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Journal of Scleroderma and Related Disorders logoLink to Journal of Scleroderma and Related Disorders
. 2019 May 19;4(2):137–148. doi: 10.1177/2397198319838140

Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization

Hidekata Yasuoka 1, Yuen Yu Angela Tam 1, Yuka Okazaki 1,2, Yuichi Tamura 3, Koichi Matsuo 4, Carol Feghali-Bostwick 5, Tsutomu Takeuchi 1, Masataka Kuwana 1,2,✉
PMCID: PMC8922645  PMID: 35382387

Abstract

Objectives:

To investigate the systemic sclerosis–related phenotype in fos-related antigen-1 transgenic mice and its underlying mechanisms.

Methods:

Lung and skin sections of constitutive fos-related antigen-1 transgenic mice and wild-type mice were examined by tissue staining and immunohistochemistry. The tricuspid regurgitation pressure gradient was measured by transthoracic echocardiography with a Doppler technique. To assess the impact of fos-related antigen-1 expression on macrophage function, bone marrow–derived mononuclear cells were derived from mice that expressed fos-related antigen-1 under the control of doxycycline and wild-type littermates. These bone marrow–derived mononuclear cells were induced to differentiate into macrophages with or without doxycycline, and analyzed for gene and protein expression. Finally, lung explants obtained from systemic sclerosis patients and control donors were subjected to immunohistochemistry.

Results:

The lungs of fos-related antigen-1 transgenic mice showed excessive fibrosis of the interstitium and thickening of vessel walls, with narrowing lumen, in an age-dependent manner. The tricuspid regurgitation pressure gradient was significantly elevated in fos-related antigen-1 transgenic versus control mice. Increased dermal thickness and the loss of subdermal adipose tissue were also observed in the fos-related antigen-1 transgenic mice. These changes were preceded by a perivascular infiltration of mononuclear cells, predominantly consisting of alternatively activated or M2 macrophages. Overexpressing fos-related antigen-1 in bone marrow–derived mononuclear cell cultures increased the expression of M2-related genes, such as Il10, Alox15, and Arg1. Finally, fos-related antigen-1-expressing M2 macrophages were increased in the lung tissues of systemic sclerosis patients.

Conclusions:

The fos-related antigen-1 transgenic mouse serves as a genetic model of systemic sclerosis that recapitulates the major vascular and fibrotic manifestations of the lungs and skin in systemic sclerosis patients. M2 polarization mediated by the up-regulation of fos-related antigen-1 may play a critical role in the development of systemic sclerosis.

Keywords: Interstitial lung disease, macrophages, mouse model, pulmonary arterial hypertension, systemic sclerosis

Introduction

Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive fibrosis, widespread vasculopathy, and immune dysregulation. 1 An excessive and uncontrolled deposition of extracellular matrix (ECM) components, which causes a loss of function in various affected organ systems, is the end result of a complex series of interlinked vascular injuries and immune activations, 2 although the complex interplay between vasculopathy, immune cell activations, and fibrosis in this disease is still incompletely understood. Animal models are valuable for elucidating the pathogenic mechanisms of a disease and for assessing therapeutic approaches before clinical trials, but there is currently no perfect animal model of SSc that fully recapitulates the features of the human disease. 3

The fos-related antigen-2 (Fra-2) transgenic (tg) mouse is a genetic model of SSc that recapitulates the major vascular and fibrotic manifestations of the skin, lungs, and heart in SSc patients.4 –7 In the course of investigation into mechanisms regulating bone formation, one of the authors (KM) observed that mice that constitutively overexpressed another fos-related antigen-1 (Fra-1) also showed inflammation and progressive collagen deposition in the lungs. Fra-1 tg mice are known to exhibit a progressive increase in bone mass of the entire skeleton, leading to osteosclerosis. 8 Both Fra-1 and Fra-2 are members of the fos protein family, which is a component of the transcription factor activator protein-1 (AP-1) and participates in a wide range of cellular processes, including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. 9 AP-1 is composed of homodimers or heterodimers of the fos, jun, and activating transcription factor protein families, characterized as having a highly conserved dimeric basic leucine zipper DNA-binding domain. 10 AP-1 transcriptional regulation is involved in many pathologic cellular processes, including cancer, inflammation, and fibrosis. 11 , 12 AP-1 also plays a prominent role in the ECM deposition in pathologic fibrosis settings, including SSc.13 –16 In this study, we assessed the detailed SSc phenotype of Fra-1 tg mice and investigated the mechanisms responsible for its SSc phenotype.

Methods

Patients

Lung explants were obtained from five SSc patients with interstitial lung disease (ILD) and/or pulmonary arterial hypertension (PAH) who underwent lung transplantation at the University of Pittsburgh Medical Center, as described previously. 17 All patients fulfilled the 2013 classification criteria for SSc proposed by the American College of Rheumatology/European League against Rheumatism. 18 Control lungs were obtained from three normal donor lungs that were not used for transplantation. All subjects signed a consent form approved by the University of Pittsburgh institutional review boards.

Fra-1 tg mice

We used two types of female Fra-1 tg mice: mice that constitutively expressed Fra-1 (constitutive Fra-1 tg mice) and mice that expressed inducible Fra-1 under the control of doxycycline (Dox) (Dox-inducible Fra-1 tg mice). These mice were generous gifts from Dr Erwin F. Wagner, the Spanish National Cancer Research Center, Madrid, Spain. The transgene construct of the constitutive Fra-1 tg mouse was generated by fusing the full-length murine Fra-1 gene to the promotor of major histocompatibility complex class I antigen H2-Kb, to allow for the expression of Fra-1 in various cell types. 8 The Dox-inducible Fra-1 tg mice demonstrated a tight, Dox-dependent regulation of the ectopic Fra-1 gene expression. 19 Ectopic Fra-1 expression under the control of Dox was confirmed to be similar to that of the constitutive tg mice, and was efficiently shut down as early as 5 days after Dox removal. Wild-type littermates were used as controls for both types of Fra-1 tg mice. All animal experimental protocols were approved a priori by the Keio University Institutional Animal Care and Use Committee.

Histological analysis

Age-matched constitutive Fra-1 tg mice and wild-type littermates were sacrificed, and the skin, heart, and lung tissues were harvested and fixed in 10% buffered formalin or snap-frozen in liquid nitrogen. In some instances, excised organs were macroscopically observed. The heart was cut in the short-axis plane, and the ratio of right-ventricle-wall thickening to left-ventricle-wall thickening plus septum thickening (RV/(LV + S)) was calculated as an indicator of right-ventricular hypertrophy, which is modified from Fulton’s index. 20 Formalin-fixed, paraffin-embedded specimens cut into 3–5 -μm sections were stained with hematoxylin and eosin or Masson’s trichrome. The severity of lung fibrosis was semi-quantitatively assessed using the Ashcroft scoring system. 21 Dermal thickness was assessed in Masson’s trichrome-stained skin sections. 5 The average of measurements from 10 randomly selected fields was used for each mouse.

For the immunostaining of tissue sections, aminoethyl carbazole (AEC) or fluorescent staining was performed as described previously. 22 , 23 The primary antibodies used were anti-CD68 monoclonal antibody (mAb) (AbD Serotec, Raleigh, NC, USA) for the detection of macrophages, anti-CD3 mAb (Thermo Fisher Scientific, Waltham, MA, USA) for T cells, and anti-CD45R/B220 mAb (BD Biosciences, San Jose, CA, USA) for B cells. Anti-α-smooth muscle actin (αSMA) and anti-CD31 mAbs (Sigma-Aldrich, St Louis, MO, USA) were also used. Negative controls were cells incubated with isotype-matched Abs to an irrelevant antigen, instead of the primary antibody. Nuclei were counter-stained with hematoxylin. In some instances, consecutive frozen sections (10 μm thick) were used for fluorescent staining. To distinguish between “classically activated” or M1 macrophages and “alternatively activated” or M2 macrophages, mannose receptor C type I CD206 and arginase-1 (in mouse) and class A scavenger receptor CD204 (in human) were used as M2 markers. 24 Specifically, the slides were incubated with an anti-CD68 mAb (AbD Serotec) and anti-CD206 polyclonal antibodies (Abcam, Cambridge, MA, USA) or anti-arginase-1 polyclonal antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), with or without anti-Fra-1 polyclonal antibodies (Santa Cruz Biotechnology), followed by an incubation with AlexaFluor® 488 and AlexaFluor® 568 species-specific IgG (Thermo Fisher Scientific). For the staining of human samples, formalin-fixed, paraffin-embedded sections (6-µm thick) were incubated with an anti-CD68 mAb (Agilent Technologies Inc, Santa Clara, CA) and anti-CD204 polyclonal antibodies (TranGenic Inc, Fukuoka, Japan), with or without anti-Fra-1 antibody (Santa Cruz Biotechnology). Nuclei were counter-stained with TO-PRO3 (Life Technologies, Grand Island, NY, USA). The slides were examined with a confocal laser fluorescence microscope (Fluoview FV1000: Olympus, Tokyo, Japan). To determine the number of cells staining positive for a given marker, at least 10 fields per mouse were evaluated at high-power field (HPF; ×400).

Trans-thoracic echocardiography

Mice were anesthetized by 1.5% isoflurane inhalation and were anchored to a platform in the supine position. Short axis echocardiography and Doppler echocardiographic measurements were made using the Vevo 660 system (VisualSonics, Toronto, Ontario, Canada) with a 600 series real-time microvisualization scanhead probe. 25 The velocity of tricuspid regurgitation jet was measured, and the tricuspid regurgitation pressure gradient (TRPG) was calculated based on a simplified Bernoulli equation. RV/(LV + S) was used to assess the degree of right ventricular hypertrophy.

In vitro induction of macrophages

Macrophages were derived in vitro in cultures of bone marrow–derived cells (BMDCs) as described previously 26 with some modifications. In brief, bone marrow cells were isolated by flushing the femurs of Dox-inducible Fra-1 tg and control mice with phosphate buffered saline (PBS), and the BMDCs were purified by density gradient centrifugation using Lymphosepar II (ImmunoBiological Laboratories, Takasaki, Japan) at 400 g for 30 min. The buffy-coat layer was harvested and rinsed with PBS, and pelleted by centrifugation at 300 g for 10 min. The cells were resuspended in complete medium consisting of RPMI 1640, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (25 mM), L-glutamine (2 mM), 10% fetal bovine serum (FBS), penicillin (100 µ/mL), and streptomycin (100 µg/mL) at 6 × 105 cells/mL, and cultured in 12-well plates with macrophage colony-stimulating factor (M-CSF) (100 µ/mL) (Peprotech, Rocky Hill, NJ, USA), in the presence or absence of Dox (1 μg/mL) (Sigma-Aldrich). The medium was changed every 3 days to fresh medium containing the same supplements except that the FBS concentration was reduced to 0.1%. On day 7, the adherent cells were harvested by incubating them with 2 mM ethylenediaminetetraacetic acid in PBS on ice for 30 min.

Semi-quantitative and quantitative polymerase chain reaction (PCR)

The mRNA expression was examined using reverse transcription-PCR. Briefly, total RNA was isolated from cultured cells using an RNeasy MiniKit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. One microgram of RNA was reverse transcribed into cDNA with oligo(dT)12-18 primers (Takara Bio, Shiga, Japan), and subjected to PCR. The genes examined included the M1 markers Tnfa and Il12; M2 markers Il10, Alox15, Fizz1, and Ym1; 27 and an internal control Beta-actin (Supplemental Table). After amplification, the samples were separated by electrophoresis on a 2% agarose gel, and the bands were visualized with ethidium bromide. The intensities of the bands were quantified using Image J software (National Institute of Health, Bethesda, MA, USA) and normalized to that of Beta-actin. In some experiments, mRNA expression was quantitatively measured using TaqMan® real-time PCR system (Applied Biosystems, Foster City, CA, USA) using 18 S rRNA for standardization. Specific primers and probes for type I collagen alpha 1 and 2 (co1a1 and col1a2) were purchased from Applied Biosystems.

Quantification of collagen in lung tissue

The collagen deposition in lung tissue was analyzed quantitatively by Sircol collagen dye-binding assay (Biocolor, Newtownabbey, UK).

Detection of anti-nuclear antibody (ANA)

Indirect immunofluorescence was conducted using plasma diluted at 1:20 for detection of ANA using Fluoro HEPANA test (MBL, Nagoya, Japan), in which AlexaFluor® 488-conjugated anti-mouse IgG antibodies were used as secondary antibody.

Cytometric bead array

Cytokines and chemokines (interleukin 12 (IL-12) p70, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-10, CCL2, and IL-6) in macrophage culture supernatants were measured using a flow cytometry-based assay with the BD cytometric bead array system (BD Biosciences) combined with a mouse inflammation kit. The cells were analyzed on a FACS® Calibur flow cytometer (BD Biosciences) using CellQuest software, according to the manufacturer’s protocol.

Statistical analyses

The Mann–Whitney U test was used for unrelated, nonparametric samples. Data are expressed as the mean ± standard deviation. Values of P < 0.05 were considered statistically significant. Correlation was assessed using a single-regression model. All statistical analyses were performed using SPSS Statistics version 23 (IBM, Tokyo, Japan).

Results

Phenotype of the constitutive Fra-1 tg mice

The constitutive Fra-1 tg mice started to die after 9 weeks of age, and two-thirds of them died within 18 weeks. The survival assessment of 11 pairs of tg and wild-type littermates showed that the survival of constitutive Fra-1 tg mice was significantly worse than that of wild-type (P = 0.01) (Supplemental Figure 1A). At 10 weeks of age, an enlarged cardiovascular system, including the lungs and heart, was easily visible macroscopically in the constitutive Fra-1 tg mice, compared with wild-type littermates (Supplemental Figure 1B), suggesting that the cardiopulmonary condition was involved in the increased mortality. We confirmed that Fra-1 protein was detected in the lungs of constitutive Fra-1 tg mice, but not wild-type mice, at 5 weeks using immunofluorescence (Supplemental Figure 1C). Interestingly, Fra-1 was preferentially expressed in infiltrating mononuclear cells, compared to other cell types residing in the lung, such as epithelial cells, endothelial cells, and fibroblasts, consistent with a previous report. 28 We further examined the phenotype of the constitutive Fra-1 tg mice, focusing on features recapitulating SSc organ involvement, including ILD, PAH, and skin sclerosis. ANA was detected in none of Fra-1 tg mice or wild-type littermates.

Pulmonary interstitium

We first evaluated the lung tissues of constitutive Fra-1 tg mice and their wild-type littermates at 10 weeks after birth (Figure 1(a)). Masson-trichrome staining showed an enlarged interstitium characterized by mononuclear infiltration and excessive fibrotic change in the Fra-1 tg mice. These interstitial changes were uniform, and were observed mainly within the alveolar septa and perivascular-bronchial bundles, while fibroblastic foci or cystic changes were not detected. These characteristics were consistent with the non-specific interstitial pneumonia that is often found in SSc patients. 29 Serial evaluations of the lung tissues of constitutive Fra-1 tg mice and their wild-type littermates are shown in Figure 1(b). The interstitial changes had already started at 5 weeks, and gradually progressed over time. After 16 weeks of age, the majority of alveoli were collapsed and replaced by fibrotic tissue in the Fra-1 tg lung. The extent of lung fibrosis was semi-quantitatively assessed using the Ashcroft score, which was correlated with age (r2 = 0.87, P < 0.00003) (Figure 1(c)). Collagen deposition was increased in Fra-1 tg mice compared with wild-type littermates (5.1 ± 0.6 vs 1.8 ± 1.4 µg/100 µg weight of lung tissue, P < 0.05). In addition, mRNA expression of col1a1 and col1a2 in the lung tissue was elevated in Fra-1 tg than in wild-type mice (2.3 ± 2.9 vs 1.0 ± 0.0, 19.1 ± 18.1 vs 1.0 ± 0.0, respectively, P < 0.05 for both comparisons).

Figure 1.

Figure 1.

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Pulmonary interstitium of constitutive Fra-1 transgenic (tg) mice: (a) representative images of Masson’s trichrome-stained lung tissue obtained from constitutive Fra-1 tg and wild-type (WT) mice at 10 weeks of age. Bar denotes 1 mm. (b) Serial changes in Masson’s trichrome-stained lung tissue from constitutive Fra-1 tg and WT mice. Representative images are shown. Bar denotes 1 mm. (c) Correlation between Ashcroft score and age in nine Fra-1 tg mice.

Pulmonary vasculature

The pulmonary arteries of 10-week-old constitutive Fra-1 tg mice showed prominent thickening of the vessel walls and narrowing of the lumen, compared with their wild-type littermates (Figure 2(a)). As shown in Figure 2(b), detailed examination of the vascular structure revealed a mixture of various degrees of vascular changes, including concentric laminar intimal thickening (A), medial hypertrophy (B), adventitial thickening with intimal and medial thickening (C), and concentric and eccentric non-laminar thickening (D), but no plexiform lesions, dilated/angiomatoid lesions, or vasculitis. These obstructive vascular changes were consistent with the histologic features of PAH in SSc patients. 30 Evaluation of the vasculature of constitutive Fra-1 tg and wild-type mice over time revealed significant perivascular mononuclear cell infiltrates in the Fra-1 tg mice at 5 weeks of age that were gradually replaced by accumulated ECM (Figure 2(c)). Concentric laminar intimal thickening and medial hypertrophy became apparent at 10 weeks of age or later. Consecutive lung sections of 10-week-old constitutive Fra-1 tg mice showed that the thickened pulmonary arterial walls were composed mainly of accumulated ECM and α-SMA-positive cells, which were likely to be smooth muscle cells and myofibroblasts, while the endothelium was lined with a single layer of CD31-positive endothelial cells (Figure 2(d)). Finally, the neo-muscularization of arterioles, which was indicated by α-SMA staining, was specifically detected in lung tissues from Fra-1 tg mice at 10 weeks (Figure 2(e)).

Figure 2.

Figure 2.

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Pulmonary vasculature of constitutive Fra-1 transgenic (tg) mice. (a) Representative images of pulmonary arteries in lung tissue from Fra-1 tg and wild-type (WT) mice at 10 weeks of age. Tissue sections were subjected to Masson’s trichrome staining. Bar denotes 200 µm. (b) Characteristic histologic findings of pulmonary arteries in 10-week-old Fra-1 tg mice, including (a) concentric laminar intimal thickening, (b) medial hypertrophy, (c) adventitial thickening, and (d) concentric and eccentric non-laminar thickening. Lung tissues were stained with Masson’s trichrome. Each bar denotes 100 µm. (c) Serial changes in Elastica van Gieson-stained lung tissue from constitutive Fra-1 tg and WT mice. Representative images of pulmonary arteries are shown. Bar denotes 100 µm. (d) Serial lung sections from 10-week-old Fra-1 tg and WT mice, stained with Elastica van Gieson (EVG), Masson’s trichrome (MT), anti-αSMA, or anti-CD31. Representative images of similar findings obtained from four pairs of tg and WT mice are shown. Bar denotes 100 µm. (e) Representative findings of the neo-muscularization of arterioles (detected by αSMA staining, arrows) in lung tissue from 10-week-old Fra-1 tg mice. Representative images of similar findings obtained from four pairs of tg and WT mice are shown. Bars denote 1 mm (left) and 100 µm (right), respectively.

To examine whether the histologic findings of pulmonary arterial narrowing resulted in functional consequences, trans-thoracic echocardiography was conducted in four pairs of 10-week-old constitutive Fra-1 tg and wild-type mice. As shown in Supplemental Figure 2A, the tricuspid regurgitation jet was clearly visible in the Tra-1 tg mice, but not in wild-type mice, and the TRPG was significantly elevated in Fra-1 tg mice compared to their wild-type littermates (0.49 ± 0.37 vs 0.05 ± 0.03 mm Hg, P < 0.01). In addition, the RV/(LV + S) was significantly greater in the Fra-1 tg mice than in the wild-type controls (0.40 ± 0.09 vs 0.12 ± 0.03, P < 0.002) (Supplemental Figure 2B). These findings indicated that the histologic features of the damaged pulmonary arteries were associated with a functional impairment of pulmonary circulation, consistent with PAH.

Skin

We examined the histology of the skin obtained from the back of 10-week-old constitutive Fra-1 tg and wild-type mice. Representative skin sections are shown in Figure 3(a). Compared with the wild-type controls, the Fra-1 tg mice showed a thicker dermis, with increased perivascular mononuclear cell infiltrates and ECM accumulation. Notably, the subdermal adipose tissue was almost completely lost in the Fra-1 tg skin. Fra-1 tg mice showed a time-dependent increase in dermal thickness starting as early as 5 weeks. The dermis was significantly thicker in the constitutive Fra-1 tg mice compared to wild-type littermates (216 ± 26 vs 118 ± 22 µm, P < 0.05) (Figure 3(b)). These skin histologic features resemble the skin histology of SSc patients. 31

Figure 3.

Figure 3.

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Skin of constitutive Fra-1 transgenic (tg) mice. (a) Representative images of skin sections from 10-week-old Fra-1 tg mice and wild-type (WT) littermates. Sections were stained with hematoxylin and eosin or Masson’s trichrome. Double-headed arrows indicate the thickness of the dermis. Scale bar denotes 100 µm. (b) Dermal thickness in Fra-1 tg and WT mice. Statistical comparison was made using the Mann–Whitney U test.

Evaluation of infiltrating mononuclear cells in the lungs of constitutive Fra-1 tg mice

The histologic features of the lungs and skin from constitutive Fra-1 tg mice began with infiltration of mononuclear cells, followed by the accumulation of ECM, and resultant structural remodeling. To identify the types of infiltrating mononuclear cells, we performed immunohistochemistry for macrophages, T cells, and B cells in the lung tissues from four pairs of Fra-1 tg and wild-type mice (Figure 4(a) and (b)). There were significantly more infiltrating macrophages in the constitutive Fra-1 tg than in wild-type lung (40 ± 10 vs 21 ± 7/HPF, P < 0.05), whereas there was no difference in the number of T or B cells. As shown in Figure 4(c), virtually all of the macrophages in the lungs expressed Fra-1. There were significantly more CD206-positive M2 macrophages in the Fra-1 tg compared to wild-type mice (27 ± 10 vs 3 ± 1/HPF, P < 0.05), although the number of CD206-negative macrophages was comparable (11 ± 8 vs 16 ± 11/HPF). As a result, the M2/M1 ratio was significantly higher in the Fra-1 tg mice than in their wild-type littermates (4.7 ± 0.9 vs 0.2 ± 0.1, P < 0.001). Most of the macrophages infiltrating the lungs of the Fra-1 tg mice expressed M2-associated arginase-1 (Figure 4(d)). These findings collectively indicated that M2 polarization is a characteristic of the constitutive Fra-1 tg mice.

Figure 4.

Figure 4.

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Evaluation of infiltrated mononuclear cells in the lungs of constitutive Fra-1 transgenic (tg) mice. (a) Lung sections from Fra-1 tg and wild-type (WT) mice were stained for CD68 (macrophages), CD3 (T cells), and B220 (B cells). Bar denotes 100 µm. (b) The number of infiltrating macrophages, T cells, and B cells in four pairs of Fra-1 tg and WT mice. Results are shown as cells per high power field (HPF). Statistical comparisons were made using the Mann–Whitney U test. (c) Detection of M2 macrophages in the lungs from Fra-1 tg and WT mice, determined by the co-expression of CD68 (green), CD206 (blue), and Fra-1 (red). Representative images of similar findings obtained from four pairs of tg and WT mice are shown. Bar denotes 100 µm. (d) Detection of M2 macrophages in the lungs from Fra-1 tg and WT mice, determined by examining the co-expression of CD68 (red), arginase-1 (Arg1, green), and TO-PRO3 (blue). Representative images of similar findings obtained from four pairs of tg and WT mice are shown. Bar denotes 100 µm.

Role of the Fra-1 overexpression in M2 polarization

To examine whether the Fra-1 overexpression contributed to the promotion of M2 differentiation, we used Dox-inducible Fra-1 tg mice. BMDCs derived from the Dox-inducible Fra-1 tg and control mice were cultured under macrophage differentiation conditions in the presence or absence of Dox. The recovered macrophages were then subjected to gene expression analysis, and the culture supernatants were used to measure cytokines and chemokines. As shown in Figure 5(a), significant increases in the gene expression of M2 markers Il10, Alox15, and Arg1 were observed in the macrophages derived from the Dox-inducible Fra-1 tg mice in the presence versus the absence of Dox (1.60 ± 0.21 vs 0.50 ± 0.49; 0.59 ± 0.13 vs 0.11 ± 0.09; 0.31 ± 0.01 vs 0.12 ± 0.01, respectively, P < 0.05 for all comparisons), although there was no difference in the gene expression of the M1 markers Tnfa and Il12. Additional M2-related genes, including Fizz1 and Ym1, also exhibited a trend toward increased expression in the macrophages derived from Dox-inducible Fra-1 tg mice in the presence versus the absence of Dox. Finally, in the presence of Dox, the culture supernatants of macrophages derived from Dox-inducible Fra-1 tg mice contained more IL-10 than those derived from control mice (Figure 5(b)), but the levels of the M1-associated IL-12p70, IFN-γ, and TNF-α were comparable.

Figure 5.

Figure 5.

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Role of Fra-1 up-regulation in M2 polarization determined by in vitro culture of bone marrow–derived mononuclear cells (BMDCs). BMDCs derived from doxycycline (Dox)-inducible Fra-1 transgenic (tg) and control mice were cultured under macrophage-differentiation conditions in the presence or absence of Dox. Four pairs of tg and WT mice were used. (a) Expression levels of genes associated with M1 and M2 macrophages in BMDCs from four pairs of Dox-inducible Fra-1 tg and control mice cultured in the presence or absence of Dox. Gene expression levels were detected by semi-quantitative polymerase chain reaction (PCR). *P < 0.05 by Mann–Whitney U test. (b) Expression levels of cytokines and chemokines associated with M1 or M2 macrophages in the culture supernatants of BMDCs derived from Dox-inducible Fra-1 tg and control mice cultured under macrophage-differentiation conditions in the presence of Dox. Statistical comparison was made using the Mann–Whitney U test.

Fra-1 expression in infiltrating macrophages in lung tissues from SSc patients

Lung tissue sections obtained from five SSc patients with ILD or PAH and three healthy controls were immunostained for evaluation of Fra-1-expressing M2 macrophages. As shown in Figure 6(a), Fra-1-expressing M2 macrophages, which were identified as cells expressing CD68 (blue), CD204 (green), and Fra-1 (red) simultaneously, were increased in the representative lung tissue from a patient with SSc, but not in the tissue from a healthy control. There were significantly more Fra-1-expressing M2 macrophages in lung tissues of SSc patients compared to donor controls (22 ± 4 vs 1 ± 1/HPF, P < 0.05) (Figure 6(b)).

Figure 6.

Figure 6.

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Fra-1 expression in infiltrated macrophages in the lung tissue from SSc patients with interstitial lung disease and/or pulmonary arterial hypertension. (a) Representative images of lung-tissue sections from a patient with SSc and a healthy control, stained for CD68 (blue), CD204 (green), and Fra-1 (red). Bar denotes 100 µm. (b) The number of cells positive for CD68, CD204, and Fra-1 in five patients with SSc and three healthy controls (HC). Results are shown as cells per high power field (HPF). Statistical comparisons were made using the Mann–Whitney U test.

Discussion

We demonstrated that Fra-1 tg mice spontaneously develop progressive fibrosis and occlusive vasculopathy of the lungs, leading to death by 18 weeks of age. These animals also develop progressive skin thickening. Thus, this animal model recapitulates the major vascular and fibrotic manifestations of the skin and lungs seen in SSc patients,4 –7 and should be included in the genetic models of SSc, despite lack of autoantibody production. The phenotype of Fra-1 tg mice is principally similar to that of mice that constitutively expressed Fra-2, another Fos family protein.13 –16

In constitutive Fra-1 tg mice, the infiltration of macrophages, especially those with the M2 phenotype, into the lungs preceded vascular remodeling and the accumulation of ECM, which led to distortion of the organ structure. These observations suggest that the infiltration of M2 macrophages is critical for promoting the vascular remodeling and excessive fibrosis. In this regard, accumulating evidence indicates that monocytes and macrophages have important roles in initiating and/or perpetuating the pathogenic process of SSc. 24 Specifically, the perivascular infiltration of macrophages has been detected in SSc skin, lung, and other tissues, especially in the early disease phase. 32 A variety of CD14+ monocyte subpopulations are increased in the peripheral blood of SSc patients, including type I collagen-producing monocytes, 33 CXCR4+ circulating cells with monocytic and endothelial features, 34 monocytic pro-angiogenic hematopoietic cells, 35 monocytes expressing high levels of versican and CCL2, 36 and tissue inhibitor of metalloproteinases 1-expressing monocytes. 37 In addition, a recent study examining the lung tissue of SSc patients with ILD found prominent infiltrates of fibrocytes, 38 which originated from precursors within the circulating CD14+ monocytes. 39 Therefore, the pathogenic process of SSc is likely to be triggered by the recruitment of circulating monocytes to affected sites, where they acquire profibrotic properties, that is, the production of a variety of profibrotic growth factors, cytokines, and chemokines, to stimulate resident mesenchymal cells, and their own trans-differentiation into ECM-producing cells.39 –41

In the Fra-1 tg mice, it is likely that M2 polarization contributes to the development of the SSc phenotype. Monocytes and macrophages were first reported to have a pivotal role in removing microbes by phagocytosis or by secreting cytokines, but recent studies have shown that their physiological and pathological roles are variable, leading to their classification into pro-inflammatory M1 and anti-inflammatory/pro-fibrotic M2 types.42 –44 Originally, M2 was implicated in the development of T helper 2 effector responses and in the suppression of M1 responses, but M2 activation also leads to the continuous production of pro-fibrotic mediators that promote myofibroblast proliferation and activation of the epithelial- or endothelial-mesenchymal transition, resulting in excessive fibrosis. 43 In addition, M2 regulates ECM turnover by modulating expression of matrix metalloproteinase (MMP) family members and the tissue inhibitors of MMPs. 45 Recently, the importance of M2 in the pathogenesis of SSc has been increasingly recognized. For example, it was shown that macrophages in the early SSc skin express M2 markers, including CD163 and CD204. 46 In a phase II clinical trial of the anti-IL-6 receptor biologic tocilizumab in early, active patients with diffuse cutaneous SSc, tocilizumab treatment resulted in the down-regulation of M2-associated genes in the skin, a sustained reduction in circulating CCL18, an M2-associated chemokine, and an improvement in skin sclerosis. 47

We clearly showed using Dox-inducible Fra-1 tg mice that overexpressing Fra-1 promotes M2 polarization. In this regard, in dextran sulfate sodium-induced colitis and bone fracture mouse models, macrophages overexpressing Fra-1 show an impaired production of pro-inflammatory cytokines, including TNF-α and IL-12, because Fra-1 is a negative regulator of NF-κB-mediated stress responses. 48 , 49 Moreover, Fra-1 tg mice are sensitive to the diffuse lung injury induced by gefinitib combined with lipopolysaccharide, which is mediated through a massive accumulation of macrophages in association with the overproduction of several chemokines, including CCL2. 28 Furthermore, a recent in vitro study reported that Fra-1 upregulation in RAW264.7 macrophages induces an M2 phenotype, which is characterized by the secretion of high levels of IL-10, CCL2, CCL22, and Arg-1 and decreased expressions of IL-12p35, TNF-α, and iNOS. 50 Taken together, these findings suggest that Fra-1 is an important inducer of M2 macrophages. Interestingly, CCL2, a soluble mediator that activates M2 macrophages, is a known target gene downstream of Fra-1. 28

Previous studies 4 , 8 , 51 , 52 and our report have indicated that broad expression of Fra-1 or Fra-2 in the constitutive transgenic mice show similar phenotype, including increased bone mass and generalized fibrosis, although direct comparison has never been conducted. Since, in Fra-1 tg mice, expression of other AP-1 components including Fra-2 was not altered, 8 suggesting that expression of Fra-1 and Fra-2 is principally independent. The diverse cellular processes mediated by AP-1 family members in response to various physiological and pathogenic stimuli have generally been attributed to the nature of activation of Jun and Fos family members, their dimeric composition, and the duration of the subsequent induction of downstream genes; 9 , 10 however, the exact relevance of dimeric composition composed of either Fra-1 or Fra-2 to the cellular processes is largely undefined. Further studies to examine impact of Fra-1 and Fra-2 overexpression on AP-1 activity and resultant functional consequence in SSc phenotype is necessary.

A temporal relationship between cancer and SSc onset has been widely recognized, especially in SSc patients with anti-RNA polymerase III antibodies. 53 Since AP-1 is involved also in the development, propagation, and invasion of cancer, 9 Fra-1 tg mouse model might be useful for evaluating this connection. However, there was no tumor formation or production of SSc-related autoantibody in Fra-1 tg mice by age of SSc phenotype occurrence.

Conclusion

The Fra-1 tg mouse model is a new genetic model of SSc and recapitulates the major vascular and fibrotic manifestations of the skin and lungs in SSc patients. M2 polarization mediated by the up-regulation of Fra-1 may play a critical role in the development of the SSc phenotype. The use of this animal model will help clarify the roles of M2 macrophages in the pathogenesis of SSc.

Supplemental Material

Supplemental_Figure_1 – Supplemental material for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization
Click here for additional data file. (270KB, jpg)

Supplemental material, Supplemental_Figure_1 for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization by Hidekata Yasuoka, Yuen Yu Angela Tam, Yuka Okazaki, Yuichi Tamura, Koichi Matsuo, Carol Feghali-Bostwick, Tsutomu Takeuchi and Masataka Kuwana in Journal of Scleroderma and Related Disorders

Supplemental_Figure_2 – Supplemental material for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization
Click here for additional data file. (261.3KB, jpg)

Supplemental material, Supplemental_Figure_2 for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization by Hidekata Yasuoka, Yuen Yu Angela Tam, Yuka Okazaki, Yuichi Tamura, Koichi Matsuo, Carol Feghali-Bostwick, Tsutomu Takeuchi and Masataka Kuwana in Journal of Scleroderma and Related Disorders

Supplemental_Table_1-final – Supplemental material for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization
Click here for additional data file. (65.7KB, docx)

Supplemental material, Supplemental_Table_1-final for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization by Hidekata Yasuoka, Yuen Yu Angela Tam, Yuka Okazaki, Yuichi Tamura, Koichi Matsuo, Carol Feghali-Bostwick, Tsutomu Takeuchi and Masataka Kuwana in Journal of Scleroderma and Related Disorders

Acknowledgments

We thank Drs Yasunari Takada and Ichiro Takada, and Ms Mari Fujiwara for their assistance with the mouse experiments. We also thank Dr Hiroshi Takei for assistance in Sircol assay.

Footnotes

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: This project was supported by funding from the Kan Research Institute Inc, and from a research grant for intractable diseases from the Japanese Ministry of Health, Labour and Welfare.

Supplemental material: Supplemental material for this article is available online.

ORCID iD: Masataka Kuwana Inline graphic https://orcid.org/0000-0001-8352-6136

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental_Figure_1 – Supplemental material for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization
Click here for additional data file. (270KB, jpg)

Supplemental material, Supplemental_Figure_1 for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization by Hidekata Yasuoka, Yuen Yu Angela Tam, Yuka Okazaki, Yuichi Tamura, Koichi Matsuo, Carol Feghali-Bostwick, Tsutomu Takeuchi and Masataka Kuwana in Journal of Scleroderma and Related Disorders

Supplemental_Figure_2 – Supplemental material for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization
Click here for additional data file. (261.3KB, jpg)

Supplemental material, Supplemental_Figure_2 for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization by Hidekata Yasuoka, Yuen Yu Angela Tam, Yuka Okazaki, Yuichi Tamura, Koichi Matsuo, Carol Feghali-Bostwick, Tsutomu Takeuchi and Masataka Kuwana in Journal of Scleroderma and Related Disorders

Supplemental_Table_1-final – Supplemental material for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization
Click here for additional data file. (65.7KB, docx)

Supplemental material, Supplemental_Table_1-final for Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization by Hidekata Yasuoka, Yuen Yu Angela Tam, Yuka Okazaki, Yuichi Tamura, Koichi Matsuo, Carol Feghali-Bostwick, Tsutomu Takeuchi and Masataka Kuwana in Journal of Scleroderma and Related Disorders

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