Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

Bone marrow mesenchymal stem cells (BMSCs) from Wistar rats were purchased from Cyagen Biosciences, Inc., China. The cells were cultured in BMSC medium (Cyagen Biosciences, China) supplemented with 10% FBS (Cyagen Biosciences, China), 1% antibiotic drugs (streptomycin and penicillin; Sparkjade Biotechnology, China), and 1% glutamine. The complete medium was changed every 2 days until the density of the adherent cells reached approximately 80%. The rat alveolar macrophage cell line NR8383 was purchased from Wuhan Pricella Biotechnology Co., Ltd., China. The cells were cultured in Ham's F-12K medium (Wuhan Pricella Biotechnology Co., Ltd., China) supplemented with 20% FBS (Wuhan Pricella Biotechnology Co., Ltd., China) and 1% antibiotic drugs (streptomycin and penicillin; Sparkjade Biotechnology, China). The complete medium was changed every 3 days until the cell density reached approximately 90%, and the cells were passaged at a 1:2 ratio.

NR8383 cells (8×10⁵) were cultured in 12-well plates and cotreated with LPS (100 ng/mL) (Sigma, USA) and IFN-γ (20 ng/mL) (PeproTech, USA) for 24 h to induce macrophage polarization and establish M1 macrophages (NR8383^M1 cells). NR8383^M1 cells were collected and washed three times with PBS and then directly cocultured with BMSCs at ratios of 1:1, 1:2, 1:5, and 1:10 (BMSCs: NR8383^M1 cells). NR8383 cells were collected after 24 hours for further analysis.

To each well of a six-well plate, 500 µl of TRIzol (Thermo Fisher, USA) was added for lysis of the cells, or the tissue samples were lysed by adding 1 mL of TRIzol per 50 g. Chloroform was added at a ratio of 5:1 (vol.) TRIzol:chloroform, and the mixture was centrifuged (12,000 rpm, 15 min, 4 °C), precipitated with isopropanol, washed with 75% alcohol, and dried. NanoDrop (Thermo scientific,USA) was used to measure the RNA concentration, and the A260/OD280, A260/OD230, and RNA concentration were recorded. Reverse transcription was performed using a Hifair^® III 1st Strand cDNA Synthesis Kit (YEASEN, China), and quantitative real-time PCR (qPCR) was carried out using Hieff^® qPCR SYBR^® Green Master Mix (No Rox) (YEASEN, China). The relative expression levels of the target genes were calculated according to the 2^^-ΔΔCt method. The primer sequences are listed in Table 1.

Whole-cell proteins were prepared by lysing cells in RIPA buffer (Sparkjade Biotechnology, China) supplemented with protease inhibitors (Beijing Solarbio Science & Technology Co., Ltd., China). The total protein concentration was determined using a BCA protein assay kit (Sparkjade Biotechnology, China). After denaturation at 95°C for 10 min, the protein samples were separated by gel electrophoresis and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)). The primary antibody was removed by washing the membrane three times in TBST, after which the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (SA00001-15, SA00001-2, Proteintech, China) for 1 h. After washing three times in TBST, the target proteins were visualized with enhanced chemiluminescence reagents (YEASEN, China).

The cells were collected and blocked with an Fc receptor blocker (1 μg/1×10⁶ cells; BD, USA) for 15 min at 4°C. The samples were fixed and permeabilized using the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend, USA) for 20 minutes. Intracellular staining was performed with APC-anti-CD68 (sc-20060, Santa Cruz Biotechnology, USA), PE-anti-iNOS (sc-7271, Santa Cruz Biotechnology, USA), FITC-anti-CD206 (sc-58986, Santa Cruz Biotechnology, USA) or FITC-anti-Arg-1 (sc-81154, Santa Cruz Biotechnology, USA) for 30 minutes at room temperature in the dark. Flow cytometry analysis was performed on a CytoFLEX flow cytometer (Beckman Coulter, USA), and data analysis was performed using CytExpert software.

Following fixation with 4% paraformaldehyde for 30 min, the cells were permeabilized with 1% Triton X-100 (prepared in PBS; Sparkjade Biotechnology, China) for 20–30 min and blocked with goat serum (Sparkjade Biotechnology, China) for 1 h at room temperature. The samples were then incubated with primary antibodies overnight at 4°C, followed by incubation with appropriate fluorophore-conjugated secondary antibodies for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI (1 μg/mL) for 20 min. The localization of the protein and its expression in the cells were observed using a fluorescence microscopy.

CD200 small interfering RNA (siCD200) and negative control siRNA (NC) were designed by Guangzhou Ruibo Biotechnology Co., Ltd. (China). BMSCs (2×10⁵) were cultured in 6-well plates without dual antibodies but containing 10% FBS medium. Transfection was performed with transfection complexes containing siCD200 at a concentration of 50 nM, according to the manufacturer's protocol.

NR8383 cells (1.5×10⁶) were plated into 6-well plates for 12 h. After pretreatment with SP600125 (JNK inhibitor; Sparkjade Biotechnology, China) at concentrations of 5 μM and 10 μM for 30 min, the NR8383 cells were cotreated with LPS (100 ng/mL) and IFN-γ (20 ng/mL). The cells were collected after 24 hours for further analysis.

Sixty SPF-grade adult male Wistar rats weighing approximately 220 g were purchased from Beijing Viton Lihua Laboratory Animal Technology Co. The rats were kept in a cleanroom at Yi Shengyuan Gene Technology (Tianjin) Co., Ltd., at a constant temperature of 24 ± 1°C. Adequate food and water were provided, and the animals were fasted for 1 day before sampling. The animal experiment was approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd. (Approval number YSY-DWLL-2022236).

The smoke generator used in this study was independently developed by the School of Disaster and Emergency Medicine of Tianjin University and can be placed in small animals for one-to-one nasal inhalation for modelling (9). The temperature of the smoke generator was set to 37°C, and the set value of the oxygen concentration was 19~21% (the air compressor provided oxygen to prevent suffocation of the animal due to lack of oxygen). After ignition, when the CO concentration in the smoke furnace was stable at approximately 3500 ppm, the rats were fixed on the immobilizer with the nose and mouth forwards for modelling. During the modelling process, the smoke concentration could be adjusted using the valve under the instrument to stabilize the mean value. After 25 min, the rats were removed and fully exposed to the air, and their response and respiratory rate were observed.

Adult male rats were randomly assigned to the control group, SII model group, SII+BMSCs treatment group, SII+BMSCs^NC treatment group, or SII+BMSCs^siCD200 treatment group. At 12 h after smoke injury modelling, 200 μL of PBS (control group, SII model group), 200 μL of the BMSC suspension (SII+BMSCs treatment group, containing 1×10⁶ cells), 200 μL BMSC^NC suspension (SII+BMSCs^NC treatment group, containing 1×10⁶ cells), or 200 μL BMSC^siCD200 suspension (SII+BMSCs^siCD200 treatment group, containing 1×10⁶ cells) was injected via the tail vein. After transplantation, the animals were returned to the cage for further rearing, and six animals from each group were sampled on Days 1 and 3 after injection.

Lung tissue samples were taken from each group of rats, and surface blood was washed in saline, drained, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (HE) to assess lung injury. The lung injury score was graded according to the histologic scoring criteria proposed by Oishi H et al. (20).

The lung tissue wet-dry weight ratio (wet/dry ratio) was used to assess pulmonary oedema. The middle lobe of the right lung was resected, the surface water was absorbed by filter paper, and the lobe was weighed, dried in an oven at 65°C for approximately 72 h (until the weight no longer changed), and weighed again. The ratio of the weight of the lung tissue before and after drying was used to determine the wet/dry ratio of the lung tissue.

Place the rat in a supine position, and a gavage needle was inserted into the trachea to bluntly isolate the upper lobe of the right lung, which was ligated using sutures. The alveolar lavage fluid was recovered by injecting 4 mL of saline into the lungs through the gavage needle, repeatedly aspirating 3 times, and placing the fluid on ice. An appropriate amount of alveolar lavage fluid was collected and diluted with BCA to measure the protein concentration.

The BALF was collected and centrifuged at 3000 rpm for 15 min at room temperature. The concentrations of IL-1β, IL-6, TNF-α and TGF-β were determined using ELISA kits (Nanjing Jiancheng, China) according to the manufacturer's protocol.

SPSS 25.0 and GraphPad Prism 8.0 were used for the statistical data analysis. Normally distributed continuous variables are expressed as means ± standard deviations (SDs), and unpaired two-tailed t tests were used to compare two groups of variables, whereas more than two groups were analyzed via one-way ANOVA followed by Dunnett's multiple comparisons analysis or two-way ANOVA followed by Tukey's multiple comparisons analysis. P values less than 0.05 were considered to indicate statistical significance.

The results of the qPCR assay revealed a significant increase in the mRNA expression of iNOS, a marker of M1 macrophages, in the LPS/IFN-γ group compared with that in the control group at 6 h, 12 h, and 24 h, with a peak at 6 h (Figure 1A). The WB results revealed that iNOS and CD86 expression in the LPS/IFN-γ group gradually increased over time compared with the control group and peaked at 24 h (Figure 1B). The FCM results also revealed that the percentage of iNOS⁺ cells (M1-type cells) gradually increased over time and peaked at 24 h (Figures 1C, D). The gene expression levels of M1-type cytokines (TNF-α, IL-6, and IL-1β) followed the same trend as the changes in the gene expression level of iNOS, with the levels of all cytokines peaking at 6 h and then decreasing (Figure 1E). Therefore, combined stimulation of alveolar macrophages (AMs) with 100 ng/mL LPS and 20 ng/mL IFN-γ for 24 h successfully established an in vitro M1-type cell model.

M1 macrophages (NR8383^M1 cells) were cocultured with BMSCs at ratios of 1:1, 1:2, 1:5 and 1:10 BMSCs: NR8383^M1 cells. The results revealed that BMSCs at different ratios of coculture significantly decreased the percentage of iNOS⁺ cells (M1-type cells) compared to the NR8383^M1 group, with the most pronounced decreases in the groups with coculture ratios of 1:2 and 1:5. BMSCs significantly increased the percentage of M2-type (CD206⁺) cells in all but the 1:10 group, with the most significant increase in the percentage of M2-type cells in the group with a coculture ratio of 1:2 (Figures 2A-C). Moreover, as shown in Figure 2D, BMSCs at different coculture ratios significantly reduced the M1/M2 ratio, with the lowest M1/M2 ratio in the group with a coculture ratio of 1:2. These findings suggest that the BMSCs in this coculture system are more capable of inducing the M2-type polarization of alveolar macrophages, and therefore, a 1:2 ratio was chosen for subsequent experiments.

First, an immunofluorescence assay revealed that more than 80% of the BMSCs were positive for CD200 expression on the cell surface, which was localized mainly in the cell membrane and cytoplasm, indicating that the rat BMSCs expressed the antigen CD200 on the surface (Figure 3A). The percentage of CD200R⁺ cells in the NR8383 cell line reached 98.85 ± 1.11% according to the FCM assay (Figure 3B).

To further investigate the role of CD200 in the regulation of alveolar macrophage polarization by BMSCs, we used siRNA transfection to inhibit CD200 expression in BMSCs. Immunofluorescence assays revealed that CY3-positive siRNA-transfected BMSCs presented obvious red fluorescence, and the transfection efficiency was approximately 50% at 24 h. The transfection efficiency increased significantly, and the fluorescence intensity obviously increased after 48 h, indicating that the siRNA could be successfully transfected into the rat BMSCs (Figure 3C). After 48 h of transfection, the CD200 gene expression levels in the siRNA-01, siRNA-02 and siRNA-03 groups were significantly lower than those in the NC group (Figure 3D), while the protein expression levels were the same as the gene expression levels, with inhibition rates of 54%, 43% and 77%, respectively, and the knockdown effect was most obvious in the siRNA-03 group (Figure 3E). Therefore, siRNA-03 was selected for subsequent experiments based on the above experimental results.

Finally, we investigated the effect of CD200 knockdown on alveolar macrophage polarization by BMSCs. Consistent with the results of previous experiments, compared with the control group, NR8383 cells were polarized towards the M1 phenotype after being treated with LPS/IFN-γ and were partially transformed to the M2 phenotype after 24 h of coculture with BMSCs, which resulted in a decrease in the percentage of iNOS⁺ cells (M1-type), an increase in the percentage of Arg-1⁺ cells (M2-type), and a significant decrease in the ratio of iNOS⁺/Arg-1⁺ (M1/M2) (Figures 4A-D). Compared with the BMSCs^NC group, the inhibitory effect of BMSCs on iNOS⁺ cells was significantly attenuated by siCD200 transfection, while the percentage of Arg-1⁺ cells was significantly reduced, and the iNOS⁺/Arg-1⁺ ratio (M1/M2) was significantly increased (Figures 4A-D). However, the percentage of iNOS⁺ cells and the M1/M2 cell ratio were significantly lower in the BMSCs^siCD200 group than in the NR8383^M1 group. These findings suggest that CD200 knockdown partially affects the effect of BMSCs on macrophage polarization, whereas other modalities are also involved in the regulation of macrophage polarization by BMSCs.

Moreover, we further tested macrophage polarization-related marker molecules and cytokines, and the results were consistent with the above results. Compared with those in control cells, the M2-type markers Arg-1 and CD206 and the anti-inflammatory cytokine TGF-β were significantly decreased in NR8383 cells after LPS/IFN-γ stimulation (Figures 4E-H). Moreover, the protein expression levels of the M1-type marker iNOS and the proinflammatory cytokine IL-1β were significantly increased (Figures 4I, J). After coculture, BMSCs significantly promoted the polarization of M1 macrophages to M2 macrophages, downregulated the expression level of the proinflammatory factor IL-1β and upregulated the expression level of the anti-inflammatory factor TGF-β. siCD200 transfection effectively attenuated the ability of BMSCs to promote the conversion of M1 macrophages to M2 macrophages, and downregulated the effect of BMSCs on alveolar macrophage polarization. These findings suggest that the CD200-CD200R pathway is involved in the immunomodulatory effects of BMSCs on alveolar macrophages.

Compared with that in control cells, the expression level of iNOS in NR8383 cells was significantly greater after treatment with LPS/IFN-γ, and the macrophages were polarized towards the M1 phenotype. Moreover, compared with that in the control group, the level of phosphorylated JNK significantly increased in a time-dependent manner after LPS/IFN-γ treatment, whereas the level of phosphorylated P38 did not significantly change (Figures 5A, B). These findings suggest that the JNK-MAPK pathway may be activated and involved in the polarization of NR8383 cells towards the M1 phenotype. Next, the JNK inhibitor SP600125 was used to determine whether the M1 polarization of NR8383 cells was mediated via JNK-MAPK signaling. As shown in Figures 5C, D, compared with those in the M1 group, the expression levels of iNOS in both different inhibitor groups were significantly lower in a dose-dependent manner, and the inhibitory effect was most obvious in the 10 μM SP600125 group. Flow cytometry results also demonstrated that SP600125 treatment significantly reduced the percentage of iNOS⁺ macrophages (M1-type) compared to the M1 group, with a more pronounced effect observed at 10 μM SP600125. (Figures 5E, F). These findings suggest that the JNK-MAPK pathway is involved in the LPS/IFN-γ-induced polarization of NR8383 cells towards the M1 phenotype.

To investigate whether the regulation of alveolar macrophage polarization by BMSCs via CD200 is related to JNK-MAPK signaling, we observed the effect of CD200 on JNK activity. The results revealed that BMSCs coculture significantly inhibited JNK phosphorylation and that JNK-MAPK signal activation was suppressed, whereas CD200 knockdown significantly attenuated the inhibitory effect of BMSCs on the activity of JNK signaling compared with that in the BMSCs^NC group (Figures 5G, H). These findings suggest that BMSCs inhibit LPS/IFN-γ-induced JNK-MAPK signal activation via CD200-CD200R, which in turn contributes to the M2-type polarization of alveolar macrophages.

HE staining of lung tissue revealed that the SII model group exhibited pathological changes, such as alveolar effusion, interstitial thickening, destruction of some alveolar structures, fusion of adjacent alveoli with each other to form larger vesicles, and inflammatory cell infiltration, which were consistent with ALI pathological changes (Figure 6A). The lung damage score was significantly lower than that of the SII model group at both 1 and 3 days after treatment with BMSCs, which was manifested by a reduction in the area of solid lung lesions and a decrease in the degree of septal thickening, but the degree of oedema was still more obvious. Compared with the SII+BMSCs^NC group, the SII+BMSCs^siCD200 group presented significantly greater lung injury scores, characterized by pronounced alveolar structural fusion and increased pulmonary consolidation (Figures 6B, C).

The lung wet/dry ratios of the lung tissues in the SII model group were significantly greater than those in the control group on Days 1 and 3, accompanied by significant pulmonary oedema. The wet/dry ratio after BMSC transplantation was significantly lower than that in the SII model group, suggesting that BMSCs effectively attenuated SII-induced pulmonary oedema. There was a trend towards an increase in the lung tissue wet/dry ratio in the SII+BMSCs^siCD200 group compared with the SII+BMSCs^NC group, but the difference was not significant (Figures 6D, E). The total protein concentration in the BALF is shown in Figures 6F, G. On both Days 1 and 3, BMSCs significantly reduced the total protein concentration in the alveolar lavage fluid of rats with SII, whereas CD200 knockdown attenuated this effect, increasing the protein concentration. The total protein concentrations in the BALF were also significantly higher in the SII+BMSCs^siCD200 group than in the SII+BMSCs^NC group. These results suggest that BMSCs attenuate smoke inhalation-induced pulmonary oedema and have a therapeutic effect on SII, whereas CD200 knockdown attenuates the therapeutic effect of BMSCs.

As shown in Figures 7A-D, the percentage of iNOS⁺ cells (M1-type) in alveolar lavage fluid was significantly greater in the SII+PBS group than in the control group. One day after BMSC transplantation, the percentage of iNOS⁺ cells (M1-type) was significantly lower and the percentage of Arg-1⁺ cells (M2-type) was significantly higher, whereas M1/M2 was significantly lower than that in the SII+PBS group. Compared with that in the SII+BMSCs^NC group, the percentage of iNOS⁺ (M1-type) cells was significantly greater in the SII+BMSCs^siCD200 group, whereas there was no significant difference in the percentage of Arg-1⁺ (M2-type) cells between the two groups (Figures 7B, C). In addition, the downregulation of the M1/M2 ratio was weaker in the SII+BMSCs^siCD200 group than in the SII+BMSCs^NC group, with a statistically significant difference between the two groups (Figure 7D). These results are consistent with in vitro data showing that CD200 knockdown attenuates the regulation of alveolar macrophage-to-M2-type cell transformation by BMSCs.

The level of M1/M2 macrophage polarization in the alveolar lavage fluid 3 d after the transplantation of BMSCs was essentially the same as that at 1 d, as shown in Figures 7E-H. Compared with those in the SII+BMSCs^NC group, the percentage of iNOS⁺ (M1-type) cells, the percentage of CD206⁺ (M2-type) cells, and the M1/M2 ratio in the SII+BMSCs^siCD200 group were significantly different. Moreover, the percentage of iNOS⁺ (MI-type) cells and the ratio of M1/M2-type cells were significantly lower in the SII+BMSCs^siCD200 group than in the SII+PBS group, suggesting that the knockdown of CD200 partially affects the effect of BMSCs on macrophage polarization. Therefore, we demonstrated that BMSCs influence the inflammatory response by promoting macrophage polarization towards anti-inflammatory M2 macrophages in SII. The effect of BMSCs on alveolar M1/M2 macrophage polarization was significantly attenuated but not completely eliminated after CD200 was knocked down. In addition to the CD200-CD200R pathway, paracrine secretion may also be related to the regulatory effect of BMSCs.

Compared with those in the control group, the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the SII+PBS group were significantly higher, in the BALF on Days 1 and 3, whereas the level of the anti-inflammatory cytokine TGF-β was significantly lower (Figure 8). Transplantation of BMSCs significantly downregulated the levels of IL-1β, IL-6, and TNF-α while promoting TGF-β expression. This is related to the fact that BMSCs regulate the M1/M2 immune balance of alveolar macrophages and contribute to macrophage polarization towards anti-inflammatory M2 macrophages. At 1 d after BMSC transplantation, CD200 knockdown did not significantly affect the expression of inflammation-related cytokines, whereas at 3 d after transplantation, the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were significantly greater and the level of the anti-inflammatory factor TGF-β was significantly lower in the SII+BMSCs^siCD200 group than in the SII+BMSCs^NC group. These findings suggest that CD200 knockdown attenuates the immunosuppressive effects of BMSCs on pro-/anti-inflammatory cytokines.