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NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression

Open AccessPublished:May 20, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100810
      Pulmonary fibrosis is a progressive lung disease often occurring secondary to environmental exposure. Asbestos exposure is an important environmental mediator of lung fibrosis and remains a significant cause of disease despite strict regulations to limit exposure. Lung macrophages play an integral role in the pathogenesis of fibrosis induced by asbestos (asbestosis), in part by generating reactive oxygen species (ROS) and promoting resistance to apoptosis. However, the mechanism by which macrophages acquire apoptosis resistance is not known. Here, we confirm that macrophages isolated from asbestosis subjects are resistant to apoptosis and show they are associated with enhanced mitochondrial content of NADPH oxidase 4 (NOX4), which generates mitochondrial ROS generation. Similar results were seen in chrysotile-exposed WT mice, while macrophages from Nox4−/− mice showed increased apoptosis. NOX4 regulated apoptosis resistance by activating Akt1-mediated Bcl-2-associated death phosphorylation. Demonstrating the importance of NOX4-mediated apoptosis resistance in fibrotic remodeling, mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages exhibited increased apoptosis and were protected from pulmonary fibrosis. Moreover, resolution occurred when Nox4 was deleted in monocyte-derived macrophages in mice with established fibrosis. These observations suggest that NOX4 regulates apoptosis resistance in monocyte-derived macrophages and contributes to the pathogenesis of pulmonary fibrosis. Targeting NOX4-mediated apoptosis resistance in monocyte-derived macrophages may provide a novel therapeutic target to protect against the development and/or progression of pulmonary fibrosis.

      Keywords

      Abbreviations:

      AEC (alveolar epithelial cell), ASK1 (apoptosis signal-regulating kinase 1), BAD (Bcl-2-associated death), BMDM (bone-marrow-derived macrophage), Csf1r (colony-stimulating factor 1 receptor), IPF (idiopathic pulmonary fibrosis), MDM (monocyte-derived macrophage), MMVF (man-made vitreous fiber), NOX4 (NADPH oxidase 4), PKB (protein kinase B), RAM (resident alveolar macrophage), ROS (reactive oxygen species)
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      NADPH oxidase 4 (NOX4) is a constitutively active enzyme whose primary functions is to generate ROS, particularly hydrogen peroxide (
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      ). NOX4 has been shown to be highly expressed in AECs from IPF subjects, and NOX4-mediated ROS generated by AECs has been implicated in inducing the apoptosis seen in these cells (
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      A key role for NOX4 in epithelial cell death during development of lung fibrosis.
      ). Nox4 has been reported to mediate the apoptosis resistance of IPF lung fibroblasts by inhibiting the transcription factor, nuclear factor erythroid-derived 2-like 2 (Nrf2), promoting redox imbalance, and myofibroblast senescence (
      • Hecker L.
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      ). Recent data shows that apoptosis resistance occurs in fibrotic lung macrophages (
      • Larson-Casey J.L.
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      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ). We recently showed that NOX4 is crucial for lung macrophage profibrotic polarization to mediate fibrotic repair after asbestos exposure (
      • He C.
      • Larson-Casey J.L.
      • Davis D.
      • Hanumanthu V.S.
      • Longhini A.L.F.
      • Thannickal V.J.
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      NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.
      ), but it is not known if NOX4 regulates apoptosis resistance in lung macrophages or if macrophage NOX4 is required for the progression of fibrotic repair.

      Results

      Mitochondrial NOX4 expression is associated with lung macrophage apoptosis resistance

      To understand the role of apoptosis resistance in lung macrophages, we isolated lung macrophages from normal and asbestosis subjects. Normal subjects showed an increase in TUNEL positive cells compared with lung macrophages isolated from asbestosis subjects (Fig. 1A). Lung macrophages from asbestosis subjects showed significantly reduced caspase-3 activity compared with normal subjects (Fig. 1B). Because NOX4 is crucial for lung macrophage profibrotic polarization and profibrotic macrophages are associated with disease progression due to their prolonged survival (
      • He C.
      • Larson-Casey J.L.
      • Davis D.
      • Hanumanthu V.S.
      • Longhini A.L.F.
      • Thannickal V.J.
      • Gu L.
      • Carter A.B.
      NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.
      ,
      • Larson-Casey J.L.
      • Vaid M.
      • Gu L.
      • He C.
      • Cai G.Q.
      • Ding Q.
      • Davis D.
      • Berryhill T.F.
      • Wilson L.S.
      • Barnes S.
      • Neighbors J.D.
      • Hohl R.J.
      • Zimmerman K.A.
      • Yoder B.K.
      • Longhini A.L.F.
      • et al.
      Increased flux through the mevalonate pathway mediates fibrotic repair without injury.
      ,
      • Larson-Casey J.L.
      • Deshane J.S.
      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ,
      • Larson-Casey J.L.
      • Murthy S.
      • Ryan A.J.
      • Carter A.B.
      Modulation of the mevalonate pathway by Akt regulates macrophage survival and development of pulmonary fibrosis.
      ), we determined if NOX4 was expressed in lung macrophage mitochondria from subjects with asbestosis. NOX4 was increased 14-fold in the mitochondria from lung macrophages isolated from asbestosis subjects compared with normal subjects (Fig. 1C). Similarly, mitochondrial NOX4 expression was significantly increased in WT mice with asbestos-induced lung fibrosis (Fig. 1, D and E).
      Figure thumbnail gr1
      Figure 1Mitochondrial NOX4 expression is associated with lung macrophage apoptosis resistance. BAL was performed on normal (n = 5) and asbestosis (n = 4) subjects. A, TUNEL staining, B, caspase-3 activity, C, quantification of NOX4 immunoblot analysis (inset). Mice were exposed to MMVF or chrysotile asbestos and were euthanized 21 days later. D, immunoblot analysis in mitochondrial fraction and E, quantification of immunoblot in D in WT mice (n = 3). F, TUNEL staining, and G, caspase-3 activity in WT (n = 4) and Nox4−/− mice (n = 4). Bar in A and G = 100 μm. ∗p < 0.05; ∗∗∗p < 0.0001. Values shown as mean ± SEM. Two-tailed t-test statistical analysis was utilized for B, C, and E. One-way ANOVA followed by Tukey's multiple comparison test was utilized for G.
      To more directly link NOX4 to apoptosis resistance, chrysotile-exposed WT mice revealed a lack of TUNEL staining, while there was increased TUNEL staining in macrophages from Nox4−/− mice exposed to MMVF and chrysotile (Fig. 1F). Lung macrophages from chrysotile-exposed WT mice had decreased caspase-3 activity, whereas caspase-3 activity was increased in Nox4−/− mice (Fig. 1G). These data demonstrate that NOX4 has a critical role in mediating apoptosis resistance in lung macrophages.

      Mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages are protected from pulmonary fibrosis

      To understand the role of monocyte-derived macrophages (MDMs) in fibrosis development, mice were exposed to chrysotile for 0, 5, 10, 15, and 21 days. Increased recruitment of MDMs and a reduction of resident alveolar macrophages (RAMs) were evident in mice 10 days after chrysotile exposure (Fig. 2, AC and S1A). The number of RAMs decreased, while MDMs increased in a time-dependent manner. Similarly, CCL2 levels in the BAL fluid were significantly increased in 10 days after exposure to chrysotile with maximal levels seen at day 21 (Fig. 2D).
      Figure thumbnail gr2
      Figure 2Mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages are protected from pulmonary fibrosis. WT mice were exposed to chrysotile and BAL was harvested on day 0, 5, 10, 15, and 21. A, representative flow cytometry plots of resident alveolar macrophages (RAM, CD45+CD11b+/−Ly6GCD64+Ly6cSiglec Fhi) and monocyte-derived macrophages (MDM, CD45+CD11b+/−Ly6GCD64+Ly6cSiglec Flow). Number of flow sorted B, RAMs (n = 4–5) and C, MDMs (n = 4–5). D, CCL2 levels in BAL fluid (n = 4–5). WT and Nox4−/−Lyz2-cre mice were exposed to MMVF or chrysotile. BAL was performed on day 21. E, Representative flow cytometry plots of RAMs and MDMs. Number of flow sorted F, RAMs (n = 5) and G, MDMs (n = 5). H, CCL2 levels in BAL fluid (n = 5). I, Caspase-3 activity in RAMs (n = 5) and J, MDMs (n = 5). ∗∗p < 0.001; ∗∗∗p < 0.0001. Values shown as mean ± SEM. One-way ANOVA followed by Tukey's multiple comparison test was utilized.
      The conditional deletion of Nox4 in macrophages (Nox4−/−Lyz2-cre) abrogated the increase in MDMs after chrysotile exposure, and RAMs remained increased similar to the level seen in the MMVF control (Fig. 2, EG). BAL fluid from Nox4−/−Lyz2-cre mice showed significantly reduced CCL2 levels, suggesting a potential mechanism for the reduced MDM recruitment in the chrysotile-exposed mice (Fig. 2H).
      We questioned which monocyte/macrophage cell subset had NOX4-mediated apoptosis resistance during fibrosis. RAMs from chrysotile-exposed WT mice displayed apoptosis resistance, while Nox4−/−Lyz2-cre RAMs showed significantly greater caspase-3 activity (Fig. 2I). Chrysotile exposure significantly reduced caspase-3 activity in MDMs from WT mice, and Nox4−/−Lyz2-cre MDMs showed activity similar to WT controls (Fig. 2J). The absence of NOX4-mediated apoptosis resistance in MDMs protected Nox4−/−Lyz2-cre mice from pulmonary fibrosis (Fig. S1B). In contrast, the Masson's trichrome staining showed dense collagen deposition in chrysotile-exposed WT mice. The histological findings were confirmed biochemically by hydroxyproline assay (Fig. S1C). These observations suggest that NOX4 expression in MDMs mediates apoptosis resistance that has a critical role in fibrotic repair.

      NOX4 mediates apoptosis resistance via phosphorylation of BAD

      To determine the mechanism by which NOX4 mediates apoptosis resistance, we focused on downstream signaling of NOX4. We determined if Akt1 is modulated by NOX4 in lung macrophages. The mitochondrial localization of NOX4 was induced in macrophages treated with chrysotile asbestos or in macrophages expressing NOX4. The combination increased expression further (Fig. 3A). NOX4 expression correlated with Akt1 activation, as chrysotile exposure and NOX4 expression induced the activation of Akt1. The opposite was seen in macrophages with NOX4 silenced, and there was no effect with chrysotile exposure (Fig. 3B). Likewise, bone-marrow-derived macrophages (BMDM) isolated from Nox4−/− mice showed an absence of Akt1 activation (Fig. 2C).
      Figure thumbnail gr3
      Figure 3NOX4 mediates apoptosis resistance via phosphorylation of BAD. A, immunoblot analysis of macrophages (THP-1 cells) expressing empty or NOX4 exposed to chrysotile. B, immunoblot analysis of macrophages (THP-1 cells) transfected with scramble or NOX4 siRNA. C, immunoblot analysis of bone-marrow-derived macrophages isolated from WT and Nox4−/− mice and exposed to MMVF or chrysotile. D, Caspase-3 activity in macrophages (THP-1 cells) expressing empty or NOX4 treated with chrysotile (n = 5). Macrophages (THP-1 cells) expressing empty or NOX4 were transfected with scramble or Akt1 siRNA. E, immunoblot analysis and F, caspase-3 activity (n = 5). G, immunoblot analysis and H, caspase-3 activity of macrophages (THP-1 cells) expressing empty or Akt1CA that were transfected with scramble or NOX4 siRNA. I, immunoblot analysis of macrophages expressing empty or NOX4 treated with chrysotile. Macrophages (MH-S cells) expressing empty or NOX4 were transfected with scramble or Bad siRNA. J, immunoblot analysis and K, caspase-3 activity (n = 3). L, representative immunoblot analysis from flow sorted RAMs (n = 5) and MDMs (n = 5) from WT and Nox4−/−Lyz2-cre mice were exposed to MMVF or chrysotile. BAL was performed on normal (n = 4) and asbestosis (n = 5) subjects. M, immunoblot analysis, quantification of N, NOX4, O, p-Akt1, and P, p-BAD immunoblots in M were conducted. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001. Values shown as mean ± SEM. Two-tailed t-test statistical analysis was utilized for N-P. One-way ANOVA followed by Tukey's multiple comparison test was utilized for D, F, H, K.
      Because NOX4 overexpression diminishes caspase-3 activity in macrophages (Fig. 3D), we questioned if this was mediated in an Akt-dependent manner. Overexpression of NOX4 induced Akt1 activation and Bcl-2-associated death (BAD) phosphorylation at Ser136 (Fig. 3E). BAD phosphorylation was mediated by Akt1 as silencing Akt1 in macrophages expressing NOX4 showed an absence of BAD phosphorylation. Unphosphorylated BAD increased caspase-3 activity in macrophages with Akt1 silenced (Fig. 3F). Further demonstrating these effects were mediated by Akt1 activation, Nox4 silencing inhibited Akt1 activation and BAD phosphorylation (Fig. 3G); however, macrophages expressing constitutively active Akt1 with Nox4 silenced led to Akt1 activation and phosphorylation of BAD. Akt1-mediated phosphorylation of BAD inhibited caspase-3 activity (Fig. 3H). Implicating the importance of Akt1 activation, we showed that phosphatase and tensin homologue (PTEN) expression was reduced in chrysotile-exposed macrophages (Fig. S2). Moreover, phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) was similarly reduced.
      Validating that NOX4 mediated apoptosis resistance via BAD phosphorylation, we found that the proapoptotic proteins, Bax and Bak, had cytoplasmic localization in macrophages expressing NOX4 or exposed to chrysotile (Fig. 3I). To confirm the relationship of BAD to resistance of apoptosis, NOX4 mediated activation of Akt1 in macrophages with Bad silenced (Fig. 3J), and the absence of BAD expression reduced caspase-3 activity (Fig. 3K).
      To determine the biological relevance of BAD in vivo, the absence of NOX4 in MDMs from Nox4−/−Lyz2-cre mice was associated with absent Akt1 activation and BAD phosphorylation, whereas chrysotile-exposed WT mice showed increased Akt1 activation and BAD phosphorylation (Fig. 3L). In humans, asbestosis subjects showed increased NOX4 and activation of Akt1, which correlated with a significant increase in BAD phosphorylation in lung macrophages compared with normal volunteers (Fig. 3, MP). These data strongly suggest that NOX4 mediates apoptosis resistance via phosphorylation of BAD in an Akt1-dependent manner.

      NOX4-mediated mitochondrial ROS is required for apoptosis resistance

      Because mtROS in lung macrophages is linked to fibrotic repair (
      • Murthy S.
      • Ryan A.
      • He C.
      • Mallampalli R.K.
      • Carter A.B.
      Rac1-mediated mitochondrial H2O2 generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1.
      ,
      • Osborn-Heaford H.L.
      • Ryan A.J.
      • Murthy S.
      • Racila A.M.
      • He C.
      • Sieren J.C.
      • Spitz D.R.
      • Carter A.B.
      Mitochondrial Rac1 GTPase import and electron transfer from cytochrome c are required for pulmonary fibrosis.
      ,
      • He C.
      • Murthy S.
      • McCormick M.L.
      • Spitz D.R.
      • Ryan A.J.
      • Carter A.B.
      Mitochondrial Cu,Zn-Superoxide Dismutase mediates pulmonary fibrosis by augmenting H2O2 generation.
      ,
      • Larson-Casey J.L.
      • Deshane J.S.
      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ), we measured ROS generation in macrophages from chrysotile-exposed mice. WT mice exposed to chrysotile had increased H2O2 generation in the lung macrophages, whereas the H2O2 generation in macrophages from chrysotile-exposed Nox4−/− mice was below control levels (Fig. 4A). Confirming that mtROS generation regulated NOX4-mediated apoptosis resistance, we isolated BMDM from WT and Nox4−/− mice. MitoTEMPO reduced mtROS generation from chrysotile-exposed BMDM from WT mice to control levels, and mtROS generation from Nox4−/− BMDM showed greater reduction regardless of treatment (Fig. 4B). MitoTEMPO treatment reduced NOX4 expression, Akt1 activation, and BAD phosphorylation in chrysotile-exposed BMDM from WT mice, whereas NOX4, activated Akt1, and phosphorylated BAD were essentially absent in Nox4−/− BMDM (Fig. 4C). NOX4-meditaed mtROS generation led to a significant reduction in caspase-3 activity in WT BMDM, and MitoTEMPO treatment increased caspase-3 activity to control levels (Fig. 4D). BMDM from Nox4−/− mice showed increased caspase-3 activity compared with WT, and neither chrysotile nor MitoTEMPO altered the activity. These data suggested that macrophage NOX4 modulated apoptosis resistance by regulating mtROS to mediate Akt1 phosphorylation of BAD at Ser136.
      Figure thumbnail gr4
      Figure 4NOX4-mediated mitochondrial ROS is required for apoptosis resistance. A, H2O2 generation was measured in BAL cells isolated from WT (n = 5) and Nox4−/− (n = 5) mice were exposed to MMVF or chrysotile. Bone-marrow-derived macrophages were isolated from WT and Nox4−/− mice and treated with vehicle or mitoTEMPO (10 μM, 16 h) and MMVF or chrysotile. B, Mitochondrial H2O2 (n = 3), C, immunoblot analysis, and D, caspase-3 activity (n = 6) were measured. ∗∗∗p < 0.0001. Values shown as mean ± SEM. One-way ANOVA followed by Tukey's multiple comparison test was utilized.

      Macrophage Nox4 regulates progression of dysregulated fibrotic repair

      To determine the role of macrophage NOX4 in apoptosis resistance during progression of fibrotic remodeling, Nox4 was conditionally deleted in mice after fibrosis was established. Mice harboring a conditional deletion of Nox4 in MDMs were generated using a tamoxifen-inducible Cre driven by the colony-stimulating factor 1 receptor (Csf1r) promoter (Nox4−/−Csf1rMeriCreMer). We verified that fibrosis was evident in Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice 12 days after exposure to chrysotile (Fig. 5, A and B). In subsequent experiments, tamoxifen was administered to MMVF- and chrysotile-exposed Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice starting at day 12. Chrysotile-exposed Nox4fl/fl mice had a reduction in the number of RAMs compared with MMVF-exposed Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice (Fig. 5, C and D). The reduction of RAMs was associated with a significant increase of MDMs in chrysotile-exposed Nox4fl/fl mice, while Nox4−/−Csf1rMeriCreMer mice showed an absence of MDM recruitment (Fig. 5, C and E).
      Figure thumbnail gr5
      Figure 5Macrophage Nox4 regulates progression of dysregulated fibrotic repair. Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice were harvested 12 days exposure to MMVF or chrysotile. A, representative histology of lung sections with Masson's trichrome staining (n = 3) and B, hydroxyproline analysis of homogenized lung (n = 3). Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice were exposed to MMVF or chrysotile. Mice were subjected to tamoxifen injections (75 mg/kg, i.p. daily) for 5 consecutive days beginning at day 12. BAL was performed on day 21. C, representative flow cytometry plots of RAMs and MDMs. Number of flow sorted D, RAMs (n = 4–5) and E, MDMs (n = 4–5). F, immunoblot analysis and caspase-3 activity in G, RAMs (n = 4–5) and H, MDMs (n = 4–5). I, representative histology of lung sections with Masson's trichrome staining (n = 4–5) and J, hydroxyproline analysis of homogenized lung (n = 4–5). ∗∗∗p < 0.0001. Bar = 500 μm in A and I. Values shown as mean ± SEM. Two-tailed t-test statistical analysis was utilized for B. One-way ANOVA followed by Tukey's multiple comparison test was utilized for D-E, G-H, J.
      Tamoxifen administration depleted NOX4 expression in FACS-sorted MDMs from Nox4−/−Csf1rMeriCreMer mice, while RAMs retained NOX4 expression (Fig. 5F). Chrysotile-exposed Nox4fl/fl mice administered tamoxifen showed an increase of NOX4 in MDMs compared with RAMs. The absence of NOX4 in MDMs from Nox4−/−Csf1rMeriCreMer mice resulted in an absence in Akt1 activation and phosphorylation of BAD. There was no significant difference in caspase-3 activity in RAMs from Nox4fl/fl mice and Nox4−/−Csf1rMeriCreMer mice (Fig. 5G). MDMs showed a significant decrease in caspase-3 activity in chrysotile-exposed Nox4fl/fl mice, whereas it was significantly increased in Nox4−/−Csf1rMeriCreMer mice (Fig. 5H).
      To determine the impact of NOX4 regulating lung macrophage profibrotic polarization, we analyzed profibrotic cytokines in BAL fluid from Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice. BAL fluid isolated 12 days after chrysotile exposure (prior to tamoxifen injection) showed increased PDGF-BB and IL-10 levels in BAL fluid from both strains (Fig. S3, A and B). Tamoxifen-induced deletion of Nox4 reduced PDGF-BB and IL-10 levels near control levels, while Nox4fl/fl mice showed a further increase 21 days after chrysotile exposure. We then determined if NOX4 regulates lung macrophage profibrotic polarization via Akt1-mediated BAD phosphorylation. Arginase activity was increased in macrophages expressing NOX4, and exposure to chrysotile increased the activity further (Fig. S3C). The silencing of Bad in macrophages significantly reduced arginase activity below control levels, implicating the importance of this pathway in the regulation of profibrotic polarization.
      The deletion of Nox4 mediated resolution in established fibrosis. MMVF-exposed Nox4fl/fl and Nox4−/−Csf1rMeriCreMer mice showed normal lung architecture. Chrysotile-exposed Nox4−/−Csf1rMeriCreMer showed resolution of collagen deposition in the lungs compared with architectural distortion in the chrysotile-injured Nox4fl/fl mice (Fig. 5I). Tamoxifen-induced deletion of Nox4 in Nox4−/−Csf1rMeriCreMer mice with established fibrosis led to significantly reduced hydroxyproline content to the level seen in the MMVF-exposed Nox4fl/fl mice (Fig. 5J). These results demonstrated that NOX4 regulated MDM apoptosis resistance and reversed fibrotic remodeling. These observations suggest that targeting macrophage NOX4 in pulmonary fibrosis may reverse disease progression.

      Discussion

      NOX4 has been implicated in many fibrotic diseases, including the skin (
      • Spadoni T.
      • Svegliati Baroni S.
      • Amico D.
      • Albani L.
      • Moroncini G.
      • Avvedimento E.V.
      • Gabrielli A.
      A reactive oxygen species-mediated loop maintains increased expression of NADPH oxidases 2 and 4 in skin fibroblasts from patients with systemic sclerosis.
      ), liver (
      • Sancho P.
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      • Crosas-Molist E.
      • Roncero C.
      • Fernandez-Rodriguez C.M.
      • Pinedo F.
      • Huber H.
      • Eferl R.
      • Mikulits W.
      • Fabregat I.
      NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.
      ), pancreas (
      • Masamune A.
      • Watanabe T.
      • Kikuta K.
      • Satoh K.
      • Shimosegawa T.
      NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells.
      ), kidney (
      • Sedeek M.
      • Callera G.
      • Montezano A.
      • Gutsol A.
      • Heitz F.
      • Szyndralewiez C.
      • Page P.
      • Kennedy C.R.
      • Burns K.D.
      • Touyz R.M.
      • Hebert R.L.
      Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: Implications in type 2 diabetic nephropathy.
      ), heart (
      • Cucoranu I.
      • Clempus R.
      • Dikalova A.
      • Phelan P.J.
      • Ariyan S.
      • Dikalov S.
      • Sorescu D.
      NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts.
      ), and lung (
      • He C.
      • Larson-Casey J.L.
      • Davis D.
      • Hanumanthu V.S.
      • Longhini A.L.F.
      • Thannickal V.J.
      • Gu L.
      • Carter A.B.
      NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.
      ,
      • Hecker L.
      • Logsdon N.J.
      • Kurundkar D.
      • Kurundkar A.
      • Bernard K.
      • Hock T.
      • Meldrum E.
      • Sanders Y.Y.
      • Thannickal V.J.
      Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance.
      ,
      • Carnesecchi S.
      • Deffert C.
      • Donati Y.
      • Basset O.
      • Hinz B.
      • Preynat-Seauve O.
      • Guichard C.
      • Arbiser J.L.
      • Banfi B.
      • Pache J.C.
      • Barazzone-Argiroffo C.
      • Krause K.H.
      A key role for NOX4 in epithelial cell death during development of lung fibrosis.
      ). Within the lung, NOX4 has been shown to be increased in myofibroblasts, alveolar epithelial cells, and macrophages to promote the development of pulmonary fibrosis. Studies showed TGF-β1 regulates NOX4 expression in IPF lung fibroblasts to mediate mtROS production (
      • Hecker L.
      • Vittal R.
      • Jones T.
      • Jagirdar R.
      • Luckhardt T.R.
      • Horowitz J.C.
      • Pennathur S.
      • Martinez F.J.
      • Thannickal V.J.
      NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury.
      ). NOX4 modulates myofibroblast activation by regulating Nrf2 to influence fibroblast apoptosis resistance (
      • Hecker L.
      • Logsdon N.J.
      • Kurundkar D.
      • Kurundkar A.
      • Bernard K.
      • Hock T.
      • Meldrum E.
      • Sanders Y.Y.
      • Thannickal V.J.
      Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance.
      ). Similarly, AECs stimulated with TGF-β1 showed increased NOX4 expression to regulate ROS generation (
      • Carnesecchi S.
      • Deffert C.
      • Donati Y.
      • Basset O.
      • Hinz B.
      • Preynat-Seauve O.
      • Guichard C.
      • Arbiser J.L.
      • Banfi B.
      • Pache J.C.
      • Barazzone-Argiroffo C.
      • Krause K.H.
      A key role for NOX4 in epithelial cell death during development of lung fibrosis.
      ). NOX4-dependent ROS was necessary for TGF-β1-induced AEC injury and apoptosis. We showed that NOX4 is crucial for lung macrophage profibrotic polarization and fibrotic remodeling (
      • He C.
      • Larson-Casey J.L.
      • Davis D.
      • Hanumanthu V.S.
      • Longhini A.L.F.
      • Thannickal V.J.
      • Gu L.
      • Carter A.B.
      NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.
      ). Nox4−/− macrophages failed to polarize to the profibrotic phenotype and had reduced mtROS production. Contrary to fibroblasts and AECs, NOX4 regulated TGF-β1 expression in macrophages by mediating mtROS. Here we show that NOX4 regulates apoptosis resistance in macrophages by a redox-dependent mechanism.
      During fibrotic remodeling in the lung, AECs often undergo apoptosis. Studies show that activation of the Fas-Fas ligand pathway in AECs promoted pulmonary fibrosis (
      • Hagimoto N.
      • Kuwano K.
      • Miyazaki H.
      • Kunitake R.
      • Fujita M.
      • Kawasaki M.
      • Kaneko Y.
      • Hara N.
      Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen.
      ). Apoptotic, hyperplastic epithelial cells are present in IPF subjects and exhibit caspase-3 activation and inhibition of Bcl-2 (
      • Plataki M.
      • Koutsopoulos A.V.
      • Darivianaki K.
      • Delides G.
      • Siafakas N.M.
      • Bouros D.
      Expression of apoptotic and antiapoptotic markers in epithelial cells in idiopathic pulmonary fibrosis.
      ). ER stress in AECs has been shown to predispose the lung to fibrotic remodeling in experimental models (
      • Lawson W.E.
      • Cheng D.S.
      • Degryse A.L.
      • Tanjore H.
      • Polosukhin V.V.
      • Xu X.C.
      • Newcomb D.C.
      • Jones B.R.
      • Roldan J.
      • Lane K.B.
      • Morrisey E.E.
      • Beers M.F.
      • Yull F.E.
      • Blackwell T.S.
      Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs.
      ). IPF fibroblasts exhibit apoptosis resistance via increased expression of Bcl-2 (
      • Zhou Y.
      • Huang X.
      • Hecker L.
      • Kurundkar D.
      • Kurundkar A.
      • Liu H.
      • Jin T.H.
      • Desai L.
      • Bernard K.
      • Thannickal V.J.
      Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis.
      ), while other studies indicate that IPF fibroblasts are also resistant to Fas-mediated apoptosis (
      • Tanaka T.
      • Yoshimi M.
      • Maeyama T.
      • Hagimoto N.
      • Kuwano K.
      • Hara N.
      Resistance to Fas-mediated apoptosis in human lung fibroblast.
      ). The role of apoptosis in macrophages has been controversial. Bleomycin has been shown to induce apoptosis in alveolar macrophages treated ex vivo (
      • Hamilton Jr., R.F.
      • Li L.
      • Felder T.B.
      • Holian A.
      Bleomycin induces apoptosis in human alveolar macrophages.
      ). Administration of apoptotic macrophages in the lungs of rats promoted macrophage infiltration and apoptosis, impaired efferocytosis, and induced development of pulmonary fibrosis (
      • Wang L.
      • Scabilloni J.F.
      • Antonini J.M.
      • Rojanasakul Y.
      • Castranova V.
      • Mercer R.R.
      Induction of secondary apoptosis, inflammation, and lung fibrosis after intratracheal instillation of apoptotic cells in rats.
      ). Recent data implicated that apoptosis resistance occurs in lung macrophages and is associated to the profibrotic polarization of macrophages (
      • Larson-Casey J.L.
      • Deshane J.S.
      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ). IPF lung macrophages require mitophagy for the removal of dysfunctional mitochondria, which induces apoptosis resistance. Implicating the importance of macrophages in fibrosis, several studies demonstrated that promoting apoptosis of recruited lung macrophages protects mice from lung fibrosis (
      • Misharin A.V.
      • Morales-Nebreda L.
      • Reyfman P.A.
      • Cuda C.M.
      • Walter J.M.
      • McQuattie-Pimentel A.C.
      • Chen C.I.
      • Anekalla K.R.
      • Joshi N.
      • Williams K.J.N.
      • Abdala-Valencia H.
      • Yacoub T.J.
      • Chi M.
      • Chiu S.
      • Gonzalez-Gonzalez F.J.
      • et al.
      Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.
      ,
      • McCubbrey A.L.
      • Barthel L.
      • Mohning M.P.
      • Redente E.F.
      • Mould K.J.
      • Thomas S.M.
      • Leach S.M.
      • Danhorn T.
      • Gibbings S.L.
      • Jakubzick C.V.
      • Henson P.M.
      • Janssen W.J.
      Deletion of c-FLIP from CD11b(hi) macrophages prevents development of bleomycin-induced lung fibrosis.
      ). Here we show that NOX4 mediates apoptosis resistance in MDMs by regulating BAD in a redox-dependent manner (Fig. 6). Apoptosis resistance in RAMs does not require BAD phosphorylation, supporting the notion that RAMs do not contribute to fibrosis. We cannot rule out that apoptosis in RAMs may occur via other proteins or signaling pathways, as NOX4 has been shown to alter Bcl-2 expression (
      • Jacobsen K.
      • Bertran-Alamillo J.
      • Molina M.A.
      • Teixido C.
      • Karachaliou N.
      • Pedersen M.H.
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      • Gimenez-Capitan A.
      • Drozdowskyj A.
      • Viteri S.
      • Larsen M.R.
      • Lassen U.
      • et al.
      Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer.
      ). Moreover, modulating NOX4-mediated macrophage apoptosis in established fibrosis results in the resolution of asbestos-induced pulmonary fibrosis.
      Figure thumbnail gr6
      Figure 6Macrophage Nox4 regulates fibrotic progression in monocyte-derived macrophages. Asbestos exposure promotes the mitochondrial localization of NOX4, which mediates mitochondrial ROS (mtROS) to activate Akt1. The phosphorylation of BAD at Ser136 by Akt1 mediated apoptosis resistance in monocyte-derived macrophages and increased fibrotic remodeling.
      NOX4 has been shown to regulate Akt activation in the kidney, vasculature, and heart (
      • Yan F.
      • Wang Y.
      • Wu X.
      • Peshavariya H.M.
      • Dusting G.J.
      • Zhang M.
      • Jiang F.
      Nox4 and redox signaling mediate TGF-beta-induced endothelial cell apoptosis and phenotypic switch.
      ,
      • Zhao Q.D.
      • Viswanadhapalli S.
      • Williams P.
      • Shi Q.
      • Tan C.
      • Yi X.
      • Bhandari B.
      • Abboud H.E.
      NADPH oxidase 4 induces cardiac fibrosis and hypertrophy through activating Akt/mTOR and NFkappaB signaling pathways.
      ,
      • Gorin Y.
      • Ricono J.M.
      • Kim N.H.
      • Bhandari B.
      • Choudhury G.G.
      • Abboud H.E.
      Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells.
      ). Akt, also known as protein kinase B (PKB), is a serine/threonine protein kinase mediating cell growth, metabolism, and ROS production. The prosurvival signaling by Akt is associated with many human cancers, diabetes, neurodegenerative disorders, and Akt is upregulated in various fibrotic tissues (
      • Palmieri M.
      • Pal R.
      • Nelvagal H.R.
      • Lotfi P.
      • Stinnett G.R.
      • Seymour M.L.
      • Chaudhury A.
      • Bajaj L.
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      • Bremner L.
      • Saleem U.
      • Tse D.Y.
      • Sanagasetti D.
      • Wu S.M.
      • Neilson J.R.
      • et al.
      mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases.
      ,
      • Cho H.
      • Mu J.
      • Kim J.K.
      • Thorvaldsen J.L.
      • Chu Q.
      • Crenshaw 3rd, E.B.
      • Kaestner K.H.
      • Bartolomei M.S.
      • Shulman G.I.
      • Birnbaum M.J.
      Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta).
      ,
      • Campa C.C.
      • Silva R.L.
      • Margaria J.P.
      • Pirali T.
      • Mattos M.S.
      • Kraemer L.R.
      • Reis D.C.
      • Grosa G.
      • Copperi F.
      • Dalmarco E.M.
      • Lima-Junior R.C.P.
      • Aprile S.
      • Sala V.
      • Dal Bello F.
      • Prado D.S.
      • et al.
      Inhalation of the prodrug PI3K inhibitor CL27c improves lung function in asthma and fibrosis.
      ,
      • Xu Y.
      • Mizuno T.
      • Sridharan A.
      • Du Y.
      • Guo M.
      • Tang J.
      • Wikenheiser-Brokamp K.A.
      • Perl A.T.
      • Funari V.A.
      • Gokey J.J.
      • Stripp B.R.
      • Whitsett J.A.
      Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis.
      ). Within the fibrotic lung, Akt has been shown to be increased in IPF fibroblasts (
      • Xia H.
      • Diebold D.
      • Nho R.
      • Perlman D.
      • Kleidon J.
      • Kahm J.
      • Avdulov S.
      • Peterson M.
      • Nerva J.
      • Bitterman P.
      • Henke C.
      Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.
      ), and single-cell RNA sequencing has identified Akt activation in IPF epithelial cells, including basal cells (
      • Yang E.
      • Zha J.
      • Jockel J.
      • Boise L.H.
      • Thompson C.B.
      • Korsmeyer S.J.
      Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death.
      ). While we have shown that Akt1 is activated in lung macrophages from subjects with IPF (
      • Larson-Casey J.L.
      • Deshane J.S.
      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ), NOX4 has not previously been connected to the regulation of Akt1 activation in humans with pulmonary fibrosis.
      BAD regulates apoptosis by interacting with Bcl-2 and Bcl-XL (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
      ). While Bcl-2 and Bcl-XL promote apoptosis resistance by binding to Bax and preventing the release of cytochrome c from the mitochondria, the binding of BAD with Bcl-2 or Bcl-XL sequesters these proteins in the cytoplasm to induce apoptosis. Akt specifically phosphorylates BAD on serine136 (equivalent of the human serine 99 residue) leading to the inability of BAD to heterodimerize with Bcl-2 or Bcl-XL (
      • von Harsdorf R.
      • Li P.F.
      • Dietz R.
      Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis.
      ), thus permitting them to bind to Bax to block apoptosis.
      While mtROS is linked to apoptosis and apoptosis resistance, the relationship of NOX4 and apoptosis in macrophages is not known. Our studies show that NOX4-mediated apoptosis resistance in monocyte-derived macrophages was redox-dependent. Our results contrast those found in cardiac myocytes, where ROS induced the translocation of BAD to the mitochondria and its binding with Bcl-2 to provoke apoptosis (
      • Cho H.
      • Mu J.
      • Kim J.K.
      • Thorvaldsen J.L.
      • Chu Q.
      • Crenshaw 3rd, E.B.
      • Kaestner K.H.
      • Bartolomei M.S.
      • Shulman G.I.
      • Birnbaum M.J.
      Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta).
      ). Our data indicated that deletion of macrophage Nox4 in mice with established fibrosis induced resolution of the disease. Moreover, these observations provide a mechanism by which NOX4 regulates apoptosis resistance in MDMs to promote progression of fibrosis and suggest a novel therapeutic target to prevent ongoing aberrant fibrotic remodeling.

      Experimental procedures

      Human subjects

      Human BAL cells were obtained as previously described (
      • He C.
      • Murthy S.
      • McCormick M.L.
      • Spitz D.R.
      • Ryan A.J.
      • Carter A.B.
      Mitochondrial Cu,Zn-Superoxide Dismutase mediates pulmonary fibrosis by augmenting H2O2 generation.
      ) from normal subjects and asbestosis patients under an approved protocol by the Human Subjects Institutional Review Board of UAB (300001124) and the Birmingham VAMC (01670). These studies abide by the Declaration of Helsinki principles. Human BAL specimens were used for research only. All subjects provided prior written consent to participate in the study. Normal volunteers had to meet the following criteria: (1) age between 18 and 65 years; (2) no history of cardiopulmonary disease or other chronic disease; (3) no prescription or nonprescription medication except oral contraceptives; (4) no recent or current evidence of infection; and (5) lifetime nonsmoker. Asbestosis subjects had to meet the following criteria: (1) FVC (forced vital capacity) at least 50% predicted; (2) current nonsmoker; (3) no recent or current evidence of infection; (4) evidence of restrictive physiology on pulmonary function tests; and (5) usual interstitial pneumonia on high-resolution chest computed tomography. Fiberoptic bronchoscopy with bronchoalveolar lavage was performed after subjects received local anesthesia. Three subsegments of the lung were lavaged with five 20-ml aliquots of normal saline, and the first aliquot in each was discarded. The percentage of macrophages was determined by Wright–Giemsa stain and varied from 90 to 98%.

      Mice

      Animal experiments were approved by UAB Institutional Animal Care and Use Committee (21149) and were performed in accordance with NIH guidelines. Eight- to twelve-week-old male and female mice were intratracheally administered 100 μg of chrysotile asbestos or man-made vitreous fiber (MMVF), as a negative control, after being anesthetized with 3% isoflurane using a precision Fortec vaporizer. BAL was performed 21 days after exposure. WT C57BL6 mice were purchased from JAX Labs, Nox4−/− have been previously described (
      • Carnesecchi S.
      • Deffert C.
      • Donati Y.
      • Basset O.
      • Hinz B.
      • Preynat-Seauve O.
      • Guichard C.
      • Arbiser J.L.
      • Banfi B.
      • Pache J.C.
      • Barazzone-Argiroffo C.
      • Krause K.H.
      A key role for NOX4 in epithelial cell death during development of lung fibrosis.
      ), and Nox4−/−Lyz2-cre were generated by selective disruption of Nox4 gene in lineage cells of the granulocyte/monocyte lineage by crossing Nox4fl/fl mice with mice containing a Cre recombinase under control of the lysozyme M promoter as previously described (
      • He C.
      • Larson-Casey J.L.
      • Davis D.
      • Hanumanthu V.S.
      • Longhini A.L.F.
      • Thannickal V.J.
      • Gu L.
      • Carter A.B.
      NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.
      ). Nox4−/−Csf1rMeriCreMer mice were generated by crossing Nox4fl/fl mice with tamoxifen inducible Mer-iCre-Mer driven by the Csf1r promoter (FVB-Tg(Csf1r-cre/Esr1∗)1Jwp/J, stock number 019098, Jackson Laboratory). Nox4 was selectively and conditionally deleted from the monocytes/macrophages by tamoxifen-containing food or following administration of tamoxifen (75 mg/kg, i.p.) 12 days after chrysotile or MMVF exposure and continued daily for 5 consecutive days.

      Cell culture

      Human monocyte (THP-1) and mouse alveolar macrophage (MH-S) cell lines were obtained from American Type Culture Collection. Cells were maintained in RPMI 1640 media with 10% fetal bovine serum and penicillin/streptomycin supplements. All experiments were conducted in RPMI containing 0.5% serum.

      Plasmids, transfections, and small interfering RNA (siRNA)

      The pcDNA3.1-NOX4 and pUSE-Akt1CA plasmids have been previously described (
      • He C.
      • Larson-Casey J.L.
      • Davis D.
      • Hanumanthu V.S.
      • Longhini A.L.F.
      • Thannickal V.J.
      • Gu L.
      • Carter A.B.
      NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.
      ,
      • Larson-Casey J.L.
      • Murthy S.
      • Ryan A.J.
      • Carter A.B.
      Modulation of the mevalonate pathway by Akt regulates macrophage survival and development of pulmonary fibrosis.
      ). Cells were transfected using X-treme GENE 9 Transfection Reagent (Sigma) according to the manufacturer's protocol. Cells were transfected with 100 nM scramble, human Akt1, human NOX4, or mouse Bad siRNA duplex (IDT) utilizing Dharmafect 2 (Thermo Scientific) according to manufacturer's protocol. Eight hours after transfection, media was replaced, and cells were allowed to recover for 24–72 h.

      Flow cytometry

      BAL cells were blocked with 1% BSA containing TruStain fcX (anti-mouse CD16/32) antibody (101319; BioLegend), followed by staining with antibodies. Antibodies used: Rat anti-mouse CD45-PE (12–0451–82; eBiosciences), LIVE Dead-eflour506 (65–0866; Invitrogen), Rat anti-mouse CD11b-APC-Cy7 (101225; BioLegend), anti-mouse CD64-PE-Cy7 (139313; BioLegend), Rat anti-mouse Ly6G-AF700 (561236; BD), Rat anti-mouse Siglec F-APC (155507; BioLegend), and Rat anti-mouse Ly6C: eflour450 (48–5932–82; Invitrogen). Hierarchical gating strategy was used to represent the resident alveolar macrophages as CD45+CD11b+/−Ly6GCD64+Ly6cSiglec Fhi and monocyte-derived macrophages as CD45+CD11b+/−Ly6GCD64+Ly6cSiglec Flow. To determine cell number, CountBright Absolute Counting Beads (Molecular Probes) were added according to manufacturer's instructions. Briefly, counting beads were gently vortexed and 50 μl was added to stained cells in a volume of 300 μl. Counting beads were gated on forward versus linear side scatter. The following equation was used to determine cell number: (number of cell events/number of bead events) × (lot specific bead count per 50 μl/volume of sample). Data was acquired on FACSAria II or LSR II (BD Biosciences) using BD FACS DIVA software (version 8.0.1). Data was analyzed using FlowJo (FlowJo LLC) software (Version 10.5.0).

      Determination of H2O2 generation

      H2O2 production was determined fluorometrically. Cells were incubated in phenol-red free Hanks' balanced salt solution supplemented with 6.5 mM glucose, 1 mM HEPES, 6 mM sodium bicarbonate, 1.6 mM pHPA, and 0.95 μg/ml HRP. Fluorescence of pHPA-dimer was measured using a spectrofluorometer at excitation of 320 nm and emission of 400 nm (
      • Larson-Casey J.L.
      • Deshane J.S.
      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ).

      Isolation of mitochondria and cytoplasm

      Mitochondria were isolated by lysing the cells in mitochondria buffer containing 10 mM Tris, pH 7.8, 0.2 mM EDTA, 320 mM sucrose, and protease inhibitors. Lysates were homogenized using a Kontes Pellet Pestle Motor and centrifuged at 2000g for 8 min at 4 °C. The supernatant was removed and incubated at 4 °C and the pellet was lysed, homogenized, and centrifuged again. The two supernatants were pooled and centrifuged at 12,000g for 15 min at 4 °C. The pellet was washed in the mitochondrial buffer twice and then resuspended in mitochondria buffer without sucrose (
      • He C.
      • Murthy S.
      • McCormick M.L.
      • Spitz D.R.
      • Ryan A.J.
      • Carter A.B.
      Mitochondrial Cu,Zn-Superoxide Dismutase mediates pulmonary fibrosis by augmenting H2O2 generation.
      ).

      Immunoblot analysis

      Primary antibodies used: Akt1 (2938), Bad (9239), Lamin A/C (2032), Bax (2772), phospho-Akt1 (9018), phospho-Bad (Ser136) (4366), VDAC (4866) (Cell Signaling); β-actin (A5441) (Sigma); p38 (sc-7972) (Santa Cruz); NOX4 (NB110-58849) (Novus), NOX4 (109225) (Abcam); Bak (A0404) (Abclonal).

      ELISA

      CCL2 expression was determined in BALF using ELISA kits (R&D Systems) according to the manufacturer's instructions.

      TUNEL assay

      Detection of apoptosis was determined in BAL cells by TUNEL analysis, in situ Cell Death Detection Kit, TMR red (Sigma) according to the manufacturer's instructions and as previously described (
      • Larson-Casey J.L.
      • Murthy S.
      • Ryan A.J.
      • Carter A.B.
      Modulation of the mevalonate pathway by Akt regulates macrophage survival and development of pulmonary fibrosis.
      ). BAL cells were fixed with 4% paraformaldehyde in PBS, permeabilized, and counterstained with DAPI. Nikon A1 Confocal was utilized for imaging.

      Caspase-3 activity

      Caspase-3 activity was measured using EnzChek Caspase-3 Assay Kit Number 2 (Molecular Probes) according to the manufacturer's protocol. Cells were lysed in 1 × lysis buffer, subjected to a freeze–thaw cycle, centrifuged to remove cellular debris, and loaded into individual microplate wells. The 2× reaction buffer with substrate was immediately added to the samples, and fluorescence was measured (excitation/emission 496/520 nm). Activity was normalized to protein concentration in each sample. A supplied inhibitor was used as a negative control in all experiments (
      • Larson-Casey J.L.
      • Murthy S.
      • Ryan A.J.
      • Carter A.B.
      Modulation of the mevalonate pathway by Akt regulates macrophage survival and development of pulmonary fibrosis.
      ).

      Hydroxyproline assay

      Lung tissues were dried to a stable weight and acid hydrolyzed with 6N HCl for 24 h at 110 °C. Samples were resuspended in 1.5 ml phosphate-buffered saline followed by incubation at 60 °C for 1 h. Samples were centrifuged at 13,000 rpm, and the supernatant was taken for hydroxyproline analysis by using chloramine-T. Hydroxyproline concentration was normalized to the dry weight of the tissue (
      • Larson-Casey J.L.
      • Deshane J.S.
      • Ryan A.J.
      • Thannickal V.J.
      • Carter A.B.
      Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis.
      ).

      Statistical analysis

      Statistical comparisons were performed using a Student's t-test when only two groups of data are presented, or one-way ANOVA with a Tukey's post hoc test. All statistical analysis was expressed as ±SEM and p < 0.05 was considered to be significant. GraphPad Prism statistical software was used for all analysis.

      Data availability

      All data are contained within the article.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      Support for the Comprehensive Flow Cytometry Core provided by National Institutes of Health P30AR048311 and National Institutes of Health P30AI27667.

      Author contributions

      J. L. L.-C. and A. B. C. conceptualization; J. L. L.-C., L. G., J. K., and A. D. data curation; J. L. L.-C. and L. G. formal analysis; J. L. L.-C., L. G., J. K., A. B. C., and A. D. investigation; J. L. L.-C. and A. B. C. methodology; J. L. L.-C. and A. B. C. writing-original draft; J. L. L.-C, L. G., and A. B. C. resources; A. B. C. supervision; A. B. C. funding acquisition; A. B. C. validation; A. B. C. project administration; A. B. C. writing-review and editing.

      Funding and additional information

      Research reported in this publication was supported by the National Institutes of Health grants 2R01 ES015981-13 and P42 ES027723-2 and Department of Veteran Affairs Merit Review 1 I01 CX001715-2 to A. B. C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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