inflammatory cytokines and growth factors in lung tissue lysates of mice subjected to VILI. We found that VILI-induced elevation in IL-6, IL-1β, and TNF-α levels in the lung tissue lysates peaked between days 0 and 1 and returned to the baseline on day 7 after VILI. In contrast, the concentrations of VEGF and TGF-β in lung tissue lysates decreased immediately after VILI, but progressively increased and remained at a higher level than the baseline on day 14 after VILI. The findings of VEGF expression in lung tissue following VILI are different from those of VEGF expression in BALF and serum reported in our previous study [18]. However, these findings are consistent with the results obtained in the oleic-acid-induced ALI of Guo et al. [32], where the expression of VEGF increased in BALF and serum but decreased in the lung tissue [32]. Our findings can be explained by a proposed mechanism of VEGF downregulation in lung tissue during ALI, in which alveolar epithelial cells and endothelial cells, the constitutive source of VEGF in the lungs, are reduced in number due to direct lung injury [33, 34].

The augmented expression of VEGF and TGF-β in the injured lungs highlighted the roles played by VEGF and TGF-β in post-VILI lung repair. Proteins belonging to epidermal growth factor and fibroblast growth factor families are involved in repairing injured lung epithelium [4]. VEGF, besides its well-known angiogenetic properties, acts as a potent lung epithelial mitogen involved in resolving lung injury [35–38]. Studies have suggested that VEGF plays an important role in recovery from ALI, but the findings remain debatable [33, 39–42]. The ambiguous results may be due to the variation in VEGF obtained from different sites at different time points after ALI. This implies that the effects of VEGF vary with the affected cells in the lungs and depend on the time points evaluated after ALI. VEGF receptor signaling is required for the maintenance of alveolar structures in normal rat lungs, and also regulates the proliferation and apoptosis of injured alveolar epithelial cells in an autocrine or paracrine manner [7, 43, 44]. Acute respiratory distress syndrome (ARDS) is the most severe form of ALI. Medford et al. [45] found that VEGF expression was significantly upregulated in late ARDS after day 7 compared to both normal subjects as well as early ARDS within 48 hours. Pneumonia is one of the direct causes of ARDS. Strouvalis et al. [46] showed an association between increased concentration of circulating VEGF and resolution of ventilator-associated pneumonia (VAP). The findings of their animal-based experiments provide evidence that VEGF plays a role in the resolution of lung injury starting from 5 days post- lung injury. In the present study, we analyzed the expression of VEGF and the number of Ki67-positive cells in the alveolar epithelium of lung sections and found a strong positive correlation between VEGF expression and the proliferation of alveolar epithelial cells during VILI resolution. The results suggest that VEGF may contribute to alveolar reconstruction in the injured lungs during healing from VILI. Taken together, these findings suggest a dual role for VEGF in VILI. The increased concentration of VEGF in BALF and serum contribute to progressive lung injury in the early phase of VILI, but the elevated expression of VEGF in the lung tissue facilitates lung repair during the late recovery phase. Like VEGF, the contribution of TGF-β in lung repair is both complex and controversial but is more suggestive of failed repair and pulmonary fibrosis [47]. Our work on TGF-β is ongoing, and its involvement is yet to be fully elucidated.

As previously mentioned, alveolar epithelial cells, as a primary source of pulmonary VEGF, are destroyed during the initial phase of ALI. This results in decreased expression of VEGF in the injured lung tissue [20, 33]. In the recovery period following VILI, we found that VEGF increased progressively, and lung-recruited Ly6C^+low monocytes showed a similar trend in VEGF expression. Monocytes and macrophages belong to the mononuclear phagocyte system and circulating monocytes are the precursors of pulmonary macrophages [48, 49]. During inflammation, Ly6C^+high monocytes are rapidly recruited to sites of injury where they extravasate and differentiate into macrophages [9]. In injured tissue, inflammation and phagocytosis of apoptotic cells prompt macrophages to synthesize and release VEGF, which is crucial for tissue repair after injury [38, 50, 51]. Steinberg et al. [52] showed that in ARDS, a progressive increase in alveolar macrophages is associated with patient survival, and Eric D et al. [53] discovered that clinical outcomes depend on distinct alveolar macrophage transcriptional programs. David et al. [39] reported that VEGF levels in alveolar macrophages and epithelial lining fluid (ELF) from patients with ARDS were significantly less than those from at-risk subjects, and the increased VEGF levels in the ELF at day 4 were associated with recovery. These findings suggest that macrophages could be an essential source of VEGF and contribute to tissue repair in the injured lungs. Monocytes, like macrophages, can also produce VEGF [54]. In this study, we found that the temporal expression of pulmonary VEGF proteins was consistent with that of VEGF mRNA in lung-recruited monocytes, and that depletion of monocytes suppressed the restoration of pulmonary VEGF during resolution of VILI. These findings suggest that lung-recruited monocytes, mainly expressing Ly6C^+low, are a cellular source of pulmonary VEGF in the injured lungs and may drive healing process via secreting VEGF to promote epithelial repair.

There are several notable limitations to the present study. First, some of the experimental mice have died before being sacrificed. The cause of death may have been excessive inflammation and lung injury. This suggests that the concentrations of inflammatory cytokines in the injured lung tissue may have been underestimated. Second, there is a lack of direct evidence of a causal relationship between increased pulmonary VEGF and post-VILI proliferation of alveolar epithelial cells. We did not directly suppress the expression of pulmonary VEGF to examine the influence on the number of Ki67-positive cells in the alveolar epithelium during recovery from VILI. Since VEGF plays a crucial role in angiogenesis, systemic VEGF inhibition may produce unpredictable confounding effects on the estimation. In addition, there is no effective and selective method to precisely silence or inhibit the expression of VEGF in the lungs. Thus, further studies are required to resolve this issue. Third, the subset of monocytes expressing Ly6C^+high or Ly6C^+low and contributing to the restoration of pulmonary VEGF in injured lungs was not characterized. Due to the lack of an available method to deplete monocytes selectively in vivo, it is difficult to elucidate the differential effects of monocytes with different phenotypes on the restoration of pulmonary VEGF. However, our findings provide credible evidence that lung-recruited monocytes expressing Ly6C^+low are the predominant cells involved in resolving lung injury.

Conclusion

During recovery from VILI, the time courses of pulmonary growth factors are different from those of inflammatory cytokines. Pulmonary VEGF level remains higher than the baseline and is associated with pulmonary epithelial proliferation. During VILI, monocytes participate in two distinct phases. Lung-recruited Ly6C^+high monocytes dominate during the initiation of VILI, but Ly6C^+low monocytes become more abundant during the recovery phase following VILI. Monocytes are a cellular source of VEGF and contribute to post-VILI restoration of pulmonary VEGF. Thus, lung-recruited monocytes and pulmonary VEGF may play crucial roles in post-VILI lung repair.

Supporting information

S1 Fig. Flow chart depicting animal experiment design.

The numbers of animals used in the different experimental groups and the numbers of animals died during mechanical ventilation and recovery.

(TIF)

Click here for additional data file.^{(649.3KB, tif)}

S1 Text. Detailed illustrations of the statistical analysis.

(DOCX)

Click here for additional data file.^{(137.3KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by the Chang Gung Medical Foundation of Taiwan (grant no. CMRPG6C0231 and CMRPG6C0232) and CKL received the grants. The URL of the foundation website is https://www.cgmh.org.tw/en/. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0248959.r001

Decision Letter 0

Heinz Fehrenbach

18 Sep 2020

PONE-D-20-27843

Roles of lung-recruited monocytes and pulmonary vascular endothelial growth factor (VEGF) in resolving ventilator-induced lung injury (VILI)

PLOS ONE

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We look forward to receiving your revised manuscript.

Kind regards,

Heinz Fehrenbach

Academic Editor

PLOS ONE

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3. Please provide the specific sequences of th eprimers used in the PCR analysis.

4. At this time, we ask that you please provide scale bars on the microscopy images presented in Figures 1 and 4, and refer to the scale bar in the corresponding Figure legend.

5. Please provide the product numbers and any lot numbers of the antibodies purchased for immunohistochemistry in your study.”

6. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(a) Please state the number of mice used in the study

(b) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment)

(c) Please describe the post-operative care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.

Thank you for your attention to these requests.

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8. Please ensure your methods are described in sufficient detail for others to replicate the study. Specifically, please provide the following:

i) Please provide the names of the ELISA kits used to quantify pulmonary growth factors and cytokines.

ii) Please include the Methods described in the appendix in the main manuscript body."

9. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting

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Additional Editor Comments:

Please revise and re-submit as soon as possible so that I can invite the reviewers.

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PLoS One. 2021 Mar 19;16(3):e0248959. doi: 10.1371/journal.pone.0248959.r002

Author response to Decision Letter 0

22 Nov 2020

Dear Dr. Heinz Fehrenbach,

Thank you for your letter and the opportunity to revise our paper in PLOS ONE. We appreciate your insightful comments on revising the Materials & Methods of the paper.

I have included the comments immediately after this letter and responded to them individually, indicating exactly how we addressed each concern or problem and describing the changes we have made. The revisions have been approved by all four authors, and I have again been chosen as the corresponding author. The changes are marked in red in the paper as you requested, and the marked and unmarked versions of the revised manuscript have been uploaded to the online submission system.

In response to your comments on the Materials & Methods section, we have described the number of animals per group for each experiment and the number of replications in each experiment. In addition, we have included in the main text detailed descriptions of the methods, which were originally presented in the supplementary information.

We hope the revised manuscript will better suit PLOS ONE but are happy to consider further revisions, and we thank you for your continued interest in our research.

Sincerely,

Chung-Sheng Shi

Graduate Institute of Clinical Medicine Sciences, College of Medicine, Chang Gung University,

Editor comments, Journal Requirements, Author Responses and Manuscript Changes

Editor Comments:

Before inviting expert reviewers, I checked your manuscript and noticed that there are neither details about the number of animals per group and experiments nor about the number of relications of experiments. Please include these data in the Materials & Methods section as well as in the figure legends. In addition, you may wish to include detailed descriptions of the methods, which you give in the supplement, directly into the main text.

Response:

We have rewritten and detailed the number of animals per group in each experiment and the number of replications for each experiment in the Materials and Methods section. In addition, we have moved the detailed descriptions of the methods initially presented in the supplementary information directly into the Materials and Methods section of the main text.

Journal Requirements

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

Response:

We have reformatted our manuscript and file names according to the journal style requirements.

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. While you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure that your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter the referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

Response:

We have had our manuscript professionally edited by Editage.

We have uploaded a copy with red text highlighting the changes and a clean copy as the new manuscript file.

3. Please provide the specific sequences of the primers used in the PCR analysis.

Response:

The primer sequences used for RT-PCR analysis of VEGF mRNA have been included in the Materials and Methods section (page 12, line 201). The primer sequences of VEGF and GADPH (endogenous control) are as follows:

VEGF forward: 5’-ATCATGCGGATCAAACCTCACCA-3’;

VEGF reverse: 5’-TCACCGCCTTGGCTTGTCACAT-3’;

GAPDH forward 5’-CTCACTGGCATGGCCTTCCGTGT-3’,

GAPDH reverse: 5’-GATGTCATCATATTTGGCAGGT-3’.

4. At this time, we ask that you please provide scale bars on the microscopy images presented in Figures 1 and 4, and refer to the scale bar in the corresponding Figure legend.

Response:

We have added scale bars to the microscopy images in Figures 1 and 4 (page 15, line 251 and page 19, line 342). The scale bars represent 200 �m, as described in the corresponding figure legends.

5. Please provide the product numbers and any lot numbers of the antibodies purchased for immunohistochemistry in your study.”

Response:

We have edited the Materials and Methods section and detailed the product numbers and lot numbers of the antibodies used for immunohistochemistry analysis in our study (page 8, line 122).

6. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(a) Please state the number of mice used in the study

(b) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment);

(c) Please describe the postoperative care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.

Thank you for your attention to these requests.

Response:

(a) We have described the number of animals per group for each experiment and the number of replications for each experiment in the Materials and Methods section (page 7, line 112; page 9, line 136; page 10, line 156; page 10, line 167 and page 13, line 211).

(b) and (c)

We have edited the Methods section and described the animal welfare (page 6, line 80) and post-treatment (page 7, line 107).

For the animal welfare, all experimental protocols using animals were performed following the Guide to the Care and Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital, Chiayi, Taiwan (Approval number: 2012102301 (September 21, 2013)). All animal experiments were performed in the Animal Center of Chang Gung Memorial Hospital at Chiayi, and mice were housed following IACUC, national, and international animal welfare protocols. The center introduced the AAALAC certification system in 2013 and had obtained AAALAC certification in July 2014 (No: TTQ08384). To optimize environmental conditions for reducing mouse stress on animal welfare, animals were housed in a room with constant temperature (20–25°C) and humidity (40–60%) under a 12‐h light/dark cycle, mouse cages with enrichment toys were limited to 5 mice per cage, animals were given free access to food and water, and the center was maintained in low noises and vibrations.

For the postoperative care, the extubated mice received post-treatment care following the IACUC guidelines. We checked the appearance of mice every day and measured the body weight of mice twice/week. Besides, a full‐time veterinarian would take care of animal health and well-being until they were sacrificed with an overdose of anesthesia on days 1, 3, 7, or 14 after VILI.

Response:

We have detailed the anesthesia of the animals, including the drug, type, and dosage of anesthesia (page 7, line 97), during ventilation (page 7, line 103), and for sacrifice (page 7, line 105). Briefly, mice were anesthetized using intraperitoneal Zoletil 50 (80 mg/kg; Tiletamine-Zolazepam, Virbac, Carros CEDEX, France) and received a single intravenous tail vein injection of 20 ng LPS (O111B4; Sigma-Aldrich, St Louis, MO, USA) immediately before HTV ventilation. The mice were intubated and ventilated with an SAR-830 series small animal ventilator with multi-animal setups (CWE Inc., Ardmore, PA, USA) under room air and general anesthesia with regular intraperitoneal injections of Zoletil 50 (10 mg/kg/h) for 4 h. After ventilation, the mice were sacrificed immediately (day 0) or extubated after spontaneous breath resumption and sacrificed on day 1, 3, 7, or 14 after VILI by an overdose of anesthesia (Zoletil 50, 50 mg/kg).

8. Please ensure that your methods are described in sufficient detail for others to replicate the study. Specifically, please provide the following:

i) Please provide the names of the ELISA kits used to quantify pulmonary growth factors and cytokines.

ii) Please include the Methods described in the appendix of the main manuscript body."

Response:

We have edited the Materials and Methods section and described the names and lot numbers of the ELISA kits used in the quantification of pulmonary growth factors and cytokines (page 10, line 161). In addition, we have moved the detailed descriptions of the methods initially presented in the supplement, directly into the Materials and Methods section of the main text.

Response:

We have carefully read PLOS ONE's expectations for statistical reporting and SAMPL guidelines for general guidance on statistical reporting and ensured the description of the statistical analysis section complies with the essential principle. For instance, the name and version of statistical software, the form of summarized data, total sample and group sizes for each analysis, the method used for each analysis, and the alpha level for defining statistical significance has been detailed in the Methods section (page 13, line 222). Besides, detailed illustrations of the statistical analysis are provided in the supporting information S1 Text.

10. PLOS requires an ORCID iD for the corresponding author in the Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in the Editorial Manager. To do this, go to update my information (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your editorial manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ.

Response:

The ORCID iD of the corresponding author, Chung-Sheng Shi, has been edited, linked, and validated in the online submission system of the Editorial Manager.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(28.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0248959.r003

Decision Letter 1

Heinz Fehrenbach

5 Jan 2021

PONE-D-20-27843R1

Roles of lung-recruited monocytes and pulmonary vascular endothelial growth factor (VEGF) in resolving ventilator-induced lung injury (VILI)

PLOS ONE

Dear Dr. Shi,

Two expert reviewers from the field raised a number of points that have to be addressed when revising the manuscript. In particular, all details such as animal numbers and handling, outcome of the experiments, statistical tests used as well as methodological issues have to be included so that the study can be fully appreciated by our readers. Please submit a detailed point-by-point response to all comments along with your revised manuscript. In addition, I suggest that you get a native speaker involved in editing the English language.

Please submit your revised manuscript by Feb 19 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Heinz Fehrenbach

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The resubmitted manuscript entitled „Roles of lung-recruited monocytes and pulmonary vascular endothelial growth factor (VEGF) in resolving ventilator-induced lung injury (VILI)” by Chung-Sheng Shi et al. describes the role of monocytes and the release of VEGF in a model of VILI. The authors described

All points of the reviewer are properly edited the statistics and described the different statistical models in supplement. All numbers of the experiments were mentioned and statistical variances shown as mean � SD. The text body was well corrected and most of the typos removed. There are some miner points I would like to address.

Minor points:

1. In figure 4B, day 1 the authors should check if the magnification is really the same as in the other histological images. It seems to be a higher magnification. Please clarify.

2. The correlation between the pulmonary VEGF and alveolar epithelial barrier one side and the role of monocytes on the other side are two possible explanations for the changes of VEGF during VILI. However, the authors work out the differences more detailed in the discussion.

3. In the discussion section (line 378-381) the authors compared the bacterial infection in ventilated patients with their two-hit model of LPS and ventilation. The author should clarify that there is a substantial difference between bacterial infection and LPS treatment in terms of cytokine profiles

Reviewer #2: General comments.

Please, clearly decribe how many animals were used in each Group. How many animals entered a Group, how many survived VILI, how many were sacrificed early, etc. Discuss, speculate how this may have impacted on the results? Did animals with an excessive Inflammation die early? Did you check?

Consider a figure, flow chart.

Please, clearly decribe the ventilation used to induce VILI.

Please, clearly describe the anima handling after the experiment. Did the mice receive pain medication, etc.

The statistics used may not be appropriate. You used parametric tests despite the low n. The use of non-parametric test would be more cautious. Use non-parametric tests or explain. Did you check for normal distribution in all groups?

Avoid nonsensical phrases: The results reveal, etc.

Always state the measurement unit.

Line 79

Animals and two-hit model of VILI

The description of the animal model is insufficient.

Clearly describe each hit of the two hit model, e.g.:

The first hit consisted of ……..

The second hit ……..

Then, give a detailed report of the experimental procedure in the correct chronological order.

i. anesthesia

ii. LPS-injection

iii. Intubation

iv. Ventilation

v. Extubation

Specify, the ventilation used to induce VILI besides tidal volume and respiratory rate further: inspiration time, I:E, resulting peak pressure and duration of ventilation!

Whan did you stop ventilation. How and when as a mouse extubated? Did you extubate right from VILI ventilation? What kind of ventilation did you used to wean anesthesia.

Line 92:

….water, and the center was maintained in low noises and vibrations.

Sounds awkward. Rephrase

Line 112:

The number of animals used per group was as follows: normal control (n = 6), day 0 (n = 6 ), day 1 (n = 6), day 3 (n = 6), day 7 (n = 12, 6 of them for the study of depletion of pulmonary monocytes), and day 14 (n = 12, 6 for the study of depletion of pulmonary monocytes).

This sounds to good. Not one animal died? How many animals did you loose during induction of VILI. How many animals entered each group and how many remained over the following days? What was the selection process to euthanize an animal prematurely?

It needs to be discussed in the discussion section in how far that may have altered the results.

Line 117:

How were the lungs inflated – manually? With a defined pressure/volume?

Line 215:

How were the mice sedated for this injection. Did you need to ventilate them?

Line 222:

You used parametric tests despite the small number of animals. Did you check for normal distribution? Non-parametric tests and Spearman correlation may be the more conservative strategy.

Line 245:

C57BL/6 mice received i.v. with 20 ng of LPS and high-stretch mechanical ventilation that produced two-hit VILI.

Sentence does not make sense. Rephrase.

Line 253:

….; n = 3-5, ….

What does that n mean? It does not match the group size given in the method section?

This refers to all other n = ..... in the figure descriptions.

Line 262:

Avoid nonsencical phrases like: The results revealed ……

Line 281:

..SD; n = 4-5,

What is this n?

General comment:

What is a background level of something in mice not held under experimental conditions?

If you mean the first measured level before the intervention you may want to use the term baseline. Do you mean the level in the control animals at that specific time point?

Consider using concentration instead of level whenever appropriate.

Line 310:

ELISA of inflammatory cytokines and growth factors…..

Specify, lung tissue concentration, plasma concentration?

Line 323

….from the background level of 252.8 � 25.9 to 77.3 � 7.3 on…..

What is the measurement unit? This refers to the entire paragraph!

Line 328

…. level of 87 � 5 to 48 � 5 per 100 alveoli on….

87 what? 87 cells per 100 alveoli?

Line 354

….VILI (0.19 � 0.05; p = 0.009, Fig 5) a…

Unit?

Line 379

….. activated by an underlying bacterial infection.

Rephrase. Bacterial infection is just one of many activators.

Line 391

The expression of Gr1 (Ly6C) on monocytes is closely linked to their divergent roles in inflammatory tissue.

Avoid nonsensical phrases like: linked ….to divergent roles.

Line 386

…VILI, contrary to inflammatory cytokines.

That is not contrary. VEGF increases while inflammation fades or vice versa. Rephrase.

Line 386

VEGF expression in lung tissue is associated with the proliferation of alveolar epithelial cells.

That is an assumption or quote of former works.

First state the main findings. Then, refer to existing knowledge. Then, present a conclusion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2021 Mar 19;16(3):e0248959. doi: 10.1371/journal.pone.0248959.r004

Author response to Decision Letter 1

19 Feb 2021

Chung-Sheng Shi

Chang Gung University

Graduate Institute of Clinical Medicine Sciences

No. 259, Wenhua 1st Rd

Guishan Dist., Taoyuan City 33302, Taiwan

csshi@mail.cgu.edu.tw

Dr. Heinz Fehrenbach

Academic Editor

PLOS ONE

February 17, 2021

Subject: Revision and resubmission of manuscript PONE-D-20-27843

Dear Dr. Heinz Fehrenbach,

Thank you for your letter and the opportunity to revise our paper in PLOS ONE again. In response to the reviewers’ comments, we have detailed point-by-point responses to all comments along with our revised manuscript.

I have included the comments immediately after this letter and responded to them individually, indicating exactly how we addressed each concern or problem and describing the changes we have made. Besides, we have had our manuscript professionally edited by Editage again. All four authors have approved the revisions, and I have again been chosen as the corresponding author. The changes are marked in red in the paper as you requested, and the marked and unmarked versions of the revised manuscript have been uploaded to the online submission system.

We hope the revised manuscript will better suit PLOS ONE but are happy to consider further revisions, and we thank you for your continued interest in our research.

Sincerely,

Chung-Sheng Shi

Graduate Institute of Clinical Medicine Sciences, College of Medicine, Chang Gung University,

Author Responses to Reviewers and Manuscript Changes

Response to Reviewers

Thank you for your precious comments and advice. Those comments are valuable and helpful for revising and improving our paper and the critical guiding significance to our research. We have studied words carefully and have made corrections, which we hope meet with approval. Revised portions are marked in red on the paper. The major corrections in the paper and the responses to the reviewer’s comments are as following:

Response:

Thank you for your summary. We appreciate your efforts in reviewing our manuscript. We have revised the manuscript accordingly. Our point-by-point responses are detailed below:

Minor points:

1. In figure 4B, day 1 the authors should check if the magnification is really the same as in the other histological images. It seems to be a higher magnification. Please clarify.

Response:

We have confirmed that the magnification of the day 1 image in figure 4B is the same as that of other images. The alveoli in this image may be damaged and expanded due to VILI, so it is why the alveoli' size in this image is different from others. However, to respect the inflammatory cells, the size is the same.

Response:

Thank you for your comments. We are very grateful for your affirmation.

Response:

Thank you for your precious comments and advice. We have rewritten the first paragraph (lines 407-416) and discussed the substantial difference between bacterial infection and LPS treatment in the second paragraph (lines 417-428). In the paragraph, we have discussed the substantial difference of the time-course expression of inflammatory cytokines between bacterial infection and LPS treatment and cited new references (line 423).

Reviewer #2: General comments.

Consider a figure, flow chart.

Response:

Thank you for your precious comments and advice. We have made a flow chart in supporting information S1 Fig, shown below, to illustrate the numbers of animals used in the different experimental groups and the numbers of animals that died during mechanical ventilation and recovery. For some of the experimental mice died before being sacrificed. The cause of death may have been excessive inflammation and lung injury. This suggests that the concentrations of inflammatory cytokines in the injured lung tissue may have been underestimated. We have stated the situation as a limitation of the study in the discussion section (lines 541-545).

S1 Fig. Flow chart depicting animal experiment design.

Please, clearly decribe the ventilation used to induce VILI.

Response:

Thank you for your advice. We have rewritten the section of materials and methods and described the detail of the ventilation set for inducing VILI (lines 102-111).

Please, clearly describe the anima handling after the experiment. Did the mice receive pain medication, etc.

Response:

Thank you for your careful review. We have described the detail of the animal handling after the experiment in the section of materials and methods ( lines 111-115). The experimental mice did not receive pain medication after the experiment for no tracheostomy creation for intubation and no other wound to be produced by the experimental procedures.

Response:

Thank you for your precious comments and advice. We have rechecked the normality of the data by using Shapiro-Wilk test and used parametric or non-parametric tests as appropriate. For comparisons between two groups, data were analyzed by unpaired Mann–Whitney U test. For comparisons between multiple groups, parametric (one-way ANOVA with Bonferroni’s or Dunnett’s post hoc test) or non-parametric test (the Kruskal-Wallis test with Bonferroni correction) was used. Besides, we used Spearmen’s correlation to evaluate the relationship between the IHC intensity of VEGF and the number of Ki67-positive cells in the alveolar epithelium of lung sections (lines 231-236). Detailed illustrations of the statistical analysis are provided in the supporting information S1 Text.

Avoid nonsensical phrases: The results reveal, etc.

Response:

Thank you for your advice. We have rewritten the phrases (lines 275 and 379).

Always state the measurement unit.

Response:

Thank you for your careful review and advice. We have rechecked and made sure that the measurement units have been stated. Besides, for the statements of the IHC staining intensity and mRNA expression, we have presented with relative intensities and fold, respectively.

Line 79

Animals and two-hit model of VILI

The description of the animal model is insufficient.

Clearly describe each hit of the two hit model, e.g.:

The first hit consisted of ……..

The second hit ……..

Then, give a detailed report of the experimental procedure in the correct chronological order.

i. anesthesia

ii. LPS-injection

iii. Intubation

iv. Ventilation

v. Extubation

Specify, the ventilation used to induce VILI besides tidal volume and respiratory rate further: inspiration time, I:E, resulting peak pressure and duration of ventilation!

Whan did you stop ventilation. How and when as a mouse extubated? Did you extubate right from VILI ventilation? What kind of ventilation did you used to wean anesthesia.

Response:

Thank you for your precious comments and advice. We have rewritten and detailed the two-hit model's description in the section of materials and methods (lines 95-111).

Line 92:

….water, and the center was maintained in low noises and vibrations.

Sounds awkward. Rephrase

Response:

Thank you for your comment. We have rewritten the paragraph (lines 89-93).

Line 112:

It needs to be discussed in the discussion section in how far that may have altered the results.

Response:

Thank you for your precious comments and advice. We have detailed in a flow chart, shown in S1 Fig, to illustrate the numbers of animals used in the different experimental groups and the numbers of animals that died during mechanical ventilation and recovery. For some of the experimental mice died before being sacrificed. The cause of death may have been excessive inflammation and lung injury. This suggests that the concentrations of inflammatory cytokines in the injured lung tissue may have been underestimated. We have stated the situation as a limitation of the study in the discussion section (lines 541-545).

Line 117:

How were the lungs inflated – manually? With a defined pressure/volume?

Response:

Thank you for your careful review. We have rewritten the section of materials and methods. For histopathology and immunohistochemistry analysis, mouse lungs were manually inflated and fixed by injecting 10% buffered formalin and embedded in paraffin (line 123).

Line 215:

How were the mice sedated for this injection. Did you need to ventilate them?

Response:

Thank you for your careful review. We have rewritten and detailed the description of mouse anesthesia in the experiment of “Depletion of pulmonary monocytes” ( lines 225-226 and 228). The experimental mice received injection with Clophosome-A in the tail vein after anesthesia with intraperitoneal Zoletil 50. During the experiment, the mice remained with spontaneous breath and did not receive mechanical ventilation.

Line 222:

You used parametric tests despite the small number of animals. Did you check for normal distribution? Non-parametric tests and Spearman correlation may be the more conservative strategy.

Response:

Thank you for your precious comments and advice. We have rechecked the normality of the data and used Spearman correlation to assess association (line 236).

Line 245:

C57BL/6 mice received i.v. with 20 ng of LPS and high-stretch mechanical ventilation that produced two-hit VILI.

Sentence does not make sense. Rephrase.

Response:

Thank you for your comment. We have rewritten the sentence (lines 258-259).

Line 253:

….; n = 3-5, ….

What does that n mean? It does not match the group size given in the method section?

This refers to all other n = ..... in the figure descriptions.

Response:

Thank you for your careful review. We have correct the typing error in the section of materials and methods ( line 143) and removed the wrong description in the section of results (line 254). Detailed illustrations of the statistical analysis are provided in the supporting information S1 Text.

Line 262:

Avoid nonsencical phrases like: The results revealed ……

Response:

Thank you for your advice. We have rewritten the sentence (line 275).

Line 281:

..SD; n = 4-5,

What is this n?

Response:

Thank you for your careful review. For flow cytometric analysis, the number of animals used per group was as follows: normal control (n = 4), day 0 (n = 4), day 1 (n = 4), day 3 (n = 4), day 7 (n = 5), and day 14 (n = 5), which has been stated in the section of materials and methods. We have correct the descriptions in the legend of Fig 2 (lines 295 and 299).

General comment:

What is a background level of something in mice not held under experimental conditions?

If you mean the first measured level before the intervention you may want to use the term baseline. Do you mean the level in the control animals at that specific time point?

Consider using concentration instead of level whenever appropriate.

Response:

Thank you for your precious comments and advice. We have corrected the descriptions to use the term “baseline” instead of “background level” and “concentration” instead of “level” accordingly.

Line 310:

ELISA of inflammatory cytokines and growth factors…..

Specify, lung tissue concentration, plasma concentration?

Response:

Thank you for your careful review. We have detailed the description to clarify that the ELISA samples were obtained from the lung tissue of mice (line 328).

Line 323

….from the background level of 252.8 � 25.9 to 77.3 � 7.3 on…..

What is the measurement unit? This refers to the entire paragraph!

Response:

Thank you for your careful review and comments. We have correct the measurement unit as the relative staining intensity (lines 341, 345, 354, and 368).

Line 328

…. level of 87 � 5 to 48 � 5 per 100 alveoli on….

87 what? 87 cells per 100 alveoli?

Response:

Thank you for your careful review. We have correct the description of the unit (lines 348 and 352).

Line 354

….VILI (0.19 � 0.05; p = 0.009, Fig 5) a…

Unit?

Response:

Thank you for your careful review. We have correct the description of the unit (lines 380, 381, and 383).

Line 379

….. activated by an underlying bacterial infection.

Rephrase. Bacterial infection is just one of many activators.

Response:

Thank you for your precious comments and advice. We have rewritten the sentence (line 417).

Line 391

The expression of Gr1 (Ly6C) on monocytes is closely linked to their divergent roles in inflammatory tissue.

Avoid nonsensical phrases like: linked ….to divergent roles.

Response:

Thank you for your careful review and comments. We have rewritten the sentence (lines 430-431).

Line 386

…VILI, contrary to inflammatory cytokines.

That is not contrary. VEGF increases while inflammation fades or vice versa. Rephrase.

Response:

Thank you for your precious comment and advice. We have rewritten the paragraph (lines 411-412).

Line 386

VEGF expression in lung tissue is associated with the proliferation of alveolar epithelial cells.

That is an assumption or quote of former works.

First state the main findings. Then, refer to existing knowledge. Then, present a conclusion.

Response:

Thank you for your precious comments and advice. We have rewritten the description that the main finding that the VEGF expression in lung tissue is positively related to the expression of Ki67, a proliferation marker, in alveolar epithelial cells (lines 412-414). We have also discussed the finding and possible VEGF roles in post-injury lung referred from other studies (lines 485-512). Finally, we conclude that pulmonary VEGF may play a crucial role in post-VILI lung repair (line 568).

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PLoS One. doi: 10.1371/journal.pone.0248959.r005

Decision Letter 2

Heinz Fehrenbach

9 Mar 2021

Roles of lung-recruited monocytes and pulmonary vascular endothelial growth factor (VEGF) in resolving ventilator-induced lung injury (VILI)

PONE-D-20-27843R2

Dear Dr. Shi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

All points of the reviewer are properly addressed. The statistics methods and conclusions are valid.

The interpretation of the LPS response in the VILLI-model was distinguished from an a bacterial infection in the discussion section.

In addition the experimental model is described in more detail, which approved the manuscript. Even, the flowchart for the experimental design is very helpful.

Reviewer #2: Thank you for your detailed and lengthy response to all of my questions.

It is my humble opinion, that the article has markedly improved.

**********

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PLoS One. doi: 10.1371/journal.pone.0248959.r006

Acceptance letter

Heinz Fehrenbach

11 Mar 2021

PONE-D-20-27843R2

Roles of lung-recruited monocytes and pulmonary vascular endothelial growth factor (VEGF) in resolving ventilator-induced lung injury (VILI)

Dear Dr. Shi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Prof. Dr. Heinz Fehrenbach

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Flow chart depicting animal experiment design.

The numbers of animals used in the different experimental groups and the numbers of animals died during mechanical ventilation and recovery.

(TIF)

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S1 Text. Detailed illustrations of the statistical analysis.

(DOCX)

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Roles of lung-recruited monocytes and pulmonary Vascular Endothelial Growth Factor (VEGF) in resolving Ventilator-Induced Lung Injury (VILI)

Chin-Kuo Lin

Tzu-Hsiung Huang

Cheng-Ta Yang

Chung-Sheng Shi

Roles

Abstract

Introduction

Materials and methods

Animals and two-hit model of VILI

Histopathology and immunohistochemistry analysis

Preparation of lung single cells and flow cytometric analysis

Quantitation of pulmonary growth factors and cytokines

Isolation of lung-recruited monocytes

RT-PCR analysis of VEGF mRNA

Depletion of pulmonary monocytes

Statistical analysis

Results

Abundance of inflammatory cells in lung tissues following VILI

Fig 1. Time course of lung inflammatory cell response following VILI as determined from histological analysis.

Phenotype of monocytes recruited to the lungs during the recovery phase following VILI

Fig 2. Phenotype and time course of post-VILI levels of lung-recruited monocytes as determined from flow cytometric analysis.

Time courses of pulmonary growth factor and inflammatory cytokine expressions during recovery from VILI

Fig 3. Time courses of pulmonary inflammatory cytokine and growth factor levels following VILI.

Correlation between pulmonary VEGF and alveolar epithelial repair

Fig 4. Immunocytochemistry for VEGF and Ki67 in lung sections of mice following VILI.

Contribution of monocytes to pulmonary VEGF production during recovery from VILI

Fig 5. Time course of VEGF mRNA expression in lung-recruited monocytes following VILI.

Fig 6. Pulmonary VEGF protein levels in mice without and with depletion of monocytes on days 7 and 14 after VILI.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Heinz Fehrenbach

Roles

Author response to Decision Letter 0

Decision Letter 1

Heinz Fehrenbach

Roles

Author response to Decision Letter 1

Decision Letter 2

Heinz Fehrenbach

Roles

Acceptance letter

Heinz Fehrenbach

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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