NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression

Jennifer L Larson-Casey, Linlin Gu, Jungsoon Kang, Ashish Dhyani, A Brent Carter, Jennifer L Larson-Casey, Linlin Gu, Jungsoon Kang, Ashish Dhyani, A Brent Carter

Abstract

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: NOX4; apoptosis resistance; monocyte-derived macrophages; pulmonary fibrosis.

Conflict of interest statement

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

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
Mitochondrial 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.
Figure 2
Figure 2
Mice 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+/−Ly6G−CD64+Ly6c−Siglec Fhi) and monocyte-derived macrophages (MDM, CD45+CD11b+/−Ly6G−CD64+Ly6c−Siglec 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.
Figure 3
Figure 3
NOX4 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.
Figure 4
Figure 4
NOX4-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.
Figure 5
Figure 5
Macrophage 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.
Figure 6
Figure 6
Macrophage 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.

References

    1. Attfield M.D., Wood J.M., Antao V.C., Pinheiro G.A. Changing patterns of pneumoconiosis mortality--United States, 1968-2000. MMWR Morb. Mortal Wkly. Rep. 2004;53:627–632.
    1. Guidotti T.L., Miller A., Christiani D., Wagner G., Balmes J., Harber P., Brodkin C.A., Rom W., Hillerdal G., Harbut M., Green F.H.Y. Diagnosis and initial management of nonmalignant diseases related to asbestos. Am. J. Respir. Crit. Care Med. 2004;170:691–715.
    1. Yang M., Wang D., Gan S., Fan L., Cheng M., Yu L., Wang B., Li W., Ma J., Zhou M., Chen W. Increasing incidence of asbestosis worldwide, 1990-2017: Results from the Global Burden of Disease study 2017. Thorax. 2020;75:798–800.
    1. King T.E., Jr., Bradford W.Z., Castro-Bernardini S., Fagan E.A., Glaspole I., Glassberg M.K., Gorina E., Hopkins P.M., Kardatzke D., Lancaster L., Lederer D.J., Nathan S.D., Pereira C.A., Sahn S.A., Sussman R. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N. Engl. J. Med. 2014;370:2083–2092.
    1. Richeldi L., du Bois R.M., Raghu G., Azuma A., Brown K.K., Costabel U., Cottin V., Flaherty K.R., Hansell D.M., Inoue Y., Kim D.S., Kolb M., Nicholson A.G., Noble P.W., Selman M. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N. Engl. J. Med. 2014;370:2071–2082.
    1. 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. J. Biol. Chem. 2010;285:25062–25073.
    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. J. Biol. Chem. 2012;287:3301–3312.
    1. 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. J. Biol. Chem. 2011;286:15597–15607.
    1. 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. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. J. Exp. Med. 2017;214:2387–2404.
    1. 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. Am. J. Respir. Cell Mol. Biol. 2018;58:66–78.
    1. 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. JCI Insight. 2019;4
    1. 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. Increased flux through the mevalonate pathway mediates fibrotic repair without injury. J. Clin. Invest. 2019;129:4962–4978.
    1. Gu L., Larson Casey J.L., Andrabi S.A., Lee J.H., Meza-Perez S., Randall T.D., Carter A.B. Mitochondrial calcium uniporter regulates PGC-1alpha expression to mediate metabolic reprogramming in pulmonary fibrosis. Redox Biol. 2019;26:101307.
    1. He C., Ryan A.J., Murthy S., Carter A.B. Accelerated development of pulmonary fibrosis via Cu,Zn-superoxide dismutase-induced alternative activation of macrophages. J. Biol. Chem. 2013;288:20745–20757.
    1. Murthy S., Larson-Casey J.L., Ryan A.J., He C., Kobzik L., Carter A.B. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure. FASEB J. 2015;29:3527–3536.
    1. Redente E.F., Keith R.C., Janssen W., Henson P.M., Ortiz L.A., Downey G.P., Bratton D.L., Riches D.W. Tumor necrosis factor-alpha accelerates the resolution of established pulmonary fibrosis in mice by targeting profibrotic lung macrophages. Am. J. Respir. Cell Mol. Biol. 2014;50:825–837.
    1. 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. Immunity. 2016;44:582–596.
    1. 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. Chest. 2005;127:266–274.
    1. 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. Sci. Transl. Med. 2014;6:231ra247.
    1. Nisimoto Y., Diebold B.A., Cosentino-Gomes D., Lambeth J.D. Nox4: A hydrogen peroxide-generating oxygen sensor. Biochemistry. 2014;53:5111–5120.
    1. 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. Antioxid. Redox Signal. 2011;15:607–619.
    1. Bernard K., Logsdon N.J., Miguel V., Benavides G.A., Zhang J., Carter A.B., Darley-Usmar V.M., Thannickal V.J. NADPH oxidase 4 (Nox4) suppresses mitochondrial biogenesis and bioenergetics in lung fibroblasts via a nuclear factor erythroid-derived 2-like 2 (Nrf2)-dependent pathway. J. Biol. Chem. 2017;292:3029–3038.
    1. 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. J. Biol. Chem. 2014;289:36204–36219.
    1. 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. Arthritis Rheumatol. 2015;67:1611–1622.
    1. Sancho P., Mainez J., 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. PLoS One. 2012;7
    1. Masamune A., Watanabe T., Kikuta K., Satoh K., Shimosegawa T. NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2008;294:G99–G108.
    1. 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. Am. J. Physiol. Renal Physiol. 2010;299:F1348–F1358.
    1. 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. Circ. Res. 2005;97:900–907.
    1. 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. Nat. Med. 2009;15:1077–1081.
    1. 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. Am. J. Respir. Cell Mol. Biol. 1997;17:272–278.
    1. 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. Proc. Natl. Acad. Sci. U. S. A. 2011;108:10562–10567.
    1. 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. J. Clin. Invest. 2013;123:1096–1108.
    1. Tanaka T., Yoshimi M., Maeyama T., Hagimoto N., Kuwano K., Hara N. Resistance to Fas-mediated apoptosis in human lung fibroblast. Eur. Respir. J. 2002;20:359–368.
    1. Hamilton R.F., Jr., Li L., Felder T.B., Holian A. Bleomycin induces apoptosis in human alveolar macrophages. Am. J. Physiol. 1995;269:L318–L325.
    1. 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. Am. J. Physiol. Lung Cell Mol. Physiol. 2006;290:L695–L702.
    1. Jacobsen K., Bertran-Alamillo J., Molina M.A., Teixido C., Karachaliou N., Pedersen M.H., Castellvi J., Garzon M., Codony-Servat C., Codony-Servat J., Gimenez-Capitan A., Drozdowskyj A., Viteri S., Larsen M.R., Lassen U. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer. Nat. Commun. 2017;8:410.
    1. 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. Cell Death Dis. 2014;5
    1. 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. Circulation. 2015;131:643–655.
    1. 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. Am. J. Physiol. Renal Physiol. 2003;285:F219–F229.
    1. Palmieri M., Pal R., Nelvagal H.R., Lotfi P., Stinnett G.R., Seymour M.L., Chaudhury A., Bajaj L., Bondar V.V., Bremner L., Saleem U., Tse D.Y., Sanagasetti D., Wu S.M., Neilson J.R. mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases. Nat. Commun. 2017;8:14338.
    1. Cho H., Mu J., Kim J.K., Thorvaldsen J.L., Chu Q., Crenshaw E.B., 3rd, 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) Science. 2001;292:1728–1731.
    1. 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. Inhalation of the prodrug PI3K inhibitor CL27c improves lung function in asthma and fibrosis. Nat. Commun. 2018;9:5232.
    1. 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. JCI Insight. 2016;1
    1. 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. J. Exp. Med. 2008;205:1659–1672.
    1. 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. Cell. 1995;80:285–291.
    1. 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. Cell. 1997;91:231–241.
    1. von Harsdorf R., Li P.F., Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation. 1999;99:2934–2941.

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel