Multiple Death Pathways of Neutrophils Regulate Alveolar Macrophage Proliferation

Alveolar macrophage (AM) proliferation and self-renewal play an important role in the lung tissue microenvironment. However, the impact of immune cells, especially the neutrophils, on AM homeostasis or function is not well characterized. In this study, we induced in vivo migration of neutrophils into bronchoalveolar lavage (BAL) fluid and lung using CXCL1, and then co-cultured these with AMs in vitro. Neutrophils in the BAL (BAL−neutrophils), rather than neutrophils of bone marrow (BM-neutrophils), were found to inhibit AM proliferation. Analysis of publicly available data showed high heterogeneity of lung neutrophils with distinct molecular signatures of BM− and blood−neutrophils. Unexpectedly, BAL−neutrophils from influenza virus PR8-infected mice (PR8−neutrophils) did not inhibit the proliferation of AMs. Bulk RNA sequencing further revealed that co-culture of AMs with PR8−neutrophils induced IFN-α and -γ responses and inflammatory response, and AMs co-cultured with BAL−neutrophils showed higher expression of metabolism- and ROS-associated genes; in addition, BAL−neutrophils from PR8-infected mice modulated AM polarization and phagocytosis. BAL−neutrophil-mediated suppression of AM proliferation was abrogated by a combination of inhibitors of different neutrophil death pathways. Collectively, our findings suggest that multiple cell death pathways of neutrophils regulate the proliferation of AMs. Targeting neutrophil death may represent a potential therapeutic strategy for improving AM homeostasis during respiratory diseases.


Introduction
Alveolar macrophages (AMs) are lung tissue-resident plastic cells in the microenvironment of alveolar space. In contrast to other resident macrophages, AMs represent a unique population as the first line of contact with the tissue microenvironment. AMs are sentinels in the pulmonary airspaces and play important roles in the maintenance of homeostasis, tissue repair, and immune surveillance in the respiratory tract [1][2][3]. In the steady state, AMs are required for clearance of dead cells and cellular debris or release of soluble mediators, which avoid host damage and protect pulmonary function [4][5][6][7]. The absence or dysfunction of AMs can result in pulmonary alveolar proteinosis (PAP), a syndrome resulting from abnormal accumulation of alveolar surfactant in the lung [8]; in addition, ablation of AMs was also shown to increase influenza virus-induced lung damage. Therefore, maintenance of AMs is vital for lung homeostasis. In terms of immune surveillance, AMs were shown to suppress T cell proliferation, resulting in the arrest of T

Isolation and Preparation of BM-Neutrophils
BM cells were collected from tibia and femur through a 25-gage needle by flushing with cold, sterile phosphate-buffered saline (PBS). Cells were passed through a 70 µm cell filter in a 50 mL falcon tube and pelleted at 800× g for 5 min. After red blood cell lysis, the cell pellet was resuspended in 90 µL of cold MACS buffer. According to the protocol for anti-Ly6G Microbeads (Miltenyi Biotec), Ly6G + cells of BM (BM-neutrophils) were collected by positive magnetic selection using anti-Ly6G beads. The cell number was measured by 0.4% Trypan Blue (Hyclone).

AM Culture In Vitro and Co-Culture with BAL-, BM-, or PR8-Neutrophils
AMs were obtained from BAL as described previously [35]. C57BL/6 mice were sacrificed, and alveolar lavage was performed by flushing the airway for five times with 1 mL cold, sterile BAL washes via a tracheal incision. Alveolar lavages were centrifuged at 4 • C, 800× g for 5 min, and cells were obtained and purified to culture plates by adherence in complete medium (RPMI-1640 with 10% FBS and 1% Pen/Strep/glutamate, Thermo Fisher Scientific, Waltham, MA, USA); 1 × 10 5 cells per well of co-cultured and monocultured AMs were seeded in non-treated 24-well plate for 2 h at 37 • C in 5% CO 2 cell incubators. The non-adherent cells were washed off with warm PBS and cultured with a complete medium.
2.5. BMDM Culture In Vitro and Co-Culture with BAL-or BM-Neutrophils C57BL/6 mice were sacrificed, and the BM cells were collected from tibia and femur through a 25-gage needle by flushing with cold, sterile PBS. Cells were passed through a 70 µm cell filter in a 50 mL falcon tube and pelleted at 800× g for 5 min, and the pellet was suspended in ACK lysis buffer at room temperature (RT). After centrifugation at 800× g for 5 min, BM cells were resuspended and cultured with a complete medium (DMEM with 10% FBS and 1% Pen/Strep/glutamate, Thermo Fisher Scientific) supplemented with 50 ng/mL recombinant murine macrophage colony-stimulating factor (M-CSF, BioLegend) to induce differentiation into a macrophage phenotype. After 7 days, BMDMs were generated for subsequent experimentation. For co-culture of BMDMs with BAL-or BM-neutrophils, following replating of BMDMs, 1 × 10 5 cells per well of co-cultured and mono-cultured BMDMs were plated in a 24-well plate at 37 • C and 5% CO 2 cell incubators.

Phagocytosis Assay by Flow Cytometry
In accordance with the guidelines for the CellTraceTM CFSE kit (Thermo Fisher Scientific, Waltham, MA, USA), neutrophils were obtained and incubated in a proteinfree medium containing Carboxyfluorescein succinimidyl ester (CFSE) for 15 min at RT, followed by wash with a complete medium and quenching of any dye remaining in the solution. Subsequently, BAL-or PR8-neutrophils were added to AM cultures at a ratio of 1:1 for 24 h at 37 • C. The co-culture of AMs and BAL-neutrophils as control. The co-cultured cells were collected with 0.25% Trypsin-EDTA solution and processed for flow cytometry.

Co-Culture Treatment with Different Inhibitors
AMs co-cultured with BAL-neutrophils at a 1:1 ratio were treated with or without different inhibitors in medium, including G-CSF (10 ng/mL, BioLegend), Q-VD-Oph (50 µM, Selleckchem, Houston, TX, USA), NAC (1 mM, Selleckchem), and DFO (1 µM, Selleckchem), the control was co-culture treated without inhibitors. After 24 h, the cells were cultured in complete medium containing 10 ng/mL recombinant murine GM-CSF, and cells were analyzed by flow cytometry.

RNA Sequencing and Analysis
We collected AMs from AMs (alone), co-culture of AMs with BAL-neutrophils, and PR8-neutrophils. Total RNA of AMs was extracted using Trizol kit (Thermo Fisher Scientific) following the manufacturer's protocol. Two pools per genotype were used for bulk RNA-seq. After quality control, high-quality (Agilent Bioanalyzer RIN of >7.0, Agilent Technologies, Santa Clara, CA, USA) total RNA was used to generate the RNA sequencing library. cDNA synthesis, end-repair, A-base addition, and ligation of the Illumina indexed adapters were performed according to the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA, USA) and Qubit fluorometry (Invitrogen, Carlsbad, CA, USA). Paired-end libraries were sequenced on the DNBSEQ resequencing and PE 150 Kit. Base-calling was performed using DNBSEQ software (DNBSEQ A0, Beijing Genomics Institution, Shenzhen, China).
Paired-end RNA-seq reads were aligned to the mouse reference genome (GRCm38/mm10). Pre-and post-alignment quality controls, gene level raw read count, and normalized read count (i.e., FPKM) were performed using the RSeQC package (v2.3.6)(Beijing Genomics Institution, Shenzhen, China) with the NCBI mouse RefSeq gene model. For functional analysis, Gene set enrichment analysis (GSEA) was performed to identify enriched gene sets using the hallmark collection of the Molecular Signatures Database (MSigDB), containing up-and downregulated genes, and using a weighted enrichment statistic and a log2 ratio metric for ranking genes. Data were submitted to the GEO repository (GSE212080).

Quantitative RT-PCR
The total RNA from the cultured AM as indicated in the text was extracted with Total RNA purification kit (Sigma, Germany) and treated with DNase I (Invitrogen, Waltham, MA, USA). Random primers (Invitrogen) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) were used to synthesize first-strand cDNA from equivalent amounts of RNA from each sample. qPCR was performed with Fast SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). RT-PCR was conducted in duplicates in QuantStudio5 (Applied Bioscience). Results were generated with the comparative threshold cycle (Delta CT) method by normalizing to β-actin.

Statistical Analysis
Unpaired two-tailed Student's t-test (two group comparison), one-way ANOVA (multigroup comparison), Multiple t-tests (weight loss and Multiplex studies), or Log-rank (Mantel-Cox) test (survival data) were used to determine statistical significance by Graph-Pad Prism software (Prism 8, San Diego, CA, USA) and p-values < 0.05 were considered indicative of statistical significance.

BAL-Neutrophils Inhibit AM Proliferation
To investigate the role of neutrophils in AM proliferation or maintenance, we generated a model of neutrophil migration into BAL and lung in C57BL/6 mice induced by administration of recombinant CXCL1 owing to the low number of neutrophils in lung tissues. After 12 h, the percentage of neutrophils in BAL (BAL-neutrophils) reached up to 80% (data not shown), which was consistent with a previous report. Furthermore, the rCXCL1 treatment had no effect on AM and monocyte numbers, and cytokine levels [36]. Purified BAL-neutrophils were co-cultured with AMs at ratios of 1:0, 1:1, 2:1, and 8:1 for 24 h, then the Ki67 (widely used as a biomarker of cell proliferation) expression in AMs was determined with GM-CSF stimulation for 24 h. BAL-neutrophils downregulated Ki67 expression of AMs when co-cultured at a 1:1 ratio ( Figure 1A). Neutrophils of bone marrow (BM-neutrophils) were obtained by using anti-Ly6G Microbeads. However, the Ki67 expression in AMs showed no significant alteration after co-culture of BM-neutrophils with AMs at the ratios of 1:0, 1:1, 2:1, and 8:1 ( Figure 1B). To explore the difference between Cells 2022, 11, 3633 6 of 18 these two populations, BAL-and BM-neutrophils, we used a publicly available bulk RNAseq data set (GSE141745) [20] of neutrophils from lung, blood, and BM of C57BL/6 mice. Heatmap showed differentially expressed genes of neutrophils from lungs compared with neutrophils from BM or blood ( Figure 1C). GSEA of hallmark gene sets showed that lung neutrophils increased the expression of genes associated with TNF-α and TGF-β signaling, angiogenesis, P53, and inflammatory responses, but decreased the expression of G2M checkpoint, E2F targets, and mitotic spindle ( Figure 1D). Neutrophils from the lung displayed significant enrichment of senescence genes and reduction of differentiation and phagocytosis genes compared to BM-neutrophils ( Figure 1E-G). Neutrophils are generated in the bone marrow and released into the circulatory system. Neutrophils are highly motile cells, and during circulation in tissues, they gain the ability for phagocytosis or killing, and finally show aging by losing their proliferative ability [37]. These data revealed that neutrophils in the lung acquired new phenotypes and functional characteristics compared with that from BM and blood, although neutrophils are short-lived in tissues [38,39].

Neutrophils from BAL or BM Do Not Influence BMDM Proliferation
AMs have the ability for self-maintenance independent of the contribution of circulating precursors in situ. However, if AMs are depleted by lethal whole-body irradiation or inflammation, BM-derived monocytes can gain a competitive advantage and readily repopulate the AM niche, which can only differentiate into mature and fully functional AMs in the recipient lungs [40]. To probe whether neutrophils influence the proliferation of BM-derived macrophages (BMDMs), we generated BMDMs by inducing differentiation of BM cells into macrophages using recombinant murine M-CSF. The existence of BALneutrophils did not inhibit Ki67 expression of BMDMs (Figure 2A). Similarly, the Ki67 expression of AMs was also not changed in BMDMs co-cultured with neutrophils of BM ( Figure 2B). The results suggested that neutrophils, irrespective of whether these were from BAL or BM, only suppressed the proliferation of AMs, but not BMDMs.

PR8−Neutrophils Are Unable to Affect AM Proliferation
AMs are critical for the initial host response to RNA virus infection in the lung, especially influenza virus. To examine the role of neutrophils in AM self-renewal during influenza virus infection, neutrophils from BAL were sorted from PR8 infected mice (PR8-neutrophils) and then co-cultured with AMs at the ratios of 1:0, 1:1, 2:1, and 8:1. PR8-neutrophils did not inhibit Ki67 expression of AMs ( Figure 3A). To better understand the potential mechanisms by which BAL-and PR8-neutrophils regulate AM proliferation differently, we analyzed publicly available microarray data (GSE165299) of neutrophils in lung from mice on day 3 post influenza virus infection or from uninfected mice ( Figure 3B). GSEA of hallmark gene sets showed that influenza virus infected-neutrophils expressed higher levels of genes related to IFN-α and -γ responses, inflammatory responses, E2F and Myc targets, and metabolism compared with uninfected neutrophils ( Figure 3C). Moreover, the gene sets associated with monocyte chemotaxis also showed higher expression ( Figure 3D). Nevertheless, most genes expressed in uninfected neutrophils were clustered in transcriptional regulation of granulopoiesis and special markers of neutrophils ( Figure 3E). These data showed that the expression of those genes of PR8-neutrophils may cause AM activation, which then possibly lost the function for inhibiting AM proliferation. Combined with the result in Figure 1D, it seems plausible that IFN responses-associated genes or some granule genes released by BAL-neutrophils regulated AM maintenance.  of BM-derived macrophages (BMDMs), we generated BMDMs by inducing differentiation of BM cells into macrophages using recombinant murine M-CSF. The existence of BALneutrophils did not inhibit Ki67 expression of BMDMs (Figure 2A). Similarly, the Ki67 expression of AMs was also not changed in BMDMs co-cultured with neutrophils of BM ( Figure 2B). The results suggested that neutrophils, irrespective of whether these were from BAL or BM, only suppressed the proliferation of AMs, but not BMDMs.

PR8−Neutrophils Are Unable to Affect AM Proliferation
AMs are critical for the initial host response to RNA virus infection in the lung, especially influenza virus. To examine the role of neutrophils in AM self-renewal during influenza virus infection, neutrophils from BAL were sorted from PR8 infected mice (PR8neutrophils) and then co-cultured with AMs at the ratios of 1:0, 1:1, 2:1, and 8:1. PR8neutrophils did not inhibit Ki67 expression of AMs ( Figure 3A). To better understand the potential mechanisms by which BAL-and PR8-neutrophils regulate AM proliferation differently, we analyzed publicly available microarray data (GSE165299) of neutrophils in

Neutrophils Modulate AM Self-Renewal and Phagocytosis
To further analyze the underlying mechanism affecting AM proliferation, we performed bulk RNA sequencing of mono-cultured AMs and AMs co-cultured with BALor PR8-neutrophils. Several differentially expressed genes were found in three groups, especially in AMs co-cultured with PR8-neutrophils ( Figure 4A). Comparing AM (monocultured AMs) with BAL groups (AMs co-cultured with BAL-neutrophils), hallmark gene sets analysis revealed that AMs co-cultured with BAL-neutrophils tended to show increased expression of genes related to KRAS signaling, xenobiotic metabolism, and inflammatory response, but BAL-neutrophils influenced E2F targets, G2M checkpoint, mitotic spindle, apical junction and myogenesis of AMs ( Figure 4B, top). GSEA analysis showed that AMs from PR8 group (AMs co-cultured with PR8-neutrophils) induced IFN-α and -γ responses, TNF-α signaling, and inflammatory response compared with AMs of BAL group. However, AMs of the BAL group showed increased expression of cholesterol homeostasisassociated genes compared with AMs of PR8 group ( Figure 4B, bottom). These findings indicate that BAL-neutrophils may help to maintain AM homeostasis. Furthermore, AMs of the BAL group showed decreased expression of cell cycle-associated transcription genes compared with AM alone or AMs of PR8 group ( Figure 4C-D), which is consistent with the concept that BAL-neutrophils impair AM proliferation. Gene ontology analysis suggested that neutrophils interrelated chemotaxis and that granule genes were expressed in AMs of the BAL group ( Figure 4E). Collectively, these findings suggest that neutrophils of BAL probably preserved homeostasis of AMs even though AM proliferation was impaired. tered in transcriptional regulation of granulopoiesis and special markers of neutrophils ( Figure 3E). These data showed that the expression of those genes of PR8-neutrophils may cause AM activation, which then possibly lost the function for inhibiting AM proliferation. Combined with the result in Figure 1D, it seems plausible that IFN responses-associated genes or some granule genes released by BAL-neutrophils regulated AM maintenance.   (E) Enrichment plot from GSEA of infected neutrophils using gene sets for neutrophil chemotaxis and granule constituents between AMs (alone) and AMs of BAL groups. Data are presented as arithmetic mean ± SD. ns, no significant; ***, p < 0.001; **** p < 0.0001.
In this bulk RNA-seq analysis, AMs co-cultured with BAL-neutrophils showed upregulation of xenobiotic metabolism compared to AMs alone. Xenobiotic metabolism pathways are known to be involved in oxidative stress [41]; consistent with this, we found high expression of relative genes of reactive oxygen species (ROS) in AMs co-cultured with BAL-neutrophils ( Figure 5A). ROS can activate macrophages, which are usually po- (E) Enrichment plot from GSEA of infected neutrophils using gene sets for neutrophil chemotaxis and granule constituents between AMs (alone) and AMs of BAL groups. Data are presented as arithmetic mean ± SD. ns, no significant; ***, p < 0.001; **** p < 0.0001.
In this bulk RNA-seq analysis, AMs co-cultured with BAL-neutrophils showed upregulation of xenobiotic metabolism compared to AMs alone. Xenobiotic metabolism pathways are known to be involved in oxidative stress [41]; consistent with this, we found high expression of relative genes of reactive oxygen species (ROS) in AMs co-cultured with BAL-neutrophils ( Figure 5A). ROS can activate macrophages, which are usually polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2) [42,43]. M1 macrophages are induced by IFN-γ, LPS, or TNF-α and cause pro-inflammatory responses. Conversely, M2 are triggered by GM-CSF, IL-10 or IL-4, and IL-13, leading to an anti-inflammatory response [44,45]. Subsequently, we assessed whether AMs were activated by neutrophils. We found that mRNA expression of some M1-associated genes in AMs co-cultured with BAL-neutrophils was higher than that in AMs (alone), but PR8-neutrophils caused AMs to highly express M2-associated genes ( Figure 5B). Mean fluorescence intensity (MFI) of cell surface marker CD80 and CD86 was higher in AMs co-cultured with BAL-neutrophils, though AMs co-cultured with PR8-neutrophils showed increased expression of CD71 and CD206 ( Figure 5C) [46]. This is consistent with the results of other studies in which high GM-CSF level of AMs were shown to induce M1 to M2 switch after influenza A virus infection [47]. In addition, phagocytotic capacity is a classical functional property of macrophages by which they actively engulf unwanted material, dead cells, and debris during maintenance of homeostasis, tissue injury, and infection [48,49]. Usually, M2 macrophages exhibit high phagocytic activity. Herein, the heatmap and Quantitative Realtime-PCR showed that AMs of the PR8 group had more differentially expressed phagocytosis-associated genes than AMs alone or AMs of BAL group ( Figure 5D,E). We also measured AM phagocytotic capacity using CFSE-labeled neutrophils in the co-culture system. AMs were found to engulf more CFSE-labeled PR8neutrophils than BAL-neutrophils ( Figure 5F). These data indicate that BAL-neutrophils not only influence AM proliferation, but also affect the phagocytosis.

Cell Death of BAL-Neutrophils Inhibits AM Proliferation
Neutrophils are short-lived immune cells. Spontaneous death of neutrophils may take place at any moment of homeostasis and inflammation, and various cell death pathways are involved, including apoptosis, necrosis, pyroptosis, autophagy, and ferroptosis. For example, aging neutrophils were shown to accumulate ROS, negatively regulate phosphatidylinositol 3,4,5-trisphosphate/Akt signaling and activate pan-caspase-mediated pathways [50,51]. Autophagy regulates degranulation, ROS and inflammatory responses of neutrophils [52]. In this study, AMs and neutrophils were co-cultured for more than 24 h, and neutrophil death may have possibly occurred. Influenza virus infection was shown to result in the apoptosis of neutrophils and the formation of NETs [29,30]; however, Figure 3A suggests that the signaling pathways of apoptosis and NETosis for neutrophils were not major factors for inhibiting AM proliferation. Clinical data has shown that G-CSF is an effective drug to treat neutropenia [53]. In addition, Q-VD-Oph was shown to be a pan-caspase inhibitor of neutrophil death. In a previous study [54], G-CSF, Q-VD-Oph (Quinoline-Val-Asp-Difluorophenoxymethylketone, a pan-caspase inhibitor), NAC (N-acetyl cysteine, an antioxidant mediator), and DFO (deferoxamine mesylate, an iron chelator) were applied alone or in combination to target the neutrophil death pathways. None of the inhibitors when used alone significantly increased Ki67 expression of AMs co-cultured with BAL-neutrophils, but their combined usage increased Ki67 expression of AMs ( Figure 6). These data suggested that the cell death of BAL-neutrophils impacted AM proliferation, and that multiple death pathways were involved in this phenomenon.
ity. Herein, the heatmap and Quantitative Realtime-PCR showed that AMs of the PR8 group had more differentially expressed phagocytosis-associated genes than AMs alone or AMs of BAL group ( Figure 5D,E). We also measured AM phagocytotic capacity using CFSE-labeled neutrophils in the co-culture system. AMs were found to engulf more CFSElabeled PR8-neutrophils than BAL-neutrophils ( Figure 5F). These data indicate that BALneutrophils not only influence AM proliferation, but also affect the phagocytosis.

Discussion
AM maintenance via proliferation and self-renewal is important for lung tissue development in situ and for host protection during infection. Previous studies have identified a few genes that are required for the development and homeostasis of AMs. GM-CSF and TGF-β were shown to be important for the differentiation of fetal monocytes into AMs and for the postnatal maturation of AMs [55,56]. Furthermore, AMs interact with T cells, DCs, AECs, type 2 innate lymphoid cells (ILC2s), and alveolar type 2 cells (AT2s) via crosstalk between cytokine signaling pathways in order to respond to damage to the lung microenvironment [9,11,17,57,58]. However, the role of neutrophils in AM maintenance is not clear. In the present study, neutrophils isolated from BAL were found to influence AM proliferation, and multiple death pathways of neutrophils were involved in this process.
Neutrophil populations are highly heterogeneous in different tissues, such as lung, blood, BM, spleen, skin, and intestine. Especially in the lung, the neutrophils are endowed

Discussion
AM maintenance via proliferation and self-renewal is important for lung tissue development in situ and for host protection during infection. Previous studies have identified a few genes that are required for the development and homeostasis of AMs. GM-CSF and TGF-β were shown to be important for the differentiation of fetal monocytes into AMs and for the postnatal maturation of AMs [55,56]. Furthermore, AMs interact with T cells, DCs, AECs, type 2 innate lymphoid cells (ILC2s), and alveolar type 2 cells (AT2s) via crosstalk between cytokine signaling pathways in order to respond to damage to the lung microenvironment [9,11,17,57,58]. However, the role of neutrophils in AM maintenance is not clear. In the present study, neutrophils isolated from BAL were found to influence AM proliferation, and multiple death pathways of neutrophils were involved in this process.
Neutrophil populations are highly heterogeneous in different tissues, such as lung, blood, BM, spleen, skin, and intestine. Especially in the lung, the neutrophils are endowed with additional biological characteristics and pro-angiogenic functions [20]. Therefore, BAL-neutrophils, but not BM-neutrophils, influence AM proliferation, probably because BALneutrophil senescence activates some pathways, such as TGF-β signaling, P53 pathway, or metabolism compared with BM-neutrophils, and those processes may be implicated in neutrophil degranulation. However, neutrophils isolated from BAL of influenza virusinfected mice did not inhibit AM proliferation, but enhanced AM phagocytosis. In addition, AMs of PR8 group showed increased expression of IFN-α and -γ response-associated genes compared with AMs of BAL group. Furthermore, the relative genes of IFN-α and -γ responses were highly expressed in PR8-neutrophils compared with uninfected neutrophils ( Figure 3C). These findings collectively suggest that AMs co-cultured with PR8-meutrophils tend to be activated by IFN responses and further regulate AM proliferation and possibly influence AM homeostasis.
However, macrophages also have other phenotypes and functions based on the tissue environment [59]. Firstly, to better understand macrophage physiology, BMDMs are commonly used as an in vitro experimental model for immunological studies [60]. In this study, neutrophils isolated from BAL or BM were not found to affect the proliferation of BMDMs. One potential reason is the high proliferation rate of BMDMs, and that AMs differ from other macrophages with respect to their tissue specificities. However, due to the limitations of studying other macrophages' isolation and culture, it is unclear whether BAL-neutrophils affect other macrophage proliferation. Hence, we conclude only that BALneutrophils only inhibit AM proliferation, but not BMDMs. Secondly, external stimulation can modify macrophage metabolism from the M1 phenotype in the inhibitory state to the M2 phenotype in the repair state [61,62]. In addition, NETs of neutrophils may induce macrophage polarization to M1 phenotype via release of pro-inflammatory cytokines during acute and chronic diseases [32,63]. According to our bulk RNA-seq data, AMs cocultured with BAL-neutrophils showed increased expression of inflammation-associated genes compared with AM alone. In addition, the levels of M1 markers CD80 and CD86 were higher than those in AM alone. We speculate that cell death participates in regulating AM proliferation during co-culture of AMs and BAL-neutrophils.
Neutrophils play crucial roles in regulating innate and adaptive immune responses in steady state and disease. Usually, the half-life of neutrophils is less than 24 h. Neutrophil death can lead to the release of their cytotoxic components, which may harm the host and cause inflammatory or autoimmune disease. For example, granulocyte transfusion and G-CSF have been used to treat neutropenia induced by bacterial or fungal infections. Nevertheless, this therapeutic option is limited by the rapid death of neutrophils [64]. Global cell depletion of neutrophils following influenza virus infection leads to a more severe inflammatory response and increases disease severity [65]. However, depletion of a small number of neutrophils is conducive to antiviral response [66]. In our study, AMs and neutrophils were co-cultured for more than 24 h in all experiments, and neutrophil death was observed (data not shown). According to Fan et al.'s study [54], we chose some inhibitors to treat co-culture of AMs and BAL-neutrophils. For example, Q-VD-OPh is pan-caspase inhibitor and has an effect on preventing apoptosis involved by caspase 3, 7, 8, 9, 10 and 13 [67,68]. NAC can decrease apoptosis of resting neutrophils [69], and ameliorates neutrophil functions during acute pancreatitis. DFO is a slow iron chelator, which can inhibit neutrophil ROS and NETs [70]. In Figure 6, we found that none of these inhibitors when used alone significantly increased Ki67 expression in AMs in co-culture, but usage of a combination of cell death inhibitors further confirmed the involvement of multiple death pathways in neutrophil cell death. In the steady state, programmed cell death of neutrophils contributes to the maintenance of AM homeostasis. Different signaling pathways participate in the different stages of neutrophil degranulation [71], and BAL-neutrophils are probably in a different degranulation stage from that of BMand PR8-neutrophils, which may explain the inhibitory effect of BAL-neutrophils on AM proliferation. Notwithstanding, we observed that PR8-neutrophils isolated from the influenza virus infection model lost the inhibitory effect on AM proliferation; however, PR8-neutrophils stimulated AM activation and further impacted AM homeostasis. Thus, targeting neutrophil cell death is a potential therapeutic approach for AM maintenance in some respiratory diseases.

Conclusions
In summary, neutrophils from different tissues in healthy or infected mice have different characteristics. Our data show that the life and death of neutrophils play an important role in the proliferation and homeostasis of tissue-resident macrophages in pulmonary airspaces, especially with respect to the various modes of death. Combined usage of different inhibitors of neutrophil death may potentially recover AM proliferation and regulate AM homeostasis.