Statins and pulmonary fibrosis: the potential role of NLRP3 inflammasome activation

Abstract

Rationale: The role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) in the development or progression of interstitial lung disease (ILD) is controversial.

Objectives: To evaluate the association between statin use and ILD.

Methods: We used regression analyses to evaluate the association between statin use and interstitial lung abnormalities (ILA) in a large cohort of smokers from COPDGene. Next, we evaluated the effect of statin pretreatment on bleomycin-induced fibrosis in mice and explored the mechanism behind these observations in vitro.

Measurements and main results: In COPDGene, 38% of subjects with ILA were taking statins compared with 27% of subjects without ILA. Statin use was positively associated in ILA (odds ratio, 1.60; 95% confidence interval, 1.03-2.50; P = 0.04) after adjustment for covariates including a history of high cholesterol or coronary artery disease. This association was modified by the hydrophilicity of statin and the age of the subject. Next, we demonstrate that statin administration aggravates lung injury and fibrosis in bleomycin-treated mice. Statin pretreatment enhances caspase-1-mediated immune responses in vivo and in vitro; the latter responses were abolished in bone marrow-derived macrophages isolated from Nlrp3(-/-) and Casp1(-/-) mice. Finally, we provide further insights by demonstrating that statins enhance NLRP3-inflammasome activation by increasing mitochondrial reactive oxygen species generation in macrophages.

Conclusions: Statin use is associated with ILA among smokers in the COPDGene study and enhances bleomycin-induced lung inflammation and fibrosis in the mouse through a mechanism involving enhanced NLRP3-inflammasome activation. Our findings suggest that statins may influence the susceptibility to, or progression of, ILD. Clinical trial registered with www.clinicaltrials.gov (NCT 00608764).

Trial registration: ClinicalTrials.gov NCT00608764.

Figure 1.

Statin use is associated with interstitial lung abnormalities (ILA). (A) Odds ratios (OR) for the association between individual statins (arranged in order of increasing hydrophilicity as measured by decreasing logD) and ILA. ORs and 95% confidence intervals (CIs) are represented by boxes (with their size proportional to the sample size) and bars, respectively. The overall association between statin use and ILA is represented by a diamond. The upper limit of the 95% CI for the association between pravastatin and ILA is greater than 10 (95% CI, 1.99–10.70). (B) OR for the association between statins and specific radiologic features. Black boxes (with their size proportional to the sample size) and bars represent ORs and 95% CIs for the association between statins overall and specific radiologic features. Green boxes and bars represent ORs and 95% CIs for the association between lipophilic statins and specific radiologic features. Blue boxes and bars represent ORs and 95% CIs for the association between hydrophilic statins and specific radiologic features. The association between statin use (including all statins, lipophilic, and hydrophilic statins) and ILA in general is represented by diamonds. The upper limits of the 95% CIs for the association between hydrophilic statins and bronchiectasis and honeycombing are greater than 10 (95% CI, 1.78–11.40, and 95% CI, 0.89–19.70, respectively). (C) Axial volumetric chest computed tomographic (CT) images of the hemithorax representing specific radiologic findings of ILA present in COPDGene subjects on statin medications. 1: Nondependent ground-glass present in a subject taking simvastatin. 2: Nonemphysematous cysts present in a subject taking lovastatin. 3: Centrilobular nodules present in a subject taking rosuvastatin. 4: Nondependent reticular markings present in a subject taking atorvastatin. 5: Traction bronchiectasis present in a subject taking rosuvastatin. 6: Honeycombing present in a subject taking pravastatin. (D) OR for the association between statins and ILA stratified by age (including subjects aged 45–55 yr [n = 415], subjects aged 55–65 yr [n = 451], and in subjects >65 yr old [n = 490]). Black boxes (with their size proportional to the sample size) and bars represent ORs and 95% CIs for the association between statins overall and ILA stratified by age.

Figure 2.

Statin increases bleomycin-induced lung inflammatory response and fibrotic changes in mice. (A) Sections of paraffin-embedded lung tissue from mice with different treatments were stained with hematoxylin and eosin (original magnification ×200). Bar, 20 μm, Day 14; BLM = bleomycin; CTL = control; S+B = pravastatin + bleomycin; STA = pravastatin. (B) Semiquantitative histopathology score was shown. (C) Sections of paraffin-embedded lung tissue from mice treated with different treatments were stained with Masson trichrome (original magnification ×400). Bar, 40 μm, Day 14; BLM = bleomycin; CTL = control; S+B = pravastatin + bleomycin; STA = pravastatin. (D) Pulmonary collagen deposition was quantified and expressed as micrograms of hydroxyproline per left lung. (E and F) Concentration of IL-1β and IL-18 in bronchoalveolar lavage fluid (BALF) at Day 7 was measured by ELISA. (G and H) Concentration of IL-1β and IL-18 in lung homogenates was measured by ELISA. (I) Lung homogenates were analyzed by immunoblotting for IL-1β and caspase-1. BLM = mice received with intraperitoneal injection of phosphate-buffered saline (PBS) and intratracheal instillation of bleomycin; CTL = mice received with intraperitoneal injection of PBS and intratracheal instillation of PBS; S+B = mice received with intraperitoneal injection of pravastatin and intratracheal instillation of bleomycin; STA = mice received with intraperitoneal injection of pravastatin and intratracheal instillation of PBS. Day 7: CTL, n = 5; STA, n = 5; BLM, n = 8; S+B, n = 9. Day 14: CTL, n = 5; STA, n = 5; BLM, n = 11; S+B, n = 11. *P < 0.05 compared with CTL group. ^#P < 0.05 compared with BLM group.

Figure 3.

Statin enhances NLRP3 inflammasome activation in macrophages. (A and B) Pravastatin pretreatment enhances the secretion of IL-1β and IL-18 in macrophages stimulated with LPS and adenosine triphosphate (ATP). J774A.1 macrophages were pretreated with pravastatin or vehicle (phosphate-buffered saline) for 24 hours and then incubated with LPS (500 ng/ml) for 4 hours, followed by stimulation with ATP (5 mM) for 1 hour. Secretion of IL-1β and IL-18 into the media was measured by ELISA. *P < 0.01 versus cells treated with LPS and ATP. (C) Pravastatin pretreatment increases caspase-1 activation. J774A.1 macrophages were pretreated with pravastatin (10 μM) for 24 hours and then incubated with LPS (500 ng/ml) for 4 hours, followed by stimulation with ATP (5 mM) for 15 minutes. Cell lysates were analyzed by immunoblotting for caspase-1. (D and E) Bone marrow–derived macrophages from caspase-1^−/− mice were pretreated with pravastatin (10 μM) for 24 hours and then incubated with LPS (200 ng/ml) for 4 hours, followed by stimulation with ATP (5 mM) for 1 hour. Secretion of IL-1β and IL-18 was analyzed by ELISA. *P < 0.01 versus caspase-1^−/− cells treated with LPS and ATP. (F–H) NLRP3 inflammasome is involved in the role of pravastatin on caspase-1 activation. Bone marrow–derived macrophages from NLRP3^−/− mice were pretreated with pravastatin (10 μM) for 24 hours and then incubated with LPS (200 ng/ml) for 4 hours, followed by stimulation with ATP (5 mM) for 15 minutes (H) or 1 hour (F and G). Secretion of IL-1β and IL-18 was analyzed by ELISA. *P < 0.01 versus NLRP3^−/− cells treated with LPS and ATP. Cell lysates were analyzed by immunoblotting for caspase-1. (I) Pravastatin increases interaction of NLRP3 inflammasome-associated molecules. J774A.1 macrophages were pretreated with pravastatin (10 μM) for 24 hours and then incubated with LPS (500 ng/ml) for 4 hours, followed by stimulation with ATP (5 mM) for 15 minutes. Cell lysates were analyzed for interaction of NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) or NLRP3 and pro–caspase-1 by immunoprecipitation. WT = wild type.

Figure 4.

Statin pretreatment increases mitochondrial reactive oxygen species (mtROS) generation. (A) LPS-primed J774A.1 macrophages were stained with MitoSOX for 15 minutes before stimulation with adenosine triphosphate (ATP) in the absence or presence of pravastatin, and then analyzed by flow cytometric analyses. Representative histograms are shown. CTL = control; NC = negative control—no staining. (B) Relative mean fluorescence intensity (MFI) of MitoSOX was represented. *P < 0.01 versus untreated cells. ^#P < 0.05 versus cells stimulated with LPS and ATP. (C) MitoTracker was used to show mitochondria, MitoSOX red was used to show ROS, and DAPI was used to show the nuclei of the cells. MitoSOX red labeled ROS was shown in mitochondria and was increased by pravastatin pretreatment, compared with LPS-ATP treatment only. (D) J774A.1 macrophages were pretreated with MitoTEMPO (500 μM) 1 hour before LPS treatment in the absence or presence of pravastatin. Level of mtROS in cells was analyzed by MitoSOX labeling. (E and F) Macrophages were pretreated with MitoTEMPO 1 hour before LPS treatment in the absence or presence of statin, followed by ATP stimulation for 1 hour. Cytokine secretion was analyzed by ELISA. *P < 0.01 versus cells treated with LPS and ATP. ^#P < 0.01 versus statin-pretreated cells stimulated with LPS and ATP.