甲状腺激素通过改善上皮线粒体功能抑制小鼠肺纤维化

TH blunts lung fibrosis in two murine models

Systemic administration of T4 (100μg/kg) at days 10, 12, 14, 16, after bleomycin administration significantly blunted fibrosis but caused significant increases in serum T3 levels (Supplementary Fig. 1). To avoid the side effects of systemic administration of T4 and maximize therapeutic efficacy we focused on the pulmonary delivery of the physiologically active hormone T3 by aerosol. Aerosolized T3 (40 μg/Kg) therapy, given every other day starting at the established fibrosis stage (days 10–20 after bleomycin), caused significant decreases in hydroxyproline levels (2.25-fold, P < 0.05) (Fig. 2a), and histologic evidence of fibrosis (Fig. 2b) without affecting serum T3 levels (Fig. 2b). The effects were comparable to those observed with oral administration of pirfenidone (100 mg/kg) or nintedanib (60 mg/kg), the two drugs currently approved by the US FDA for the treatment of human IPF¹⁵ (Fig. 2a,b).

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Figure 2

Aerosolized T3 blunts established fibrosis in two murine models of lung fibrosis. (a) Effects of aerosolized T3, nintedanib or pirfenidone on lung hydroxyproline content. Data presented as box-and-whisker plots are from one of two independent experiments with similar results with horizontal bars representing mean hydroxyproline content per lung set (μg/gr) + SEM, *P < 0,001. (b) Masson’s Trichrome staining of representative lung sections (n = 5) from each group of treated mice. Scale bars, 50 μm. (c) Aerosolized T3 administration effects on T3 serum levels. Data are presented as box-and-whisker plots with horizontal bars representing mean T3 serum levels (ng/ml) + SEM, P = 0.7. (d) Pressure–volume-loops (PV-loops) of mice (n = 5/group) treated with aerosolized T3 following intratracheal challenge with bleomycin or normal saline, P < 0.001. (e) Kaplan-Meier plot survival plots of mice treated with aerosolized T3, pirfenidone, nintedanib or equivalent volume of vehicle following intratracheal challenge with double dose of bleomycin or normal saline, n = 10 mice/group. (f) T3 effects on hydroxyproline content in mice in the following groups: Saline – transgene not induced, Dox+vehicle - transgene induced animals treated with vehicle, DOX+T3 – transgene induced and animals treated with aerosolized T3. Data are presented as box-and-whisker plots with horizontal bars representing mean hydroxyproline content per lung set (μg/gr) + SEM, *P = 0.035. (g) Masson’s Trichrome staining of representative lung sections (n = 5) in the triple transgenic (CC10-rtTA-tTS-TGF-β₁) in the groups from f. Scale bars, 50 μm. The statistical test used was one-way ANOVA with Student-Newman-Keuls post-hoc test for pairwise comparisons, (a) (F=38.6, df=54), (c) (F=0.6, df=56), (d) (F=11.9, df=44), (f) (F=4.5, df=14)

Aerosolized T3 therapy reversed the significant reduction in static lung compliance induced by bleomycin, reflecting a functional improvement in respiratory mechanics and reduction in fibrosis (Fig. 2d). Similar functional benefits were observed with pirfenidone and nintedanib treatment (data not shown). To observe the effects of aerosolized T3 on survival we used a higher dose of bleomycin (3.0 U/kg). In contrast to pirfenidone and nintedanib, aerosolized T3 caused a substantial survival benefit with significant reduction in mortality from 90% in the untreated group to 20% in the T3 treated arm (Fig. 2e). This survival benefit suggested that T3 potentially limited the extent of acute lung injury caused by high dose bleomycin in addition to its anti-fibrotic effects. To assess the effects of aerosolized T3 in another animal model of experimental lung fibrosis, we used the triple transgenic mice in which a CC10 promoter drives the expression of active TGFβ1 in response to doxycycline treatment¹⁶. Aerosolized T3 therapy, given every other day, at days 10–20 after doxycycline treatment resulted in significantly lower pulmonary fibrosis as assessed by lower hydroxyproline levels (1.15-fold, P < 0.05) and Masson Trichrome staining compared to mice treated with doxycycline and vehicle (Fig. 2f,g).

TH reverses bleomycin-induced mitochondrial abnormalities

As previously observed¹¹ AECIIs in bleomycin-treated mice exhibited swollen mitochondria, with severely damaged electron-lucent cristae and disrupted membranes and significantly reduced numbers of normal looking mitochondria, while aerosolized T3 reversed these changes (Fig. 3a,b). Aerosolized T3 also reversed many of the functional abnormalities induced by bleomycin in AECIIs. Specifically, primary AECIIs isolated on day 21 from the lungs of bleomycin-treated animals (AECBs) exhibited significantly lower mitochondrial membrane potential than AECIIs from mice treated with aerosolized T3 after bleomycin (AECBT3s) or AECIIs isolated from uninjured lungs (AECTRLs) (1.4- and 1.5-fold, respectively) (Fig. 3c). AECBs exhibited significantly decreased oxygen consumption rate (OCR) compared to AECTRLs (3-fold) that was restored to baseline levels in AECBT3s (Fig. 3d). In vitro, bleomycin treatment caused significant declines in the mitochondrial bioenergetic profile of primary human small airway epithelial cells (SAECs), primary mouse lung AECIIs and mouse lung epithelial cells (MLE12), but T3, added 4 hours after bleomycin, reversed this decline. SAECs, AECIIs and MLE12 that were treated with T3 after bleomycin exhibited significantly higher mitochondrial membrane potential (MMP, 1.2-, 1.3-, and 1.3-fold, respectively) (Fig 3e–g) and oxygen consumption rate (OCR, 2.1-, 1.5- and 6.9-fold, respectively) (Fig. 3h–j) compared to cells treated with vehicle after bleomycin. A detailed analysis of the mitochondrial bioenergetic profiles in lung epithelial cells, both in vivo (isolated AECIIs) and in vitro (SAECs, AECIIs, MLE12) showed similar differences between T3-treated groups and controls (Supplementary Fig 2).

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Figure 3

TH treatment restores bleomycin-induced mitochondrial abnormalities in alveolar epithelial cells. (a) Representative transmission electron microscopy (TEM) images (n = 16, 19 and 13, respectively) of AECIIs from mice treated with saline (upper panel), bleomycin (3.0U/kg) (middle panel) or bleomycin + aerosolized T3 (40μg/kg) (lower panel). Black arrows indicate damaged and swollen mitochondria with severely disrupted electron-lucent cristae, arrow heads indicate normal appearing mitochondria. Boxed regions are shown enlarged in the next column. Scale bars, 2 μM (left panels), 1 um (middle panels), 500 nm (right panels). (b) Quantitative analysis of the percentage of normal mitochondria per cell/group. Bars represent mean score + SEM, *P < 0,001. (c) Mitochondrial function in AECIIs cultured from animals treated with saline, bleomycin or bleomycin + T3. Data are presented as box-and-whisker plots with horizontal bars representing mean MMP levels (green/red fluorescence ratio) + SEM, *P < 0,001. (d) Oxygen consumption rate (OCR, pmol/min) was measured under basal conditions followed by addition of oligomycin, FCCP, rotenone and antimycin as indicated, *P < 0.001). (e–g) Green/red fluorescence ratio as a readout of MMP in primary human small airway epithelial cells (SAECs) (e), primary mouse AECIIs (f) and mouse lung epithelial cells (MLE12) (g) exposed to bleomycin or PBS and then treated with T3 or vehicle. Data are presented as box-and-whisker plots with horizontal bars representing mean MMP levels + SEM, *P < 0.001, P = 0.003 and P < 0.001, respectively). (h–j) Oxygen consumption rate (OCR, pmol/min) of the category of cells indicated in e–g as measured under basal conditions followed by addition of oligomycin (0.25μM), FCCP (1 μM), as well as rotenone and antimycin (1 μM), as indicated. (k–m) Effects of in vitro T3 treatment on mitochondrial biogenesis in the same category cells indicated in e–g as measured by the levels of Cytochrome c oxidase subunit IV (COX-IV) and Succinate Dehydrogenase Complex Flavoprotein subunit A (SDHA). Data are presented as box-and-whisker plots with horizontal bars representing mean COX-IV/SDHA ratio + SEM, *P < 0.001. (n) Immunoblot analysis of markers of autophagy (LC3BI, II, p62, PINK1) and mitochondrial biogenesis (PPARGCA1) in the same category of cells indicated in e–g. Immunoblot gels were cropped and uncropped images of the immunoblot gels are in Supplementary Figure 4. The statistical test used was One-way ANOVA with Student-Newman-Keuls post-hoc test for pairwise comparisons (b) (F=34.5, df=50), (c) (F=38.6, df=23) (d) (F=91.4, df=42), (e) (F=7.04, df=94) (f) (F=5.15, df=83) (g), (F=35.3, df=95), (k) (F=39.6, df=88), (l) (F=30.1, df=74), (m) (F= 74.6, df= 87).

To assess the effects of T3 treatment on mitogenesis we measured the Cytochrome c oxidase subunit IV (COX-IV) and Succinate Dehydrogenase Complex Flavoprotein subunit A (SDHA) ratio as previously described¹⁷. Epithelial cells treated with bleomycin exhibited a significantly lower COX-IV/SDHA ratio compared to vehicle treatment. Bleomycin treated cells that were also treated with T3 exhibited ratios similar to controls. This effect was observed in the three cell types (Fig. 3k–m, 1.4-, 1.2-, 1.4-fold, respectively). Epithelial cells treated with bleomycin exhibited significantly lower protein expression of PPARGC1A, a transcriptional co-activator of mitochondrial metabolism and biogenesis, compared to vehicle treated controls, but in cells treated with T3 following bleomycin PPARGC1A expression was similar to controls (Fig. 3n). T3 treatment after bleomycin was associated with significant higher levels of PINK1. Changes in autophagic flux, as indicated by altered autophagy protein light chain 3 (LC3B) and p62 protein levels were consistently seen in SAECs but not in AECIIs or MLE12 (Fig. 3n).

TH suppresses mitochondria-regulated apoptosis in AECIIs

After showing that TH opposes bleomycin-induced mitochondrial function abnormalities in primary mouse epithelial cells in vivo and in both human and mouse epithelial cells in vitro, we aimed to determine the effects of TH treatment on mitochondria-regulated apoptotic pathways. Immunoblot analyses revealed that exposure of AECIIs to bleomycin led to higher protein expression of the pro-apoptotic mediator BCL-2 associated X protein (BAX) and lower expression of the anti-apoptotic mediator BCL-xL (Fig. 4a). Importantly, bleomycin promoted the intracellular movement of BAX from the cytosol to mitochondria in SAECs, as indicated by co-localization of BAX with mito-tracker in the immunofluorescence analysis (Fig. 4b). BAX upregulation and mitochondrial translocation were associated with enhanced apoptotic activity as revealed by increased number of TUNEL-positive cells (Fig. 4c,d). This observation was verified in vivo by immunohistochemistry analysis showing increased TUNEL staining in the alveolar epithelium in mouse lungs challenged with bleomycin (Fig. 4e,f). T3 treatment reversed the BAX/BCL-xL ratio (Fig. 4a), attenuated BAX translocation to mitochondria (Fig. 4b) and consequently diminished bleomycin-induced apoptotic injury in AECs, both in vitro and in vivo, as indicated by significant reductions in TUNEL-positive cells (Fig. 4c–e).

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Figure 4

TH attenuates mitochondria-regulated apoptosis in lung epithelial cells. (a) Immunoblot analysis of markers of mitochondria-cell apoptosis (BAX, BCL-xL). Each lane represents a biological replicate and 2 experiments were done in each case. Immunoblot gels were cropped, and uncropped images of the immunoblot gels are in Supplementary Fig 4. (b) Immunofluorescence analysis for Mito-Tracker (red cationic dye that stains active mitochondria) and BAX (green) in SAECs after bleomycin or PBS exposure and treatment with or without T3. Localization of BAX with mito-tracker is indicated in yellow. Boxed regions are shown enlarged at lower left panels. Scale bars, 50μm, insets: 10 μm. (c,d) Immunofluorescence (c) and quantitative analysis (d) of double positive SAECs (TUNEL/DAPI). Data are presented as box-and-whisker plots with horizontal bars representing mean % percentage of double positive cells + SEM. One-way ANOVA (F=366.7, df=15) with Student-Newman-Keuls post-hoc test for pairwise comparisons, *P < 0.001. (e,f) Immunohistochemistry (e) and quantitative analysis (f) of TUNEL-positive cells in lung samples derived from mice challenged bleomycin or saline and then treated with T3 or vehicle at days 10–20. Data are presented as box-and-whisker plots with horizontal bars representing mean % percentage of double positive cells + SEM, *P < 0.001. Scale bars, 100μm. The statistical test used was one-way ANOVA (F=33.3, df=15) with Student-Newman-Keuls post-hoc test for pairwise comparisons.

IPF Tissues

Lung tissue samples were obtained through the University of Pittsburgh Health Sciences Tissue Bank and Yale university Pathology Tissue service, which were surgical remnants of biopsies or lungs explanted from subjects with IPF who underwent pulmonary transplantation.

Human subject approval

All studies have been approved by the Institutional Review Board at the University of Pittsburgh and Yale School of Medicine.

RNA extraction

Total RNA was extracted from 30 – 50 mg of frozen lung tissue in 700 uL of Qiazol (Lysis buffer, Qiagen, Valencia, CA) according to the manufacturer’s instructions. The purity of the RNA was verified using NanoDrop at 260 nm and the quality of the RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent, Technologies, Santa Clara, CA).

Quantitative Real-Time PCR

Gene expression was determined by TaqMan® (Life Technologies, Thermo Scientific Inc. Rockford IL, USA) according to manufacturer’s instruction. β-glucuronidase (Gusb) was employed as an internal standard control and the specific primers and probes were all obtained from Life Technologies (Thermo Scientific Inc. Rockford IL, USA). Each reaction was performed in triplicate. Relative gene expression was normalized to a value of 1.0 for the unstimulated control group. Control reactions without cDNA or RNA were done as a negative control. Fold change was calculated taking the mean of the controls as the baseline.

Protein extraction and western blot analysis

Proteins from tissues or cultured cells were extracted, transferred onto PVD-membranes, hybridized overnight with appropriate primary antibodies (PPARGC1A - sc-13067 and ab-54481, DIO2-sc-98716, BCL-xL- ab32124, PINK1-ab23707, LC3B-ab51520, p62/SQSTM1-ab56416, BAX-ab32503 and ab-53154, β-actin-sc-47778) obtained from Santa Cruz Biotechnology, CA (sc), or Abcam, Cambridge, UK (ab) and visualized using the Gel Doc XR+ System (Biorad, Lab. Inc. Life Science, USA), according to manufacturer’s protocol.

DIO2 activity

The DIO2 assay was established previously based on the release of radioiodide from the ¹²⁵I^- labelled substrate (100000 cpm-5′-¹²⁵I of T4-Sigma, MO) as previously described⁵². Briefly, the mixture containing 25 ml tissue homogenate in 0·1mM-potassium phosphate buffer (pH 7·0) and 1mM-EDTA, and 650 ml of substrate for a final concentration of 1 nM-T4 (Sigma St Louis, MO), 20mM-cofactor diothiothreitol and 1mM-propylthiouracil (pH 7.0) was incubated at 37°C for 1 h. The reactions were stopped by the addition of 50 ml ice-cold 5% bovine serum albumin followed by 350 ml 10% ice-cold TCA, and mixtures were centrifuged at 4000 g for 20 min. The supernatant was further purified by cation exchange chromatography using 1·6ml Dowex 50 W-X2 (100–200 mesh; Sigma). The iodide was then eluted twice with 1ml 10% glacial acetic acid and counted in a g-counter. Enzymatic activity was expressed in fmol¹²⁵I^-released/h per mg protein. Enzymatic activity was expressed in fmol¹²⁵I^-released/h per mg protein.

Animal studies approval

All animal studies were conducted in accordance with the NIH guidelines for humane treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC), Yale University (2014-11592).

Experimental models of lung fibrosis

All animal models were performed twice and data was jointly analyzed. All animals treated were included in the analysis. Interventions were not blinded, but analysis of animal samples was. a) Bleomycin-model:C57Bl/6, 9–12 weeks-old, female mice were purchased (Taconic Biosciences, Hudson, NY). Knockout mice (Dio2^−/−), Ppargc1a^−/− and Pink1^−/− of C57/BL6 background were obtained from Jackson Lab (Bar-Harbor, ME). As previously published^11,53 genetic deletion of either PINK1 or PPARGC1A does not produce any endogenous lung phenotype and is not associated with derangements of lung physiology. Ppargc1a^−/− and Pink1^−/− mice are not embryonically lethal, they produce litters of appropriate Mendelian size, are fertile and healthy and present with no gross morphology abnormalities. Mice were anesthetized by placing them in a chamber having paper towels soaked with 40% isoflurane solution diluted with 1,2-propanediol. Mice were randomly assigned to either intratracheal 1.5 U/kg of bleomycin (Hospira, IL) or equivalent volume (50 μL) of 0.9% normal saline was administered intratracheally as previously described^54,55. To test therapeutic efficacy of TH in bleomycin-induced established fibrosis we used intraperitoneal T4 (T2376-Sigma Aldrich, 100μg/kg), or aerosolized T3 (T2877-Sigma Aldrich, 40 μg/kg). T4 was administered systemically at days 10, 12, 14, 16 and mice were sacrificed on day 19, following bleomycin-challenge. Aerosolized T3 was administered every other day at days 10–20 and mice were sacrificed on day 21. Dose regimens were based on previously published protocols^24,56. Equivalent volumes of normal saline 0.9% were used as controls in all approaches. For survival analysis we used a double dose of bleomycin (3.0 U/kg) and administered aerosolized T3 (40 μg/kg) or normal saline 0.9% at days at days 10–20 (established fibrosis) following challenge and survival data was collected at day 21, respectively. Mice were randomly assigned to receive either pirfenidone (100 mg/kg) or nintedanib (60 mg/kg) or vehicle (0.9% saline) via oral gavage, as previously described⁵⁷, at days 10–20 (on a daily basis) following bleomycin administration and mice were sacrificed at day 21. Aerosolized delivery of T3 was performed following a standardized protocol. Briefly: T3 was diluted to a final concentration of 40 μg/kg in 6 ml of PBS and suspension was aerosolized using a conventional aerosol nebulizer (Omron) throughout a chamber that allowed simultaneous exposure of 8 mice for 30 minutes until mist stopped forming in the nebulizer chamber. b) TGF-β1-induced lung fibrosis: Inducible lung targeted TGF-β1-overexpressing triple transgenic mice (CC10-rtTA-tTS-TGF-β₁₎ generated as previously described¹⁶ were used. Briefly: A triple transgenic system based on the tetracycline-controlled transcriptional suppressor (tTS) and the reverse tetracycline transactivator (rtTA) was constructed. In this system, the CC10 promoter constitutively drives the expression of rtTA and tTS in a lung-specific fashion. In the absence of dox, tTS binds to and actively suppresses the expression of the tet-O–regulated TGF-β1 transgene. In the presence of dox, tTS is released allowing the activating, dox binding rtTA to bind to the tet-O and activate transgene expression. As expected induced TGF-β₁ – overexpression upon addition of doxycycline water on a daily basis (days 0–20) caused airway and parenchymal fibrotic response as assessed by increased lung collagen deposition indicated by hydroxyproline levels and Masson Trichrome staining. Aerosolized T3 (40 μg/kg) or normal saline 0.9% was administered every other day on days 10–20 following addition of doxycycline and mice where sacrificed on day 21. c) Sobiterome therapeutic protocol: Sobiterome was provided by the laboratory of Dr. Scanlan as previously described^21,58. C57Bl/6, 9–12 weeks-old, female mice were randomly assigned to be challenged with 1.5 U/kg of bleomycin or equivalent volume of normal saline 0.9% at day 0. Mice were then randomly assigned to treatment with 5 mg/kg of sobiterome diluted in 50 μL of normal saline 0.9% or vehicle (equivalent volume of normal saline 0.9%) administered by oral gavage at days 10, 12, 14, 16 and 18 following bleomycin administration and mice were sacrificed on day 21.

In-vivo respiratory mechanics (Flexi-Vent system-SCIREQ)

To evaluate in-vivo respiratory mechanics as an assessment tool for therapeutic efficacy we applied the Flexi-Vent computer-controlled piston-ventilator system according to manufacturer’s protocol. Briefly: Mice were anesthetized with ketamine and then mice tracheotomized and ventilated using the Flevi-Vent system with an average breathing frequency of 150 breaths/min. During the maximal vital capacity (MVC) perturbation, the lungs were inflated to a standard pressure of +30 cmH₂O and then deflated to determine MVC. Maximal pressure–volume-loops (PV-loops) between TLC (+30 cmH₂O) and FRC (2.8 cmH₂0) were finally generated to obtain static compliance (Cst) of the respiratory system. In every animal, each manoeuver was repeated until three acceptable measurements (coefficient of determination ≥0.95) were recorded. The average of three acceptable measurements was calculated.

Histology and immunohistochemistry

Tissue sections (4 μm) were stained with Masson Trichrome (collagen/connective tissue), two slices per animal, and two animals per group, as previously described. Immune staining for DIO2 (ab77481, Abcam, Cambridge, UK) or PPARGC1A (ab54481, Abcam, Cambridge, UK) was performed after paraffin removal, hydration, and blocking, following the recommendation of the manufacturer and described by us⁵⁹. Sections were incubated overnight at 4°C with the primary antibody (diluted 1:100 in PBS) and during 1 hour at room temperature with the secondary antibodies (Sigma, USA). The sections were counterstained with hematoxylin. The primary antibody was replaced by non-immune serum for negative controls.

Hydroxyproline assay

Lung hydroxyproline was analyzed with hydroxyproline colorimetric assay kit from Biovision (Milpitas, CA) following manufacturer’s instruction, as previously described⁶⁰. Data are expressed as μg of hydroxyproline/right lung.

Measurement of T3 serum levels

Serum from mouse peripheral blood was collected by BD vacutainers, and serum total total triiodothyronine T₃ levels were measured T3 ELISA kits (ABIN2685558, Antibodies-Online, Atlanta, GA), following the manufacturer’s instructions.

Isolation of primary alveolar epithelial cells (AECIIs)

Primary AECIIs were isolated from mice treated with 0.9% saline, or bleomycin (1.5U/kg), or bleomycin + aerosolized T3 (40μg/kg), using a modified protocol of a previously published method⁶¹. AECIIs were identified as population with CD45/CD32/CD16/CD11b-ve,SSc^hi cells. Using this protocol, a purity exceeding 80% and viability of 90% was obtained. Isolated cells were used for functional assays.

Cell culture experiments, immunofluorescence staining and assays of apoptosis

Human small airway epithelial cells (SAECs) were purchased from Lonza, Allendale, NJ (LOT: 000470903). Primary mouse AECIIs were isolated from the lungs of unchallenged mice. Mouse lung epithelial cell line (MLE12) was purchased from ATCC (CRL-2110). Cells were seeded into 96-well plates (40.000/well), exposed to bleomycin (15 mU/ml) or PBS for 4 hours and then treated with T3 (15 ng/ml) or vehicle control for 8 hours. Immunofluorescence staining was performed using Mito-Tracker (Thermo Fisher Scientific, Waltham, MA), a cationic dye that stains active mitochondria and PPARGC1A (ab54481, Abcam, Cambridge, UK), according to manufacturer’s instructions. Detection of apoptotic cells was performed with TUNEL-assay using the in situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IA, USA, Catalog No 11684795910). DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. The human lung adenocarcinoma cell line A549 (ATCC^® CCL-185™, ATCC, Manassas, VA), negatively tested for mycoplasma, was cultured in DMEM supplemented with 10% fetal bovine serum. Cells were pre-incubated with either dronedarone 10 μM diluted in 0.04% DMSO or vehicle control (0.04% DMSO) for 24 hours and then treated with T3 (15 ng/ml) or vehicle (normal saline 0.9%) for 24 hrs.

Extracellular Flux Technology

The oxygen consumption rates (OCR) of primary mouse AECIIs, SAECs, and MLE-12 cells were measured by using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), as previously described⁶². All assays were performed using a seeding density of 60,000 cells/well in 200μl of DMEM in a XF96 cell culture microplate (Seahorse Bioscience). After the cells were switched to unbuffered DMEM supplemented with 2 mM sodium pyruvate and 20mM carnosine 1h prior to the beginning of the assay and maintained at 37 °C. OCR was measured after sequentially adding to each well 25μl of oligomycin (an ATP-synthase inhibitor), FCCP (a protonophore) and rotenone (inhibitors of complex I and III), to reach working concentrations of 1μg/ml, 1μM and 0.5μM respectively. OCR is reported in picomoles/minute/60,000 cells.

Determination of ATP production

Total ATP was determined using an ATP Fluorometric Assay Kit (BioVision, San Francisco, CA) as previously described⁶². Briefly, harvested cultured cells were lysed with a lysis buffer followed by centrifugation at 10.000g for 2 min, at 4°C. The level of ATP was determined by mixing 20 μl of the supernatant with 100 μl of luciferase reagent which catalyzed the light production from ATP and luciferin. Reactions were performed in 6-well plates at a seeding density of 10⁶ cells/well. Luminance was measured by a monochromator microplate reader. Total ATP levels were expressed as nmol/mg protein.

Mitochondrial membrane potential (MMP)

The changes in relative MMP (ΔΨm) were measured using the JC-10 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolocarbocyanine iodide) molecular probe, as previously described⁶².

Transmission Electron microscopy (TEM)

For TEM, mice were perfused with 4% Paraformaldehyde (PFA) and lung samples were dissected out in 2% PFA. These were fixed in 2.5% gluteraldehyde/2% PFA in 0.1M sodium cacodylate buffer pH7.4 for 1 hour, followed by rinsing in PBS buffer then post fixed in 1% osmium tetroxide. They were en-bloc stained in 2% aqueous uranyl acetate then rinsed and dehydrated in an ethanol series followed by resin infiltration Embed 812 (Electron Microscopy Sciences) and baked overnight at 60 C. Hardened blocks were cut using a Leica UltraCut UC7. 60nm sections were collected on formvar/carbon coated grids and contrast stained using 2% uranyl acetate and lead citrate. Grids were viewed FEI TencaiBiotwin TEM at 80Kv. Images were taken using Morada CCD and iTEM (Olympus) software. At least 10 cells from low- and high-magnification images (×11,500, ×26,000) were used to count the number of mitochondria per AECII (identified by the presence of lamellar bodies).

Mitobiogenesis assay

Human SAECs, primary (AECIIs) isolated from the mouse lungs and MLE-12 were seeded in 96-well microplates containing 100 μL of culture medium. Cells were first exposed to bleomycin (10mU/ml) diluted in PBS or vehicle control (PBS) for 4 hours and then were treated with T3 (15ng/ml) or vehicle control for 8 hours. The levels of two mitochondrial proteins were measured simultaneously in each well by using a colorimetric ELISA-kit, according to manufacturer’s protocol (MitoBiogenesis™ In-Cell ELISA Kit- Abcam-ab110217)¹⁷. The two proteins are each subunits of a different oxidative phosphorylation enzyme complex, one protein being subunit I of Complex IV (Cytochrome c oxidase subunit-COX-IV), which is mitochondrial-DNA-encoded, and Succinate Dehydrogenase Complex Flavoprotein subunit A-SDHA) which is a 70kDa subunit of Complex II and nuclear-DNA-encoded.

Immunogold staining protocol

The grids containing tissue samples were placed section side down on drops of 1% hydrogen peroxide for 5 minutes, rinsed and blocked for nonspecific binding on 3% bovine serum albumin in Tris buffered saline containing 1% Triton-X for 30 minutes. Grids were incubated with a primary antibody anti-rabbit thyroid hormone receptor alpha-1 and beta (PA1-211A and PA1-213A, respectively Thermo Fisher, Pierce) 1:500 overnight, rinsed in TBS then incubated with the secondary antibody 10nm protein A gold (University of Utrecht, Cell Microscopy Core, Netherlands) for 30 minutes. The grids were well rinsed in PBS, fixed using 1% gluteraldehyde for 5mins, rinsed again, dried and heavy metal contrasted using 2% aqueous uranyl acetate and lead citrate.

The work was in part supported by US National Institute of Health (NIH) grants R01HL095397, R01HL127349 (N.K.), R01HL1109233, R01HL125250 (E.L.H.), RO1DK65055 (A.C.B.), RO1DK52798 (T.S.S.), Flight Attendant Medical Research Institute (P.J.L.), American Lung Association Award RT 350419, Marie Sklodowska/Curie, ERS/EU-RESPIRE 2-8860-2015 grant (A.T.). American Lung Association Award RG-415350 (A.S.), Pulmonary Fibrosis Foundation (PPF) Albert Rose Established Investigator Award 415245 (G.Y.)

A.T., G.Y., and N.K., are inventors on a pending patent on use of thyroid hormone as an antifibrotic agent entitled: “Novel Methods of Treating or Preventing Fibrotic Lung Diseases” - OCR 6368 - 047162-7029P1 (00219). N.K., consulted Biogen Idec, Boehringer Ingelheim, Numedii, MMI, and Pliant and has an ongoing collaboration with MiRagen, all outside the submitted work. E.L.H., consulted for Boehringer Ingelheim and held grant funding from Sanofi and Promedior, all outside the submitted work.