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. 2012 Apr;38(3):124-34.
doi: 10.3109/01902148.2012.658148.

Telomerase deficiency does not alter bleomycin-induced fibrosis in mice

Amber L Degryse  1 , Xiaochuan C Xu, J Luke Newman, Daphne B Mitchell, Harikrishna Tanjore, Vasiliy V Polosukhin, Brittany R Jones, Frank B McMahon, Linda A Gleaves, John A Phillips 3rd, Joy D Cogan, Timothy S Blackwell, William E Lawson
Affiliations

Telomerase deficiency does not alter bleomycin-induced fibrosis in mice

Amber L Degryse et al. Exp Lung Res. 2012 Apr.

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by interstitial lung infiltrates, dyspnea, and progressive respiratory failure. Reports linking telomerase mutations to familial interstitial pneumonia (FIP) suggest that telomerase activity and telomere length maintenance are important in disease pathogenesis. To investigate the role of telomerase in lung fibrotic remodeling, intratracheal bleomycin was administered to mice deficient in telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC) and to wild-type controls. TERT-deficient and TERC-deficient mice were interbred to the F6 and F4 generation, respectively, when they developed skin manifestations and infertility. Fibrosis was scored using a semiquantitative scale and total lung collagen was measured using a hydroxyprolinemicroplate assay. Telomere lengths were measured in peripheral blood leukocytes and isolated type II alveolar epithelial cells (AECs). Telomerase activity in type II AECs was measured using a real-time polymerase chain reaction (PCR)-based system. Following bleomycin, TERT-deficient and TERC-deficient mice developed an equivalent inflammatory response and similar lung fibrosis (by scoring of lung sections and total lung collagen content) compared to controls, a pattern seen in both early (F1) and later (F6 TERT and F4 TERC) generations. Telomere lengths were reduced in peripheral blood leukocytes and isolated type II AECs from F6 TERT-deficient and F4 TERC-deficient mice compared to controls. Telomerase deficiency in a murine model leads to telomere shortening, but does not predispose to enhanced bleomycin-induced lung fibrosis. Additional genetic or environmental factors may be necessary for development of fibrosis in the presence of telomerase deficiency.

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Conflict of interest statement

DECLARATION OF INTEREST

All of the authors confirm that they have no competing or conflicting interests regarding the investigations and results outlined in this manuscript. All authors are employees of Vanderbilt University. In addition, Drs. Blackwell and Lawson are also supported by the Department of Veterans Affairs.

Figures

Figure 1
Figure 1
F1 TERC deficient and F1 TERT deficient mice had similar levels of lung fibrosis fibrosis following intratracheal bleomycin compared to their respective littermate controls. A) Semiquantitative scoring of lung fibrosis from 8–10 week old F1 TERC deficient (F1 TERC −/−) and F1 littermate wild type controls (F1 TERC +/+) at 3 weeks following 0.04 units of intratracheal bleomycin. N = 6 for F1 TERC −/− and 6 for F1 TERC +/+. Experiment survival was 6/6 for F1 TERC −/− and 6/6 for F1 TERC +/+. B) Semiquantitative scoring of lung fibrosis from 8–10 week old F1 TERT deficient (F1 TERT −/−) and F1 littermate wild type controls (F1 TERT +/+) at 3 weeks following 0.04 units of intratracheal bleomycin. N = 5 for F1 TERT −/− and 5 for F1 TERT +/+. Experiment survival was 5/5 for F1 TERT −/− and 5/5 for F1 TERT +/+. C) Semiquantitative scoring of lung fibrosis from 8–12 month old F1 TERC −/− and F1 TERC +/+ littermate controls at 2 weeks following 4 biweekly repetitive doses of 0.04 units of intratracheal bleomycin. N = 9 for F1 TERC −/− and 10 for F1 TERC +/+. Experiment survival was 9/14 for F1 TERC −/− and 10/14 for F1 TERC +/+.
Figure 2
Figure 2
F4 TERC deficient (F4 TERC −/−) and F6 TERT deficient (F6 TERT −/−) mice did not develop spontaneous lung fibrosis. A) Schematic illustrating breeding strategy to develop F4 TERC deficient and F6 TERT deficient mice. Trichrome blue stained lung sections from B) F4 TERC deficient, C) F6 TERT deficient, and D) wild type (WT) control mice. Magnification x200.
Figure 3
Figure 3
F4 TERC deficient, F6 TERT deficient, and wild type control mice had similar levels of lung fibrosis following intratracheal bleomycin. A–F) Trichrome blue stained lung sections from A,D) F4 TERC deficient, B,E) F6 TERT deficient, and C,F) wild type (WT) control mice. Magnification: A–C x40; D–F x200. G) Semiquantitative scoring of lung fibrosis from 8–10 week old F4 TERC deficient (F4 TERC −/−), F6 TERT deficient (F6 TERT −/−), and wild type (WT) control mice at 3 weeks following 0.04 units of bleomycin. N=11 for F4 TERC−/−, 11 for F6 TERT −/−, and 13 for WT. Experiment survival was 11/13 for F4 TERC−/−, 11/13 for F6 TERT −/−, and 13/15 for WT. H) Semiquantitative scoring of lung fibrosis from 8–10 week old F4 TERC −/−, F6 TERT −/−, and WT control mice at 3 weeks following 0.08 units of bleomycin. N=5 for F4 TERC −/−, 9 for F6 TERT −/−, and 7 for WT. Experiment survival was 5/8 for F4 TERC −/−, 9/12 for F6 TERT −/−, and 7/10 for WT. I) Semiquantitative scoring of lung fibrosis and J) total lung collagen content as assessed by a microplate hydroxyproline assay from 8–12 month old F4 TERC −/−, F6 TERT −/−, and WT control mice at 3 weeks following 0.04 units of bleomycin (bleo) or saline. N=9 for F4 TERC −/− saline treated, F6 TERT −/− saline treated, WT saline treated, and F4 TERC −/− bleomycin treated; 12 for F6 TERT −/− bleomycin treated; and 10 for WT bleomycin treated. Experiment survival post bleomycin was 9/10 for F4 TERC −/−, 12/13 for F6 TERT −/−, and 10/10 for WT. For collagen data in J, p<0.05 between individual genotype columns for saline versus bleo. K) Semiquantitative scoring of lung fibrosis from 8–10 week old F4 TERT −/− and WT controls at 2 weeks following 8 biweekly repetitive doses of 0.04 units of intratracheal bleomycin. N = 12 for F4 TERT −/− and 9 for WT. Experiment survival was 12/14 for F4 TERT −/− and 9/11 for WT.
Figure 3
Figure 3
F4 TERC deficient, F6 TERT deficient, and wild type control mice had similar levels of lung fibrosis following intratracheal bleomycin. A–F) Trichrome blue stained lung sections from A,D) F4 TERC deficient, B,E) F6 TERT deficient, and C,F) wild type (WT) control mice. Magnification: A–C x40; D–F x200. G) Semiquantitative scoring of lung fibrosis from 8–10 week old F4 TERC deficient (F4 TERC −/−), F6 TERT deficient (F6 TERT −/−), and wild type (WT) control mice at 3 weeks following 0.04 units of bleomycin. N=11 for F4 TERC−/−, 11 for F6 TERT −/−, and 13 for WT. Experiment survival was 11/13 for F4 TERC−/−, 11/13 for F6 TERT −/−, and 13/15 for WT. H) Semiquantitative scoring of lung fibrosis from 8–10 week old F4 TERC −/−, F6 TERT −/−, and WT control mice at 3 weeks following 0.08 units of bleomycin. N=5 for F4 TERC −/−, 9 for F6 TERT −/−, and 7 for WT. Experiment survival was 5/8 for F4 TERC −/−, 9/12 for F6 TERT −/−, and 7/10 for WT. I) Semiquantitative scoring of lung fibrosis and J) total lung collagen content as assessed by a microplate hydroxyproline assay from 8–12 month old F4 TERC −/−, F6 TERT −/−, and WT control mice at 3 weeks following 0.04 units of bleomycin (bleo) or saline. N=9 for F4 TERC −/− saline treated, F6 TERT −/− saline treated, WT saline treated, and F4 TERC −/− bleomycin treated; 12 for F6 TERT −/− bleomycin treated; and 10 for WT bleomycin treated. Experiment survival post bleomycin was 9/10 for F4 TERC −/−, 12/13 for F6 TERT −/−, and 10/10 for WT. For collagen data in J, p<0.05 between individual genotype columns for saline versus bleo. K) Semiquantitative scoring of lung fibrosis from 8–10 week old F4 TERT −/− and WT controls at 2 weeks following 8 biweekly repetitive doses of 0.04 units of intratracheal bleomycin. N = 12 for F4 TERT −/− and 9 for WT. Experiment survival was 12/14 for F4 TERT −/− and 9/11 for WT.
Figure 4
Figure 4
Lung inflammation as assessed by bronchoalveolar lavage (BAL) cell counts was similar among F4 TERC deficient (F4 TERC −/−), F6 TERT deficient (F6 TERT −/−), and wild type (WT) control mice at 2 weeks following bleomycin. Graphs depict total BAL A) leukocytes, B) macrophages, C) neutrophils, and D) lymphocytes. N = 7 for F4 TERC −/−, 5 for F6 TERT −/−, and 8 for WT.
Figure 5
Figure 5
Telomere lengths were shortened in F4 TERC deficient (F4 TERC −/−) and F6 TERT deficient (F6 TERT −/−) mice compared to wild type (WT) controls. A) Real time PCR telomere lengths from peripheral blood leukocytes. N = 14 for F4 TERC −/−, 13 for F6 TERT −/−, and 9 for WT. *p<0.01 compared to other columns. B) Real time PCR telomere lengths from isolated type II AECs. N = 3 for F4 TERC −/−, 4 for F6 TERT −/−, and 4 for WT. * p<0.01 compared to other columns.
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