Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-β1 (TGF-β1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF.

Objectives: We aimed to define an association between mtDNA and fibroblast responses in IPF.

Methods: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-β1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects.

Measurements and main results: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-β1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival.

Conclusions: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

Keywords: biomarkers; interstitial lung disease; mechanotransduction; mitochondria.

Figure 1.

Transforming growth factor-β1 (TGF-β1)–stimulated normal human lung fibroblasts (NHLFs) show altered metabolism and high concentrations of extracellular mitochondrial DNA. (A and B) NHLFs stimulated with 5 ng/ml of TGF-β1 for 7 days (right) demonstrated the previously reported increase in glycolysis relative to unstimulated cells (left) as measured by significant elevations in (A) extracellular acidification rate (ECAR) and (B) ratio of ECAR to oxygen consumption rate (OCR). Data are presented as mean ECAR (mpH/min) and mean (±SEM) ratio of ECAR to OCR, respectively. (C) A standard curve was developed from serial dilutions of a commercially available plasmid containing the sequence of the human MT-ATP6 gene. (D) Relative to supernatants obtained from NHLFs cultured with normal medium (left), the mean MT-ATP6 copy number is significantly increased in the supernatant of NHLFs stimulated with 5 ng/ml of TGF-β1 for 7 days (right). Data are presented graphically as log base 10 of the raw values (MT-ATP6 copies per microliter of supernatant) with mean ± SEM. A graph including the raw values is presented in Figure E2D. CT = cycle threshold.

Figure 2.

Transforming growth factor-β1 (TGF-β1) stimulation of normal human lung fibroblasts (NHLFs) reduces mitochondrial mass without affecting cell viability. (A) NHLFs were stimulated with 5 ng/ml of TGF-β1 for 7 days, at which point mitochondrial mass was determined using polymerase chain reaction–based comparison of DNA derived from mitochondria (assessed by the MT-ATP6 gene) and the genome (measured by the β-actin gene). Relative to unstimulated cells (left), there was a significant decline in the mean ratio of mitochondrial DNA (mtDNA) to genomic DNA (gDNA) in TGF-β1–treated cells (right). Data are presented as the mean (±SEM) fold change in ratio of mtDNA to gDNA. (B) Cell counts in NHLF cultures stimulated with (right) and without (left) 5 ng/ml of TGF-β1 for 7 days were unchanged across both conditions. Data are presented as mean (±SEM) fold change in cell count. (C) Assessment of viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed similar fold changes in absorbance at a wavelength of 540 nm between NHLFs stimulated with (right) and without (left) 5 ng/ml of TGF-β1 for 7 days. Data are presented as the mean (±SEM) fold change in absorbance. (D) Stimulation of NHLFs with 0.5 ng/ml of mtDNA for 48 hours significantly increased α-smooth muscle actin (α-SMA) expression, relative to β-actin, by NHLFs. Data are presented as mean (±SEM) α-smooth muscle actin expression relative to β-actin.

Figure 3.

Direct contact with stiff surfaces phenocopies exposure to transforming growth factor-β1 in normal human lung fibroblasts (NHLFs). Measurements of cellular metabolism revealed enhanced aerobic glycolysis in NHLFs grown on the 20-kPa hydrogels for 7 days (right) compared with cells grown on the 1-kPa hydrogels (left), as evidenced by significantly elevated (A) extracellular acidification rate (ECAR) and (B) ECAR/oxygen consumption rate (OCR) ratio. Data are shown as mean (±SEM) ECAR (mpH/min) and mean (±SEM) ratio of ECAR/OCR, respectively. (C) After 7 days, relative to supernatant obtained from NHLFs seeded on the 1-kPa hydrogel (left), there was a significant increase in MT-ATP6 concentration in the supernatant of NHLFs seeded on the 20-kPa hydrogel (right). Data are presented graphically as log base 10 of the raw values (MT-ATP6 copies per microliter of supernatant) with mean (±SEM). A graph including the raw values is presented in Figure E3B. NHLFs grown on the 20-kPa hydrogel (right) demonstrated no significant change in (D) cell counts or (E) viability based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay compared with cells grown on the 1-kPa hydrogel (left) for 7 days. Data are presented as mean (±SEM) fold change in cell count and mean (±SEM) fold change in absorbance, respectively.

Figure 4.

Idiopathic pulmonary fibrosis (IPF) fibroblasts exhibit enhanced glycolysis and increased extracellular mitochondrial DNA. Compared with normal human lung fibroblasts (NHLFs) (left), IPF fibroblasts (right) displayed enhanced aerobic glycolysis, as measured by a significantly elevated (A) extracellular acidification rate (ECAR) and (B) ECAR/oxygen consumption rate (OCR) ratio. Data are presented as mean (±SEM) ECAR (mpH/min) and mean (±SEM) ratio of ECAR/OCR, respectively. (C) Relative to samples obtained from NHLFs (left), a significant increase in MT-ATP6 concentration was detected in supernatants from IPF fibroblasts (right). Data are presented graphically as log base 10 of the raw values (MT-ATP6 copies per microliter of supernatant) with mean (±SEM). A graph including the raw values is presented in Figure E4B. (D) Cell counts were unchanged between NHLFs (left) and IPF fibroblasts (right). Data are presented as mean (±SEM) fold change in cell count. (E) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated no significant differences in the viability of NHLFs (left) and IPF fibroblasts (right). Data are presented as mean (±SEM) fold change in absorbance.

Figure 5.

Mitochondrial DNA is elevated in the lungs and plasma of patients with idiopathic pulmonary fibrosis (IPF). (A) Immunohistochemistry-based detection of TOM20 in IPF lung tissues (relative to control samples) revealed strong detection in fibrotic regions of lung in cells that also expressed α-smooth muscle actin (α-SMA). Top left: Normal lung stained with TOM20 shows detection in macrophages (brown). Top right: IPF lung stained with TOM20 shows detection in fibrotic regions (brown). Images are counterstained with hematoxylin, and scale bar is 50 μm. Bottom left: Combined immunofluorescence for α-SMA (red) and TOM20 (green) on IPF lung tissue. Slides are counterstained with 4′,6-diamidino-2-phenylindole (blue nuclear stain), and images include a 50-μm scale bar. Bottom right: Enlargement of the boxed area shows codetection of TOM20 and α-SMA in spindle-shaped cells with the appearance of fibroblasts. (B) Relative to control lung tissue, there were a significantly higher percentage of TOM20-positive cells expressing α-SMA in the IPF lung. Data are presented as mean (±SEM) percentage of TOM20 cells that also express α-SMA. (C) Relative to bronchoalveolar lavage (BAL) samples obtained from subjects without parenchymal lung disease (left), the median concentration of MT-ATP6 was significantly increased in BAL samples obtained from IPF subjects (right). Data are presented graphically as log base 10 of the raw values of MT-ATP6 copies per microliter of BAL with median value and interquartile range. A graph including the raw values is presented in Figure E5A. (D) Relative to plasma specimens obtained from aged-matched control subjects (left), median concentration of MT-ATP6 in both the Pittsburgh (middle) and Yale (right) IPF cohorts was significantly increased. Data are presented graphically as log base 10 of the raw values of MT-ATP6 copies per microliter of plasma with median value and interquartile range. A graph including the raw values is presented in Figure E6A.

Figure 6.

Excessive plasma mitochondrial DNA is predictive of all-cause mortality in two independent cohorts. Kaplan-Meier plot for all-cause mortality reveals a significant survival benefit for subjects with a plasma MT-ATP6 concentration greater than or equal to 3,614.24 copies per microliter (top line) relative to those with a plasma MT-ATP6 concentration greater than 3,614.24 copies per microliter (bottom line). These findings were (A) derived from the Pittsburgh idiopathic pulmonary fibrosis (IPF) cohort and (B) validated in the Yale IPF cohort. Following adjustments for covariates of age, sex, race, FVC percent predicted, adjusted diffusion capacity for carbon monoxide percent predicted, and GAP index score, the cutpoint of 3,614.24 copies per microliter remained a strong predictor of all-cause mortality in (C) the Pittsburgh IPF cohort and (D) the Yale IPF cohort. CI = confidence interval; HR = hazard ratio.

Figure 7.

Post-treatment plasma mitochondrial DNA concentrations are reduced in patients who respond to pirfenidone. Relative to subjects who did not respond to pirfenidone (nonresponders; right), as demonstrated by a greater than 10% reduction in FVC percent predicted after 1 year of treatment, subjects who responded to pirfenidone by retaining stable FVC after 1 year of treatment (responders; left) showed a significant decline in their change in median plasma MT-ATP6 concentration, as compared with their baseline MT-ATP6 concentration, after 3 months of therapy. Data are presented graphically as change in median with interquartile range of MT-ATP6 copies per microliter of plasma.