Simvastatin up-regulates adenosine deaminase and suppresses osteopontin expression in COPD patients through an IL-13-dependent mechanism

Chronic obstructive pulmonary disease (COPD) is driven by chronic inflammation and tissue remodeling process in response to noxious particles or gases [1, 2]. Currently, there are no effective therapies that alter disease progression and improve survival [2] due to the complexity of signaling pathways that maintain chronic inflammation and tissue destruction [3].

Adenosine plays a key role in airway inflammation and remodeling in COPD [4]. Extracellular adenosine was produced upon cell damage to balance tissue repair against excessive airway remodeling in COPD [5‐10]. Conversely, in prolonged or repeated tissue injury, chronic adenosine elevation can activate signaling pathways that promote tissue injury [5, 9]. Extracellular adenosine production is regulated by ecto-5'-nucleotidase (CD73); the rate-limiting step that converts AMP to adenosine [11]. CD73 expression and activity is markedly increased in patients with severe COPD, suggesting the high production of adenosine [3]. Adenosine can either interact with adenosine receptors or be transported into cytosol via facilitated nucleoside transporters [12]. Adenosine can be deaminated to inosine by adenosine deaminase (ADA) [13]. Adenosine is upregulated in IL-13-transgenic mice through the suppression of ADA activity and mRNA accumulation [14]. The synergistic effect of adenosine and IL-13 may contribute to the severity of airway inflammation and fibrosis in COPD [14]. Suppression of ADA activity together with CD73 up-regulation promotes adenosine production in the COPD lungs [3]. The association between defective ADA activity and COPD was demonstrated in ADA-deficient mice [15]. The pathological features of COPD was prevented and reversed by lowering adenosine levels with exogenous PEG-ADA [14‐17].

Osteopontin (OPN) can function both as a matrix protein and a pleiotropic cytokine. OPN expression is regulated by IL-13 that can be induced by cigarette smoke extract [18, 19]. OPN accumulation in smokers correlates with the degree of airflow limitation [20]. Sputum OPN is significantly higher in COPD patients than in healthy smokers in agreement with the extent of emphysema [21]. In COPD lungs, OPN is primarily localized in alveolar macrophages and to a lesser extent in epithelial cells, T cells and fibroblasts [22]. Both adenosine and OPN are involved in COPD pathogenesis. ADA deficiency causes OPN-dependent neutrophilia and alveolar air-space enlargement [22]. Increasing adenosine signaling in severe COPD is directly associated with increased OPN transcripts [3, 22]. Therefore, the simultaneous inhibition of OPN as well as adenosine might be an additional strategy for prevention of COPD progression in particular deterioration of lung function caused by airway fibrosis.

Statins inhibit the synthesis of the cholesterol isoprenoid intermediates farnesylpyrophosphate (FPP) and geranylgeranyl pyro-phosphate (GGPP) [23]. Statins suppressed OPN mRNA and protein expression in an ovarian cancer cell line due to several diverse homologous cis-acting consensus sequences in human OPN promoters [24, 25]. In addition, this suppression may result from statin-depleted isoprenoids [26] that affects the expression of OPN [27]. However, the effect of simvastatin in the context of IL-13-regulated adenosine and OPN in COPD patients is not known.

We have demonstrated that simvastatin reversed IL-13-suppressed ADA activity in COPD patients that leads to the down-regulation of adenosine signaling and therefore inhibits OPN expression through the direct inhibition of IL-13-activated STAT6 pathway. Simvastatin-restored ADA activity positively correlated with the magnitude of changes in pre-bronchodilator FEV₁. Inhibition of IL-13 may reverse the imbalance between ADA and OPN in COPD and therefore may prevent COPD progression.

Flow of subjects through the study, demographic data, sputum cytology in response to simvastatin were previously described (Table 1) [29]. Pre-BD FEV₁ values were positively correlated with baseline ADA but negatively correlated with OPN (r_s = 0.76; p < 0.001, r_s = −0.75; p < 0.001, respectively) (Fig. 1a, 1b).

We have demonstrated for the first time that simvastatin enhances ADA expression while inhibiting osteopontin via inhibition of STAT6-dependent IL-13 signaling. In addition to ADA enhancement, simvastatin suppressed CD73 expression, both of which resulted in increasing sputum inosine. Our study confirmed the diametrically opposed role of OPN and ADA in patients with COPD. Osteopontin were inversely correlated, whereas ADA levels were directly correlated with pre-BD FEV₁. Simvastatin-restored ADA activity was associated with improving pre-BD FEV₁. Our mechanistic exploration in vitro suggested that CSE enhanced IL-13 that induced OPN but suppressed ADA in COPD-derived MDM via the STAT6-dependent pathway whereas in healthy-derived MDM, CSE suppressed IL-13, resulting in the reverse effect to OPN and ADA transcription. Simvastatin inhibited CSE-enhanced IL-13 in a dose-dependent manner and subsequently decreased STAT6 phosphorylation, leading to the reciprocal induction of ADA and inhibition of OPN. In addition, simvastatin did not affect adenosine receptor expression and had no inhibitory effects on IL-13, OPN, and ADA in CSE-treated MDM from healthy subjects.

Statins have strong immune-modulating effects in both the systemic and pulmonary inflammation, which could have substantial benefits in patients with COPD [33]. Anti-inflammatory effects of statins are mediated through inhibition of key inflammatory and remodeling pathways [33]. This is supported by the recent study showing that statins significantly reduced airway and pulmonary neutrophils in stable COPD [34]. Our previous study also demonstrated that statin therapy could suppress IL-17 and restore IL-10 in COPD although this was associated with reduction in alveolar macrophages but not neutrophils as previously explained [29]. Therefore, statins may attenuate progressive decline in lung function although there have been very few studies investigating signaling molecules simultaneously involved in both inflammatory and fibrogenic activities including OPN and IL-13.

IL-13 polymorphism is associated with an increased risk of COPD [35‐37]. Alveolar or extrapulmonary macrophages produce IL-13 in response to rhinovirus or parasitic infection in mice [38, 39]. CSE may induce IL-13 producing murine macrophage M2 polarization in mice, either through its direct effect on JAK/STAT3 or indirectly through mast cell-associated mechanisms [40, 41]. In addition, a previous study showed that RSV infection enhanced cigarette smoke-induced IL-13 gene expression, but not IL-13 release, in mice although the cellular source of IL-13 was not identified [42]. CSE induces IL-13 secretion by macrophages in mice. However, there are very few data addressing the ability of CSE to induce an IL-13 response in human macrophages from patients with COPD. We have demonstrated that CSE markedly induced IL-13 response with STAT6 activation in MDM from COPD patients. However, this was not the case for the effect of CSE on IL-13 in healthy-derived MDM that revealed in reverse, possibly preventing healthy MDM from the establishment of IL-13/OPN axis. Therefore, CSE-stimulated COPD-derived MDM could be used in our study to investigate the effects of simvastatin on CSE-induced IL-13-mediated OPN and ADA expression.

OPN induces airway neutrophilic inflammation and airway remodeling [22] and its expression is increased in patients with COPD [3, 21]. Simvastatin may suppress pro-inflammatory and induce anti-inflammatory cytokines in COPD [29]. Furthermore, simvastatin inhibits IL-13 inducible OPN gene in airway epithelial cells. However, whether IL-13 regulates OPN expression and whether simvastatin affected this in patients with COPD is unknown. Therefore, the mechanistic understanding of how OPN accumulation is induced by IL-13 and how simvastatin mediates its inhibitory effects on IL-13-induced OPN accumulation in COPD patients may provide a new therapeutic target in future. The present study is the first to investigate whether CSE induces OPN transcription in an IL-13-dependent manner and showed that silencing of IL-13 or STAT6 with siRNA resulted in the down-regulation of CSE-enhanced induction of OPN and the up-regulation of CSE-suppressed expression of ADA. Furthermore, our findings confirmed and extended the previous data that in addition to inhibition of CSE–induced increase in OPN expression [18, 27], simvastatin also inhibited CSE-induced increase in IL-13 production by MDM from patients with COPD in a dose-dependent manner. In addition, the effect of simvastatin on CSE-induced OPN expression could be overcome by the addition of exogenous IL-13, confirming that inhibition of CSE-induced OPN by simvastatin was mediated through IL-13 suppression. Simvastatin reduces IL-13 levels with varying doses in a mouse asthma and bleomycin-induced pulmonary fibrosis model [43, 44]. However, our result is in contradiction to previous studies in Th2-biased animal models in that simvastatin induced Ym1 in dendritic cells which resulted in the augmentation of IL-13 release from Th2 cells [45]. This leads us to speculate that there are varying effects of simvastatin on IL-13 which are dependent upon specific cell types and types of inflammation.

Several studies have shown that IL-13 is strongly induced by adenosine in ADA knockout mice during an inflammatory and remodeling response [14, 30, 31]. During this response, IL-13 causes a progressive accumulation of adenosine and inhibits ADA activity and mRNA accumulation [14]. Our study demonstrates the inhibitory effect of IL-13 on ADA in patients with COPD, and that simvastatin treatment reverses IL-13-suppressed ADA in CSE-treated MDM from COPD patients. Restoration of ADA as well as inhibition of CD73 in the airways of COPD patients by simvastatin could be the mutually beneficial conversion of adenosine to inosine as exemplified by an increase in sputum inosine levels. However, we could not exclude the possibility that the reversal of ADA suppression may diminish the IL-13-induced increase in adenosine [14] and the possibility that this may be enhanced by simvastatin-inhibited IL-13 in our COPD patients.

IL-13 and adenosine stimulate each another in an amplification pathway that may contribute to progression and/or chronicity of IL-13, which at least in part is augmented by increased adenosine receptor expession [14]. A₁R, A_2BR and A₃R are elevated in the lungs of IPF models. Previous data suggested that there was elevated expression of A_2BR in macrophages in COPD lung specimens [3]. Persistent activation of A_2BR signaling maintains inflammatory mediators, such as IL-6, IL-8, IL-13 and OPN [3], [46‐48]. In contrast, another study demonstrated elevation of A_2AR and A₃R but down-regulation of A_2BR transcripts [49]. The discrepancy between both studies may be related to differences in disease severity and smoking status. The present study has demonstrated that simvastatin failed to suppress adenosine receptors. This results indicates that the inhibitory effects of simvastatin on IL-13 and OPN response is mediated through down-regulation of adenosine signaling resulting from modulation of adenosine rather than its receptors.

Our results were in the agreement with other previous studies showing the beneficial effects of statins [50‐52] but differed from the results of the recent study (STATCOPE) demonstrating no benefit [53]. A plausible explanation for the discrepancy between STATCOPE and other previous studies including our study is the inclusion of a large percentage of COPD patients with coexisting overt cardiovascular disease who are ‘statin users’ and ‘non-users’, but would benefit from statin therapy. These comorbid phenotypes of COPD are likely to express a poor prognosis due to one or a combination of undertreated pulmonary inflammation, unrecognized systemic inflammation or subclinical cardiovascular disease. These comorbid conditions are strongly associated with an increased risk of hospitalization with acute exacerbations and greater mortality [54‐56]. Therefore, there is possibility that many patients with COPD who have not been prescribed with statins in other previous studies do badly from undertreatment, a hypothesis mentioned by STATCOPE investigators to explain this discordance [53]. Another explanation for the STATCOPE findings is that COPD patients with coexisting clinical and subclinical cardiovascular disease underlying acute exacerbations has been all but excluded [57, 58].

The present study has demonstrated the involvement of IL-13 in CSE-induced OPN and simultaneously suppressed ADA in MDM and the consistent imbalance between IL-13-regulated OPN and ADA in patients with COPD. The balance toward IL-13-induced OPN can be reversed by simvastatin treatment. However, whether the reversal would provide the long-term benefit in lung function decline for patients with COPD required further study.

This study provides the better understanding of the molecular mechanisms underlying the effects of simvastatin on ADA/OPN balance in COPD patients. Simvastatin treatment induces ADA and inhibits OPN expression in the airways and monocyte-derived macrophages from COPD patients. This effect is mediated through the direct inhbition of IL-13 activated STAT6 pathway which drives the imbalance between ADA and OPN towards the latter.