Our findings in a large cohort of smokers demonstrate that statin use may increase the risk of developing radiographic evidence of ILD including findings characteristic of pulmonary fibrosis. This risk may be modified by the hydrophilicity of statin and the age of the subject. In support of our clinical findings, we demonstrate that statin use exacerbates bleomycin-induced lung fibrosis in mice. Further, our study demonstrates that statin pretreatment increases mtROS in stimulated cells, resulting in increased NLPR3 inflammasome-mediated immune responses.
Our data provide support to evidence from numerous case reports (15
) suggesting that statins may cause ILD. Although prior case-control studies limited to the association between statin use and idiopathic pulmonary fibrosis alone (29
) have not demonstrated significant associations, these studies are limited by small sample size (29
) and the potential for selection bias in control subjects (29
), and include cases selected by diagnostic codes alone (30
). In contrast, our study includes the CT characterization of a large cohort (>2,100 subjects), and presents associations between statins and ILA scored by multiple readers masked to information about current medication use.
Although our data, and those of others (30
), support an association between cardiovascular disease the development of fibrotic lung disease, several lines of evidence suggest that cardiovascular disease alone is unlikely to entirely explain our findings: (1
) the association between statin use and ILA is independent of presence of cardiovascular disease and additional medications commonly prescribed for cardiovascular disease; (2
) although hydrophilic statin users in COPDGene were at greater risk for ILA, this was not coupled with an increased report of coronary artery disease (in fact, users of hydrophilic statin were slightly less likely to report coronary artery disease compared with users of lipophilic statin); and (3
) we demonstrate experimentally that statin use can exacerbate lung fibrosis in mice.
Comparable with our clinical findings, our results in mice indicate that statin administration enhances bleomycin-induced caspase-1–mediated immune response in the lung. Moreover, we show that enhanced activation of caspase-1 correlates with an increase in fibrotic change in the lung treated with bleomycin and pravastatin. The effect of statin administration on cytokine secretion was exerted on upstream steps of NLRP3 based on the following observations: (1
) NLRP3 deficiency completely impaired the effect of statin pretreatment on IL-1β and IL-18 secretion; (2
) formation of NLRP3 inflammasome induced by LPS and ATP was increased by statin pretreatment; and (3
) the effect of statin on the cytokine secretion was inhibited by glyburide, which suppresses the activation pathway upstream of the NLRP3 inflammasome but downstream of P2X7 receptor. These results suggest that statins target the activation pathway upstream of NLRP3 inflammasome, and further implicate activation of the NLRP3 inflammasome in fibrotic lung disease (31
Although mtROS are important for various mitochondrial functions including biosynthesis of many molecules and catabolic pathways, it has been shown that excess mtROS generation hyperactivates immune responses (20
). Our data show that statin administration increases immune responses in our inflammasome-activating models; however, it is still unclear how statins enhance mtROS in the stimulated macrophages. Of note, in blocking the synthesis of cholesterol, statins block the synthesis of ubiquinone, which is essential in mitochondrial electron transport (34
). Although not all studies demonstrate that statins increase mtROS (35
), some of these discrepancies may be explained by different stimuli and differences in tissue specificity (35
Our findings contrast with two previous reports that suggest that statins could ameliorate bleomycin-induced lung injury (10
). These studies differ in the type of statin and the dose of bleomycin used (Ou and coworkers used simvastatin and instilled bleomycin 15- to 20-fold higher [0.3 U/10 g] than standard dosing for such experiments) (37
), and in the timing of statin administration (Kim and coworkers administered pravastatin coincident with bleomycin instillation) (10
). Importantly, it should be noted that pravastatin alone did not enhance NLRP3 inflammasome activation in vitro
or aggravate the lung injury in vivo
, in the absence of proinflammatory challenge. Moreover, it may be relevant that all subjects from COPDGene were current or former smokers, because cigarette smoke alone may result in pulmonary inflammation through NLRP3 inflammasome-mediated pathways in humans (38
) and in mouse models (39
One limitation of this study is the lack of correlative data allowing us to relate in human samples the mechanisms described in the mouse. Many prior studies have demonstrated that statins contribute to the release of inflammasome-related cytokines including IL-1β and IL-18 in human peripheral blood monocytes through a caspase-1–dependent mechanism. Up-regulation of the inflammasome in response to statins is blocked by the reintroduction of downstream products of the cholesterol synthesis pathway including mevalonate and geranylgeraniol (40
). In these studies human peripheral blood monocytes frequently require a stimulus, such as Toll-like receptor ligands (e.g., LPS) to activate the inflammasome. However, in contrast to peripheral blood monocytes (or monocytic cell lines), recent evidence suggests that human alveolar macrophages may require ATP as an additional stimulus to activate the inflammasome (44
). This may have relevance to our findings because studies have demonstrated that, although smoking up-regulates extracellular ATP (45
), the regulation of intracellular ATP by statins is drug dependent (e.g., simvastatin, lovastatin, and fluvastatin result in decreases in cellular ATP levels, whereas atorvastatin, rosuvastatin, and pravastatin do not) (46
Our study has several additional limitations. First, although biopsies were not obtained in this cohort, it is important to note that biopsies on similarly ascertained cohorts of patients with ILA (47
) have demonstrated histopathologic evidence of ILD (idiopathic interstitial pneumonias in particular). Second, although we did not find evidence that the association of statin use and ILA was modified by either current use of tobacco or the intensity of tobacco smoke exposure, it is worth noting that all subjects in COPDGene have a history of at least 10 pack-years of smoking. Our group (16
) and others (17
) have previously demonstrated that smoking is associated with ILA. Therefore, it is possible that the association between statin use and ILA is limited to current and former smokers. Third, we do not have information on the duration of therapy or drug dosage in most of the patients on statins. However, it is unlikely that the variability in effective drug dosage alone explains the increased odds for ILA we demonstrate among subjects taking hydrophilic statins (at commonly prescribed doses there is a large difference in the expected cholesterol reduction between pravastatin and rosuvastatin) (27
). Fourth, although we provide experimental evidence in support of our clinical findings, further experimental work in mice exposed to tobacco smoke would be helpful to more precisely define the combined effect of smoking and statin use. Finally, although prior studies have implicated a role for the NLRP3 inflammasome in fibrotic lung disease (31
), further studies in people are required to determine the extent to which NLRP3 inflammasome activation plays a role in statin-induced ILD and ILD in general.
We urge caution in extrapolating our findings to the care of patients. Although increases in the risk of ILA, and radiologic features of pulmonary fibrosis, are causes for concern, these risks do not likely outweigh the substantial benefits of statin therapy in patients with cardiovascular disease. In addition, our findings do not rule out the possibility that statin use could benefit some patients with respiratory disease. Instead, we believe that clinicians should be aware that radiographic evidence of ILD, much like myopathy (50
), can occur in some patients on statins.