Background
The receptor for advanced glycation end-product (RAGE) is a transmembrane receptor that can bind numerous ligands, and the RAGE/ligand interaction results in cellular activation and gene transcription [
1,
2]. Although the expression of RAGE is at low levels in the majority of normal tissues, it is abundantly expressed in the normal lung, especially in type 1 pneumocytes [
3‐
5]. Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease with poor prognosis, characterized by recurrent alveolar epithelial injury followed by aberrant tissue repair and excessive matrix deposition [
6,
7]. Previous studies have shown that RAGE expression in lung tissue is reduced in IPF when compared with controls, and functional polymorphism of the RAGE gene is associated with risk of IPF [
8‐
11]. Additionally, in RAGE knockout mice, pulmonary fibrosis is caused by aging and may be further aggravated by exposure to asbestos [
12]; therefore, RAGE may play a homeostatic role in the lungs. These observations indicate that decreased expression of RAGE in the lungs is associated with the development and severity of IPF.
Alternative splicing of RAGE gene leads to the formation of endogenous secretory RAGE (esRAGE), which lacks the transmembrane domain but has an extracellular ligand-binding domain; it has anti-inflammatory properties and acts as a decoy neutralizing RAGE-ligands [
13‐
17]. Interestingly, proteomic analysis demonstrated that, compared to control subjects, the expression of esRAGE was reduced in the lung tissue of patients with IPF and not in chronic obstructive pulmonary disease (COPD) [
9]. This suggests that esRAGE may be related to the pathophysiology of IPF. On the other hand, there have been no studies investigating the association between circulatory esRAGE and the disease severity and prognosis of IPF.
This study aimed to investigate whether esRAGE concentrations in blood and bronchoalveolar lavage fluid (BALF) are associated with the prognoses of IPF, and to explore esRAGE regulatory mechanisms by focusing on RAGE gene polymorphisms. First, we measured esRAGE levels in the serum and BALF and evaluated their correlation. Second, we analyzed the association of esRAGE levels in the serum and BALF with pulmonary function parameters and prognosis in patients with IPF. Additionally, the correlation between esRAGE levels and differential cell counts in the BALF was investigated, because inflammatory cells in the lung are reported to express RAGE [
18,
19]. Finally, an exploratory analysis investigated the association of esRAGE concentrations with two RAGE gene polymorphisms: rs1800624 and rs1800625, which influenced the promoter activity of the RAGE gene [
20].
Discussion
This study demonstrated that serum esRAGE levels in IPF patients were significantly lower than those in healthy controls and that lower levels of serum esRAGE were associated with a poorer prognosis in IPF patients. Remarkably, decreased levels of BALF esRAGE were associated not only with decreased levels of serum esRAGE but also with lower DLco and poorer prognosis. These data could suggest that decreased serum levels of esRAGE may be associated with a pro-inflammatory condition of the lungs, which in turn decrease the levels of esRAGE in the BALF and result in a poor prognosis.
We found that lower levels of esRAGE in blood and BALF were independently associated with a poor prognosis in patients with IPF. RAGE-ligands such as HMGB1 and S100 proteins have been associated with the progression of IPF. In earlier reports, it has been shown that BALF and serum levels of HMGB1 are elevated in patients with IPF and that elevated serum HMGB1 levels are associated with acute deterioration and poor prognoses for IPF [
25,
26]. Additionally, Xia et al. reported that the expression of the S100 protein A4 (S100A4) is increased in mesenchymal progenitor cells in patients with IPF, and increased S100A4 expression is associated with aberrant lung fibrosis in a bleomycin-induced mouse model [
27]. Furthermore, esRAGE can act as a decoy receptor for RAGE ligands and inhibit the RAGE/ligand interaction, which may be associated with the IPF disease process [
13‐
15,
28]. Therefore, the present results extend the previous proteomic study observations of decreased esRAGE expression in the lungs of patients with IPF [
9], and indicate that decreased esRAGE may play a role in the progression of IPF.
The total pool of circulating soluble RAGE (sRAGE) consists of esRAGE and the ectodomain cell-surface RAGE released by metalloproteinases, such as matrix metalloproteinase 9 and a disintegrin and metalloprotease 10 [
29]. In patients with COPD, sRAGE is down-regulated in lung tissue, while esRAGE is not, and reduced circulatory sRAGE is associated with the decline of lung function, while esRAGE is not [
9,
30,
31]. However, this study showed that lower levels of circulatory sRAGE were associated with poor survival in patients with IPF, which was in line with the results of previous studies [
8,
32], and we confirmed the association of the lung and circulatory esRAGE levels with decreased lung function and poor prognoses. These observations indicate that sRAGE and esRAGE might play different roles in the pathophysiology of lung diseases. However, the present results support the hypothesis that the RAGE/ligand interaction is related to the pathophysiology of IPF, but further investigation is warranted to elucidate causal relationships.
Serum levels of esRAGE were significantly reduced in patients with IPF compared to control subjects. Additionally, there was a significant positive correlation between serum and BALF esRAGE concentrations. These results are in line with those of the previous study elucidating decreased esRAGE expression in the lungs in patients with IPF [
9], but its mechanism is still unknown. A possible explanation is that reduced esRAGE levels in IPF could result from lung parenchymal damage, because this study showed that decreased levels of esRAGE in BALF were related to reduced DLco, which was a sensitive indicator of the underlying parenchymal pathology [
33‐
35]. Additionally, although inflammatory cells in the lungs are a potential source of esRAGE [
18,
19], there was no correlation between esRAGE levels and differential cell counts in the BALF in this study. Another possible mechanism could be that increased RAGE/ligand interaction in the IPF lung may diminish esRAGE in the circulation [
36]. Yet another possibility is that RAGE gene polymorphisms, which modulate the expression of esRAGE, could cause the altered levels of esRAGE in IPF. In order to test this hypothesis, we investigated the association of esRAGE concentrations with two RAGE gene polymorphisms that influence promoter activity (rs1800624 and rs1800625), but there was no significant correlation [
20]. Further investigations are needed to elucidate the association between the esRAGE regulatory mechanism and lung fibrosis.
The present study had limitations. It was conducted in a single institution; therefore, the sample size was relatively small, especially with respect to procurement of BALF samples from IPF patients. Additionally, serum levels of esRAGE were significantly lower in patients with IPF than in healthy controls; however, the mean age of IPF patients was around 10 years more than that of healthy controls; further, more smokers were present among patients with IPF than controls. Although esRAGE levels in the serum and BALF were not significantly correlated with age and smoking history in this cohort (data not shown), this could have altered the results. Other studies to confirm the esRAGE circulatory prognostic cut-off level and its predictive value are necessary.
Conclusions
In conclusion, decreased esRAGE levels in both BALF and blood were associated with a poor prognosis in patients with IPF. In particular, serum levels of esRAGE may be a potential prognostic marker reflecting dysregulated RAGE/ligand interactions in the lungs, indicated by lower BALF levels of esRAGE. Finally, further studies are warranted to investigate the role of the RAGE/ligand interaction effect on the progression of IPF.
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