Introduction
Fibrotic diseases are characterized by tissue overgrowth, hardening, and/or scarring due to excessive production, deposition, and contraction of the extracellular matrix (ECM). This process usually occurs over many months and years, and can lead to organ dysfunction or death. It has been reported that immune cells, such as alternatively activated macrophages, T cells, and B cells, have been found in the tissues with fibrotic disease and that immune cells induce the production of pro-fibrotic factors and ECM [
1‐
5]. Although abnormality of the innate and adaptive immune systems induced by the increase in these immune cell numbers may contribute to the development of fibrosis, the detailed mechanism remains unclear.
Alpha2-antiplasmin (α2AP) is known to be the principal inhibitor of plasmin, resulting in the formation of a stable inactive complex, plasmin-α2AP (PAP), and inhibits fibrinolysis and ECM degradation [
6‐
8]. α2AP is synthesized in various tissues; has various functions, such as cytokine production, cell growth, and cell differentiation; and regulates angiogenesis, inflammatory response, immune modulation, tissue repair, bone formation, and brain functions [
9‐
18]. Many studies have reported that the levels of PAP in plasma are elevated in fibrotic diseases which include systemic sclerosis (SSc), diabetic nephropathy, liver cirrhosis, and rheumatoid arthritis [
19‐
22]. Recently, we showed that the expression of α2AP is elevated in fibrotic tissue [
11,
23‐
25], and α2AP induces the transforming growth factor-β (TGF-β) production through adipose triglyceride lipase (ATGL), which has been described as a member of the calcium-independent phospholipase A
2 (iPLA
2)/nutrin/patatin-like phospholipase domain-contain 2 (PNPLA2) family, and is associated with pro-fibrotic effects, such as cytokine production, myofibroblast differentiation, and ECM production [
11,
26]. α2AP also inhibits the activity of plasmin, which can directly and indirectly degrade a number of matrix proteins by activating latent metalloproteinases (MMPs) [
8] and activate hepatocyte growth factor (HGF) [
27,
28]. Furthermore, the blockade of α2AP or α2AP deficiency attenuates the development of fibrosis in humans and mice [
11,
25,
29], and the increase of α2AP expression may play a critical role in the fibrotic disease severity. However, the mechanism underlying the production of α2AP that occurs with the process of fibrosis progression remains unclear.
High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts pro-inflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). It has been reported that HMGB1 is released from immune cells, and contributes to inflammation, immune responses, myofibroblast differentiation, ECM production, and fibrosis progression [
30‐
34], and the HMGB1 inhibitor, glycyrrhizin, attenuates the development of fibrosis in the belomycin-treated mice [
35].
In the present study, we examined the mechanism underlying the production of α2AP in the development of fibrosis and showed that alternatively activated macrophage-derived HMGB1 induced α2AP production through RAGE in fibroblasts and that the blockade of IL-4 signaling by IL-4Rα neutralization suppressed these effects, resulting in the improvement of fibrotic disorder.
Discussion
Fibrotic diseases are characterized by tissue overgrowth, hardening, and/or scarring due to the excessive production, deposition, and contraction of ECM. Recently, we showed that the expression of α2AP is elevated in fibrotic tissue and that the blockade of α2AP attenuates the development of fibrosis [
11,
24,
25]. α2AP is associated with the production of pro-fibrotic factors, myofibroblast differentiation, ECM production, and plasmin inhibition [
7,
11,
26], and increased α2AP may play an important role in the development of fibrosis. Thus, we examined the mechanism underlying the production of α2AP that occurs with the development of fibrosis.
In this study, we focused on HMGB1, which is known to contribute to inflammation, immune response, myofibroblast differentiation, ECM production, and progression of fibrosis [
30‐
34], and found that HMGB1 induced pro-fibrotic changes and the production of α2AP through RAGE in fibroblasts (Fig.
1). In addition, we showed that macrophage reduction attenuated the pro-fibrotic changes and the HMGB1 and α2AP production that occurs with the process of fibrosis progression (Fig.
3). On the other hand, the pro-fibrotic changes and the HMGB1 and α2AP production were induced in bleomycin-treated T and B cell-deficient SCID mice (Fig.
2). Several studies have reported that T and B cells are not an essential requirement for the development of fibrosis [
48,
49]. These data suggest that macrophages may play a pivotal role in the process of fibrosis progression and that macrophage-regulated production of HMGB1 and α2AP may be associated with the induction and development of fibrosis.
HMGB1 is secreted by activated macrophages and is associated with the polarization of classically and alternatively activated macrophage and the promotion of cytokine production in alternatively activated macrophage [
50‐
53]. It has been reported that classically and alternatively activated macrophages are associated with the induction and development of inflammation and fibrosis [
5,
54]. In particular, alternatively activated macrophages are elevated in SSc patients [
55], and they are associated with the development of dermal fibrosis [
45,
46]. In the present study, we showed that the expression of classically and alternatively activated macrophage markers was elevated in dermal fibrosis model mice. We also showed that the blockade of IL-4Rα attenuated the pro-fibrotic changes and the increase in the expression of α2AP, HMGB1, and alternatively activated macrophage markers in dermal fibrosis model mice, but not the increase in the expression of classically activated macrophage markers. These data suggest that IL-4 signaling is associated with the increase in alternatively activated macrophage numbers and the production of HMGB1 and α2AP that occurs with the process of fibrosis progression. In addition, HMGB1 may not affect the increase in classically activated macrophage numbers in dermal fibrosis model mice, and classically activated macrophages may be not an essential requirement for the IL-4-mediated fibrosis progression. Next, we showed that IL-4-stimulated alternatively activated macrophages produced HMGB1 (Fig.
4a) and that the IL-4-stimulated alternatively activated macrophage CM induced pro-fibrotic changes and the production of α2AP through HMGB1/RAGE in fibroblasts (Fig.
4). On the other hand, IL-4 did not induce the production of HMGB1 in fibroblasts (data not shown). These data suggest that IL-4-stimulated alternatively activated macrophages produced HMGB1, and the HMGB1 subsequently induced α2AP production in fibroblasts.
The serum level of IL-4 is elevated in bleomycin-treated mice [
56,
57]. We showed that IL-4 was elevated in bleomycin-treated T and B cell-deficient SCID mice skin (Fig.
2c). IL-4 is mainly produced by T cells but is also produced by mast cells, basophils, and eosinophils [
58]. These cells may be associated with the production of IL-4, and the increase of IL-4 may cause the induction and development of fibrosis. IL-4 is also known to regulate macrophage proliferation and accumulation, and to induce collagen synthesis and fibroblast proliferation, and is associated with the development of fibrosis [
59‐
62]. Furthermore, we showed that the blockade of IL-4 signaling attenuated the pro-fibrotic changes, the increase in alternatively activated macrophage numbers, and the production of HMGB1 and α2AP that occurred with the process of fibrosis progression (Fig.
5). In addition, the IL-4Rα signaling regulates IL-4-induced STAT6 activation [
63]. We showed that the blockade of IL-4Rα attenuated the STAT6 activation in dermal fibrosis model mice. It has been reported that the inhibition of STAT6 causes the resolution of lung inflammation and fibrosis in the bleomycin-treated mice [
64], and the STAT6 pathway is associated with bone marrow-derived fibroblast activation [
65]. The attenuation of STAT6 by IL-4Rα neutralizing may contribute to the development of dermal fibrosis. These data suggest that the IL-4 signaling plays an important role on the induction and development of fibrosis, and the blockade of IL-4 signaling may be a potential target for novel therapies that prevent fibrotic diseases.
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