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Analyses of alveolar epithelial injury via lipid-related stress in mammalian target of rapamycin inhibitor-induced lung disease

Laboratory Investigation (2019) | Download Citation


Although mammalian target of rapamycin inhibitors (mTORi) are used to treat various malignancies, they frequently induce active alveolitis and dyslipidemia. Abnormal lipid metabolism affects alveolar surfactant function and results in pulmonary disorders; however, the pathophysiology of lung injury and its relationship with lipid metabolism remain unknown. We investigated the relationship between lipid metabolism and alveolar epithelial injury, focusing on peroxisome proliferator-activated receptor-γ (PPAR-γ) as a lipid stress-related factor in mTORi-induced lung injury. We clinicopathologically examined three patients with mTORi-induced lung injury. We constructed an mTORi injury mouse model using temsirolimus in mice (30 mg/kg/day), with the vehicle control and bleomycin injury groups. We also constructed a cultured alveolar epithelial cell injury model using temsirolimus (0–40 μM) in the mouse lung epithelial cell line MLE-12 and performed analysis with or without pioglitazone (PPAR-γ agonist) treatment. All three patients had dyslipidemia and lung lesions of hyperplastic pneumocytes with foamy and enlarged changes. In the mouse model, temsirolimus induced significantly higher levels of total cholesterol and free fatty acids in serum and higher levels of surfactant protein D in serum and BAL fluid with an increase in inflammatory cytokines in the lung compared to control. Temsirolimus also induced hyperplastic foamy pneumocytes with increased lipid-associated spots and larger round electron-lucent bodies compared to the control or bleomycin groups in microscopic analyses. Multiple lipid-associated spots within the cytoplasm were also induced by temsirolimus administration in MLE-12 cells. Temsirolimus downregulated PPAR-γ expression in mouse lung and MLE-12 cells but upregulated cleaved caspase-3 in MLE-12 cells. Pioglitazone blocked the upregulated cleaved caspase-3 expression in MLE-12 cells. The pathogenesis of mTORi-induced lung disease may be involved in alveolar epithelial injury, via lipid metabolic stress associated with downregulated PPAR-γ expression. Focusing on the relationship between lipid metabolic stress and alveolar epithelial injury represents a potentially novel approach to the study of pulmonary damage.

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  1. 1.

    Maroto JP, Hudes G, Dutcher JP, et al. Drug-related pneumonitis in patients with advanced renal cell carcinoma treated with temsirolimus. J Clin Oncol. 2011;29:1750–6.

  2. 2.

    Alexandru S, Ortiz A, Baldovi S, et al. Severe everolimus-associated pneumonitis in a renal transplant recipient. Nephrol Dial Transplant. 2008;23:3353–5.

  3. 3.

    Schrader J, Sterneck M, Klose H, et al. Everolimus-induced pneumonitis: report of the first case in a liver transplant recipient and review of treatment options. Transpl Int. 2010;23:110–3.

  4. 4.

    Carreño CA, Gadea M. Case report of a kidney transplant recipient converted to everolimus due to malignancy: resolution of bronchiolitis obliterans organizing pneumonia without everolimus discontinuation. Transplant Proc. 2007;39:594–5.

  5. 5.

    Saito Y, Kunugi S, Suzuki Y, et al. Granuloma-forming interstitial pneumonia occurring one year after the start of everolimus therapy. Intern Med. 2013;52:263–7.

  6. 6.

    Houde VP, Brûlé S, Festuccia WT, et al. Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue. Diabetes. 2010;59:1338–48.

  7. 7.

    Sivendran S, Agarwal N, Gartrell B, et al. Metabolic complications with the use of mTOR inhibitors for cancer therapy. Cancer Treat Rev. 2014;40:190–6.

  8. 8.

    Hutson TE, Escudier B, Esteban E, et al. Randomized phase III trial of temsirolimus versus sorafenib as second-line therapy after sunitinib in patients with metastatic renal cell carcinoma. J Clin Oncol. 2014;32:760–7.

  9. 9.

    Plantier L, Besnard V, Xu Y, et al. Activation of sterol-response element-binding proteins (SREBP) in alveolar type II cells enhances lipogenesis causing pulmonary lipotoxicity. J Biol Chem. 2012;287:10099–114.

  10. 10.

    Enzi G, Bevilacqua M, Crepaldi G. Disturbances in pulmonary gaseous exchange in primary hyperlipoproteinemias. Bull Eur Physiopathol Respir. 1976;12:433–42.

  11. 11.

    Lee SJ, Zhang J, Choi AM, et al. Mitochondrial dysfunction induces formation of lipid droplets as a generalized response to stress. Oxid Med Cell Longev. 2013;2013:327167.

  12. 12.

    Miyake Y, Sasaki S, Yokoyama T, et al. Dietary fat and meat intake and idiopathic pulmonary fibrosis: a case-control study in Japan. Int J Tuberc Lung Dis. 2006;10:333–9.

  13. 13.

    Iannello S, Cavaleri A, Camuto M, et al. Low fasting serum triglyceride and high free fatty acid levels in pulmonary fibrosis: a previously unreported finding. MedGenMed. 2002;4:5.

  14. 14.

    Baritussio A, Enzi G, Inelmen EM, et al. Altered surfactant synthesis and function in rats with diet-induced hyperlipidemia. Metabolism. 1980;29:503–10.

  15. 15.

    Müller NL, White DA, Jiang H, et al. Diagnosis and management of drug-associated interstitial lung disease. Br J Cancer. 2004;91(Suppl 2):S24–S30.

  16. 16.

    Leconte M, Nicco C, Ngô C, et al. The mTOR/AKT inhibitor temsirolimus prevents deep infiltrating endometriosis in mice. Am J Pathol. 2011;179:880–9.

  17. 17.

    Wu L, Birle DC, Tannock IF. Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts. Cancer Res. 2005;65:2825–31.

  18. 18.

    Washino S, Ando H, Ushijima K, et al. Temsirolimus induces surfactant lipid accumulation and lung inflammation in mice. Am J Physiol Lung Cell Mol Physiol. 2014;306:L1117–L1128.

  19. 19.

    Harrison JH, Lazo JS. High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis. J Pharmacol Exp Ther. 1987;243:1185–94.

  20. 20.

    Murata M, Otsuka M, Mizuno H, et al. Development of an enzyme-linked immunosorbent assay for measurement of rat pulmonary surfactant protein D using monoclonal antibodies. Exp Lung Res. 2010;36:463–8.

  21. 21.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.

  22. 22.

    Thrall RS, McCormick JR, Jack RM, et al. Bleomycin-induced pulmonary fibrosis in the rat: inhibition by indomethacin. Am J Pathol. 1979;95:117–30.

  23. 23.

    Laplante M, Sabatini DM. Regulation of mTORC1 and its impact on gene expression at a glance. J Cell Sci. 2013;126:1713–9.

  24. 24.

    Wikenheiser KA, Vorbroker DK, Rice WR, et al. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci USA. 1993;90:11029–33.

  25. 25.

    Whitsett JA, Wert SE, Weaver TE. Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. Annu Rev Med. 2010;61:105–19.

  26. 26.

    Sunaga H, Matsui H, Ueno M, et al. Deranged fatty acid composition causes pulmonary fibrosis in Elovl6-deficient mice. Nat Commun. 2013;4:2563.

  27. 27.

    Borie R, Danel C, Debray MP, et al. Pulmonary alveolar proteinosis. Eur Respir Rev. 2011;20:98–107.

  28. 28.

    Kehrer JP, Autor AP. The effect of dietary fatty acids on the composition of adult rat lung lipids: relationship to oxygen toxicity. Toxicol Appl Pharmacol. 1978;44:423–30.

  29. 29.

    Oyarzun MJ, Cabezas E, Donoso P, et al. Effects of free fatty acid infusion on rabbit pulmonary surfactant. Influence of corticosteroids. Bull Eur Physiopathol Respir. 1984;20:105–11.

  30. 30.

    Fang Y, Wang S, Zhu T, et al. Atherogenic high cholesterol/high fat diet induces TLRs-associated pulmonary inflammation in C57BL/6J mice. Inflamm Res. 2017;66:39–47.

  31. 31.

    McNeish J, Aiello RJ, Guyot D, et al. High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1. Proc Natl Acad Sci USA. 2000;97:4245–50.

  32. 32.

    Richmond VL, Chi EY. Ultrastructural observations in copper-deficient guinea-pig lung cells. Int J Exp Pathol. 1993;74:133–43.

  33. 33.

    Bedrossian CW, Warren CJ, Ohar J, et al. Amiodarone pulmonary toxicity: cytopathology, ultrastructure, and immunocytochemistry. Ann Diagn Pathol. 1997;1:47–56.

  34. 34.

    Zhang M, Xie Y, Yan R, et al. Curcumin ameliorates alveolar epithelial injury in a rat model of chronic obstructive pulmonary disease. Life Sci. 2016;164:1–8.

  35. 35.

    Kumar RK, Truscott JY, Rhodes GC, et al. Type 2 pneumocyte responses to cyclophosphamide-induced pulmonary injury: functional and morphological correlation. Br J Exp Pathol. 1988;69:69–80.

  36. 36.

    Chung MJ, Lee KS, Franquet T, et al. Metabolic lung disease: imaging and histopathologic findings. Eur J Radiol. 2005;54:233–45.

  37. 37.

    Nicholson AG, Florio R, Hansell DM, et al. Pulmonary involvement by Niemann-Pick disease. A report of six cases. Histopathology. 2006;48:596–603.

  38. 38.

    Nakatani Y, Nakamura N, Sano J, et al. Interstitial pneumonia in Hermansky-Pudlak syndrome: significance of florid foamy swelling/degeneration (giant lamellar body degeneration) of type-2 pneumocytes. Virchows Arch. 2000;437:304–13.

  39. 39.

    Yang H, Rudge DG, Koos JD, et al. mTOR kinase structure, mechanism and regulation. Nature. 2013;497:217–23.

  40. 40.

    Kim JE, Chen J. regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes. 2004;53:2748–56.

  41. 41.

    Belvisi MG, Hele DJ. Peroxisome proliferator-activated receptors as novel targets in lung disease. Chest. 2008;134:152–7.

  42. 42.

    Varisco BM, Ambalavanan N, Whitsett JA, et al. Thy-1 signals through PPARγ to promote lipofibroblast differentiation in the developing lung. Am J Respir Cell Mol Biol. 2012;46:765–72.

  43. 43.

    Burgess HA, Daugherty LE, Thatcher TH, et al. PPARgamma agonists inhibit TGF-beta induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 2005;288:L1146–L1153.

  44. 44.

    Ferguson HE, Kulkarni A, Lehmann GM, et al. Electrophilic peroxisome proliferator-activated receptor-gamma ligands have potent antifibrotic effects in human lung fibroblasts. Am J Respir Cell Mol Biol. 2009;41:722–30.

  45. 45.

    Belvisi MG, Hele DJ, Birrell MA. Peroxisome proliferator-activated receptor gamma agonists as therapy for chronic airway inflammation. Eur J Pharmacol. 2006;533:101–9.

  46. 46.

    Minhajuddin M, Fazal F, Bijli KM, et al. Inhibition of mammalian target of rapamycin potentiates thrombin-induced intercellular adhesion molecule-1 expression by accelerating and stabilizing NF-kappa B activation in endothelial cells. J Immunol. 2005;174:5823–9.

  47. 47.

    Kezic A, Becker JU, Thaiss F. The effect of mTOR-inhibition on NF-κB activity in kidney ischemia-reperfusion injury in mice. Transplant Proc. 2013;45:1708–14.

  48. 48.

    Willemsen AE, Grutters JC, Gerritsen WR, et al. mTOR inhibitor-induced interstitial lung disease in cancer patients: comprehensive review and a practical management algorithm. Int J Cancer. 2016;138:2312–21.

  49. 49.

    Chen H, Jackson S, Doro M, et al. Perinatal expression of genes that may participate in lipid metabolism by lipid-laden lung fibroblasts. J Lipid Res. 1998;39:2483–92.

  50. 50.

    Malur A, Baker AD, McCoy AJ, et al. Restoration of PPARγ reverses lipid accumulation in alveolar macrophages of GM-CSF knockout mice. Am J Physiol Lung Cell Mol Physiol. 2011;300:L73–L80.

  51. 51.

    Guthmann F, Schachtrup C, Tölle A, et al. Phenotype of palmitic acid transport and of signalling in alveolar type II cells from E/H-FABP double-knockout mice: contribution of caveolin-1 and PPARgamma. Biochim Biophys Acta. 2004;1636:196–204.

  52. 52.

    Baker AD, Malur A, Barna BP, et al. Targeted PPAR{gamma} deficiency in alveolar macrophages disrupts surfactant catabolism. J Lipid Res. 2010;51:1325–31.

  53. 53.

    Reddy RC, Keshamouni VG, Jaigirdar SH, et al. Deactivation of murine alveolar macrophages by peroxisome proliferator-activated receptor-gamma ligands. Am J Physiol Lung Cell Mol Physiol. 2004;286:L613–L619.

  54. 54.

    Malur A, Mccoy AJ, Arce S, et al. Deletion of PPAR gamma in alveolar macrophages is associated with a Th-1 pulmonary inflammatory response. J Immunol. 2009;182:5816–22.

  55. 55.

    Xiao B, Xu J, Wang G, et al. Troglitazone-activated PPARγ inhibits LPS-induced lung alveolar type II epithelial cells injuries via TNF-α. Mol Biol Rep. 2011;38:5009–15.

  56. 56.

    Schachtrup C, Malcharek S, Haitsma JJ, et al. Activation of PPARgamma reverses a defect of surfactant synthesis in mice lacking two types of fatty acid binding protein. Biochim Biophys Acta. 2008;1781:314–20.

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The authors thank Ms. A. Ishikawa, Ms. N. Kuwahara, Ms. K. Wakamasu, Ms. M. Kataoka, and Mr. T. Arai for their expert technical assistance.


This work was supported by JSPS KAKENHI Grant Number JP15K19433 (NK) and by grants-in-aid for scientific research from the Diffuse Lung Diseases Research Group (YT).

Author information


  1. Department of Analytic Human Pathology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan

    • Nariaki Kokuho
    • , Yasuhiro Terasaki
    • , Shinobu Kunugi
    • , Hirokazu Urushiyama
    • , Mika Terasaki
    •  & Akira Shimizu
  2. Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan

    • Nariaki Kokuho
    • , Yoshinobu Saito
    • , Hiroki Hayashi
    •  & Akihiko Gemma


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Correspondence to Yasuhiro Terasaki.

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