Chronic exposure to biomass ambient particulate matter triggers alveolar macrophage polarization and activation in the rat lung

Abstract The role of alveolar macrophages (AMs) in chronic obstructive pulmonary disease is unclear. We characterized the function of AMs in rats chronically exposed to biomass fuel smoke (BMF) and studied the signal pathways that regulate AMs polarization. One hundred and eighty male Sprague‐Dawley rats were divided into BMF group and clean air control (CON) group. After BMF smoke exposure for 4 days, 1 month and 6 months, the cytokine secretion and function of AMs were determined by flow cytometry, quantitative polymerase chain reaction, Western blotting and immunofluorescence. Bone marrow‐derived macrophages were cultured and exposed to particulate matter (PM) from the smoke. Exposure initially promoted pro‐inflammatory factors, but pro‐inflammatory macrophages shared features of anti‐inflammatory macrophages. Consistent with IL‐4 upregulated in bronchoalveolar lavage fluid, p‐Stat6 and peroxisome proliferator‐activated receptor γ (PPARγ) in AMs elevated at 4 days of exposure. After 6 months of exposure, CD206, TGF‐β1 and p‐Smad3 were significantly higher than the control groups. PPARγ reversed the M1 phenotype induced by PM in vitro and drove the macrophages into the M2 phenotype. Altogether, the study demonstrates the dynamic phenotype and functional changes in AMs during exposure to BMF smoke.

M1 macrophages predominate in non-allergic inflammation model, while the M2 phenotype predominates in allergic asthma. 21 COPD patients have higher levels of AMs and inflammatory mediators that contribute to the pathology of the disease. 22,23 In contrast, AMs in the bronchoalveolar lavage fluid (BALF) of COPD patients have been reported to express M2 markers. 24 Both isoforms of nitric oxide synthase (iNOS) and CD206 were shown to expressed by macrophages in the lungs of non-COPD smokers and COPD patients, indicating that macrophages in the lungs were polarized bidirectionally. 25 The function of AMs in COPD is unclear. Diversity and plasticity are characteristics of macrophages in vivo. 26 In order to maintain the homeostasis of the body, immunity constantly undergoes dynamic changes during the development and progression of the disease, as do AMs. To our knowledge, there are no published studies on dynamic changes in AMs in COPD.
In the present study, we exposed rats to biomass ambient particulate matter to study phenotypic changes and immune function of AMs in vivo, and investigate the transcriptional factors involved in activation of AMs.

| BMF smoke exposure system
Rats were exposed to smoke produced by smouldering China fir saw-dust (2 g per heating panel per session) for 2 h periods, 4 days and 5 days per week for 1 month and 6 months. The BMF smoke exposure system primarily consisted of a wood-burning unit and a whole-body exposure unit. The size of the animal exposure chamber was 265 × 205 × 140 mm (length by width by height). BMF smoke was generated by eight heating panels (500 w), which were connected in series in a wood-burning chamber. Each heating panel functioned for 20 min before the next heating panel was activated.
BMF smoke was set into the animal exposure chamber through a pump (15 L/min), while exhaust gas was pumped away by a negative pressure pump at a rate of 15 L/min, and continuous fresh air was poured into wood-burning chamber. Besides, there were two sampling ports to monitor various characteristics of PM and gas in exposure chamber.

| Particulate matter (PM) collection and extraction
PM was collected from the burning of China fir during hightemperature combustion with a moderate air supply between April 23 and May 6 of 2015 in accordance with a procedure described previously. 27 A high-volume sampler (TE-6070; Tisch Environmental) equipped with a fine particulate matter selective-inlet head(1.13 m 3 / min) was used to collect particles. Exposed filters were soaked in water for 10 min and then in dichloromethane for 4 h. The extracted solution was lyophilized and mixed. The weight of PM was defined as the increase in the weight of each filter. The PM sample was dissolved in dimethyl sulfoxide (DMSO) to a volume of 100 mg/ml, and then diluted with culture medium to yield a concentration of <0.01% DMSO.

| Sample preparation and isolation of AMs
Rats were sacrificed after 4 days, 1 month and 6 months of exposure. BALF was obtained by instilling the lungs sequentially with 8 ml ice-cold phosphate-buffered saline (PBS) four times. BALF was centrifuged to obtain cells and supernatants. The cells were suspended with 1 ml PBS and counted with a cell counter (Millipore Scepter2.0; MilliporeSigma). Cells were then plated onto six-well plates (100 × 10 4 cells/well; Corning) at 37℃ in a humidified atmosphere of 5% CO 2 . After 12 h of incubation, non-adherent cells were removed and adherent cells were used in the quantitative polymerase chain reaction (qPCR) analyses of mRNA.

| Gene expression
Total RNA was extracted and reverse-transcribed by using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio). qPCR was performed by using TB Green Premix Ex Taq (Takara Bio). The reactions were run on a CFX real-time detection system (Bio-Rad Laboratories). Table S1 lists the qPCR primer sequences.

| Histological staining
The lavaged lung (left) was then inflated with 4% formaldehyde and maintained at a pressure of 25 cmH 2 O to keep for histological assessment. Sections (5 μm) were stained with haematoxylin and eosin (HE) to assess morphological changes.

| Statistical analysis
Statistical analyses were performed using IBM SPSS 22.0 (Armonk, NY, USA), and data were expressed as mean ± standard deviation (SD). Two-group comparisons were conducted using an unpaired t test. Comparisons of more than two groups were performed using one-way ANOVA test. The Mann-Whitney U test was used to compare relative mRNA expression and CD206 mean fluorescence intensity (MFI) between experimental groups. p < 0.05 was considered significant.

| Determination of particle size distributions and gas concentrations in the exposure chamber
To measure the particle size distributions in suspension and gas concentrations as benchmarks of quality control parameters of the exposure system, we used a DustTrakⅡ aerosol detector (TSI) and a Test340 portable gas analyzer (Testo). The concentrations of particulate matter with diameters of 1, 2.5 and 10 μm (PM 1 , PM 2.5 and PM 10 ) were 27.77 ± 8.66, 28.07 ± 8.84 and 28.23 ± 8.86 mg/m 3 in the BMF exposure room, respectively (Table S2). The carbon monoxide (CO) concentration was maintained at 55.16 ± 13.77 ppm, and nitric oxide (NO) and sulphur dioxide (SO 2 ) were not detected.

| BMF smoke-induced lung morphological changes and AMs infiltration
We assessed morphometric changes in the lungs as markers of emphysema. Alveolar enlargement was calculated as the mean linear intercept (MLI), and the bronchial wall thickness was quantified by wall thickness, calculated as the total bronchial area minus the lumen area, divided by total bronchial area. BMF

| BMF smoke exposure-induced BALF cytokine expression
To investigate how BMF exposure influences pulmonary inflammation, which may affect the M1/M2 phenotype, 27 cytokines/ chemokines multiplex tests were performed ( Figure 2). We meas-

| Phenotypic characterization of AMs polarization induced by BMF smoke exposure
We used qPCR to determine the mRNA expression of a few key genes of AMs following exposure to BMF smoke (Figure 3). The result showed that nitric oxide synthase (iNOS) and IL-1β significantly ascended at 4 days of BMF smoke exposure ( Figure 3A

| BMF smoke exposure triggered signalling pathways of macrophage polarization and activation
We sought to identify the signalling pathways involved in polarization and activation of AMs underexposure, especially those involved in M2 polarization to attenuate the inflammatory response and promote tissue remodeling. 13,31 We used qPCR, Western blotting and immunofluorescence to determine the mRNA and protein levels of Stat6, Stat3, PPARγ and TGF-β1 in exposed rats. We also exam- PPARγ mRNA expression in AMs was upregulated after 4 days of exposure ( Figure S1A, p < 0.01) and declined to normal level after that ( Figure S1B,C, p = 0.66 and 0.543). PPARγ protein level in F I G U R E 1 Lung morphological changes and AMs infiltration into BALF following exposure to smoke from BMF. Scale bar: 50 μm. A, C: Small-airway wall stained with HE and statistical analysis of small-airway wall thickness. B, D: Lung tissue stained with HE and statistical analysis of mean linear intercept (MLI). E: Comparison of the total number of cells in BALF between BMF and CON groups. F: Comparison of AMs in BALF between BMF and CON groups. Data in C, D, E, and F represent the mean ± SD of a minimum number of six rats per group. *p < 0.05, **p < 0.01, significantly different from CON groups. AMs, alveolar macrophages; BALF, bronchoalveolar lavage fluid; BMF, biomass fuel smoke; CON, control lung tissue did not differ between controls and exposure groups after 1 and 6 months of BMF exposure ( Figure 4A

| PPARγ primed BMDMs exposed to PM into alternative macrophages
To study whether PPARγ reversed the M1 phenotype induced by  Figure 6D).
In addition, we found that PPARγ in BMDMs was upregulated by stimulation with IL-4 and promoted the expression of M2 markers. IL-4 stimulated PPARγ and p-STAT6 expression ( Figure 7A Figure 7C, p = 0.041).

F I G U R E 2
Smoke from BMF-induced cytokine expression in BALF. Analysis of 27 cytokine showed the levels of IL-1α, IL-1β, IL-12p70, LIX, TNFα, IL-4, EGF and VEGF. Data represent the mean ± SD of a minimum number of six rats per group. *p < 0.05, ** p < 0.01, significantly different from CON groups. BALF, bronchoalveolar lavage fluid; BMF, biomass fuel smoke; CON, control; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor

| DISCUSS ION
Indoor air pollution induced by biomass ambient particulate matter is strongly linked to the incidence and hospitalization rates of COPD. Our previous study showed that biomass ambient particulate matter retention in lung tissue-induced pulmonary inflammation, airway remodelling and alveolar cavity enlargement. [32][33][34] The chronic BMF smoke exposure model serves as a useful model to analyse how indoor air pollution promotes the progress of emphysema in lung. In this work, we demonstrated that the emergence of pro-inflammatory macrophages eventually conversed into antiinflammatory macrophages following exposure to smoke from BMF, which initiated this plasticity in AMs. We also identified the signalling pathways regulating this functional conversion of AMs and the dynamic molecular changes that drove AMs into an antiinflammatory phenotype.
Airway inflammation, airway remodelling and alveolar cavity enlargement were observed in our BMF smoke exposure model.
The early stage was characterized by airway inflammation, and the later stage was characterized by airway remodelling and alveolar cavity enlargement, in agreement with published observations in COPD models. 32 Our previous study also showed that peak expiratory flow (PEF) and forced expiratory volume in 20 milliseconds/ forced vital capacity (FEV 20 /FVC) decreased following a chronic exposure to BMF smoke, indicating lung dysfunction. 32 CD68 is a specific macrophage marker in rats, mice, and humans, and F4/80 is expressed in mature macrophages in mice. CD11b is expressed in both rats and mice, but its expression is low in AMs. 35 In the present study, we used CD68 as a marker of AMs and found that BMF

F I G U R E 3
Smoke from BMF altered the expression of genes and surface markers in AMs. A: iNOS, IL-1β and EGF mRNA expression upregulated in AMs exposed to BMF smoke for 4 days. B: TNFα mRNA expression in AMs had changed after 1 month of BMF exposure. C: iNOS, IL-1β, TNFα, TLR-2, TLR-4 and EGF mRNA expression did not change after 6 months of exposure. D, E:Comparison of CD206 expression in AMs between BMF and CON groups. F, G:Comparison of CD86 expression in AMs between BMF and CON groups. Data represent the mean ±SD of a minimum number of six rats per group. *p < 0.05, ** p < 0.01, significantly different from CON groups. AMs, alveolar macrophages; BMF, biomass fuel smoke; CON, control; EGF, epidermal growth factor exposure-induced macrophages infiltration into the lungs, which increased with the accumulation of exposure time.
Surprisingly, molecular analyses revealed that the emerging pro-inflammatory macrophages shared a feature of the antiinflammatory phenotype, which partially overlapped but was also distinct, including the co-expression of mRNA encoding of IL-1β, iNOS and PPARγ. The result also indicated that AMs produced proinflammatory factors to damage lung tissue and then skewed towards an anti-inflammatory phenotype after short-term exposure.
After 6 months of BMF smoke exposure, the protein level of TGF-β1

F I G U R E 4
Smoke from BMF exposure triggered Stat6, Stat3 and PPARγ activation. A, B: Comparison of p-Stat6, p-Stat3 and PPARγ protein expression in lung tissue between groups. C: Time-dependent activation of p-Stat6 was examined by double immunofluorescence staining of p-Stat6 (green) and CD68 (red). D: Time-dependent activation of PPARγ was examined by double Immunofluorescence staining of PPARγ (green) and CD68 (red). Scale bar: 20 μm. The values in A and B represent the mean ± SD of a minimum number of six rats per group. *p < 0.05, **p < 0.01, significantly different from CON groups. BMF, biomass fuel smoke; CON, control; PPARγ, proliferator-activated receptor γ increased, and airway and lung tissue were remodelled, resulting in the COPD pathology. The whole course from pro-inflammatory phenotype to anti-inflammatory phenotype, and then to the chronic pathology, indicated an interaction between BMF exposure and the pulmonary immune system.
Inflammation occurred in the early stage of BMF smoke exposure. Camila Oliveira da Silva and co-workers observed a dynamic change in cytokine production in mice exposed to cigarette smoke (CS), 36 involving an initial increase in TNFα and NO and a subsequent decline, although a 30 days of CS exposure increased TGF-β1 production in the lung. 36 We also found that the levels of IL-1α, IL-1β, IL-12p70, LIX as well as IL-4 in BALF increased after a shortterm exposure to BMF smoke. IL-1α and IL-1β, which are mainly produced by activated monocytes and macrophages, enhance F I G U R E 5 Smoke from BMF exposure triggered TGF-β1 pathway activation. A, B: Western blotting of TGF-β1 protein expression in lung tissue. A, C: Western blotting of protein levels of p-Smad3 and Smad3 in lung tissue. D: TGF-β1 in AMs was examined by double Immunofluorescence staining of TGF-β1 (green) and CD68 (red). E: p-Smad3 in AMs was also examined by double immunofluorescence staining of p-Smad3 (green) and CD68 (red). Scale bar: 20μm. The values in B and C represent the mean ± SD of a minimum number of six rats per group. *p < 0.05, **p < 0.01, significantly different from CON groups Dynamic changes of pro-inflammatory cytokines revealed that the most severe inflammatory injury is in the early stage of exposure.
Interestingly, levels of the Th2 cytokine IL-4 increased simultaneously to promote AMs towards the anti-inflammatory phenotype.
Levels of pro-inflammatory cytokines gradually decreased over the course of the exposure. Upregulation of IL-4 and the attenuation of inflammation were chronologically sequenced in COPD models, as seen previously. 37 Studies have reported that M2 can secrete EGF and VEGF to promote tissue repair, 17,38 suggesting that both factors are upregulated under the action of Th2 cytokine in the early stage of exposure. Shaykhiev and co-workers provided transcriptomebased evidence that smoking induces reprogramming towards M2 polarized macrophages in COPD patients, suggesting that AMs were likely involved in the pathogenesis of COPD in a non-inflammatory manner. 39 However, the clinical study did not track immune changes over time. Our study provided data on dynamic phenotype and functional changes in AMs during exposure to BMF smoke.
Macrophage polarization is controlled by signal pathway molecules, but the identity of these signal pathways is unclear in COPD.
In the present study, the anti-inflammatory phenotype was most prevalent, although it overlapped with pro-inflammatory phenotype after 4 days of exposure. We therefore focused more on signal pathways regulating the anti-inflammatory phenotype. IL-4 skews macrophages towards the M2 phenotype and activates Stat6 and Stat3.
Stat6 modulates multiple genes associated with the M2 phenotype, including CD206, arginase 1 and resistin-like α. 40 We found phosphorylation of Stat6 and Stat3 increased after 4 days of exposure, consistent with upregulation of IL-4. This supports the hypothesis that Stat6 and Stat3 signals participate in activation of M2 in the early stage of exposure.
PPARγ is an important transcription factors of M2 and primes monocytes into alternative activated macrophages. 20,41 The dynamic change of PPARγ has not been reported previously. We found that PPARγ elevated during the most severe inflammation periods, and then returned to the control level as the inflammation subsided. We also demonstrated that PPARγ was upregulated in BMDMs by IL-4 in vitro. The Th2 cytokine IL-4 is required for the development of the M2 phenotype, and IL-4-mediated signals stimulate PPARγ expression. 20,42,43 In addition, our study showed that PPARγ inhibited the activation and nuclear translocation of NFκB p65 via by inhibiting the phosphorylation of IKBα, and upregulated the expression of the M2 markers CD206 and p-STAT6, suggesting that PPARγ reversed M1 phenotype induced by PM, and drove the macrophages into the M2 phenotype rapidly in a protective manner. Drugs regulating PPARγ provide a potential interventional target in COPD; rosiglitazone was reported to inhibit cigarette smoke-induced pulmonary inflammation 44 and reduce exacerbations by attenuating pulmonary inflammation and decreasing bacterial burdens, 45 suggesting PPARγ may therefore be an effective approach in treating COPD.
TGF-β1 is another important regulatory molecule of M2, and critical for the development and maturation of AMs. TGF-β1 can also inhibit the expression of macrophage-derived inflammatory genes. 46,47 M2 participates in the development of fibrosis and contributes to disease pathogenesis. M2-derived TGF-β1 promotes tissue remodelling and wound repair by blocking the degradation of the extracellular matrix's degradation and eliciting synthesis of interstitial fibrillar collagens. 13 Besides, airway remodelling via the TGF-β1 pathway has been shown to lead to the thickening of the small-airway wall. TGF-β1 in AMs may be involved in the mechanism of airway remodelling.
Our study showed that AMs maintained an anti-inflammatory phenotype characterized by elevated CD206 and TGF-β1 markers without stimulation of Th2 cytokines after 6 months of exposure. We also found that TGF-β1 promoted CD206 expression in BMDMs in F I G U R E 7 PPARγ primed BMDMs into the M2 phenotype. A, B:Western blotting of PPARγ and p-STAT6 expression in BMDMs. C: Comparison of CD206 MFI in BMDMs between groups. The values in B and C represent the mean ± SD of six independent experiments. *p < 0.05, **p < 0.01, significantly different from control groups. BMDMs, bone marrowderived macrophages; PPARγ, proliferatoractivated receptor γ vitro. Chronic exposure to BMF smoke-induced TGF-β1 production and activated the downstream signal Smad3, which is involved in tissue remodelling. Activation of TGF-β1 in the late stage of exposure indicated the role of TGF-β1 in regulating M2 polarization and participating in lung tissue remodelling.
In summary, our study describes the dynamic phenotype and functional changes of AMs during exposure to BMF smoke, suggesting a pro-inflammatory role of AMs in the early stage of COPD and an anti-inflammatory role associated with tissue remodelling in the latter stage. Identification of more signal pathway molecules involved in AMs polarization may provide potential targets for the COPD treatments.

ACK N OWLED G EM ENTS
We thank Professor Zhongfang Wang from State Key Laboratory of Respiratory Diseases for his assistant for our experiment. We thank Liwen Bianji (Edanz) (www.liwen bianji.cn/) for editing the English text of a draft of this manuscript.

CO N FLI C T S O F I NTE R E S T
The authors declare that no conflict of interest exists.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available.