Pirfenidone modulates macrophage polarization and ameliorates radiation‐induced lung fibrosis by inhibiting the TGF‐β1/Smad3 pathway

Abstract Radiation‐induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation‐induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)‐approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X‐ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor‐β1 (TGF‐β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation‐induced expression of TGF‐β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL‐4–induced M2 macrophage polarization and IL‐13–induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase‐1 (ARG‐1), chitinase 3‐like 3 (YM‐1) and TGF‐β1. Notably, the PFD‐induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa‐B (NF‐κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation‐induced chemokine secretion in MLE‐12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF‐β1 from M2 macrophages by attenuating the activation of TGF‐β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF‐β1/Smad3.


| INTRODUC TI ON
Radiotherapy is a standardized treatment for thoracic tumours such as lung cancer, 1 oesophageal cancer, 2 malignant lymphoma 3 and breast cancer. 4 Clinical data show that the incidence of radiation-induced lung injury (RILI) is 5%-25% after radiation therapy in patients with lung cancer, followed by those with mediastinal lymphoma (5%-10%) and breast cancer (1%-5%). 5 There are two clinical manifestations of RILI: radiation pulmonary (RP) and radiation-induced lung fibrosis (RILF). To date, corticosteroids are mainly used to control RP in the clinic, while RILF leads to progressive alveolar structural disorders and irreversible pulmonary fibrous tissue remodelling, which eventually causes respiratory failure. There is currently no effective drug available to reverse RILF. 6 Macrophages are highly malleable, and their functional phenotypes depend on different microenvironments. After exposure to Toll-like receptor (TLR) ligands, macrophages experience a phenotype known as classically activated macrophages or M1 macrophages, which can produce high levels of pro-inflammatory cytokines, such as tumour necrosis factorα (TNFα), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS). In contrast, alternately activated macrophages or M2 macrophages are produced in response to cytokines such as IL-4 and IL-13 and highly express M2 macrophage-associated inflammatory factors, such as chitinase 3-like 3 (YM-1) and arginase-1 (ARG-1). 7 M1 macrophages have been shown to prevent the development of pulmonary fibrosis, and M2 macrophages are the most prominent type of macrophage in pulmonary fibrosis. 8 Therefore, the balanced transition from M1 macrophages that promote inflammation to M2 macrophages that promote fibrosis and wound healing is one of the important reasons for the development of pulmonary fibrosis after radiation.
Pirfenidone (PFD) is a multipotent pyridone analog that was discovered by the MARNAC Company in 1974. The antifibrotic effect of PFD was first found in a bleomycin-induced idiopathic pulmonary fibrosis (IPF) animal model in 1995. 9 Later, studies have shown that PFD could inhibit fibrosis by downregulating the expression of fibrogenic growth factors, inhibiting the production and release of inflammatory cytokines and reducing the occurrence of lipid peroxidation and oxidative stress injury. [10][11][12][13][14] PFD has a significant inhibitory effect on pulmonary fibrosis and fibrosis in other organs and is a broad-spectrum antifibrotic drug. A number of phase III clinical studies have shown that PFD can significantly delay the decline in forced vital capacity (FVC) in patients with IPF and significantly reduce the mortality of IPF. 15,16 Based on these data, PFD became the first drug approved by the FDA to treat IPF in 2014. The pathological and physiological process of RILF is similar to that of IPF and involves the early inflammatory reaction, lung parenchymal injury, alveolar repair and interstitial fibrosis. A previous study reported that PFD could downregulate the expression of transforming growth factor-β1 (TGF-β1) in lung tissue, leading to the inhibition of RILF progression, but the deeper mechanism has not been elucidated. 17 In the present study, we investigated the protective effects of PFD on RILF in vivo and in vitro. We demonstrated that PFD could reduce the polarization of M2 macrophages and inhibit the activation of TGF-β1/Smad3 signalling of alveolar epithelial cells (AECs) and vascular endothelial cells (VECs) by ionizing radiation. PFD was also involved in the regulation of AECs and macrophages. Thus, PFD has therapeutic potential for patients with RILF.

| Irradiation and PFD treatment
For radiation exposure, the mice were anaesthetized using sodium pentobarbital (40 mg/kg, intraperitoneally) and received a single 50 Gy dose of whole-lung X-ray delivered by a small animal radiation research platform (4 Gy/min; SSD = 333 mm; XStrahl). Pirfenidone was obtained from Beijing Kangdini Pharmaceutical Co., Ltd. and dissolved in 0.5% carboxymethyl cellulose solution (CMC, vehicle) at a concentration of 20 mg/ml, which was given orally by gavage at a dose of 300 mg/kg/day every day based on published data. 18,19

| Histology
All mice were fixed on the operating table and euthanized by femoral artery exsanguination at day 150 under sodium pentobarbital anaesthesia (40 mg/kg, intraperitoneally), and all efforts were made to minimize animal suffering. The right lung tissues were stored at −80°C for qRT-PCR and Western blot analysis, and the left lung ionizing radiation, macrophages, pirfenidone, radiation-induced lung fibrosis, transforming growth factor-β1 tissues were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Then, the lung tissues were sectioned into 4μm slices and stained with HE. Masson's trichrome was used to evaluate fibrosis based on 10 fields of view in each section. Five section was examined per lung, and five fields were randomly selected for each section. The severity of fibrosis in each field of the lung was assessed as the mean severity score using a semiquantitative grading system described by Ashcroft et al. 20 After deparaffinization in xylene, hydration with graded alcohol and antigen retrieval, the tissue sections were placed in 3% hydro- For immunohistochemical (IHC) scoring, positive reactions were defined as those showing brown signals. One section was examined per lung, and five fields were randomly selected and observed under a light microscope. The intensity was scored as follows: 0: negative; 1: weak; 2: moderate; and 3: strong. The frequency of positive cells was defined as follows: 0: less than 5%; 1: 5%-25%; 2: 26%-50%; 3: 51%-75%; and 4: greater than 75%. The IHC total score was calculated as the product of the intensity and frequency scores (0-12)  and was performed by trained scorers blind. After whole section examination, the IHC score was calculated as the mean total IHC score of all fields.

| Immunofluorescence staining of lung tissue
Immunofluorescence (IF) analysis was performed on 4μm-thick lung sections that had been dewaxed with xylene and hydrated using sequential ethanol (100%, 95%, 85% and 75%) and distilled water.
Antigen retrieval was performed by heating the sections in 0.1% sodium citrate buffer (pH 6.0).

| Cell culture
Bone marrow cells were obtained by flushing femurs from 6-to 8-week-old mice and differentiating the cells (7 days

| Macrophage polarization and PFD treatment
Pirfenidone was dissolved in dimethyl sulfoxide (DMSO, Sigma) and used at final concentrations of 1, 10, 100 or 1000 µg/ml (The DMSO was used in the 0 PFD as control.).

RAW264.7 cells and bone marrow-derived macrophages
(BMDMs) were cultured in DMEM supplemented with PFD for 24 h before treatment with chemokines to promote macrophage polarization. After 24 h of PFD incubation, the cells were stimulated with 10 ng/ml IL-4 (PeproTech) and 10 ng/ml IL-13 (PeproTech) to promote M2 polarization. After an additional 24 h, cell supernatants were collected for ELISA analysis, and the cells were washed twice with PBS and harvested in RIPA or RNAiso Plus reagent for subsequent analysis.    ; the values are the means ± SD, *p < 0.05, **p < 0.01. In Figures 3C and 5A, we run the same four subgroups, NC, PFD, RT, RT + PFD and stain YM-1, ARG-1 (in Figure 3C) and TGF-β1, p-Smad3, Smad3 (in Figure 5A). Therefore, the β-actin in Figure 3C and Figure

| Statistical analysis
Unless otherwise indicated, the data are presented as the means ± SD of independent experiments. The statistical significance of the differences between two groups was analysed with Student's t-tests. The calculations were performed using Prism software for Windows (GraphPad Software).

| Pirfenidone attenuates pulmonary fibrosis induced by whole-lung radiation
After 50 Gy of whole-lung irradiation, the skin of mice in the RT and RT + PFD groups showed obvious lesions in the irradiated area, with the colour changing from black to white ( Figure 1A). The skin lesions in the RT group were more serious than those in the RT + PFD group.
In the RT group, the lung tissues became consolidated and white, with increased weight, and the morphological changes and increased weight were alleviated by PFD treatment (Figure 1B,C).
HE staining showed that the alveolar structure of the lung tissue in the NC and PFD groups was clear, with slender alveolar walls and intact capillary walls. In the RT group, the alveolar wall was severely thickened, and the alveolar cavity became obviously small, with many fibroblast aggregates, and patchy fibrosis appeared around blood vessels and the pulmonary interstitial area. After PFD treatment, the histological changes in the lung tissues were significantly alleviated compared with those of the RT group. Masson staining showed that the structure of the lung tissues in the NC and PFD groups was normal. In contrast, the alveolar wall in the RT group showed obvious destruction, with increased collagen deposition (blue area in Figure 2A), while collagen deposition was attenuated in the RT + PFD group (Figure 2A). The expression of collagen I and collagen IV in lung tissue was examined by IHC staining and showed significantly higher expression of collagen I and collagen IV in the RT group than in the NC and PFD groups. The expression of collagen I and collagen IV in the RT + PFD group was obviously decreased ( Figure 2B).

| Pirfenidone inhibits ionizing radiationinduced M2 macrophage polarization
The IHC staining results showed that in the NC and PFD groups, the  Figure 3B).
The expression of these two proteins was the same when analysed by Western blotting ( Figure 3C).

| Pirfenidone inhibits the polarization of M2 macrophages by downregulating NF-κB p50
After RAW264.7 cells were cocultured with BMDMs in the presence of 1000 µg/ml PFD, the proliferation of both cell types was significantly inhibited. PFD at a concentration of 100 µg/ml or less had no effect on the proliferation of either cell type ( Figure 4A). PFD at a concentration of  Figure 4B). The expression of ARG-1, YM-1 and NF-κB p50 protein in RAW264.7 cells and BMDMs was measured by Western blotting and was also be inhibited by PFD ( Figure 4C). In addition, immunocytochemical staining indicated that PFD could significantly inhibit IL-4-and IL-13-induced ARG-1, YM-1 and CD163 expression ( Figure S2B).

| Pirfenidone inhibits activation of the TGF-β1/ Smad3 signal pathway in vivo and in vitro
The protein expression of TGF-β1 and p-Smad3 in lung tissues was measured by Western blotting and showed that in the RT + PFD group ( Figure 5A), the RT-induced expression levels of TGF-β1 and p-Smad3 were downregulated by PFD (the β-actin in Figure 3C and Figure 5A is the same band, because we run the same four subgroups). The cytotoxicity analysis showed that 1000 µg/ml PFD for more than 48 h significantly inhibited the proliferation of MLE-12 cells

MLE-12 cells was evaluated at 24 h after irradiation with increasing
doses of X-ray ( Figure S3A). The secretion of CCL2 and CXCL1 in the culture supernatant of MLE-12 cells was also measured at 24 h after irradiation ( Figure S3B), and the secretion of CCL2 and CXCL1 was inhibited by PFD treatment ( Figure 6A). In addition, the Transwell assay showed that the migration of RAW264.7 cells induced by the conditioned medium of irradiated MLE-12 cells was also inhibited by PFD treatment in vitro ( Figure 6C). RILF is a slow progressive process that generally occurs 6-18 months after radiation in both animals and humans, 23,24 and 15-20 Gy wholelung X-ray irradiation of mice is a common method to establish an RILF mouse model. [25][26][27] However, in our study, the mice were irradiated with 50 Gy by a small animal radiation research platform (SARRP), which is different from other small animal irradiation systems in that it satisfies all of the following requirements: high dose rate, small beam diameter and accurate dose location based on image guidance. 28 It has been reported that SARRP can improve the overall survival rate of mice by reducing lung side effects after high-precision heart irradiation. 29 Fibrosis could be seen in the lungs of mice irradiated with 30 Gy, while overt and intense fibrosis could be seen after irradiation with 60 Gy and 90 Gy. 30 Similarly, our results showed that pulmonary fibrosis in mice was severe at 150 days after 50 Gy irradiation, while orally administered 300 mg/kg PFD significantly attenuated pulmonary inflammatory infiltration and collagen accumulation. These antifibrotic effects are consistent with the results of different animal fibrosis studies. 18,31 Using a prototypical model of RILF, we confirmed that the administration of PFD could significantly inhibit ionizing radiationinduced activation of the TGF-β1/Smad3 signalling pathway. In vitro, irradiation-induced activation of the TGF-β1/Smad3 signalling pathway was significantly inhibited by PFD at concentrations less than 100 μg/ml without cytotoxicity. This finding is consistent with a recent report that PFD has a wide range of antifibrotic effects, including the inhibition of TGF-β1/Smad3 signalling. 32 can induce EMT in AECs, and this process is mainly regulated by the TGF-β1/Smad signalling pathway. 46,47 Our current study showed that only conditioned medium from RAW264.7 cells treated with IL-4 and IL-13 increased the expression of TGF-β1 and p-Smad3, and the same conditioned medium from RAW264.7 cells that had been treated with PFD (100 µg/ml) significantly suppressed TGF-β1 and p-Smad3. These findings suggested that PFD-induced inhibition of TGF-β1 secretion by M2 macrophages prevents pulmonary fibrosis by inhibiting the TGF-β1/Smad3 pathway in AECs.
Irradiated AECs can secrete a large amount of chemokines, such as CCL5, CCL2 and GM-CSF, 47 which recruit inflammatory monocytes and neutrophils to the injured site. Our results showed that PFD could significantly inhibit the expression of multiple chemokines induced by ionizing radiation in MLE-12 cells. It has been reported that PFD reduces macrophage infiltration in nephrectomized rats 48 and inhibits both IL-17A-induced macrophage migration and MCP-1-induced macrophage migration in vitro. 49 We hypothesize that PFD inhibits macrophage infiltration by reducing the secretion of chemokines from irradiated MLE-12 cells. Consistent with this hypothesis, we cultured macrophages with the supernatant from irradiated MLE-12 cells in Transwells. We found that the migration of macrophages was significantly enhanced in the presence of conditioned medium from irradiated MLE-12 cells, and PFD could impair macrophage migration to irradiated MLE-12 cell.
We have to admit that there is a deficiency in our study: although a number of animal studies have pointed out that CMC has no effect on histopathological and biochemical changes, 50-52 compared with normal saline, the mice in NC group using 0.5% CMC as control would be a better choice.
In conclusion, our findings indicated that PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF-β1/Smad3 signalling (Figure 7). A pilot study confirmed that PFD is effective in ameliorating the disability associated with RILF. 53 Our cancer centre is also conducting a multicentre phase II clinical trial (NCT03902509) on the treatment of RILI with PFD. We believe that PFD will be beneficial RILF patients in the near future.

ACK N OWLED G EM ENTS
We give our sincere gratitude to the reviewers for their valuable suggestions.

CO N FLI C T O F I NTE R E S T
None.