Introduction
Pulmonary fibrosis is an interstitial lung disease characterized by chronic inflammation and progressive fibrosis of the pulmonary interstitium (alveolar walls and septa, perivascular, perilymphatic and peribronchiolar connective tissues) [
1]. It is believed that lung inflammation initiates lung fibrosis, however, the etiological mechanism of this disease has not yet been fully elucidated [
2].
Nitric oxide (NO) is gaseous free radical, and is formed from its precursor, L-arginine, by a family of NO synthases (NOSs) with stoichiometric production of L-citrulline [
3]. NO plays an important role in maintaining respiratory homeostasis [
4],[
5]. There are three distinct isoforms of NOS, two of which are constitutive NOSs known as neuronal NOS (nNOS) and endothelial NOS (eNOS), and other is inducible NOS (iNOS). The expression of constitutive NOSs (nNOS and eNOS) has been observed in various types of pulmonary cells. For example, nNOS is expressed in neuronal cells (ganglions, trachea and bronchi), and eNOS is expressed in vascular endothelial cells and type ІІ alveolar epithelial cells in humans [
4],[
5]. On the other hand, the expression of iNOS has not been reported in quiescent cells in healthy subjects, but there have been reported that it is expressed in the airway and the lung parenchyma following stimulation by microbial endotoxins and certain proinflammatory cytokines [
4],[
5].
Free radicals, including NO, play an important role in the development of pulmonary fibrosis [
6]. In fact, increases in the expression of these NOSs in the lungs, and the plasma NOx (nitrite plus nitrate) level, a marker of NO production, have been reported in patients with pulmonary fibrosis [
7]–[
9]. The roles of the NOS system in the lungs have been evaluated using several types of animal models, and eNOS has been reported to exert a protective role in pulmonary fibrosis [
10],[
11]. Conflicting results have been reported with regard to iNOS, with some studies showing pathogenic [
12]–[
14] and protective [
15],[
16] roles for the enzyme in pulmonary fibrosis. However, because of the different roles of each NOS and the compensatory interactions among these different NOSs [
3],[
17], the assessment of the roles of NO and the NOSs themselves is difficult, and the roles of the entire NO and NOS system in pulmonary fibrosis remain to be fully elucidated.
Tsutsui
et al. have developed a mouse model in which all three NOSs were completely deleted [
3],[
17], and these triple NOS knockout (n/i/eNOS
−/−) mice demonstrated less than 3% of the normal level of NOx [
17]. The authors also reported that n/i/eNOS
−/− mice are indistinguishable from wild-type (WT) mice in terms of phenotype and develop normally with a standard increase in body weight. However, they also documented that n/i/eNOS
−/− mice are significantly hypertensive compared with WT mice and display characteristics consistent with those of nephrogenic diabetes insipidus [
17].
In this study, we investigated the essential roles of NO and the NOS system in a bleomycin (BLM)-induced pulmonary fibrosis model using the n/i/eNOS−/− mice.
Discussion
In the present study, we evaluated the roles of NO and the NOS system in pulmonary fibrosis by using mice lacking all three NO synthases, n/i/eNOS−/− mice, and showed that the lack of all NOS led to a deterioration of the fibrotic changes in the lungs of mice with BLM-induced pulmonary fibrosis. In addition, these findings were prevented by long-term treatment with a NO donor, ISDN. This is the first report to show that NO is an important factor in the progression of pulmonary fibrosis, and that NO has protective effects against BLM-induced pulmonary fibrosis.
With regard to the roles of NO in the progression of fibrosis, there have been several reports showing the protective roles of NO in cardiac [
22] and renal [
23] fibrosis using non-selective NOS inhibitors in mouse models. So far, a non-selective NOS inhibitor has been reported to worsen the mortality in a BLM-induced murine pulmonary fibrosis model [
10] and accelerated pulmonary granuloma formation in a purified protein derivative murine model [
24], another model of pulmonary fibrosis. However, because of the non-specificity of these inhibitors [
25],[
26], it is difficult to evaluate the essential roles of NO. Therefore, little has been known about the functions and roles of NO itself in pulmonary fibrosis. Concerning the role of each NOS isoform in pulmonary fibrosis, the protective effects of pulmonary fibrosis in eNOS transgenic mice [
10] and the deterioration of fibrosis in eNOS
−/− mice [
11] have also been reported. The inhibition of iNOS has been reported to suppress pulmonary fibrosis in murine model using iNOS
−/− mice [
12] and mice treated with a selective iNOS inhibitor [
12]–[
14], but there have been several conflicting reports that iNOS
−/− led to a deterioration of the progression of pulmonary fibrosis [
15],[
16]. It has been reported that the expression of nNOS was unchanged in a BLM-inhalation rat model [
27], and the role of nNOS in pulmonary fibrosis has not been fully understood.
Therefore, the role of NO in pulmonary fibrosis has been controversial, mainly because each isoform has different functions and compensatory interactions with the other isoforms [
3],[
17]. The n/i/eNOS
−/− mice provide one way to resolve the former problems of the animal models using single NOS
−/− mice or various NOS inhibitors, and we believe this murine model is an important tool for understanding the essential roles of NO [
18],[
28].
NO and the NOS system have been suggested to have both beneficial and deleterious effects on the respiratory system [
4]. These results are confusing with respect to understanding the essential role of NO. In the present study, the BLM-treated WT mice demonstrated increased NOx concentrations as well as a deterioration of fibrotic changes compared with that observed in the NS-treated WT mice, as well as increased plasma NOx levels have been reported in patients with pulmonary fibrosis [
9]. While the BLM-treated n/i/eNOS
−/− mice, despite the lack of NOx, exhibited a significant deterioration of fibrotic changes compared to the BLM-treated WT mice. In addition, the poor factors observed in the BLM-treated n/i/eNOS
−/− mice were prevented via ISDN treatment by increasing the NOx levels up to that observed in the NS-treated WT mice. Therefore, we believe that strongly reduced concentrations of NO may be associated with the progression of BLM-induced pulmonary fibrosis and that an appropriate NO concentration is required for respiratory homeostasis.
In addition, a significant body weight loss has been reported in parallel with a deterioration of pulmonary fibrosis in a BLM-treated mouse model [
29]. In the present study, the BLM-treated n/i/eNOS
−/− mice also exhibited a significant protracted course of body weight loss compared with the WT and single NOS
−/− mice.
TGF-β1 is an important pathogenic factor involved in a variety of fibroproliferative disorders, including pulmonary fibrosis [
1],[
13], and there have been several
in vitro reports that showed an increased expression of NO and a subsequent decrease of TGF-β1 due to the increase of NO production [
13],[
30]. Shibata
et al. reported an elevation of cardiac TGF-β1 expression in n/i/eNOS
−/− mice [
31], and we similarly observed upregulation of the protein levels and mRNA of pulmonary TGF-β1 in BLM-treated n/i/eNOS
−/− mice in this study. It is well known that TGF-β1 promotes the production of CTGF [
32] and collagen I [
33], which leads to the progression of pulmonary fibrosis. The subsequent increased production of CTGF and collagen I was noted in the BLM-treated n/i/eNOS
−/− mice in this study. From these results, with respect to the mechanisms underlying the antifibrotic activity induced by the absence of NO, the TGF-β1/CTGF pathway is one possible pathway involved in this process. CTGF is considered to play a critical role in the onset of fibrosis as a downstream mediator of TGF-β1 [
34], and the downregulation of the expression of CTGF mRNA by NO donors in rat mesangial cells has been previously reported [
34]. NO has also been reported to suppress the expression of CTGF by inhibiting Smad-dependent TGF-β signaling [
35]. Taken together, the deterioration of pulmonary fibrosis in the BLM-treated n/i/eNOS
−/− mice observed in this study may be explained by the above mechanism, although further studies are needed to clarify the mechanisms underlying the antifibrotic activity of NO in the setting of fibrotic lung diseases.
It is well known that increased expression levels of the proinflammatory cytokines IL-6, IL-1β and TNF-α and decreased expression levels of the anti-fibrotic cytokine IFN-γ are involved in the pathogenesis and progression of pulmonary fibrosis [
36],[
37]. Our present results are consistent with the findings of former reports, although there were no significant differences in the protein levels of IL-1β or IFN-γ. Considering the relationships between NO and the above proinflammatory cytokines, NO has been reported to be a potent inhibitor of the proinflammatory cytokine production induced by alveolar macrophages [
38],[
39]. Therefore, the increased levels of pulmonary inflammatory cytokines (IL-6, IL-1β and TNF-α) observed in the BLM-treated n/i/eNOS
−/− mice in the present study may also be explained by an increase in proinflammatory cytokine production stimulated by alveolar macrophages. Therefore, we speculate that alveolar macrophages are potent targets in the deterioration of pulmonary fibrotic changes associated with the absence of NO.
CCL-2 was upregulated in BLM-treated n/i/eNOS
−/− mice compared to BLM-treated WT mice, and therefore, the CCL-2/NO pathway was considered as an alternative pathway leading to BLM-induced pulmonary fibrosis in this study. CCL-2, also known as monocyte chemotactic protein-1 (MCP-1), belongs to the C-C chemokine superfamily of small proteins, and is considered to be a potent chemoattractant for monocytes/macrophages. Several reports have demonstrated that CCL-2 plays an important role in the development of pulmonary inflammation and fibrosis in both animal models [
40] and human studies [
41]. Previous
in vitro and
in vivo studies have shown that endothelial NO synthesis was inhibited by a non-selective NOS inhibitor and this inhibition of endothelial NO synthesis led to an increase in CCL-2 expression [
42],[
43]. The production of TGF-β1 induced by CCL-2 has also been reported
in vitro[
44], and the promotion of TGF-β1 production may be explained by the increased CCL-2 production in BLM-treated n/i/eNOS
−/− mice in this study.
In this study, the eNOS
−/− mice treated with BLM histopathologically exhibited more cellular infiltration and collagen deposition than the WT mice, although the findings of the quantitative evaluation of the fibrotic areas and collagen deposition and the analyses of the BALF did not differ significantly from those observed in the WT mice. The protective effects of eNOS against pulmonary fibrosis have been demonstrated in various studies [
10],[
11], and we believe that eNOS may also protect against the development of pulmonary fibrosis. However, it was not possible to elucidate the role of each NOS isoform in fibrotic changes compared to the WT mice based on the results of this study. These results were similar to the previous reports in models of carotid artery ligation or a high-cholesterol diet [
18],[
28]. Compensatory mechanisms involving other NOSs in terms of producing NO may explain these findings. Indeed, Morishita
et al. have revealed that the other NOSs are highly expressed in the single NOS
−/− and double NOS
−/− mice, and that NOx production is fairly well preserved in mice of those genotypes [
17]. These findings may support the importance of using a murine model lacking all three types of NOS when investigating the true functions of NO.
In conclusion, we provide the first evidence that a lack of all three NO synthases leads to the deterioration of fibrotic changes in BLM-induced pulmonary fibrosis in mice. It is speculated that NO plays an important protective role in the pathogenesis of pulmonary fibrosis.
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Competing interests
All the authors report no potential conflicts of interest.
Authors’ contributions
SN (designed experiments, performed data analysis, wrote the first draft), KY (designed experiments, performed data analysis, provided intellectual contributions), WKY (designed experiments, provided intellectual contributions), KO (provided intellectual contributions), KA (provided intellectual contributions), KY (designed experiments, provided intellectual contributions), TK (provided intellectual contributions), HI (provided intellectual contributions), YT (provided intellectual contributions), HS (provided intellectual contributions), NY (provided intellectual contributions), MT (provided intellectual contributions), HM (conceived & designed experiments, provided intellectual contributions). All authors read and approved the final manuscript.