Experimental study of the effects of pirfenidone and nintedanib on joint inflammation and pulmonary fibrosis in a rheumatoid arthritis-associated interstitial lung disease mouse model

Introduction

Background

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease with an unknown etiology (1), which affecting 0.5–1.0% of the global population. Joint pain, deformity and chronic synovial inflammation [characterized by fibroblast-like synovial (FLS)] are the characters of RA. Other changes of joint include cartilage damage, joint swelling and bone malformation. RA is a multi-system disease. It is believed that lung involvement is the most common extra-articular manifestation affecting more than 50% of RA patients (approximately 14% clinically significant, 41% asymptomatic) (2), and approximately 20% of RA patients can develop RA-associated interstitial lung disease (RA-ILD) (3). The mortality of RA-ILD patients is approximately 3 times greater than that of RA-only patients (4) with median survival time about 3–10 years upon diagnosis (5). Most standard treatments for RA, such as glucocorticoids and disease-modifying antirheumatic drugs, exhibit pulmonary toxicity and may accelerate the development of interstitial lung disease (ILD) (6). Since the pathological changes in lung of RA-ILD patient are fibrosis and scarring caused by chronic inflammation of the interstitial space, controlling of fibrosis is important in the therapy of RA-ILD (7). Usual interstitial pneumonia (UIP) is the most common histological pattern of RA-ILD (8), which is considered the most analogous to idiopathic pulmonary fibrosis (IPF). RA-ILD and IPF are associated with pulmonary fibrosis and acute exacerbations. Up-regulation of some genetic markers [interleukin-8 (IL-8), matrix metalloproteinase-2, 7, 9 and FMS-related tyrosine kinase 3 ligand] and mutations [telomerase reverse transcriptase, regulator of telomere elongation helicase, poly (A)-specific ribonuclease and surfactant associated protein C] are observed in patients with RA-ILD and IPF (9,10). These similarities support the rationale for the implementing of medication for IPF in patients with RA-ILD (11). The drugs tested mostly for RA-ILD now is the first-line medicines for IPF, pirfenidone and nintedanib. Pirfenidone is an antifibrotic agent, modulating fibrogenic growth factors (FGFs), thereby attenuating fibroblast proliferation, myofibroblast differentiation, collagen and fibronectin synthesis, and in the end deposition of extracellular matrix (ECM). This effect is mediated by suppression of transforming growth factor-β1 (TGF-β1) and other growth factors (12). Nintedanib is an inhibitor of fibroblast growth factor receptor (FGFR)-1 and vascular endothelial growth factor (VEGFR)-2, both of which are pro-angiogenic receptor tyrosine kinases. Bioinformatic study finds that mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, Janus kinase-signal transducer and activator of transcription (Jak-Stat), TGF-β, VEGF and WNT/β-catenin signalings are the main molecular pathways modulated by nintedanib (13). Existing studies show that antifibrotic effects of these two drugs are related to Jak/Stat pathways (14-16). Jak/Stat pathway is activated by a broad number of profibrotic/pro-inflammatory cytokines, like IL-6, IL-11 and IL-13, among others, which are increased in ILDs (17). The polarization of macrophages also plays an important role in the development of RA-ILD. M0 macrophages polarize toward the M1 phenotype to release factors such as tumor necrosis factor alpha (TNF-α), IL-1β, 6 and 12 to promote inflammation. In contrast, M0 macrophages polarize toward the M2 phenotype in the presence of IL-4, IL-10, IL-13, and TGF-β to promote cell growth and tissue repair. M2a macrophages is a subtype of M2-type macrophages, which can be activated by IL-4 and IL-13 and promote cell growth and tissue repair through obviously increased expression of IL-10 and TGF-β (18-21). M2 macrophages are positively correlated with pulmonary fibrosis through the secretion of IL-10, TGF-β, arginase-1 (ARG-1), mannose receptor (CD206) and scavenger receptor (CD163) (22,23). Currently, relevant researches have shown that M2 macrophage polarization can be promoted through the Jak2/Stat3 signaling pathway (24,25).

In summary, the information mentioned above seems to reveal the relationships between the interactive factors, pirfenidone and nintedanib might affect fibrosis via TGF-β-Smad-Jak/Stat-ECM molecules pathway, in which M2 macrophages modulate TGF-β expression in a by-pass way. The exact mechanism of the antifibrotic effect of pirfenidone and nintedanib in RA-ILD are still unclear. So, we designed and performed this study to further explore the mechanisms and molecular signal pathway involved.

Rationale and knowledge gap

As is mentioned above that Jak/Stat and TGF-β signalings are related to antifibrotic effects of pirfenidone and nintedanib. Meanwhile, Jak/Stat signaling pathway and macrophage polarization also play important roles in the pathogenesis of RA-ILD and FLS (1,17,26-28). Jak/Stat signaling pathway, predominantly Jak2/Stat3, is upregulated in ILD patients and animal models (17). Jak2 is not only a downstream target of TGF-β1 in fibroblasts, but also performs positive feedback to amplify TGF-β1 signaling by stimulating more TGF-β1 expression (17). M2 macrophage polarization is highly expressed in ILD, too, which is promoted by TGF-β (29). In pathological procedure of FLS, Jak2 expression is significantly increased, which activates downstream Stat3 to increase production of inflammatory cytokines and chemokines (26,30). Since pirfenidone and nintedanib were approved for the treatment of IPF and progressive fibrotic ILD, including patients with RA-ILD (31-33), The effects and mechanisms are needed to be verified by experimental studies and real world data of effectiveness and safety is required to use pirfenidone and nintedanib properly for patients with RA-ILD. It is reasonable to speculate that pirfenidone and nintedanib could attenuate pulmonary fibrosis and RA-related synovitis via TGF-β1-Jak2-macrophage polarization axis.

Objective

The aim of this study was to explore the ameliorating effects of pirfenidone and nintedanib on the pulmonary fibrosis in mice with collagen-induced RA-ILD. The effect on symptoms of RA-related arthritis were also explored. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-882/rc).

Methods

Twenty-five 8-week-old male specific pathogen-free DBA/1 mice weighing approximately 18–22 g were purchased from Beijing Charles River Co., Ltd. [Animal License No. SCXK (Beijing) 2021-0006]. Experiments were performed under a project license (No. IAC-B2019011-P-01) granted by ethics board of Greentech committee, in compliance with institutional guidelines for the care and use of animals. The mice were housed under a 12 h light/dark cycle and at a constant temperature of 22±2 ℃ with food and water available ad libitum. The mice were allowed to acclimate to these conditions for 7 days prior to initiation of the experiment. The mouse macrophage line RAW264.7 (Cat No. CL-0190) and mouse primary FLS (Cat No.CP-M323) cells were purchased from Wuhan Procell Biotechnology Co., Ltd. (Wuhan, China). Pirfenidone was nintedanib were purchased from MedChemExpress (Cat No. HY-B0673; Shanghai, China) and nintedanib was purchased from Adooq (Cat No. A10137; Nanjing, China).

Treatments of animals

Mice were randomly divided into a normal control group (control group, n=3) and a model group (n=22). Mice in the model group were injected with emulsified bovine type II collagen (bCII, 2 mg/mL) at the tail root to induce the RA-ILD model. On day 0 (0 weeks), these mice were injected with 0.1 mL of complete Freund’s adjuvant (CFA) emulsified bCII, and on day 21 (3 weeks), with 0.1 mL of incomplete Freund’s adjuvant (IFA) emulsified bCII. Two mice died during injection, 3 mice showed no joint changes, and 17 mice showed joint swelling. The 17 mice with joint swelling were divided into 3 groups based on the arthritis scores, with 5 mice in the RA-ILD group (0.1 mL CMCNa), 6 mice in the pirfenidone group (20 mg/kg), and 6 mice in the nintedanib group (60 mg/kg). The mice in the control group received 0.1 mL CMCNa. Then each group of mice was administered by gavage every other day for 5 weeks, they were anesthetized and sacrificed to collect bronchoalveolar lavage fluid (BALF), lung tissue, right knee joints (see gross flow chart in Figure 1). One mouse in the RA-ILD group died during gavage, and another mouse did not show interstitial changes in the lungs and were not included in the analysis. Between collagen induction and sacrifice, the body weights and arthritis scores of all the mice were measured (34). The arthritis scores are shown in Table 1. For a single animal, the maximum score for a single limb was 4 points, and the total score for the four limbs was 16 points.

Figure 1 Schematic diagram of the establishment of the animal model and drug administration. bCII, bovine type II collagen; CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant.

Cells and treatments

RAW264.7 cells and primary mouse FLS cells were cultured in a cell culture incubator at 37 ℃ and 5% CO₂, and experiments were conducted when the cells were 80–90% confluent. RAW264.7 cells were divided into 6 groups: the RAW264.7 control (normal culture), RAW + IL-4/IL-13 (60 ng/mL IL-4/IL-13; 12 h), RAW + IL-4/IL-13 + pirfenidone (0.5 or 1 mmol/L pirfenidone + 60 ng/mL IL-4/IL-13; 12 h), and RAW + IL-4/IL-13 + nintedanib (0.1 or 0.5 µmol/L nintedanib + 60 ng/mL IL-4/IL-13; 12 h) groups. FLS cells were divided into 6 groups: the FLS control (normal culture), FLS + TGF-β1 (10 µg/L TGF-β1), pirfenidone intervention (after 4 h of 10 µg/L TGF-β1 treatment, cells were treated with 0.5 and 1 mmol/L pirfenidone for 48 h), and nintedanib intervention (after 4 h of treatment with 10 µg/L TGF-β1, cells were treated with 0.5 and 1 µmol/L nintedanib for 48 h) groups.

Flow cytometric assessment

Phosphate buffer solution (PBS) was used to lavage the alveoli 3 times, the BALF was harvested, and the proportions of M1 macrophages and M2 macrophages were determined using flow cytometry. Antibodies for flow cytometric assessment are listed in Table S1.

Pathological assessment and immunofluorescence analysis of lung and joints

Left lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, dewaxed, dehydrated, cleared, and mounted for hematoxylin-eosin (H&E) staining and Masson staining and immunofluorescence. Pulmonary inflammation was evaluated by the Szapiel score (35) after H&E staining (Table 2), and the pulmonary fibrosis score was calculated based on Masson staining (36). Right knee joints muscle tissue were removed, fixed in 0.4% paraformaldehyde for 24 hours, decalcified with decalcification solution, and decalcified for pathologic HE staining and immunofluorescence. H&E staining analysis of the right knee joint and immunofluorescence analysis of p-Jak2 and p-Stat3 protein expression in the left lung and right knee joint. Three images were randomly selected using CaseViewer software, and the fluorescence intensity was quantified using ImageJ-win64.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the right lung tissue of mice. The purity of the RNA was determined with a ultraviolet (UV) spectrophotometer. Next, complementary DNA (cDNA) was synthesized according to the instructions of the reverse transcription kit. Then, synergetic binding reagent (SYBR) fluorescent dye was added to the PCR mixture. The PCR conditions were as follows: 95 ℃ for 30 s and 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. Then, the PCR products were monitored in real time. The mRNA levels of Jak2, Stat3 and collagen-IV (Col-IV) was calculated by the 2^−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as an internal control. The sequences of the primers used were as follows: GAPDH, F: 5'-AGGTCGGTGTGAACGGATTTG-3'; R: 3'-ACAACGGTAGTTGCTGGGG-5'; Jak2, F: 5'-AAGATGCTTTCTGGGTTGG-3'; R: 3'-GACGAGGGAGAATCTGTTACA-5'; Stat3, F: 5'-ACCTCCAGGACGACTTTGAT-3'; R: 3'-ACCTCATGCACGTTTCTGT-5'; Collagen IV, F: 5'-ATGCCCTTTCTCTTCTGCAA-3'; R: 3'-ACACCTAGCCGATAAGGAAG-5'.

Western blot

Right lung tissue, RAW264.7 cells and FLS cells were collected. Total protein was extracted and loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for electrophoresis. The proteins on the gel were transferred to a polyvinylidene difluoride membrane was incubated with primary antibodies at 4 ℃ overnight, after which the membrane was washed and incubated with secondary antibodies at 37 ℃ for 1 h. The membrane was washed again and the bands were visualized. GAPDH or β-actin was used as an internal control. ImageJ software (Image J-win64) was used to determine the grayscale value of the bands, where relative protein expression level = gray value of target protein/gray value of GAPDH or β-actin. Antibodies for western blotting are listed in Table S1.

Statistical analysis

Analyses were performed using GraphPad Prism 10.0.2 software, conforming to normal distribution. The data were expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to compare multiple groups, and intergroup comparisons were performed using a group t-test; P<0.05 was considered to indicate statistical significance.

Results

Animal model of RA-ILD and the alleviating effect of pirfenidone and nintedanib

Body weight and arthritis scores were measured in all mice between weeks 0–13 (Figure 2). The body weights of the mice in the pirfenidone and nintedanib groups were higher than those of the mice in the RA-ILD group and lower than those in the control group. The arthritis scores were lower in both the pirfenidone and nintedanib intervention groups compared with those in the RA-ILD group. There were significant differences in the visual arthritis score between the pirfenidone group and the nintedanib group before 13 weeks. Significant differences began to appear for the pirfenidone and nintedanib groups after 4 and 2 weeks of treatment, respectively, indicating that the effects of nintedanib may occur faster than those of pirfenidone (Figure 2B). In the RA-ILD group, obvious swelling, stiffness and deformation of the ankle joint and toe joint were observed, while the joint inflammation in the intervention group (pirfenidone and nintedanib) relieved (Figure 2C).

Figure 2 Body weights and arthritis scores of the mice. (A) Body weight changes of mice in each group; compared with those in the RA-ILD group (n≥3; a, P<0.05; b, P<0.01; c, P<0.001). (B) Arthritis scores change of mice in each group; compared with those in the RA-ILD group (n≥3; a, P<0.05; c, P<0.001). (C) Joint manifestations of the mice in each group: representative images of the left foot joints of the mice in the control, RA-ILD, pirfenidone and nintedanib groups. In the RA-ILD group, obvious swelling, stiffness and deformation of the ankle joint and toe joint were observed. RA-ILD, rheumatoid arthritis-associated interstitial lung disease.

To evaluate the ameliorative effects of pirfenidone and nintedanib on pulmonary fibrosis and joint inflammation in mice, pathomorphologic analyses of the lungs and knee joints were performed. H&E staining revealed thickening of the alveolar septa and inflammatory cell infiltration in the lungs of the mice in the RA-ILD group, while the lungs in the pirfenidone and nintedanib groups exhibited reduced inflammatory cell infiltration and improved alveolar structure. Mouse pulmonary inflammation scores were lower in the pirfenidone and nintedanib groups (Figure 3A,3B). Masson staining revealed a large area of blue staining in the lung tissue and alveolar septa of the mice in the RA-ILD group, indicating substantial deposition of ECM and reduced ECM deposition and fibrosis in the pirfenidone and nintedanib groups. Mouse pulmonary fibrosis scores were lower in the pirfenidone and nintedanib groups (Figure 3A,3C).

Figure 3 Pirfenidone and nintedanib alleviate lung inflammation and fibrosis in a collagen-induced RA-ILD mouse model. (A) H&E staining and Masson staining of lung tissue. H&E staining revealed thickening of the alveolar septa and inflammatory cell infiltration in the lungs of the mice in the model group and the intervention group. Masson staining revealed a large area of blue staining in the alveolar septa of the mice in the RA-ILD group, indicating increased collagen deposition in the alveolar septa. (B) Mouse pulmonary inflammation scores. (C) Mouse pulmonary fibrosis scores. n≥3. *, P<0.05; **, P<0.01. RA-ILD, rheumatoid arthritis-associated interstitial lung disease; H&E, hematoxylin-eosin.

H&E staining showed significant arthritis in the knee joints of the mice in RA-ILD group. The inflammatory reactions of arthritis were alleviated by pirfenidone and nintedanib (Figure 4A). The synovitis scores (Table 3) were lower in the pirfenidone and nintedanib groups (Figure 4B), showed that pirfenidone and nintedanib improved joint inflammation in RA-ILD mice.

Figure 4 H&E staining and synovitis scores for the knee joint. (A) H&E staining showed substantial hyperplasia of the synovial layer, inflammatory cell infiltration, collagen deposition and angiogenesis in the knee joints of the mice; bone destruction was observed in the RA-ILD group. These symptoms were alleviated in the pirfenidone and nintedanib groups. (B) Synovitis scores for the knee joints of the mice. n≥3. *, P<0.05. RA-ILD, rheumatoid arthritis-associated interstitial lung disease; H&E, hematoxylin-eosin.

Changes of M1 and M2 macrophages

The pathogenesis of RA is related to macrophage polarization, and to clarify this mechanism in this experiment, we used flow cytometry to detect macrophage polarization status in BALF. Flow cytometry of BALF was performed to determine the percentages of M1 and M2 macrophages. F4/80 was used as a marker of total macrophages, CD86 was used as a marker of M1 macrophages, and CD163 was used as a marker of M2 macrophages (Figure 5). Proportion of alveolar M1 macrophages (F4/80⁺CD86⁺) in the pirfenidone and nintedanib intervention groups showed the downward tendency without significant difference (Figure 5A,5B). The percentage of M2 macrophages was lower in the nintedanib intervention groups than in the RA-ILD group.

Figure 5 Flow cytometric analysis of the proportion of macrophages in the BALF. (A) Flow cytometric analysis of the polarization of mouse alveolar macrophages. (B) Proportion of alveolar M1 macrophages (F4/80⁺CD86⁺) in mice. (C) Proportion of alveolar M2 macrophages (F4/80⁺CD163⁺) in the BALF. n=3. ns, no statistical significance; *, P<0.05. PE, phycoerythrin; APC, allophycocyanin; RA-ILD, rheumatoid arthritis-associated interstitial lung disease; BALF, bronchoalveolar lavage fluid.

Pirfenidone and nintedanib inhibited Jak2 expression

The protein expression of p-Jak2/p-Stat3 in the lungs and knee joints was assessed using immunofluorescence, the expression of p-Jak2 and p-Stat3 increased in the lungs and knee joints of the mice in the RA-ILD group. After the intervention of pirfenidone and nintedanib, the fluorescence signals of p-Jak2 and p-Stat3 decreased significantly (Figures 6,7).

Figure 6 Immunofluorescence analysis of p-Jak2/p-Stat3 expression in the lung. (A) Expression of p-Jak2 (green) in mouse lung tissue [nucleus (blue)], in the RA-ILD group, the expression of p-Jak2 was increased. (B) Bar charts of mean density of p-Jak2 in immunofluorescence analysis. (C) Expression of p-Stat3 (red) in mouse lung tissue, in the RA-ILD group, the expression of p-Stat3 was increased. (D) Bar charts of mean density of p-Stat3 in immunofluorescence analysis. n=3. **, P<0.01; ***, P<0.005; ****, P<0.0001. RA-ILD, rheumatoid arthritis-associated interstitial lung disease.

Figure 7 Immunofluorescence analysis of p-Jak2/p-Stat3 expression in knee joint. (A) The expression of p-Jak2 (green) in the knee joints of mice in the RA-ILD group was greater than that in the pirfenidone and nintedanib groups. (B) Bar charts of mean density of p-Jak2 in immunofluorescence analysis. (C) The expression of p-Stat3 (red) in the knee joints of mice in the RA-ILD group was greater than that in the pirfenidone and nintedanib groups. (D) Bar charts of mean density of p-Stat3 in immunofluorescence analysis. n=3. ***, P<0.005; ****, P<0.0001. RA-ILD, rheumatoid arthritis-associated interstitial lung disease.

Gene expression and protein of Jak2/Stat3 and Col-IV in lung tissue were assessed by qRT-PCR and western blot. The mRNA expression of Jak2, Stat and Col-IV in the lungs in the RA-ILD group was high, and the expression levels of these genes were lower in the pirfenidone and nintedanib groups (Figure 8). Western blot analysis was performed to investigate the effects of pirfenidone and nintedanib on the expression of ECM-related proteins (Col-IV, fibronectin) and the Jak2/Stat3 and TGF-β1/Smad3 signaling pathways in the lungs. Western blot analysis revealed that the expression levels of Col-IV, fibronectin, p-Jak2/Jak2, p-Stat3/Stat3, p-Smad3/Smad3, and TGF-βR2 were greater in the RA-ILD group than in the pirfenidone and nintedanib groups (Figure 9).

Figure 8 mRNA levels of Jak2, Stat3 and Col-IV in lung tissues. Both pirfenidone and nintedanib inhibited the expression of the Jak2, Stat3 and Col-IV genes. n=3. *, P<0.05; ***, P<0.005; ****, P<0.0001. RA-ILD, rheumatoid arthritis-associated interstitial lung disease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Col-IV, collagen-IV.

Figure 9 Effects of pirfenidone and nintedanib on the expression of ECM-related proteins and the Jak2/Stat3 and TGF-β1/Smad3 signaling pathways in the lungs of mice. (A) Western blot detected Col-IV, fibronectin, p-Jak2/Jak2, p-Stat 3/Stat3, p-Smad3/Smad3, and TGF-βR2 protein expression levels in lung tissues. Pirfenidone and nintedanib inhibited the TGF-β1/Smad3 signaling pathway and down-regulated the Jak2/Stat3 signaling pathway. (B) Relative expression levels of fibronectin, Col-IV, p-Jak2, Jak2, p-Stat3, Stat3, TGF-βR2, p-Smad3 and Smad3. The grayscale values were measured three times. n=3. *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.0001. RA-ILD, rheumatoid arthritis-associated interstitial lung disease; Col-IV, collagen-IV; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ECM, extracellular matrix.

Cell experiments further explained the mechanisms involved in therapy of RA-ILD by pirfenidone and nintedanib

In order to investigate the correlation between M2 polarization and Jak2/Stat3 signaling pathway, we induced M2 polarization of RAW264.7 cells by IL-4/IL-13, and then detected the expression of related proteins by western blot after the intervention of pirfenidone and nintedanib. The levels of the M2 macrophage markers ARG-1 and CD206 in the RAW + IL-4/IL-13 model group were high, indicating the successful induction of M2 polarization (Figure 10A-10C), as well as the high expression of Jak2/Stat3 and TGF-β signaling pathway components; the expression of the above proteins was reduced in the pirfenidone and nintedanib intervention groups with different concentrations. Western blotting of animal experiment confirmed high p-Jak2, Jak2, p-Stat3, Stat3, and TGF-βR2 expression in the FLS + TGF-β1 model group, and the Jak2/Stat3 signaling pathway was inhibited after intervention with different concentrations of pirfenidone and nintedanib (Figure 11).

Figure 10 Effects of different concentrations of pirfenidone and nintedanib on p-Jak2/Jak2, p-Stat3/Stat3, TGF-βR2, CD206, and ARG-1 expression in RAW264.7 cells. (A) Protein expression in RAW264.7 cells (western blot). (B) Relative expression levels of the M2 macrophage markers ARG-1 and CD206. (C) Relative expression levels of p-Jak2, Jak2, p-Stat3, Stat3, and TGF-βR2. The grayscale values were measured three times. n=3. ns, no statistical significance; *, P<0.05; **, P<0.01; ***, P<0.005; ****, P<0.0001. CD206, cluster of differentiation 206; TGF-βR2, TGF-β receptor 2; ARG-1, arginase-1.

Figure 11 Effects of different concentrations of pirfenidone and nintedanib on p-Jak2/Jak2, p-Stat3/Stat3, and TGF-βR2 expression in FLS cells. (A) Protein expression in FLS cells (western blot). (B) Relative expression levels of p-Jak2, Jak2, p-Stat3, Stat3, and TGF-βR2. The expression of these proteins was greater in the FLS + TGF-β1 group than in the pirfenidone and nintedanib groups. Pirfenidone and nintedanib block the Jak2/Stat3 pathway through inhibition of the TGF-β1 signaling pathway. The grayscale values were measured three times. n=3. **, P<0.01; ***, P<0.005; ****, P<0.0001. FLS, fibroblast-like synovial; TGF-β1, transforming growth factor-β1; TGF-βR2, TGF-β receptor 2.

Discussion

In our study, we established a bCII-induced RA-ILD mouse model and investigated the ameliorative effects of pirfenidone and nintedanib on joint inflammation and pulmonary fibrosis, and explored the possible mechanisms. We found that pirfenidone and nintedanib blocked TGF-βR2 and the activation of Smad3, then the Jak2/Stat3 pathway. The M2 polarization of macrophages was also blocked by these drugs (significantly by nintedanib, insignificantly by pirfenidone). We speculate that pirfenidone and nintedanib insert their therapeutic effect via TGF-βR2-Smad3-Jak2/Stat3 axis, meanwhile, increased M2 macrophages amplify the inflammation and fibrosis.

Extra-articular manifestation of RA increases the mortality of RA patients. Since there are little clinical symptoms, most RA-ILD patients are diagnosed after severe impairment of lung function. Early recognition and slowing of disease progression are important (5). In our study, in the lung of the 13-week mice with RA-ILD, alveolar wall thickening, inflammatory cell infiltration and collagen deposition were reduced by pirfenidone and nintedanib. Therapeutic effects were also observed on the joint inflammation like ankle swelling, stiffness and deformation. Several clinical trials have approved the therapeutic effects of these two drugs on RA-ILD (3,32,37-40). The INBUILD trial was a phase 3 clinical trial, in which nintedanib demonstrated an improvement in patients with RA-ILD by significantly lowering the average rate of forced vital capacity (FVC) decline by 59% and reducing the risk of acute exacerbation or death (32). Patients treated with nintedanib since their initial ILD diagnosis exhibited significant improvement of lung function within 24 months of treatment (37). Nintedanib affects arthritis in murine model, reverses joint swelling and decreases the arthritis score (3). These effects of nintedanib are the same with the therapeutic effects observed in our experiments. Multiple clinical trials also demonstrated the promising potential of pirfenidone for the treatment of RA-ILD. Pirfenidone lowered the rate of FVC decline (38,40). The decrease of diffusing capacity of the lungs for carbon monoxide (DLCO%), which is a sign of falling of gas exchange, was also enhanced (39).

We reported the relief of lung fibrosis and joint injury by pirfenidone and nindatenib was via TGF-β-Jak2/Stat3 signaling pathways in this experiment. Some studies have shown that p-Jak is highly expressed in the lung of RA-ILD patients (41-43). Inhibition of the Jak2/Stat3 signaling pathway alleviated the synovitis in RA mouse (30). As a result, many Jak2 inhibitors are currently used for the treatment of RA (44). Pirfenidone is now widely used in ILD, mechanisms involved were mainly through the inhibition of Jak2/Stat3 signaling. In short, pirfenidone inhibited the expression of TGF-β1 to block Jak2 and downstream signaling, while Jak2 is not only a downstream target of TGF-β1, but also stimulates the expression of the TGF-β1 gene dynamically (45,46). These signalings are also related tightly to nintedanib. Nintedanib inhibits the progression of pulmonary fibrosis by blocking TGF-β and the activation of Smad3 in the lung of mice with RA-ILD (47). Pirfenidone and nintedanib intervention reversed the increase of the ECM components, TGF-β1/Smad3 and Jak2/Stat3 signaling pathways significantly, which was consistent with the former studies (45-47). The same phenomenon was also observed in FLS cells, further confirmed that relieving effect of pirfenidone and nintedanib was via TGF-βR2-Jak2/Stat3.

The results of flow cytometry revealed high numbers of M1 (F4/80⁺CD86⁺) and M2 (F4/80⁺CD163⁺) macrophages in BALF of the RA-ILD group. The proportions of M1 (F4/80⁺CD86⁺) and M2 (F4/80⁺CD163⁺) macrophages decreased after pirfenidone and nintedanib intervention, suggesting that these drugs have anti-inflammatory and antifibrotic effects, a finding that is consistent with the results of the former researches (24,25). RAW264.7 cells polarized towards the M2 phenotype after IL-4 and IL-13 induction, expressing highly expressed Jak2/Stat3 and TGF-β signaling pathway-related proteins. These elevated proteins were then reduced by pirfenidone and nintedanib intervention with different concentrations, suggesting that pirfenidone and nintedanib intervention may result in the inhibition of M2 macrophage polarization through TGF-β signaling pathway and the suppression of the Jak2/Stat3 signaling pathway. Former research on effect of nintedanib in RA-ILD mouse revealed that nintedanib reduced fibrosis, suppressed M2 macrophage polarization and hyperplasia of type 2 alveolar epithelial cells, which supported our results (48). In contrast to the results of our experiment, some researches got the different findings. In mice silicosis fibrosis model, bosutinib increased the number of alveolar M2 macrophages and had antifibrotic effects (49). In a hyperoxic lung injury mouse model, the number of alveolar M2 macrophages increased, but the progression of pulmonary fibrosis decreased (50). In our study, the effect of pirfenidone and nintedanib occurred 5 weeks after collagen injection, at comparably late stage. One of the explanations is that pulmonary fibrosis represents the middle and late stages of RA-ILD and the M2 hyperpolarization exacerbates the progression of pulmonary fibrosis in the late stage. Effects of M2-type polarization inhibitor drugs become evident at the late stage, too.

The proportions of M2 macrophages in the BALF of the mice in the pirfenidone and RA-ILD groups were not significantly different, while there was a significant difference between the proportions of M2 macrophages in the BALF of the mice in the nintedanib and RA-ILD groups. Based on the studies by Flaherty et al. and Noble et al. (32,51), we speculated that nintedanib may have a stronger anti-inflammatory or antifibrotic effect than pirfenidone, while pirfenidone has a slower onset of effects. Further verification through future experiments is needed. According to the label information of pirfenidone, the pharmacokinetic characters might explain this partially (see Table S2). We postulate that because of longer half-life time and less disturbance of food intake, nintedanib exists in the body longer than pirfenidone, its effect on reduction of M2 polarization is stronger at the early stage. As a result, it might take a long time to observe the obvious effect on M2 polarization of pirfenidone. A comparative study on the different effect of the drugs should be designed to further explore the mechanisms, thus provides supportive information for the therapy on RA-ILD.

Limitations of our study are: (I) the observation on effects of signaling pathway inhibitors was not performed; (II) some other key factors in the signaling pathway should be observed later in PCR and western blot; (III) samples of clinical patients should be collected and tested to further support the results in animals.

Conclusions

In summary, pirfenidone and nintedanib can relieve symptoms of arthritis and the degree of pulmonary fibrosis in a bCII-induced RA-ILD mouse model. The mechanism may be related to the inhibition of the TGF-β signaling pathway, the Jak2/Stat3 signaling pathway, and M2 polarization, thereby reducing the symptoms of arthritis and the deposition of collagen in the lungs and attenuating the extent of pulmonary fibrosis. This study provides new insight into the clinical treatment of RA and RA-ILD using pirfenidone and nintedanib. We found that the effects of pirfenidone and nintedanib on M2 macrophages polarization were different.

Acknowledgments

Thanks to Yang Yan from Chongqing Medical University for her help in the experiments.

Funding: The study was supported by Chongqing Clinical Research Center for Geriatric Diseases, Chongqing Science and Health Joint Medical Research Project (No. 2020GDRC012c), Young and Middle-aged Senior Medical Talents studio of Chongqing (grant No. ZQNYXGDR CGZS2021007), Chongqing Clinical Research Centre for Geriatric Diseases Project (No. 2020-126), 2023 Chongqing Natural Science Foundation (No. CSTB2023NSCQ-MSX0188), and Chongqing Talents (No. cstc2024ycjh-bgzxm00061).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-882/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-882/dss

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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-882/coif). X.G. is an employee of West China-Frontier PharmaTech Co., Ltd. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. IAC-B2019011-P-01) granted by the ethics board of Greentech committee, in compliance with institutional guidelines for the care and use of animals.

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