Introduction
Scleroderma (systemic sclerosis, or SSc) is a fibrotic diseases for which there is currently no approved treatment [
1]. Although the underlying causes are unknown, fibrotic disease is associated with the production and accumulation of excessive fibrous connective tissue and can be considered to arise because of an inability to appropriately terminate the normal wound repair response [
2,
3]. SSc is a prototypic multisystem and multistage fibrotic disease and is considered to be initiated by a combination of microvascular injury, inflammation, and autoimmunity, culminating in fibroblast activation and fibrosis [
3]. Histological analysis of the initial stage of scleroderma reveals perivascular infiltrates of mononuclear cells in the dermis, and these infiltrates are associated with increased collagen synthesis in the surrounding fibroblasts [
4,
5]. Thus, understanding how to control the inflammatory stage of SSc may be of benefit in controlling the progression of early-onset disease.
Microsomal prostaglandin E
2 synthases (mPGESs) are enzymes that catalyze the conversion of PGH
2 to PGE
2 [
6]. Thus far, three PGE synthases - namely cytosolic PGE synthase (cPGES), mPGES-1, and mPGES-2 - have been characterized [
6‐
8]. cPGES is localized in the cytosolic region of cells and tissues under basal conditions and is most likely to be involved in the homeostatic production of PGE
2 [
8]. mPGES-2 is also constitutively expressed in a wide variety of tissues and cell types and is synthesized as a Golgi membrane-associated protein [
9]. In contrast, mPGES-1 is induced in response to inflammation and acts downstream of cyclooxygenases [
10,
11].
mPGES-1 has been shown to be a critical mediator of inflammation, pain, angiogenesis, fever, bone metabolism, and tumorgenesis [
12‐
15]. We have previously shown that mPGES-1 expression is elevated in tissues and cells of various inflammatory diseases, including rheumatoid arthritis and osteoarthritis [
10,
11,
16,
17]. mPGES-1 null mice are resistant to chronic inflammation of joints in the models of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis [
12,
13]. We recently showed that mPGES-1 is induced during the skin wound healing process in mice [
18]. However, the expression and role of mPGES-1 in fibrogenesis are unknown.
There is no perfect mouse model that recapitulates every facet of SSc; however, the bleomycin-induced model of skin scleroderma is often used. In this model, repeated application of bleomycin, an anti-tumor antibiotic originally isolated from the fungus
Streptomyces verticillus [
19], is used to induce inflammation and subsequent fibrosis in skin [
20]. Thus, the bleomycin model of skin SSc can be used to evaluate the potential role of individual genes in the early onset (or inflammatory phase) of SSc. The aim of the present study was first to examine whether mPGES-1 shows altered expression in fibroblasts isolated either from dermal lesions of patients with SSc or from mouse skin response to bleomycin and then to assess the potential role of mPGES-1 in the early phases of SSc by subjecting mice deficient in mPGES-1 to the bleomycin model of skin scleroderma [
21].
Discussion
Since its discovery in 1999 [
6], mPGES-1 has been a target of anti-inflammatory drug therapy. mPGES-1 is induced in human synovial tissue in osteoarthritis patients and in animal models of inflammation such as full-thickness incisional models of wound healing [
18], CIA [
22], lipopolysaccharide (LPS)-induced pyresis, and adjuvant-induced arthritis [
33,
34]. Moreover, in a variety of mesenchymal cell types (including fibroblasts), mPGES-1 is induced by proinflammatory stimuli, including LPS, interleukin-1-beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) [
6,
10,
11,
17,
30,
31,
35]. These results suggest that mPGES-1 plays a key role in driving inflammation. Although a role for inflammation in fibrogenesis is well established, the
in vivo role for mPGES-1 in fibrosis has not been reported thus far.
A potent and selective inhibitor for mPGES-1 is not yet commercially available; however, mice with genetic deletion for mPGES-1 do exist, and these mice have been useful to define the in vivo role of mPGES-1. Our present study uses the bleomycin-induced model of skin fibrosis to assess whether mPGES-1 is essential for the onset of fibrosis. To provide a clinical context for our studies, we first showed that mPGES-1 protein expression was elevated in SSc skin fibroblasts. We then showed that mPGES-1 was induced in response to bleomycin in mouse skin fibroblasts in vivo.
It is largely believed that enhanced inflammatory response is necessary for fibrogenesis [
32]. Accumulating evidence indicates a critical involvement of infiltrating macrophages and T cells in the pathogenesis of SSc. High numbers of infiltrating activated macrophages and T cells have been detected in skin of patients with SSc [
36,
37] and these cells are key producers of a variety of pro-fibrotic cytokines such as transforming growth factor-beta (TGF-β), CC-chemokine ligand 2, and IL-4 and IL-17 [
38‐
40]. Therefore, we investigated the effect of mPGES-1 genetic deletion on inflammatory response by detecting macrophage infiltration in response to bleomycin treatment. mPGES-1 null mice showed marked reduction in the number of macrophages (inflammation) in response to bleomycin treatment, supporting our previous findings that mPGES-1 is a critical mediator of inflammation [
22]. In future studies, it would be very interesting to determine the different subsets of infiltrating macrophages regulated by mPGES-1 during SSc disease. In addition, it should be investigated whether and how T cells are regulated by mPGES-1 during SSc. Since this is beyond the scope of the present study, future studies need to be directed toward understanding these concepts.
After determining the effect of mPGES-1 on inflammation, we further investigated the effect of mPGES-1 deletion on the degree of skin fibrosis. mPGES-1 null mice showed a resistance to bleomycin-induced skin fibrosis, as visualized by reduced dermal thickness and collagen production. The myofibroblast is the major cell type believed to be responsible for fibrogenesis, including in SSc [
27,
41,
42]. Compared with WT mice, mPGES-1 null mice had fewer myofibroblasts in response to bleomycin injection. Our results collectively suggest that genetic deletion of mPGES-1 suppresses fibrogenesis
in vivo.
Bleomycin-induced fibrosis is an inflammation-driven model and it is well established that PGE
2, the product of mPGES-1, is one of the major proinflammatory mediators upregulated during inflammation. Given the known role of mPGES-1 in driving inflammatory responses, our results strongly suggest that mPGES-1 may play a key role in the initial, inflammatory stages of SSc. Our present study demonstrates that mice lacking mPGES-1 show resistance to bleomycin-induced fibrogenesis and is consistent with the notion that inflammation is involved with the onset of fibrosis, including SSc [
32,
43,
44]. However, it is well established that inflammation plays a biphasic role in fibrosis; for example, the inflammatory protein TNF-α plays a biphasic role in fibrogenesis by promoting the initiation/inflammatory stage of fibrogenesis but suppressing the later, fibrotic stage of fibrosis [
45‐
49]. As a specific illustration, TNF-α suppresses the ability of TGF-β to induce connective growth factor (CTGF/CCN2) in dermal fibroblasts [
45]. In this regard, it is interesting to note that PGE
2 (the only known product of mPGES-1) and iloprost (a synthetic version of prostacyclin, or PGI
2) have been repeatedly shown to exhibit antifibrotic effects in experimental models of established fibrosis, including reducing CCN2 and collagen production in normal and fibrotic dermal fibroblasts, at least in part, acting through a cAMP-mediated suppression of ERK (extracellular signal-regulated kinase) activation [
50‐
55]. Indeed, it has been hypothesized that prostacyclins limit the activation of fibroblasts following tissue injury but, in response to the original injury, may promote recruitment of inflammatory cells and lead to secondary activation of fibroblasts [
56]. Moreover, given these concerns (and consistent with our data showing that SSc fibroblasts overexpress mPGES-1), it is interesting to note that prostanoid (including PGE
2) production was greatly elevated in scleroderma cells compared with control cells and, given that excess added prostenoids reduced collagen and CCN2 overexpression in SSc fibroblasts, may act to limit further increases in collagen and CCN2 levels in these cells [
50]. Given these considerations, it is likely that although mPGES-1 may contribute to the initiation of fibrogenesis through its ability to promote inflammation, mPGES-1 may actually act to control the overexpression of profibrotic genes in established lesions [
56]. Investigation of the role of mPGES-1 in established fibrosis (for example, using the tight skin [Tsk] mouse [
57]) is beyond the scope of the present study.
Acknowledgements
The authors thank Stephane Tremblay and Frederic Pare (Osteoarthritis Research Unit, University of Montreal) for their assistance with the histological staining and histo-morphometric analyses. MRM is supported by the Joint Motion Program (JuMP) - A CIHR Training Program in Musculoskeletal Health Research and Leadership. MK is supported by the Canadian Institutes of Health Research, the Canadian Foundation for Innovation, Fonds de la Recherche en Santé du Québec, and the University of Montreal Hospital Research Centre (CR-CHUM). LJC is supported by grants from the National Institutes of Health. AL is supported by the Canadian Foundation for Innovation, the Canadian Institutes of Health Research, the Ontario Thoracic Society, the Arthritis Research Campaign, and the Reynaud's and Scleroderma Association.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MK and AL had full access to all of the data in the study, shared responsibility for the integrity of the data and the accuracy of the data analysis, and contributed to study conception and design and to analysis and interpretation of data. MRM and RM contributed to study conception and design and to acquisition, analysis, and interpretation of data. LJC contributed to study conception and design. PG-K, GP, SL, XS-w, SKP, and FK contributed to acquisition, analysis, and interpretation of data. HF, CPD, DJA and JMP contributed to analysis and interpretation of data. All authors were involved in drafting the article or revising it critically for important intellectual content and read and approved the final manuscript.