ARTICLE Telomerase treatment prevents lung profibrotic pathologies associated with physiological aging Sergio Piñeiro-Hermida1, Chiara Autilio2, Paula Martı́nez1, Fátima Bosch3, Jesús Pérez-Gil2, and Maria A. Blasco1 Short/dysfunctional telomeres are at the origin of idiopathic pulmonary fibrosis (IPF) in patients mutant for telomere maintenance genes. However, it remains unknown whether physiological aging leads to short telomeres in the lung, thus leading to IPF with aging. Here, we find that physiological aging in wild-type mice leads to telomere shortening and a reduced proliferative potential of alveolar type II cells and club cells, increased cellular senescence and DNA damage, increased Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 fibroblast activation and collagen deposits, and impaired lung biophysics, suggestive of a fibrosis-like pathology. Treatment of both wild-type and telomerase-deficient mice with telomerase gene therapy prevented the onset of lung profibrotic pathologies. These findings suggest that short telomeres associated with physiological aging are at the origin of IPF and that a potential treatment for IPF based on telomerase activation would be of interest not only for patients with telomerase mutations but also for sporadic cases of IPF associated with physiological aging. Introduction Telomeres are protective structures at the ends of eukaryotic pulmonary disease, lung cancer, and idiopathic pulmonary fi- chromosomes that are essential for chromosome stability brosis (IPF; López-Otı́n et al., 2013; Meiners et al., 2015). (Blackburn, 2001). In mammals, telomeric DNA consists of IPF affects ∼3 million people worldwide with a median sur- TTAGGG repeats bound by a six-protein complex termed vival time from diagnosis of 2–4 yr (Martinez et al., 2017; “shelterin” (Liu et al., 2004; de Lange, 2005). Telomeres shorten Richeldi et al., 2017; Raghu et al., 2018). As the major risk factor due to the incomplete replication of DNA ends with each cell for IPF is age (Raghu et al., 2011; Faner et al., 2012; Selman and division, a phenomenon known as the “end-replication” prob- Pardo, 2014), its global incidence is predicted to increase in lem (Watson, 1972; Olovnikov, 1973). Telomere shortening can association with the worldwide phenomenon of demographic be compensated through the de novo addition of telomeric re- aging (Ley and Collard, 2013). In agreement with IPF being an peats onto chromosome ends by telomerase, a reverse tran- age-associated disease, IPF patients present most of the hall- scription composed of a catalytic subunit (TERT) and an RNA marks of aging including abnormal shortening of telomeres, component (Terc) used as a template for telomere elongation genomic instability, mitochondrial dysfunction, increased oxi- (Greider and Blackburn, 1985). Critically short telomeres with dative stress, and senescence (Meiners et al., 2015; Selman and aging are considered one of primary hallmarks of aging, since Pardo, 2014; Alder et al., 2008; Demopoulos et al., 2002; Bueno they can trigger other hallmarks of aging, such as genomic et al., 2015; Korfei et al., 2013). instability, cellular senescence, and apoptosis, mitochondrial Interestingly, between 8% and 15% of familial IPF cases dysfunction and loss of the regenerative capacity of tissues are associated with mutations in telomerase or in telomere- (Blasco, 2005; Blackburn et al., 2006; López-Otı́n et al., 2013). In protective proteins (Alder et al., 2008; Armanios et al., 2007; the case of the lungs, aging causes a progressive impairment of Tsakiri et al., 2007). In agreement with defects in telomere lung function, including structural and immunological alter- maintenance and accelerated telomere shortening being at the ations that can impair the respiratory function and increase the origin of these familial IPF cases, these patients show abnor- risk of lung diseases (Lowery et al., 2013; Brandenberger and mally short telomeres (Alder et al., 2008; Armanios et al., Mühlfeld, 2017). Indeed, aging is the main risk factor for de- 2007). Mutations in telomerase genes have been observed in veloping chronic lung diseases, such as chronic obstructive 1–3% of sporadic cases of pulmonary fibrosis (Tsang et al., 2012; ............................................................................................................................................................................. 1Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre, Madrid, Spain; 2Department of Biochemistry and Molecular Biology, Research Institute “Hospital 12 de Octubre (imas12),” Complutense University, Madrid, Spain; 3Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain. Correspondence to Maria A. Blasco: mblasco@cnio.es. © 2020 Piñeiro-Hermida et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1083/jcb.202002120 1 of 20 J. Cell Biol. 2020 Vol. 219 No. 10 e202002120 Armanios and Blackburn, 2012; Armanios, 2013). Even in the length in the alveolar parenchyma and bronchi by performing absence of telomerase mutations, IPF patients are reported to an immuno–telomere quantitative FISH (Q-FISH) to measure have shorter telomeres than healthy individuals (Alder et al., telomere fluorescence intensity and the prosurfactant protein C 2008). Moreover, genome-wide association studies revealed and CC10 antibodies as markers for ATII and club cells, re- that single nucleotide polymorphisms within the Tert gene in- spectively. Immuno–telomere Q-FISH was performed on lung crease the risk of lung fibrosis (Fingerlin et al., 2013; Codd et al., sections from both Tert+/+ and G3 Tert−/− mice at different ages to 2013). Thus, mutations in telomere maintenance genes are as- measure telomeres in ATII and club cells with aging (Povedano sociated with the clinical manifestations of IPF (Alder et al., et al., 2018; Canela et al., 2007; Flores et al., 2008). We deter- 2008; Armanios et al., 2007; Tsakiri et al., 2007; Fingerlin mined both mean telomere fluorescence (mean telomere spot et al., 2013; Cronkhite et al., 2008; Chibbar et al., 2010) and intensity) as an indication of the mean telomere length and the other respiratory pathologies (Snetselaar et al., 2015; Stanley percentage of short telomeres corresponding to the 20th per- et al., 2015). centile of the telomere fluorescence intensity values of controls Short telomeres have been previously shown by us and (5-wk-old Tert+/+ mice). We found a gradual decrease in mean others to impair stem cell function and tissue regeneration telomere fluorescence in ATII and club cells with increasing age (Martı́nez and Blasco, 2011; Flores et al., 2005; Sharpless and in the lungs of Tert+/+ mice (Fig. 1, C, D, and G). Interestingly, this Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 DePinho, 2007). In the case of the lung, stem cell and pro- decrease in mean telomere fluorescence in ATII and club cells genitor compartments include bronchial SCGB1A1+ club cells was already detectable at young age in G3 Tert−/− compared with and SFTPC (surfactant protein C)+ alveolar type II (ATII) cells age-matched Tert+/+ mice, and very old Tert+/+ mice (92–125 wk) (Rawlins et al., 2009; Kotton and Morrisey, 2014; Hogan et al., showed low telomere fluorescence similar to that of young 2014). In the past, we showed that induction of telomere dys- 5-wk-old G3 Tert−/− mice (Fig. 1, C, D, and F). Also, the percentage function specifically in ATII cells was sufficient to induce pro- of short telomeres in ATII and club cells was progressively gressive and lethal pulmonary fibrosis in mice, demonstrating increased with increasing age in Tert+/+ mice and was already that dysfunctional telomeres ATII cells are at the origin of IPF elevated in G3 Tert−/− mice at young ages compared with age- (Povedano et al., 2015). We further demonstrated that the matched Tert+/+ mice (Fig. 1, E and G). Together, these results presence of short telomeres in lung cells triggered IPF in demonstrate telomere shortening with age in the lungs of wild- telomerase-deficient mice upon treatment with a low dose of the type mice, reaching at old ages values similar to those of young lung-damaging agent bleomycin, which did not induce fibrosis G3 Tert−/− mice. in wild-type mice. Interestingly, treatment with a telomerase Next, we set out to determine whether telomere shortening gene therapy that activated telomerase in the lungs was suffi- with aging in Tert+/+ mice lead to defects in the lung homeo- cient to stop the progression of fibrosis in these mice (Povedano stasis. To this end, we quantified different lung cell types pre- et al., 2018). Together, evidence from human patients and mouse viously shown to be important for lung regeneration, such as models with short telomeres indicates that short/dysfunctional ATII cells (positive for the surfactant protein C–SFTPC marker) telomeres are at the origin of fibrosis. and club cells (positive for the secretoglobin 1A1-SCGB1A1 However, it remains unknown whether physiological aging marker; Giangreco et al., 2002; Rock et al., 2011; Guha et al., also leads to short telomeres in the lung and whether this in- 2012; Desai et al., 2014). We found that physiological aging creases the risk of IPF with aging. To address this, here we study leads to a progressive decrease both in the proportion of SFTPC- telomere length and lung phenotypes both in wild-type and positive ATII cells relative to the total number of alveolar cells telomerase-deficient mice with increasing age. Furthermore, we and in the number of SCGBA1-positive club cells per epithelium address whether telomerase gene therapy could delay or prevent length both in wild-type and G3 Tert−/− mice (Fig. 1, C, H, and I). lung profibrotic phenotypes associated with physiological aging Again, G3 Tert−/− mice already showed this reduction at young in wild-type and telomerase-deficient mice. ages compared with age-matched Tert+/+ mice, and at 42–49 wk, G3 Tert−/− mice showed a reduction in ATII cells and club cells that was equivalent to very old (92–125-wk-old) Tert+/+ mice Results (Fig. 1, C, H, and I). Physiological aging leads to decreased ATII and club cells in the lungs of wild-type mice, and this is anticipated in Aging leads to fibroblast activation and collagen deposits in telomerase-deficient mice wild-type lungs and this is anticipated in telomerase-deficient We first set out to address whether physiological aging in wild- lungs type mice leads to lung pathologies. To this end, we performed One of the phenotypes associated with lung degenerative pa- a full characterization of mouse lungs at different ages (5, thologies is the presence of fibrosis. Thus, we next set out to 8–9, 42–49, and 92–125 wk of age) in both wild-type and explore the presence of profibrotic phenotypes in both Tert+/+ third-generation (G3) telomerase-deficient mice with short te- and G3 Tert−/− mice associated with physiological aging. We lomeres (Blasco et al., 1997) as a control for lung pathologies found that both Tert+/+ and G3 Tert−/− mice showed a progressive associated with accelerated telomere shortening (Fig. 1, A and B). increment with age in the expression of vimentin, a marker of We could obtain very old (92–125 wk) Tert+/+ mice, but not G3 fibroblasts in the alveolar parenchyma (Fig. 2, A and B). At 42–49 Tert−/− mice, owing to the fact that the latter have a shortened wk of age, G3 Tert−/− mice showed a significantly higher per- life span (Herrera et al., 1999a). First, we measured telomere centage of vimentin-positive area per high-power field (HPF; Piñeiro-Hermida et al. Journal of Cell Biology 2 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 1. Telomerase deficiency and natural aging in the mouse lung leads to telomere shortening and reduction in the number of ATII and club cells. (A and B) Generation of Tert+/+ and G3 Tert−/− mice and scheme of lung tissue harvesting. (A) Heterozygous Tert+/− mice were crossed to obtain Tert+/+ and G1 Tert−/− mice, and successive crosses between G1 Tert−/− and then G2 Tert−/− mice were made to generate G3 Tert−/− mice. (B) Scheme of sacrifice and collection of lungs from Tert+/+ and G3 Tert−/− mice at different time points. (C) Representative images showing a Q-FISH for telomere spot fluorescence in ATII and club cells (Cy3Tel probe [red], SFTPC- and SCGB1A1-positive cells [green]) and nuclei stained with DAPI (blue; left and center right), SFTPC (brown, red arrowheads; center left), and SCGB1A1 (brown, green arrowheads; right) immunostainings in lung sections from Tert+/+ and G3 Tert−/− at different time points. (D–I) Quantification of mean telomere fluorescence (mean spot intensity; D and F), percentage of short telomeres in ATII and club cells corresponding to the 20th percentile of the fluorescence intensity values of controls (5-wk-old Tert+/+ mice; E and G), and SFTPC-positive ATII cells (%) and SCGB1A1-positive club cells per epithelium length (millimeters; H and I). Quantifications were performed on five different alveolar areas or bronchi in a random way. Data are Piñeiro-Hermida et al. Journal of Cell Biology 3 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 expressed as mean ± SEM (n = 4 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis, and Dunn–Sidak multiple comparison test). ns, not significant. 40× field) than age-matched Tert+/+ mice and similar to those telomerase activity. Next, we studied fibroblast activation of very old (92–125-wk-old) Tert+/+ mice (Fig. 2, A and B), by analyzing the peribronchial expression of the smooth suggesting anticipation of this phenotype in the absence of muscle actin (SMA) marker. We found that the percentage of Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 2. Premature aging by accelerated telomere shortening and natural aging in the mouse lung result in increased fibroblast presence and activation and collagen deposition. (A) Representative images showing vimentin (brown; left) and SMA (brown; center left) immunostainings and Sirius Red (red; center right) and Masson (blue; right) stainings in lung sections from Tert+/+ and G3 Tert−/− mice at different time points. (B–E) Quantification of vimentin (fibroblast marker)–positive area per HPF (%; B), peribronchial SMA (fibroblast activation marker) –positive area (%; C), alveolar collagen content per HPF (%; D), and peribronchial collagen content (%; E). Quantifications were performed on five different alveolar areas or bronchi in a random way. Data are expressed as mean ± SEM (n = 4 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis, and Dunn–Sidak multiple comparison test). ns, not significant. Piñeiro-Hermida et al. Journal of Cell Biology 4 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 peribronchial SMA-positive area gradually increased with age different lung cell types with aging in both Tert+/+ and G3 Tert−/− in Tert+/+ and G3 Tert−/− mice, with G3 Tert−/− mice showing mice. To this end, we performed double immunostainings of significantly more SMA positivity at 8–9 and 42–49 wk of age whole-lung sections with the proliferation marker Ki67 and the compared with age-matched Tert+/+ mice (Fig. 2, A and C). Again, cell-specific markers SFTPC (ATII cells), SCGB1A1 (club cells), very old Tert+/+ mice (92–125 wk of age) showed the same SMA and F4/80 (alveolar macrophages [AMs]). In addition, we also positivity as 42–49-wk-old G3 Tert−/− mice (Fig. 2, A and C). In performed double immunostainings with the differentiation agreement with increased peribronchial fibroblast activation, we marker SOX2 and SCGB1A1 (club cells) to detected differentia- also found increased collagen deposits in the peribronchial areas tion of club cells both in Tert+/+ and G3 Tert−/− mice at different (Masson’s trichrome staining) and alveolar areas (Sirius Red) ages (Fig. 4 A). We found a gradual reduction in proliferating with increasing age in both Tert+/+ and G3 Tert−/− mice, with ATII cells and club cells with increasing age in Tert+/+ mice, G3 Tert−/− mice showing an anticipation of the phenotype with a which at 92–125 wk of age reached similarly low levels as 42–49- similarly high percentage of alveolar and peribronchial collagen wk-old G3 Tert−/− mice (Fig. 4, A, B, and D). We obtained similar content in 42- to 49-wk-old G3 Tert−/− mice compared with very findings in the case of F4/80-positive AMs (Fig. 4, A and C). old (92–125-wk-old) wild-type mice (Fig. 2, A, D, and E). Interestingly, we also found that differentiation of club cells (SOX2-positive club cells) progressively increased with age in Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Aging leads to increased DNA damage, apoptosis, and both Tert+/+ and G3 Tert−/− lungs, with G3 Tert−/− lungs showing senescence in wild-type lungs, and this is anticipated in significantly more differentiated Sox2-positive club cells at 8–9 telomerase-deficient mice wk old and 42–49 wk old compared with wild-type mice, which Accumulation of cells with DNA damage is associated with ag- at very old age (92–125 wk old) had similarly high levels of Sox2- ing, as well as with the presence of short telomeres in mice positive cells as 42–49-wk-old G3 Tert−/− mice (Fig. 4, A and E). (López-Otı́n et al., 2013; Herrera et al., 1999b; Samper et al., To confirm Tert deficiency in G3 Tert−/− lungs, we analyzed 2001; Bernardes de Jesus et al., 2012). A marker for double- Tert mRNA expression levels in total lung extracts of young stranded DNA breaks and dysfunctional telomeres is phospho- (5 wk) and older (61–72 wk) Tert+/+ and G3 Tert−/− mice. As ex- rylation of γ-H2AX (D’adda et al., 2003; Takai et al., 2003). Here, pected, Tert mRNA expression was negligible in young and we set out to quantify γ-H2AX–positive cells in the lungs of old G3 Tert−/− mice compared with age-matched Tert+/+ mice Tert+/+ and G3 Tert−/− mice at different ages. We observed in- (Fig. 4 F). Additionally, since macrophages have been recognized creased γ-H2AX–positive cells per HPF with age in the alveolar to play a significant role in IPF pathogenesis (Zhang et al., 2018), parenchyma both in Tert+/+ and G3 Tert−/− mice, with G3 Tert−/− we assessed mRNA expression of markers of M1 (Tnf, Il1b, and mice showing an anticipation of the phenotype at all ages and Il6) and M2 (Il4, Il10, and Il13) macrophages in total lung extracts with 42–49-wk-old G3 Tert−/− mice reaching levels similar to from young and older Tert+/+ and G3 Tert−/− mice (Fig. 4, G–L). In very old (92–125-wk-old) wild-type mice (Fig. 3, A and B). Next, general, a significant increase in the expression of these markers we determined apoptosis in lung alveolar parenchyma as mea- was anticipated in G3 Tert−/− compared with Tert+/+ mice (Fig. 4, sured by presence of activated caspase-3 (C3)–positive cells. G–L). These results were supported by analysis of TNF, inter- Again, we found an increased number of C3-positive cells with leukin (IL)-6, IL-4, and IL-10 protein levels in lung homogenates age in both Tert+/+ and G3 Tert−/− mice, with G3 Tert−/− mice (Fig. 4, M–P). In general, protein levels of these cytokines were showing an anticipation of the phenotype at all ages and with 42- significantly increased in young and old G3 Tert−/− mice com- to 49-wk-old G3 Tert−/− mice reaching levels similar to very old pared with Tert+/+ mice. Remarkably only IL-10 protein levels (92–125-wk-old) wild-type mice (Fig. 3, A and C). increased with the age in both G3 Tert−/− and Tert+/+ mice (Fig. 4, Finally, we also measured presence of alveolar p16-positive M–P). cells, which are associated with senescence. Notably, we found an increase in p16-positive cells with age in the lungs of Tert+/+ and Impaired pulmonary surfactant activity in aged wild-type G3 Tert−/− mice (Fig. 3, A and D). Interestingly, both Tert+/+ and lungs is anticipated in telomerase-deficient mice G3 Tert−/− mice showed an increased number of senescent mac- We next set out to address the potential age-derived biophysical rophages (F4/80-p16–positive cells) with aging, with 42–49-wk- changes in lung surfactant activity in both Tert+/+ and G3 Tert−/− old G3 Tert−/− mice showing the same percentage of macrophages mice. To do so, we tested the biophysical properties of pulmo- positive for p16 as very old (92–125-wk-old) wild-type mice (Fig. 3, nary surfactant isolated from bronchoalveolar lavages in a cap- A and E). The percentages of alveolar senescent cells positive for tive bubble surfactometer (Autilio and Pérez-Gil, 2019). the macrophage marker F4/80 were 85.72% (8–9-wk-old Tert+/+ The adsorption and postexpansion adsorption (Fig. 5 A) into mice), 88.24% and 89.59% (42–49-wk-old Tert+/+ and Tert−/− mice, air–liquid interfaces of surfactant were both significantly better respectively), and 73.18% (92–125-wk-old Tert+/+ mice), suggesting in the youngest Tert+/+ mice (4–7 wk old) compared with G3 that most of the alveolar senescent cells were macrophages. Tert−/− mice of several ages. However, the difference in surface active properties disappeared with increasing age of Tert+/+ Aging leads to decreased proliferation of ATII and club cells in mice, from 23 wk on. Interestingly, 71–72-wk-old Tert+/+ mice wild-type lungs, and this is anticipated in the absence of showed a further impairment in surfactant properties during telomerase dynamic cycles when compared with younger Tert+/+ mice (4–7 or As we observed increased DNA damage and senescence with 23–34 wk old; Fig. 5 B). Once subjected to breathing-like con- aging in total lung cells, we next addressed proliferation of ditions, physiological aging affected the capability of surfactant to Piñeiro-Hermida et al. Journal of Cell Biology 5 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 3. Induced-telomere shortening and natural aging increase DNA damage, apoptosis, and macrophage senescence in the mouse lung. (A) Representative immunostainings for alveolar γ-H2AX (brown, green arrowheads; left), activated C3 (brown, orange arrowheads; center left), p16 (brown, blue arrowheads; center right), and F4/80 and p16 (purple and brown, respectively; red arrowheads indicate double F4/80-p16-positive AMs; right) in Tert+/+ and G3 Tert−/− mice at different time points. (B–D) Quantification of alveolar γ-H2AX–positive (B) and C3- positive (C), and p16-positive (D) cells, and double F4/80-p16-positive AMs (%; E). Quantifications were performed on five different alveolar areas in a random way. Data are expressed as mean ± SEM (n = 4 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis, and Dunn–Sidak multiple comparison test). ns, not significant. achieve γ values <9 mN/m under the conditions tested, leading to The content in surfactant proteins SP-B and SP-C also tended high relative area of compression (Fig. 5 B) and low stability of to decrease in G3 Tert−/− mice with increasing age (Fig. S1 B). As interfacial films (Fig. S1 A). This reduced surfactant activity was described for both healthy mice (Mahavadi et al., 2010) and anticipated in G3 Tert−/− mice that already showed an early neonates without lung disease (Autilio et al., 2020), the levels of worsening in the relative area of compression at 4–7 wk of age SP-B and SP-C at the extracellular lavage exhibit a substantial (Fig. 5 B). Similarly, at 23 wk of age, G3 Tert−/− mice presented a variability in the control group. Moreover, the extreme hydro- significantly higher minimum γ when compared with Tert+/+ phobicity of SP-B and SP-C and their strong association with mice of the same age (Fig. 5 B). lipids typically make their Western blot (WB) bands appear Piñeiro-Hermida et al. Journal of Cell Biology 6 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 4. Premature telomere shortening and natural aging reduce the proliferation of ATII cells, macrophages, and club cells and increase the differentiation of club cells. (A) Representative immunostainings for SFTPC (purple), F4/80(brown), and Ki67 (blue; orange and blue arrowheads indicate Piñeiro-Hermida et al. Journal of Cell Biology 7 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 double SFTPC-Ki67– and F4/80-Ki67–positive cells, respectively; left); SCGB1A1 (purple) and Ki67 (blue and green arrowheads indicate double SCGB1A1- Ki67–positive club cells; center), and SCGB1A1 (blue) and SOX2 (brown; red arrowheads indicate double SCGB1A1-SOX2–positive club cells; right) in Tert+/+ and G3 Tert−/− mice at different time points. (B–E) Quantification of SFTPC-Ki67–positive (B), F4/80-Ki67–positive (C), SCGB1A1-Ki67–positive (D), and SCGB1A1- SOX2–positive (E) cells. (F–L) Changes in total lung mRNA expression levels of Tert (F) and total lung mRNA expression of Tnf (G), Il1b (H), and Il6 (I; pro- inflammatory markers) and Il4 (J), Il10 (K), and Il13 (L; anti-inflammatory markers) normalized to 18S expression, and (M–P) protein levels of TNF (M), IL-6 (N), IL-4 (O), and IL-10 (P) assessed in lung homogenates from young (5 wk) and older (61–72 wk) Tert+/+ and G3 Tert−/− mice. Quantifications were performed on five different alveolar areas or bronchi in a random way. Data are expressed as mean ± SEM (n = 4 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis, and Dunn–Sidak multiple comparison test). ns, not significant. blurred, especially in the case of SP-C. In spite of this limitation, confirms a worsening in surfactant biophysical properties dur- we could detect a significantly lower amount of the two proteins ing physiological aging regardless of Tert presence, since sur- in the lung fluid at 23–34 wk of age, becoming only a trend when factant proteins are required to stabilize the respiratory surface 71–72-wk-old Tert+/+ and G3 Tert−/− mice were compared. This and facilitate breathing mechanics. Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 5. Physiological aging leads to impaired surfactant activity which is partially restored by telomerase gene therapy in both wild-type mice and telomerase-deficient mice. (A and B) Captive bubble results from Tert+/+ and G3 Tert−/− mice with increasing weeks of age. (C and D) Captive bubble results from 71–72-wk-old Tert+/+ and G3 Tert−/− mice treated with AAV9-empty or AAV9-Tert. (A and C) Top: Surface tension obtained 1 s after applying surfactant onto the air bubble (initial adsorption). Bottom: Surface tension obtained 1 s after the bubble expansion (postexpansion [PE] adsorption). (B and D) Top: Representative γ-relative area isotherms obtained under breathing-like dynamic cycles for each group tested. Bottom: Minimum and maximum γ along with the relative area of compression obtained during the 20th cycle. White bars represent Tert+/+ mice. Gray bars represent G3 Tert−/− mice. Data are expressed as mean ± SEM (n = 5–6 animals per group). *, P < 0.05; **, P < 0.01. Between different ages for each condition: One-way ANOVA with post hoc Dunn–Sidak multiple comparison test. Between different conditions within the same age: following a Shapiro–Wilk normality test, two-sample unpaired t test, or Mann–Whitney test when appropriate. γ, surface tension; max, maximum; min, minimum. Piñeiro-Hermida et al. Journal of Cell Biology 8 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Telomerase gene therapy prevents lung fibrotic-like decreased percentage of vimentin-positive area per HPF com- phenotypes in wild-type and telomerase-deficient mice pared with the corresponding cohorts treated with the null We previously showed that activation of telomerase in the vector (Fig. 7, A and B). We also detected a significant decrease in lung using adeno-associated serotype 9 vector (AAV9)–Tert gene fibroblast activation by reduced percentage of peribronchial therapy vectors was sufficient to stop the progression of lung SMA-positive area in both Tert+/+ and G3 Tert−/− mice treated fibrosis in a mouse model of lung fibrosis owing to the presence with AAV9-Tert compared with the corresponding cohorts of short telomeres and a sublethal dose of the lung-damaging treated with the null vector (Fig. 7, A and C). Finally, we also agent bleomycin (Povedano et al., 2018). The fact that we show observed a decreased percentage of alveolar collagen content per here that physiological aging leads to lung profibrotic pheno- HPF (Sirius Red) and peribronchial collagen content deposits types in wild-type mice that are anticipated in the context of (Masson’s trichrome staining) in both Tert+/+ and G3 Tert−/− telomerase-deficiency mice suggests that these phenotypes mice treated with AAV9-Tert compared with the corresponding originate from telomere shortening with aging and that telo- cohorts treated with the null vector (Fig. 7, A, D, and E). merase activation therapy should prevent or delay them. Thus, here we set out to test whether telomerase gene ther- Telomerase gene therapy prevents DNA damage, senescence, apy could delay or prevent lung profibrotic phenotypes associ- and apoptosis in aged wild-type and telomerase-deficient mice Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 ated with physiological aging in wild-type mice. As control, we lungs also treated with telomerase gene therapy telomerase-deficient Next, we determined the impact of AAV9-Tert gene therapy in mice with short telomeres. To this end, 27–30-wk-old Tert+/+ and rescuing DNA damage, apoptosis, and senescence in Tert+/+ and G3 Tert−/− mice received an intravenous injection of either G3 Tert−/− lungs treated with either AAV9-Tert or AAV9-null virus AAV9-Tert or AAV9-null virus particles, and these mice were particles (Fig. 8). We found that both Tert+/+ and G3 Tert−/− mice followed until their end point (week 21; Fig. 6 A). First, we treated with AAV9-Tert showed significantly decreased numbers measured total lung mRNA expression of Tert by quantitative of γ-H2AX–positive cells per HPF in the lung parenchyma com- PCR (qPCR) to confirm Tert overexpression in these mice. We pared with the corresponding cohorts treated with the null vector found that both Tert+/+ and G3 Tert−/− mice treated with AAV9- (Fig. 8, A and B), indicating decreased DNA damage upon telo- Tert showed significantly increased Tert mRNA levels 21 wk after merase treatment. Similarly, we detected significantly decreased treatment compared with the corresponding mice treated with numbers of activated caspase3-positive cells in the alveolar pa- the AAV9-null vectors (Fig. 6 B). Next, we determined telomere renchyma of both Tert+/+ and G3 Tert−/− lungs treated with AAV9- length in the alveolar parenchyma and bronchi of these mice using Tert compared with those treated with the null vector (Fig. 8, A immuno–telomere Q-FISH to evaluate the mean telomere fluores- and C). Interestingly, increased senescence as detected by p16- cence (mean telomere spot intensity) and the percentage of short positive cells specifically in the case of AMs was also rescued in telomeres in the indicated cell types. We found that both Tert+/+ and both Tert+/+ and G3 Tert−/− lungs treated with AAV9-Tert com- G3 Tert−/− mice treated with AAV9-Tert showed increased mean pared with those treated with the null vector (Fig. 8, A, D, and E). telomere fluorescence and significantly decreased percentage of Finally, by performing double immunostainings with the short telomeres in ATII and club cells compared with the corre- proliferation marker Ki67 and the specific markers SFTPC (ATII sponding mice treated with the AAV9-null vectors (Fig. 6, C–F and I). cells), SCGB1A1 (club cells), and F4/80 (AMs), we observed that Next, we set out to study whether AAV9-Tert treatment proliferation of ATII cells, club cells, and AMs was significantly rescued the numbers of ATII cells and club cells in the lung by increased in both Tert+/+ and G3 Tert−/− lungs treated with performing immunostainings with SFTPC and SCGB1A1, re- AAV9-Tert compared with those treated with the null vector spectively. We found that both Tert+/+ and G3 Tert−/− mice (Fig. 9, A–D). Interestingly, the number of SOX2-positive dif- treated with AAV9-Tert showed significantly increased numbers ferentiating club cells was also significantly reduced in Tert+/+ of ATII and club cells (Fig. 6, G–I), indicating a recovery of lung and Tert−/− lungs upon telomerase gene therapy (Fig. 9, A and E). cellularity. The increased number of ATII cells in wild-type mice Finally, to address whether treatment with telomerase gene upon treatment with AAV9-Tert was confirmed by increased therapy also prevented expression of proinflammatory and anti- transcriptional levels of different surfactant genes produced by inflammatory markers, we determined mRNA expression of Tnf, ATII cells, including Sftpa1, Sftpb, and Sftpd, compared with Il1b, Il6, Il4, Il10, and Il13 in total lung extracts from Tert+/+ and AAV9-null–treated wild-type mice (Fig. 6, J–L). In the case of G3 Tert−/− mice (Fig. 9, F–K). We observed significantly de- G3 Tert−/− mice treated with AAV9-Tert, we observed a tendency creased expression of these markers in both Tert+/+ and toward an increase in the expression of these genes, but this was G3 Tert−/− mice treated with AAV9-Tert compared with those not statistically significant (Fig. 6, J–L). treated with the null vector (Fig. 9, F–K). These findings were also supported by decreased protein levels of TNF, IL-6, IL-4, and Telomerase gene therapy prevents fibrotic-like phenotypes in IL-10 in lung homogenates from Tert+/+ and G3 Tert−/− mice upon aged wild-type and telomerase-deficient mice treatment with AAV9-Tert (Fig. 9, L–O). Next, we set out to address whether treatment with telomerase gene therapy also prevented fibrotic phenotypes with aging in Telomerase gene therapy prevents age-related impairment of both Tert+/+ and G3 Tert−/− mice. We found that both Tert+/+ and surfactant activity in wild-type and telomerase-deficient mice G3 Tert−/− mice treated with AAV9-Tert showed significantly Finally, we addressed whether treatment with telomerase gene decreased numbers of fibroblasts in the lung, as indicated by a therapy improves age-related pulmonary surfactant inactivation Piñeiro-Hermida et al. Journal of Cell Biology 9 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 6. Telomerase gene therapy in the mouse lung results in telomere elongation and increased number of ATII and club cells. (A) Experimental protocol for the administration of AAV9-Tert and AAV9-empty virus. 27–30-wk-old Tert+/+ and G3 Tert−/− mice were administered with 2E12vg of AAV9-Tert Piñeiro-Hermida et al. Journal of Cell Biology 10 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 and AAV9-empty virus by intravenous tail injection. Animals were sacrificed 21 wk later for lung tissue harvesting. (B) Total lung mRNA levels of Tert nor- malized to 18S expression in AAV9-empty– and AAV9-Tert–treated animals. (C–H) Quantification of mean telomere fluorescence (mean spot intensity; C and E), percentage of short telomeres in ATII and club cells corresponding to the 20th percentile of the fluorescence intensity values of controls (5-wk-old Tert+/+ mice; D and F), and SFTPC-positive ATII cells (%) and SCGB1A1-positive club cells per epithelium length (millimeters) in AAV9-Tert– and AAV9-empty–treated mice (G and H). (I) Representative images showing a Q-FISH for telomere spot fluorescence in ATII and club cells (Cy3Tel probe [red], SFTPC- and SCGB1A1- positive cells [green], and nuclei stained with DAPI [blue]; left and center right), and SFTPC (brown, red arrowheads; center left) and SCGB1A1 (brown, green arrowheads; right) immunostainings in lung sections from Tert+/+ and G3 Tert−/− mice treated with the AAV9-Tert (upper panels) or AAV9-empty (lower panels) virus. (J–L) Changes in total lung mRNA expression levels of the surfactant markers Sftpa1, Sftpc, and Sftpd normalized to 18S expression. Quantifications were performed on five different alveolar areas or bronchi in a random way. Data are expressed as mean ± SEM (n = 4 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis, and Dunn–Sidak multiple comparison test). ns, not significant. in both Tert+/+ and G3 Tert−/− mice. To that end, we compared the amount of SP-B in Tert+/+ mice (Fig. S2 B), with a tendency to activity of surfactant obtained from 71- to 72-wk-old G3 Tert−/− decrease SP-C content. This may be due to a therapy-mediated and Tert+/+ mice treated with AAV9-empty or AAV9-Tert. We rebalance of surfactant homeostasis that is more evident when found that telomerase gene therapy significantly improved endogenous Tert is also present. Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 surfactant interfacial properties of old mice. G3 Tert−/− mice treated with AAV9-Tert showed an enhanced surfactant ad- sorption with reduced relative area of compression during dy- Discussion namic cycles (Fig. 5, C and D) when compared with the animals In spite of the recent introduction of two new antifibrotic drugs treated with AAV9-empty. Similarly, surfactant from Tert+/+ mice for the treatment of IPF, pirfenidone and nintedanib, there is treated with telomerase exhibited a significant decrease in relative still no cure for IPF, a devastating chronic progressive interstitial area of compression (Fig. 5 D) compared with AAV9-empty group. lung disease with a poor prognosis, with a median survival from Interestingly, telomerase gene therapy did not change SP-B the time of diagnosis of <4 yr (Richeldi et al., 2017; Martinez and SP-C content in G3 Tert−/− mice but significantly reduced the et al., 2017; Raghu et al., 2018; Somogyi et al., 2019). Thus, Figure 7. AAV9-Tert treatment reduces fibroblast presence and activation and collagen deposition in the mouse lung. (A) Representative images of vimentin (brown; left) and SMA (brown; center left) immunostainings and Sirius Red (red; center right) and Masson (blue; right) stainings in lung sections from Tert+/+ and G3 Tert−/− mice treated with the AAV9-Tert (upper panels) or AAV9-empty (lower panels) virus. (B–E) Quantification of vimentin-positive (fibroblast marker) area per HPF (%; B), peribronchial SMA-positive (fibroblast activation marker) area (%; C), alveolar collagen content per HPF (%; D), and peribronchial collagen content (%; E). Quantifications were performed on five different alveolar areas or bronchi in a random way. Data are expressed as mean ± SEM (n = 4 animals per group). **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis and Dunn–Sidak multiple comparison test). ns, not significant. Piñeiro-Hermida et al. Journal of Cell Biology 11 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 8. Telomerase gene therapy ameliorates DNA damage, apoptosis, and macrophage senescence in the mouse lung. (A) Representative im- munostainings for alveolar γ-H2AX (brown, green arrowheads; left), activated C3 (brown, orange arrowheads; center left), p16 (brown, blue arrowheads; center right), and F4/80 and p16 (purple and brown, respectively; red arrowheads indicate double F4/80-p16–positive AMs; right) in lungs of Tert+/+ and G3 Tert−/− mice treated with the AAV9-Tert (upper panels) or AAV9-empty (lower panels) virus. (B–E) Quantification of alveolar γ-H2AX–positive (B), C3-positive (C), and p16-positive (D) cells and double F4/80-p16–positive AMs (%; E). Quantifications were performed on five different alveolar areas in a random way. Data are expressed as mean ± SEM (n = 4–5 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis and Dunn–Sidak multiple comparison test). ns, not significant. additional hypotheses on the origin of IPF are necessary to drive 2014). Telomere shortening with age is considered one of the new, more effective therapeutic approaches. Indeed, pirfeni- primary hallmarks of aging, and the presence of critically short done and nintedanib show anti-inflammatory and antifibrotic telomeres owing to mutations in telomerase genes is associated activity and slow IPF progression, but they are not curative with familial cases of IPF (Alder et al., 2008; Armanios et al., (Ahluwalia et al., 2014; King et al., 2014; Hunninghake, 2014; 2007; Tsakiri et al., 2007; Fingerlin et al., 2013; Cronkhite et al., Karimi-Shah and Chowdhury, 2015). We have recently demon- 2008; Chibbar et al., 2010). A role for short/dysfunctional telo- strated that AAV9-Tert therapy prevents the progression of meres at the origin of IPF is also supported by genetically pulmonary fibrosis in a mouse model of pulmonary fibrosis modified mouse models with mutations in telomerase or telo- owing to presence of short telomeres (Povedano et al., 2018), thus mere maintenance genes (Povedano et al., 2015; Liu et al., 2018). demonstrating that short telomeres are at the origin of pulmonary In particular, we previously showed that a low bleomycin dose fibrosis in mice and that elongation of short telomeres by telo- that does not induce pulmonary fibrosis in wild-type mice merase can prevent progression of the disease. Remarkably, we synergizes with short telomeres to trigger full-blown progress- did not find any deleterious effects of AAV9-Tert gene therapy in ive pulmonary fibrosis in telomerase-deficient mice (Povedano mice. In particular, we previously reported that AAV9-Tert gene et al., 2015). Similarly, induction of acute telomere dysfunction therapy resulted in a significant increase in median and maxi- by abrogation of an essential telomere-protective protein, TRF1, mum life span accompanied by an amelioration of various age- specifically in ATII cells of the lung, is sufficient to induce associated phenotypes, including delayed cancer (Bernardes de progressive and lethal IPF in mice (Povedano et al., 2015). Thus, Jesus et al., 2012). Moreover, we recently demonstrated AAV9- both in human patients and in mouse models, short/dysfunc- Tert therapy did not lead to an increase in cancer incidence or tional telomeres can induce IPF. Furthermore, ATII cells have earlier onset of cancer in mice (Muñoz-Lorente et al., 2018). been identified as a relevant cell type at the origin of the disease In this regard, cellular aging is likely to be at the origin of IPF, (Povedano et al., 2015). as suggested by the fact that the incidence of IPF increases with To date, however, it remains uncertain whether physiological age (Raghu et al., 2011; Faner et al., 2012; Selman and Pardo, aging also leads to significant telomere shortening in the lungs Piñeiro-Hermida et al. Journal of Cell Biology 12 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure 9. Treatment with AAV9-Tert restores the proliferation of AT II cells, macrophages, and club cells and reduces the differentiation of club cells. (A) Representative immunostainings for SFTPC (purple), F4/80 (brown), and Ki67 (blue; orange and blue arrowheads indicate double SFTPC-Ki67– and F4/80-Ki67–positive cells, respectively; left); SCGB1A1 (purple) and Ki67 (blue; green arrowheads indicate double SCGB1A1-Ki67–positive club cells; center); and SCGB1A1 (blue) and SOX2 (brown; red arrowheads indicate double SCGB1A1-SOX2–positive club cells; left) in lungs of Tert+/+ and G3 Tert−/− mice treated with the AAV9-Tert (upper panels) or AAV9-empty (lower panels). (B–E) Quantification of SFTPC-Ki67–positive (B), F4/80-Ki67–positive (C), SCGB1A1- Ki67–positive (D), and SCGB1A1-SOX2–positive (E) cells. (F–K) Changes in total lung mRNA expression levels of Tnf (F), Il1b (G), and Il6 (H; proin- flammatory markers) and Il4 (I), Il10 (J), and Il13 (K; anti-inflammatory markers) normalized to 18S expression and (L–O) protein levels of TNF (L), IL-6 (M), IL-4 (N), and IL-10 (O) assessed in lung homogenates from AAV9-empty– and AAV9-Tert–treated animals. Quantifications were performed on five different alveolar Piñeiro-Hermida et al. Journal of Cell Biology 13 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 areas or bronchi in a random way. Data are expressed as mean ± SEM (n = 4–5 animals per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Shapiro–Wilk, Kruskal–Wallis, and Dunn–Sidak multiple comparison test). ns, not significant. and could be at the origin of a number of cases of sporadic IPF. In Pardo and Selman, 2017; Campisi, 2013). In particular, the effects this regard, patients with sporadic IPF show shorter telomeres observed on ATII and club cells in Tert−/− mice could be self- than normal healthy individuals (Tsang et al., 2012; Armanios autonomous and/or a consequence of a different cellular envi- and Blackburn, 2012; Armanios, 2013), and mice also show ronments. Indeed, senescent cells secrete inflammatory cyto- shorter telomeres in the lungs with increasing age (Birch et al., kines, chemokines, growth factors, and remodeling proteins that 2015). Here, we report that physiological aging in wild-type mice have autocrine and paracrine effects on the tissue microenvi- leads to telomere shortening in SCGB1A1-positive club cells ronment (Acosta et al., 2008; Kuilman et al., 2008; Parikh et al., and SFTPC-positive ATII cells, a phenomenon that is anticipated 2019). As previously described by us, telomerase-deficient mice in telomerase-deficient mice (Tert−/− mice). In addition, we ob- secret higher cytokine levels than wild-type mice (Mosteiro serve both reduced numbers and reduced proliferation of club et al., 2016). cells and ATII cells with aging in wild-type mice, and again It is of particular interest that the majority of senescent cells Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 this phenotype is anticipated in telomerase-deficient mice. were found to be AMs that have a crucial role in maintaining the These findings suggest that telomere shortening associated with correct surfactant homeostasis, preventing the accumulation of physiological aging can impair the stemness of bronchial club already used surfactant at the air–liquid interface with a limited cells and ATII cells, which was previously shown to have epi- biophysical performance (Autilio and Pérez-Gil, 2019). An im- thelial stem/progenitor properties (Giangreco et al., 2002; pairment of surfactant activity has been already proposed as an Rawlins et al., 2009; Rock et al., 2011; Guha et al., 2012; Hogan early marker of fibrosis in a model of bleomycin-induced fi- et al., 2014; Kotton and Morrisey, 2014; Desai et al., 2014). Thus, brosis in mice (Lopez-Rodriguez et al., 2016). It is therefore these results presented here support the notion that telomere conceivable that the intrinsic deficiency in operative surfactant, shortening with aging can impair the ability of stem cells to both as a consequence of deactivated material that is not prop- regenerate tissues (Blasco, 2007; Flores et al., 2008). erly cleared by aged macrophages and the lack of enough Further supporting this, we find here a significant increase of amounts of newly synthetized surfactant by a reduced popula- differentiating SOX2-positive club cells with aging, which again tion of type II cells, leads to a progressively impaired pulmonary was anticipated in the telomerase-deficient mice. Sox2 has been mechanics contributing ultimately to reduction in lung tissue previously shown a marker of differentiation of club cells elasticity and its abnormal remodeling. (Tompkins et al., 2009), which is increased in bleomycin- Of relevance, we detected significantly increased collagen induced pulmonary fibrosis in mice (Chuang et al., 2018), as deposits with increasing age in wild-type lungs suggestive of well as in patients suffering from IPF (Plantier et al., 2011). early stages of IPF, which was anticipated in telomerase-deficient Interestingly, we also find here that mouse aging results in a mice, again highlighting the role of short telomeres in the origin reduced proliferation of the lung resident AMs and increased of these pathological findings. This is interesting, because pre- expression of M1 and M2 macrophage markers. Tissue-resident vious studies have failed to detect lung fibrosis in the telomerase- AMs are the most numerous immune cells present in the lung deficient mouse model (Liu et al., 2007; Lee et al., 2009; Degryse and have a key role in lung homeostasis by protecting the lung et al., 2012). during tissue damage (Higashimoto et al., 1993; Boyd et al., 2012; Together, these molecular, cellular, biophysical, and patho- Hussell and Bell, 2014; Byrne et al., 2015; Wong et al., 2017; logical findings support that telomere shortening associated Angelidis et al., 2019). Specifically, M1 macrophages contribute with wild-type mouse aging could be at the origin of fibrotic-like to tissue injury after excessive production of proinflammatory pathologies in mice. Here, we further demonstrate this hy- mediators (e.g., TNF, IL-1B, and IL-6), and M2 macrophages lead pothesis by rescuing these lung phenotypes at the molecular, to resolution of inflammation and tissue repair upon anti- cellular, biophysical, and pathological levels using an AAV9- inflammatory cytokine activation (e.g., IL-4, IL-10, and IL-13; based telomerase (Tert) gene therapy strategy, which has been Zhang et al., 2018; Liu et al., 2019). M2 macrophages were re- previously shown by us to transduce lung cells, including ATII ported to promote myofibroblast differentiation and are asso- cells, and elongate telomeres (Povedano et al., 2018), as well as to ciated with pulmonary fibrogenesis (Hou et al., 2018). In this block the progression of IPF in a mouse model of IPF which sense, overexpression of IL-6 and IL-10 were reported to impact combined presence of short telomeres owing to telomerase de- the polarization of profibrotic M2 macrophages (Sun et al., 2011; ficiency and damage to the lungs using a low dose of bleomycin Ayaub et al., 2017). In addition, IL-4 and IL-13 are required for that did not induce IPF in wild-type mice (Povedano et al., 2015). the maintenance of pulmonary fibrosis (Jakubzick et al., 2003). In particular, treatment of old wild-type mice with AAV9-Tert In line with shorter telomeres, wild-type mouse lungs show rescued the proliferative defects of club cells, ATII cells, and increased DNA damage, senescence, and apoptosis, together resident AMs and rescued senescence, apoptosis, DNA damage, with an impairment in surfactant biophysical activity with in- and pulmonary surfactant activity, as well as all signs of fibrosis, creasing age, and this is anticipated in telomerase-deficient in aged wild-type mice. mice. Previous authors have also suggested a role for senes- In summary, here we demonstrate that telomerase gene cence in IPF (Selman and Pardo, 2014; Spagnolo et al., 2015; therapy prevents the onset of lung degenerative and profibrotic Piñeiro-Hermida et al. Journal of Cell Biology 14 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 pathologies associated with physiological aging in wild-type and Lung tissue collection and preparation telomerase-deficient mice. These findings contribute to a better Before tissue collection, animals were euthanized by intraperi- understanding of the importance of TERT as a potential target toneal injection of 10 µl/g of a ketamine/xylazine anesthetic for future therapeutic approaches in IPF, especially in patients in combination in saline (100:10 mg/kg, respectively). Following whom pulmonary fibrosis is associated with short telomeres. dissection, lungs from Tert+/+ and G3 Tert−/− mice of the natural aging groups were harvested, fixed in 4% formaldehyde, and subsequently embedded in paraffin for histopathology and im- Materials and methods munohistochemistry. In the case of Tert+/+ and G3 Tert−/− mice Mice and ethical statement destined for the telomerase gene therapy experiments, only the Tert heterozygous mice were generated as previously described lefts lungs were processed for histopathology and immunohis- (Liu et al., 2000) and backcrossed to >98% C57/BL6 background. tochemistry, and right lung lobes were separated and snap fro- Tert+/− mice were intercrossed to generate Tert+/+ and first- zen in liquid nitrogen and stored at −80°C for qPCR. generation (G1) homozygous Tert−/− knockout mice. Second- generation (G2) Tert−/− mice were generated by successive Telomere Q-FISH analysis breeding of G1 Tert−/− and then G3 Tert−/− mice by crosses be- After deparaffinization and rehydration, tissues underwent Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 tween G2 Tert−/− mice (Fig. 1 A). Finally, Tert+/+ and G3 Tert−/− antigen retrieval in 10 mM sodium citrate buffer, and per- mice were used for aging experiments for which lungs of these meabilization was performed in PBS 0.5% Triton X-100 for 3 h. mice where collected at different time points (Fig. 1 B). These Next, tissues were washed three times for 5 min each in PBS 1X, mice were also used for telomerase gene therapy assays (Fig. 6 fixed in 4% formaldehyde for 5 min, washed three times for A). Experimental groups were composed by male (70%) and 5 min each in PBS, and dehydrated in a 70–90%–100% ethanol female (30%) mice in the case of molecular, cellular and path- series (5 min each). Following air drying the slides, 30 µl of the ological characterization. In addition, only male mice were used telomere probe mix (10 mM TrisCl, pH 7.2, 25 mM MgCl2, 9 mM for the biophysical characterization. It should be noted that no citric acid, 82 mM Na2HPO4, 50% deionized formamide [Sigma- significant differences were found between sexes in any of the Aldrich], 0.25% blocking reagent [Roche], and 0.5 µg/ml Telo- experiments performed (data not shown). All animals were bred meric PNA probe [Panagene]) was added, and slides were and maintained under specific pathogen–free conditions in incubated for 3 min at 85°C and then 1 h at room temperature in laminar flow caging at the Spanish National Cancer Centre an- a wet chamber in the dark. Slides were washed twice for 15 min imal facility in accordance with the recommendations of the each in 10 mM TrisCl (pH 7.2) and 0.1% BSA in 50% formamide Federation of European Laboratory Animal Science Associations. and then three times for 5 min each in TBS 0.08% Tween 20. The All animal procedures were approved by the CNIO Institutional tissues were then washed three times for 5 min each in PBS 0.1% Animal Care and Use Committee and the Ethics Committee for Triton X-100, blocked with 10% donkey serum in PBS 0.1% Research and Animal Welfare. Triton X-100 for 1 h, and incubated overnight at 4°C with rabbit prosurfactant protein C (AB3786, 1:200; EMD Millipore) and Viral particle production and treatment mouse CC10 (clone E-11, sc-365992, 1:200; Santa Cruz Biotech- Viral vectors were generated by triple transfection of HEK293 nology) antibodies. After three washes with PBS 0.1% Triton X- cells and purified as previously described (Matsushita et al., 100, tissues were incubated with secondary antibodies for 1 h, 1998; Ayuso et al., 2010). Cells were cultured in roller bottles washed three times for 5 min each in PBS 1X, and then mounted (Corning) in DMEM supplemented with 10% FBS to 80% con- with Vectashield mounting media with DAPI (Vector Laborato- fluence and cotransfected with a plasmid carrying the expres- ries). Confocal images (40× magnification, 2× zoom, NA 1.25) sion cassette flanked by the AAV2 viral inverted terminal were acquired as stacks using an SP5-WLL confocal microscope repeats, a helper plasmid carrying the AAV rep2 and cap9 genes, (Leica Microsystems), and maximum projection images were and a plasmid carrying the adenovirus helper functions (plas- created with the LAS AF 2.7.3.9723 software. Telomere signal mids kindly provided by K.A. High, Children’s Hospital of intensity was quantified using Definiens Developer Cell software Philadelphia, Philadelphia, PA). The expression cassettes were version XD 64 2.5. For each analysis, three visual fields per under the control of the cytomegalovirus promoter and con- section were scored. tained a SV40 polyA signal for eGFP, and the cytomegalovirus promoter and the 39-untranslated region of the Tert gene as Histopathological analyses and immunostaining polyA signal for Tert. AAV9 particles were purified using two Paraffin-embedded lungs were cut into 3-µm sections for his- cesium chloride gradients, dialyzed against PBS, filtered, and topathological evaluation or immunohistochemistry. Sirius Red stored at −80°C until use. Then, 27–30-wk old Tert+/+ and (Sigma-Aldrich) and Masson’s trichrome (Dako) stainings were G3 Tert−/− mice were administered with 2E12vg of AAV9-Tert performed following manufacturer guidelines for the quantifi- and AAV9-empty virus particles in a volume of 100 µl 0.001% cation of alveolar and peribronchial collagen content (%). Rabbit Pluronic F-68 in PBS 1X by a single intravenous tail injection vimentin (clone D21H3, 5741, 1:50; Cell Signaling Technology) (Fig. 6 A). Pluronic F-68 was used to prevent aggregations of the and mouse SMA (clone 1A4, IR611, 1:4; Dako and Agilent virus particles (Foust et al., 2009). Animals were then sacrificed Technologies) antibodies were used for the determination of 21 wk after the administration of the AAV9-Tert and AAV9- vimentin and peribronchial SMA-positive areas (%). Rabbit empty virus particles. prosurfactant protein C (AB3786, 1:100; EMD Millipore) and goat Piñeiro-Hermida et al. Journal of Cell Biology 15 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 CC10 (clone T-18, sc-9772, 1:1,000; Santa Cruz Biotechnology) AACGAAGAACA-39 and Il4-R, 59-CGAAAAGCCCGAAAGAGTC-39 antibodies were used to determine the number of SFTPC- (GenBank accession no. NM_021283.2); Il10-F, 59-GCACTACCA positive cells to total cell numbers (%) and the number of AAGCCACAAGG-39 and Il10-R, 59-TAAGAGCAGGCAGCATAGCA-39 SCGB1A1-positive cells per epithelium length (millimeters). Rat (GenBank accession no. NM_ 010548.2); Il13-F, 59-GCCTCCCCG F4/80 antibody (clone A3-1, MCA497, 1:20; AbD Serotec, and Bio- ATACCAAAAT-39 and Il13-R, 59-CTTCCTCCTCAACCCTCCTC-39 Rad) was used to determine the number of F4/80-positive (GenBank accession no. NM_008355.3); Sftpa1-F, 59-CCATCG macrophages. Mouse phospho-histone H2A.X (Ser139; clone CAAGCATTACAAAG-39 and Sftpa1-R, 59-CACAGAAGCCCCATC JBW301, 05–636, 1:20; EMD Millipore), rabbit cleaved C3 CAG-39 (GenBank accession no. NM_023134.4); Sftpb-F, 59-CTG (Asp175; 9661, 1:300; Cell Signaling Technology), and rat p16 CTGCTTCCTACCCTCTG-39 and Sftpb-R, 59-ATCCTCACACTCTTG (clone 33B; CNIO Monoclonal Antibodies Core Unit, Madrid, GCACA-39 (GenBank accession no. NM_147779.2); Sftpd-F, 59- Spain) antibodies were used to assess the level of DNA damage TGAGAATGCTGCCATACAGC-39 and Sftpd-R, 59-GAATAGACC (%) of SFTPC-positive ATII cells (SFTPC/H2AX) and the apo- AGGGGCTCTCC-39 (GenBank accession no. NM_009160.2); ptosis and senescence rates (%) of SFTPC-positive ATII cells and Rn18s-F, 59-ATGCTCTTAGCTGAGTGTCCCG-39 and Rn18s-R, 59- F4/80-positive macrophages (SFTPC/F4/80/C3, SFTPC/p16, and ATTCCTAGCTGCGGTATCCAGG-39 (GenBank accession no. NR_ F4/80/p16). Rabbit Ki-67 (clone D3B5, 12202, 1:50; Cell Signaling 003278.3). Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Technology) and rabbit SOX2 (clone C70B1, 3728, 1:75; Cell Sig- naling Technology) antibodies served to assess the level of pro- Pulmonary surfactant isolation and activity liferation (%) of SFTPC- and SCGB1A1-positive ATII and club cells Surfactant was collected from bronchoalveolar lavages of mice and F4/80-positive macrophages (SFTPC/F4/80/Ki67 and lungs as described before (Autilio et al., 2018). In detail, bron- SCGB1A1/Ki67) and the degree of differentiation of SCGB1A1- choalveolar lavage supernatant was ultracentrifuged (100,000 g, positive club cells (SCGB1A1/SOX2). All images (40× magnifica- 4°C, 1 h) and diluted with 5 mM Tris buffer containing 150 mM tion, 2× optical zoom, NA 1.20) were acquired using the optical NaCl (5 mM; pH 7; Sigma-Aldrich) to reach a concentration of microscope Provis AX70 (Olympus) connected to a camera 10 mg/ml of phosphatidylcholine (PC). PC was tested in tripli- Olympus PM-C35DX. Olympus cellSens Entry 1.18 software was cate using an enzymatic colorimetric method (Spinreact; St. used to acquire images. Histological quantifications were per- Esteve de Bas). formed on five different alveolar areas or five different bronchi The interfacial activity of surfactant was measured in a in each case in a random way. The Fiji open-source image pro- captive bubble surfactometer as described before (Schürch et al., cessing software package (http://fiji.sc) was used to quantify 2010) upon injection of 300 nl surfactant onto the surface of the total cell numbers, epithelium length measurements, and the air bubble. Initial adsorption was measured by monitoring the percentage of DAB (color deconvolution), Sirius Red, and Mas- decay in surface tension (γ) during the first 5 min after sur- son´s trichrome positive areas. factant injection and postexpansion adsorption during the 5 min after expansion of the bubble volume from 0.05 to 0.15 ml. Then, RNA isolation, reverse transcription, and qPCR the bubble was subjected to rapid (30 cycles/min) physiological- Inferior lung lobes from AAV9-Tert or AAV9-empty injected like compression–expansion dynamics, changing its volume by animals were homogenized in TRIzol (Invitrogen), and total ∼25%, and the corresponding isotherms were built upon rep- RNA was treated with 2.72 KU/µl RNase-free DNase (Qiagen), resentation of γ versus relative area of the bubble. Surfactant purified using an RNeasy Mini Kit (Qiagen), and reverse tran- performance during cycling was evaluated by comparing mini- scribed to cDNA using SuperScript II First-Strand Synthesis mum and maximum γ and the reduction in area required to System (Invitrogen) following the manufacturer specifications. reach the minimum γ. Finally, the mechanical stability of the The quantity and quality of total RNA was assessed on a Nano- surfactant films was assessed by registering the decay in γ oc- Drop Spectrophotometer (Thermo Fisher Scientific). cDNA curring in compressed interfacial films as a consequence of the samples were then amplified by qPCR in triplicate reactions for discharge of five shocking mechanical perturbations as previ- each primer pair assayed on a 7500 Real-Time PCR Instrument ously described (Schürch et al., 2010). (Applied Biosystems) using SYBR Premix Ex Taq (Takara Bio) and the following qPCR conditions: 95°C for 2 min; 40 cycles of WB analysis 95°C for 30 s, 60°C for 30 s and 95°C for 15 s, 60°C for 1 min, 95°C WB was performed under reducing conditions as previous de- for 15 s, and 60°C for 15 s. Results were normalized using 18S scribed (Lopez-Rodriguez et al., 2016). Since surfactant proteins rRNA gene (Rn18s) as endogenous control. The following primer are the only proteins present in the complex, we normalize our pairs were used: Tert-F (Forward), 59-TGACCAGCGTGTTAGGAA data, as previously reported (Autilio et al., 2020), by loading GA-39; Tert-R (Reverse), 59 CAGGAGGAAAGGA GCCAGAG-39 onto the gel equivalent amounts of PC per sample (3 µg from (GenBank accession no. NM_009354.2); Tnf-F, 59-GCCTCTTCT isolated mice surfactant). Therefore, changes in protein com- CATTCCTGCTTG-39; Tnf-R, 59-CTGATGAGAGGGAGGCCATT-39 position have to be interpreted as connected with changes in the (GenBank accession no. NM_013693.3); Il1b-F, 59-GCAACTGTT quality of surfactant, seen as protein-to-lipid ratios. CCTGAACTCAACT-39, Il1b-R 59-ATCTTTTGGGGTCCGTCAACT39 Electrophoresis Laemmli buffer (2% SDS, 62.5 mM Tris, pH (GenBank accession no. NM_008361.3); Il6-F, 59-ACGGCCTTC 6.8, 10% glycerol, and 0.03% bromophenol blue) containing 4% CCTACTTCACA-39 and Il6-R, 59-CATTTCCACGATTTCCCAGA-39 β-mercapto-ethanol was added to insolated mice surfactant be- (GenBank accession no. NM_031168.1); Il4-F, 59-CCTCACAGC fore incubating (15 min; 90°C) and loading the samples. The Piñeiro-Hermida et al. Journal of Cell Biology 16 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 polyacrylamide gel (16%) was run for ∼1 h, and proteins were Salud Carlos III. C. Autilio and J. Pérez-Gil acknowledge funding transferred onto polyvinylidene fluoride membranes with a from the Spanish Ministry of Science and Innovation (RTI2018- semi-dry system (1 h; 4°C) and blocked in PBS-T (100 mM 094564-B-100) and the Regional Government of Madrid (P2018/ Na2HPO4/KH2PO4-1% Tween) with 5% skim milk at room tem- NMT-4389). perature for 2 h. Membranes were then incubated overnight The authors declare no competing financial interests. with the primary human/mouse polyclonal antibodies (1:5,000, Author contributions: M.A. Blasco had the original idea and anti-mature SP-B [WRAB-48604], 1:10,000 anti-mature SP-C secured funding. M.A. Blasco, P. Martı́nez, F. Bosch, and [WRAB-76694; Seven Hills]) in PBS-T 5% milk at 4°C, washed J. Pérez-Gil supervised research. M.A. Blasco, P. Martı́nez, S. thoroughly in PBS-T, and incubated with the secondary antibody Piñeiro-Hermida, J. Pérez-Gil, and C. Autilio wrote the paper. S. (1:5,000, anti-rabbit [P0217]; Dako and Agilent) for 2 h at room Piñeiro-Hermida and C. Autilio analyzed the data and performed temperature (E3). Membranes were then developed (1 min of experiments. exposition) using a commercial ECL system (Millipore). Bands densitometry was performed by using ImageJ for Mac OS X. Submitted: 21 February 2020 Revised: 23 June 2020 ELISAs Accepted: 17 July 2020 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Superior right lung lobes were homogenized in RIPA Buffer (Thermo Fisher Scientific) containing a protease-phosphatase inhibitor mixture (Roche), and total protein concentration was References determined with the Pierce BCA Protein Assay Kit (Thermo Acosta, J.C., A. O’Loghlen, A. Banito, M.V. Guijarro, A. Augert, S. Raguz, M. Fisher Scientific). Cytokine levels were assessed in homogenized Fumagalli, M. Da Costa, C. Brown, N. Popov, et al. 2008. Chemokine lung tissue lysates using mouse IL-4, IL-10, IL-13, and TNF signaling via the CXCR2 receptor reinforces senescence. Cell. 133: 1006–1018. https://doi.org/10.1016/j.cell.2008.03.038 Quantikine ELISA Kits (R&D Systems), following the manu- Ahluwalia, N., B.S. Shea, and A.M. Tager. 2014. New therapeutic targets in facturer’s specifications, normalized to total lung protein levels. idiopathic pulmonary fibrosis. Aiming to rein in runaway wound- healing responses. Am. J. Respir. Crit. Care Med. 190:867–878. https:// doi.org/10.1164/rccm.201403-0509PP Statistics Alder, J.K., J.J.L. Chen, L. Lancaster, S. Danoff, S.C. Su, J.D. Cogan, I. Vulto, M. Statistical analyses were accomplished using SPSS Statistics Xie, X. Qi, R.M. Tuder, et al. 2008. Short telomeres are a risk factor for Software v21 for Windows (IBM). Following a Shapiro–Wilk idiopathic pulmonary fibrosis. Proc. Natl. Acad. Sci. USA. 105: 13051–13056. https://doi.org/10.1073/pnas.0804280105 normality test, either a one-way ANOVA test or a Kruskal– Angelidis, I., L.M. Simon, I.E. Fernandez, M. Strunz, C.H. Mayr, F.R. Greiffo, Wallis test were used, and then the post hoc Dunn–Sidak G. Tsitsiridis, M. Ansari, E. Graf, T.M. Strom, et al. 2019. An atlas of multiple test was performed for multiple comparisons. Ac- the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nat. Commun. 10:963. https://doi.org/10.1038/s41467-019 cording to the sample distribution, either a Mann–Whitney or -08831-9 unpaired t test was used to compare Tert+/+ and G3 Tert−/− Armanios, M.. 2013. Telomeres and age-related disease : how telomere biol- mice of the same age or 71–72-wk-old mice with and without ogy informs clinical paradigms. J. Clin. Invest. 123:996–1002. https://doi .org/10.1172/JCI66370 telomerase therapy. Results are shown as mean values ± SEM. Armanios, M., and E.H. Blackburn. 2012. The telomere syndromes. Nat. Rev. For all analyses, a P value < 0.05 was considered statistically Genet. 13:693–704. https://doi.org/10.1038/nrg3246 significant. Armanios, M.Y., J.J.L. Chen, J.D. Cogan, J.K. Alder, R.G. Ingersoll, C. Markin, W.E. Lawson, M. Xie, I. Vulto, J.A. Phillips, III, et al. 2007. Telomerase mutations in families with idiopathic pulmonary fibrosis. N. Engl. Online supplemental material J. Med. 356:1317–1326. https://doi.org/10.1056/NEJMoa066157 Fig. S1 shows that the interfacial stability of surfactant material Autilio, C., and J. Pérez-Gil. 2019. Understanding the principle biophysics concepts of pulmonary surfactant in health and disease. Arch. Dis. Child. from Tert+/+ mice under compression of the air–liquid interface Fetal Neonatal Ed. 104:F443–F451. https://doi.org/10.1136/archdischild is significantly reduced after 71–72 wk of physiological aging. -2018-315413 Fig. S2 shows that telomerase therapy does not affect membrane Autilio, C., M. Echaide, D. De Luca, and J. Pérez-Gil. 2018. Controlled hypo- thermia may improve surfactant function in asphyxiated neonates with stability but apparently reduces the content of SP-B in Tert+/+ or without meconium aspiration syndrome. PLoS One. 13. e0192295. 71–72-wk-old mice. https://doi.org/10.1371/journal.pone.0192295 Autilio, C., M. Echaide, S. Shankar-Aguilera, R. Bragado, D. Amidani, F. Sal- omone, J. Pérez-Gil, and D. De Luca. 2020. Surfactant Injury in the Early Phase of Severe Meconium Aspiration Syndrome. Am. J. Respir. Cell Mol. Acknowledgments Biol. rcmb.2019-0413OC. https://doi.org/10.1165/rcmb.2019-0413OC Research in the Blasco Lab is funded by the Fundación Botı́n and Ayaub, E.A., A. Dubey, J. Imani, F. Botelho, M.R.J. Kolb, C.D. Richards, and K. Fundación Banco Santander (Spain); Instituto de Salud Carlos Ask. 2017. Overexpression of OSM and IL-6 impacts the polarization of pro-fibrotic macrophages and the development of bleomycin-induced III, Spanish Ministry of Science and Innovation (DTS17/ 00152), lung fibrosis. Sci. Rep. 7:13281. https://doi.org/10.1038/s41598-017-13511-z cofunded European Regional Development Fund (ERDF); Span- Ayuso, E., F. Mingozzi, J. Montane, X. Leon, X.M. Anguela, V. Haurigot, S.A. ish Estate Research Agency, Spanish Ministry of Science and Edmonson, L. Africa, S. Zhou, K.A. High, et al. 2010. High AAV vector purity results in serotype- and tissue-independent enhancement of Innovation (project RETOS SAF2017-82623-R), cofunded by transduction efficiency. Gene Ther. 17:503–510. https://doi.org/10.1038/ ERDF and RyPSE-CM Programme (B2017/BMD-3770), Commu- gt.2009.157 nity of Madrid, cofunded by the European Social Fund and ERDF. Bernardes de Jesus, B., E. Vera, K. Schneeberger, A.M. Tejera, E. Ayuso, F. Bosch, and M.A. Blasco. 2012. Telomerase gene therapy in adult and old The CNIO, certified as Severo Ochoa Excellence Centre, is sup- mice delays aging and increases longevity without increasing cancer. ported by the Spanish Government through the Instituto de EMBO Mol. Med. 4:691–704. https://doi.org/10.1002/emmm.201200245 Piñeiro-Hermida et al. Journal of Cell Biology 17 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Birch, J., R.K. Anderson, C. Correia-Melo, D. Jurk, G. Hewitt, F.M. Marques, Faner, R., M. Rojas, W. Macnee, and A. Agustı́. 2012. Abnormal lung aging in N.J. Green, E. Moisey, M.A. Birrell, M.G. Belvisi, et al. 2015. DNA chronic obstructive pulmonary disease and idiopathic pulmonary fi- damage response at telomeres contributes to lung aging and chronic brosis. Am. J. Respir. Crit. Care Med. 186:306–313. https://doi.org/10.1164/ obstructive pulmonary disease. Am. J. Physiol. Lung Cell. Mol. Physiol. rccm.201202-0282PP 309:L1124–L1137. https://doi.org/10.1152/ajplung.00293.2015 Fingerlin, T.E., E. Murphy, W. Zhang, A.L. Peljto, K.K. Brown, M.P. Steele, J.E. Blackburn, E.H.. 2001. Switching and signaling at the telomere. Cell. 106: Loyd, G.P. Cosgrove, D. Lynch, S. Groshong, et al. 2013. Genome-wide 661–673. https://doi.org/10.1016/S0092-8674(01)00492-5 association study identifies multiple susceptibility loci for pulmonary Blackburn, E.H., C.W. Greider, and J.W. Szostak. 2006. Telomeres and telo- fibrosis. Nat. Genet. 45:613–620. https://doi.org/10.1038/ng.2609 merase: the path from maize, Tetrahymena and yeast to human cancer Flores, I., M.L. Cayuela, and M.A. Blasco. 2005. Effects of telomerase and and aging. Nat. Med. 12:1133–1138. https://doi.org/10.1038/nm1006-1133 telomere length on epidermal stem cell behavior. Science. 309: Blasco, M.A.. 2005. Mice with bad ends: mouse models for the study of te- 1253–1256. https://doi.org/10.1126/science.1115025 lomeres and telomerase in cancer and aging. EMBO J. 24:1095–1103. Flores, I., A. Canela, E. Vera, A. Tejera, G. Cotsarelis, and M.A. Blasco. 2008. https://doi.org/10.1038/sj.emboj.7600598 The longest telomeres: a general signature of adult stem cell compart- Blasco, M.A.. 2007. Telomere length, stem cells and aging. Nat. Chem. Biol. 3: ments. Genes Dev. 22:654–667. https://doi.org/10.1101/gad.451008 640–649. https://doi.org/10.1038/nchembio.2007.38 Foust, K.D., E. Nurre, C.L. Montgomery, A. Hernandez, C.M. Chan, and B.K. Blasco, M.A., H.W. Lee, M.P. Hande, E. Samper, P.M. Lansdorp, R.A. DePinho, Kaspar. 2009. Intravascular AAV9 preferentially targets neonatal and C.W. Greider. 1997. Telomere shortening and tumor formation by neurons and adult astrocytes. Nat. Biotechnol. 27:59–65. https://doi.org/ mouse cells lacking telomerase RNA. Cell. 91:25–34. https://doi.org/10 10.1038/nbt.1515 .1016/S0092-8674(01)80006-4 Giangreco, A., S.D. Reynolds, and B.R. Stripp. 2002. Terminal bronchioles Boyd, A.R., P. Shivshankar, S. Jiang, M.T. Berton, and C.J. Orihuela. 2012. Age- harbor a unique airway stem cell population that localizes to the Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 related defects in TLR2 signaling diminish the cytokine response by bronchoalveolar duct junction. Am. J. Pathol. 161:173–182. https://doi alveolar macrophages during murine pneumococcal pneumonia. Exp. .org/10.1016/S0002-9440(10)64169-7 Gerontol. 47:507–518. https://doi.org/10.1016/j.exger.2012.04.004 Greider, C.W., and E.H. Blackburn. 1985. Identification of a specific telomere Brandenberger, C., and C. Mühlfeld. 2017. Mechanisms of lung aging. Cell terminal transferase activity in Tetrahymena extracts. Cell. 43:405–413. Tissue Res. 367:469–480. https://doi.org/10.1007/s00441-016-2511-x https://doi.org/10.1016/0092-8674(85)90170-9 Bueno, M., Y.C. Lai, Y. Romero, J. Brands, C.M. St Croix, C. Kamga, C. Corey, Guha, A., M. Vasconcelos, Y. Cai, M. Yoneda, A. Hinds, J. Qian, G. Li, L. Dickel, J.D. Herazo-Maya, J. Sembrat, J.S. Lee, et al. 2015. PINK1 deficiency J.E. Johnson, S. Kimura, et al. 2012. Neuroepithelial body microenvi- impairs mitochondrial homeostasis and promotes lung fibrosis. J. Clin. ronment is a niche for a distinct subset of Clara-like precursors in the Invest. 125:521–538. https://doi.org/10.1172/JCI74942 developing airways. Proc. Natl. Acad. Sci. USA. 109:12592–12597. https:// Byrne, A.J., S.A. Mathie, L.G. Gregory, and C.M. Lloyd. 2015. Pulmonary doi.org/10.1073/pnas.1204710109 macrophages: key players in the innate defence of the airways. Thorax. Herrera, E., E. Samper, and M.A. Blasco. 1999a. Telomere shortening in 70:1189–1196. https://doi.org/10.1136/thoraxjnl-2015-207020 mTR−/− embryos is associated with failure to close the neural tube. Campisi, J.. 2013. Aging, cellular senescence, and cancer. Annu. Rev. Physiol. EMBO J. 18:1172–1181. https://doi.org/10.1093/emboj/18.5.1172 75:685–705. https://doi.org/10.1146/annurev-physiol-030212-183653 Herrera, E., E. Samper, J. Martı́n-Caballero, J.M. Flores, H.W. Lee, and M.A. Canela, A., P. Klatt, and M.A. Blasco. 2007. Telomere length analysis. In Bi- Blasco. 1999b. Disease states associated with telomerase deficiency ap- ological Aging. Methods in Molecular Biology. Vol. 371. T.O. Tollefsbol, pear earlier in mice with short telomeres. EMBO J. 18:2950–2960. editor. Humana Press, Totowa, NJ. 45–72., https://doi.org/10.1007/978-1 https://doi.org/10.1093/emboj/18.11.2950 -59745-361-5_5 Higashimoto, Y., Y. Fukuchi, Y. Shimada, K. Ishida, M. Ohata, T. Furuse, C. Chibbar, R., J.A. Gjevre, F. Shih, H. Neufeld, E.G. Lemire, D.A. Fladeland, and Shu, S. Teramoto, T. Matsuse, E. Sudo, et al. 1993. The effects of aging D.W. Cockcroft. 2010. Familial interstitial pulmonary fibrosis: a large on the function of alveolar macrophages in mice. Mech. Ageing Dev. 69: family with atypical clinical features. Can. Respir. J. 17:269–274. https:// 207–217. https://doi.org/10.1016/0047-6374(93)90024-L doi.org/10.1155/2010/591523 Hogan, B.L.M., C.E. Barkauskas, H.A. Chapman, J.A. Epstein, R. Jain, C.C.W. Chuang, H.M., L.I. Ho, M.H. Huang, K.L. Huang, T.W. Chiou, S.Z. Lin, H.L. Su, Hsia, L. Niklason, E. Calle, A. Le, S.H. Randell, et al. 2014. Repair and and H.J. Harn. 2018. Non-canonical regulation of type i collagen regeneration of the respiratory system: complexity, plasticity, and through promoter binding of sox2 and its contribution to ameliorating mechanisms of lung stem cell function. Cell Stem Cell. 15:123–138. pulmonary fibrosis by butylidenephthalide. Int. J. Mol. Sci. 19. E3024. https://doi.org/10.1016/j.stem.2014.07.012 https://doi.org/10.3390/ijms19103024 Hou, J., J. Shi, L. Chen, Z. Lv, X. Chen, H. Cao, Z. Xiang, and X. Han. 2018. M2 Codd, V., C.P. Nelson, E. Albrecht, M. Mangino, J. Deelen, J.L. Buxton, J.J. macrophages promote myofibroblast differentiation of LR-MSCs and Hottenga, K. Fischer, T. Esko, I. Surakka, et al; CARDIoGRAM consor- are associated with pulmonary fibrogenesis. Cell Commun. Signal. 16:89. tium. 2013. Identification of seven loci affecting mean telomere length https://doi.org/10.1186/s12964-018-0300-8 and their association with disease. Nat. Genet. 45:422–427–e2. https:// Hunninghake, G.M.. 2014. A new hope for idiopathic pulmonary fibrosis. N. doi.org/10.1038/ng.2528 Engl. J. Med. 370:2142–2143. https://doi.org/10.1056/NEJMe1403448 Cronkhite, J.T., C. Xing, G. Raghu, K.M. Chin, F. Torres, R.L. Rosenblatt, and Hussell, T., and T.J. Bell. 2014. Alveolar macrophages: plasticity in a tissue- C.K. Garcia. 2008. Telomere shortening in familial and sporadic pul- specific context. Nat. Rev. Immunol. 14:81–93. https://doi.org/10.1038/ monary fibrosis. Am. J. Respir. Crit. Care Med. 178:729–737. https://doi nri3600 .org/10.1164/rccm.200804-550OC Jakubzick, C., E.S. Choi, B.H. Joshi, M.P. Keane, S.L. Kunkel, R.K. Puri, and D’adda, F., D. Fagagna, P.M. Reaper, L. Clay-Farrace, H. Fiegler, P. Carr, T. C.M. Hogaboam. 2003. Therapeutic attenuation of pulmonary fibrosis Von Zglinicki, G. Saretzki, N.P. Carter, and S.P. Jackson. 2003. A DNA via targeting of IL-4- and IL-13-responsive cells. J. Immunol. 171: damage checkpoint response in telomere-initiated senescence. Nature. 2684–2693. https://doi.org/10.4049/jimmunol.171.5.2684 426:194–198. https://doi.org/10.1038/nature02118 Karimi-Shah, B.A., and B.A. Chowdhury. 2015. Forced vital capacity in idio- de Lange, T.. 2005. Shelterin: the protein complex that shapes and safeguards pathic pulmonary fibrosis--FDA review of pirfenidone and nintedanib. human telomeres. Genes Dev. 19:2100–2110. https://doi.org/10.1101/gad N. Engl. J. Med. 372:1189–1191. https://doi.org/10.1056/NEJMp1500526 .1346005 King, T.E., Jr., W.Z. Bradford, S. Castro-Bernardini, E.A. Fagan, I. Glaspole, Degryse, A.L., X.C. Xu, J.L. Newman, D.B. Mitchell, H. Tanjore, V.V. Polosu- M.K. Glassberg, E. Gorina, P.M. Hopkins, D. Kardatzke, L. Lancaster, khin, B.R. Jones, F.B. McMahon, L.A. Gleaves, J.A. Phillips, III, et al. et al; ASCEND Study Group. 2014. A phase 3 trial of pirfenidone in 2012. Telomerase deficiency does not alter bleomycin-induced fibrosis patients with idiopathic pulmonary fibrosis. N. Engl. J. Med. 370: in mice. Exp. Lung Res. 38:124–134. https://doi.org/10.3109/01902148 2083–2092. https://doi.org/10.1056/NEJMoa1402582 .2012.658148 Korfei, M., D. von der Beck, I. Henneke, P. Markart, C. Ruppert, P. Mahavadi, B. Demopoulos, K., D.A. Arvanitis, D.A. Vassilakis, N.M. Siafakas, and D.A. Ghanim, W. Klepetko, L. Fink, S. Meiners, et al. 2013. Comparative pro- Spandidos. 2002. MYCL1, FHIT, SPARC, p16(INK4) and TP53 genes teome analysis of lung tissue from patients with idiopathic pulmonary associated to lung cancer in idiopathic pulmonary fibrosis. J. Cell. Mol. fibrosis (IPF), non-specific interstitial pneumonia (NSIP) and organ do- Med. 6:215–222. https://doi.org/10.1111/j.1582-4934.2002.tb00188.x nors. J. Proteomics. 85:109–128. https://doi.org/10.1016/j.jprot.2013.04.033 Desai, T.J., D.G. Brownfield, and M.A. Krasnow. 2014. Alveolar progenitor and Kotton, D.N., and E.E. Morrisey. 2014. Lung regeneration: mechanisms, ap- stem cells in lung development, renewal and cancer. Nature. 507: plications and emerging stem cell populations. Nat. Med. 20:822–832. 190–194. https://doi.org/10.1038/nature12930 https://doi.org/10.1038/nm.3642 Piñeiro-Hermida et al. Journal of Cell Biology 18 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Kuilman, T., C. Michaloglou, L.C.W. Vredeveld, S. Douma, R. van Doorn, C.J. Pardo, A., and M. Selman. 2017. Fibroblast Senescence and Apoptosis. Ajrcmb. Desmet, L.A. Aarden, W.J. Mooi, and D.S. Peeper. 2008. Oncogene-induced 56:145–146. senescence relayed by an interleukin-dependent inflammatory network. Parikh, P., S. Wicher, K. Khandalavala, C.M. Pabelick, R.D. Britt, Jr., and Y.S. Cell. 133:1019–1031. https://doi.org/10.1016/j.cell.2008.03.039 Prakash. 2019. Cellular senescence in the lung across the age spectrum. Lee, J., R. Reddy, L. Barsky, J. Scholes, H. Chen, W. Shi, and B. Driscoll. 2009. Am. J. Physiol. Lung Cell. Mol. Physiol. 316:L826–L842. https://doi.org/10 Lung alveolar integrity is compromised by telomere shortening in .1152/ajplung.00424.2018 telomerase-null mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 296:L57–L70. Plantier, L., B. Crestani, S.E. Wert, M. Dehoux, B. Zweytick, A. Guenther, and https://doi.org/10.1152/ajplung.90411.2008 J.A. Whitsett. 2011. Ectopic respiratory epithelial cell differentiation in Ley, B., and H.R. Collard. 2013. Epidemiology of idiopathic pulmonary fi- bronchiolised distal airspaces in idiopathic pulmonary fibrosis. Thorax. brosis. Clin. Epidemiol. 5:483–492. https://doi.org/10.2147/CLEP.S54815 66:651–657. https://doi.org/10.1136/thx.2010.151555 Liu, Y., B.E. Snow, M.P. Hande, D. Yeung, N.J. Erdmann, A. Wakeham, A. Itie, Povedano, J.M., P. Martinez, J.M. Flores, F. Mulero, and M.A. Blasco. 2015. D.P. Siderovski, P.M. Lansdorp, M.O. Robinson, et al. 2000. The telo- Mice with Pulmonary Fibrosis Driven by Telomere Dysfunction. Cell merase reverse transcriptase is limiting and necessary for telomerase Rep. 12:286–299. https://doi.org/10.1016/j.celrep.2015.06.028 function in vivo. Curr. Biol. 10:1459–1462. https://doi.org/10.1016/S0960 Povedano, J.M., P. Martinez, R. Serrano, Á. Tejera, G. Gómez-López, M. Bo- -9822(00)00805-8 badilla, J.M. Flores, F. Bosch, and M.A. Blasco. 2018. Therapeutic effects Liu, D., M.S. O’Connor, J. Qin, and Z. Songyang. 2004. Telosome, a mam- of telomerase in mice with pulmonary fibrosis induced by damage to malian telomere-associated complex formed by multiple telomeric the lungs and short telomeres. eLife. 7. e31299. https://doi.org/10.7554/ proteins. J. Biol. Chem. 279:51338–51342. https://doi.org/10.1074/jbc eLife.31299 .M409293200 Raghu, G., H.R. Collard, J.J. Egan, F.J. Martinez, J. Behr, K.K. Brown, T.V. Liu, T., M.J. Chung, M. Ullenbruch, H. Yu, H. Jin, B. Hu, Y.Y. Choi, F. Ishi- Colby, J.F. Cordier, K.R. Flaherty, J.A. Lasky, et al; ATS/ERS/JRS/ALAT Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 kawa, and S.H. Phan. 2007. Telomerase activity is required for Committee on Idiopathic Pulmonary Fibrosis. 2011. An official ATS/ bleomycin-induced pulmonary fibrosis in mice. J. Clin. Invest. 117: ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence- 3800–3809. https://doi.org/10.1172/JCI32369 based guidelines for diagnosis and management. Am. J. Respir. Crit. Liu, Y.Y., Y. Shi, Y. Liu, X.H. Pan, and K.X. Zhang. 2018. Telomere shortening Care Med. 183:788–824. https://doi.org/10.1164/rccm.2009-040GL activates TGF-β/Smads signaling in lungs and enhances both lipopol- Raghu, G., M. Remy-Jardin, J.L. Myers, L. Richeldi, C.J. Ryerson, D.J. Lederer, ysaccharide and bleomycin-induced pulmonary fibrosis. Acta Pharma- J. Behr, V. Cottin, S.K. Danoff, F. Morell, et al; American Thoracic So- col. Sin. 39:1735–1745. https://doi.org/10.1038/s41401-018-0007-9 ciety, European Respiratory Society, Japanese Respiratory Society, and Liu, G., H. Zhai, T. Zhang, S. Li, N. Li, J. Chen, M. Gu, Z. Qin, and X. Liu. 2019. Latin American Thoracic Society. 2018. Diagnosis of idiopathic pul- New therapeutic strategies for IPF: Based on the “phagocytosis- monary fibrosis An Official ATS/ERS/JRS/ALAT Clinical practice secretion-immunization” network regulation mechanism of pulmonary guideline. Am. J. Respir. Crit. Care Med. 198:e44–e68. https://doi.org/10 macrophages. Biomed. Pharmacother. 118. 109230. https://doi.org/10 .1164/rccm.201807-1255ST .1016/j.biopha.2019.109230 Rawlins, E.L., T. Okubo, Y. Xue, D.M. Brass, R.L. Auten, H. Hasegawa, F. López-Otı́n, C., M.A. Blasco, L. Partridge, M. Serrano, and G. Kroemer. 2013. Wang, and B.L.M. Hogan. 2009. The role of Scgb1a1+ Clara cells in the The hallmarks of aging. Cell. 153:1194–1217. https://doi.org/10.1016/j.cell long-term maintenance and repair of lung airway, but not alveolar, .2013.05.039 epithelium. Cell Stem Cell. 4:525–534. https://doi.org/10.1016/j.stem Lopez-Rodriguez, E., C. Boden, M. Echaide, J. Perez-Gil, M. Kolb, J. Gauldie, .2009.04.002 U.A. Maus, M. Ochs, and L. Knudsen. 2016. Surfactant dysfunction Richeldi, L., H.R. Collard, and M.G. Jones. 2017. Idiopathic pulmonary fibrosis. during overexpression of TGF-β1 precedes profibrotic lung remodeling Lancet. 389:1941–1952. https://doi.org/10.1016/S0140-6736(17)30866-8 in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 310:L1260–L1271. https:// Rock, J.R., C.E. Barkauskas, M.J. Cronce, Y. Xue, J.R. Harris, J. Liang, P.W. doi.org/10.1152/ajplung.00065.2016 Noble, and B.L.M. Hogan. 2011. Multiple stromal populations contribute Lowery, E.M., A.L. Brubaker, E. Kuhlmann, and E.J. Kovacs. 2013. The aging to pulmonary fibrosis without evidence for epithelial to mesenchymal lung. Clin. Interv. Aging. 8:1489–1496. https://doi.org/10.2147/CIA.S51152 transition. Proc. Natl. Acad. Sci. USA. 108:E1475–E1483. https://doi.org/ Mahavadi, P., M. Korfei, I. Henneke, G. Liebisch, G. Schmitz, B.R. Gochuico, P. 10.1073/pnas.1117988108 Markart, S. Bellusci, W. Seeger, C. Ruppert, et al. 2010. Epithelial stress Samper, E., J.M. Flores, and M.A. Blasco. 2001. Restoration of telomerase and apoptosis underlie Hermansky-Pudlak syndrome-associated in- activity rescues chromosomal instability and premature aging in Terc−/ terstitial pneumonia. Am. J. Respir. Crit. Care Med. 182:207–219. https:// − mice with short telomeres. EMBO Rep. 2:800–807. https://doi.org/10 doi.org/10.1164/rccm.200909-1414OC .1093/embo-reports/kve174 Martı́nez, P., and M.A. Blasco. 2011. Telomeric and extra-telomeric roles for Schürch, D., O.L. Ospina, A. Cruz, and J. Pérez-Gil. 2010. Combined and in- telomerase and the telomere-binding proteins. Nat. Rev. Cancer. 11: dependent action of proteins SP-B and SP-C in the surface behavior and 161–176. https://doi.org/10.1038/nrc3025 mechanical stability of pulmonary surfactant films. Biophys. J. 99: Martinez, F.J., H.R. Collard, A. Pardo, G. Raghu, L. Richeldi, M. Selman, J.J. 3290–3299. https://doi.org/10.1016/j.bpj.2010.09.039 Swigris, H. Taniguchi, and A.U. Wells. 2017. Idiopathic pulmonary fi- Selman, M., and A. Pardo. 2014. Revealing the pathogenic and aging-related brosis. Nat. Rev. Dis. Primers. 3:17074. https://doi.org/10.1038/nrdp.2017 mechanisms of the enigmatic idiopathic pulmonary fibrosis. an integral .74 model. Am. J. Respir. Crit. Care Med. 189:1161–1172. https://doi.org/10 Matsushita, T., S. Elliger, C. Elliger, G. Podsakoff, L. Villarreal, G.J. Kurtzman, .1164/rccm.201312-2221PP Y. Iwaki, and P. Colosi. 1998. Adeno-associated virus vectors can Sharpless, N.E., and R.A. DePinho. 2007. How stem cells age and why this be efficiently produced without helper virus. Gene Ther. 5:938–945. makes us grow old. Nat. Rev. Mol. Cell Biol. 8:703–713. https://doi.org/10 https://doi.org/10.1038/sj.gt.3300680 .1038/nrm2241 Meiners, S., O. Eickelberg, and M. Königshoff. 2015. Hallmarks of the Snetselaar, R., C.H.M. van Moorsel, K.M. Kazemier, J.J. van der Vis, P. Zanen, ageing lung. Eur. Respir. J. 45:807–827. https://doi.org/10.1183/09031936 M.F.M. van Oosterhout, and J.C. Grutters. 2015. Telomere length in .00186914 interstitial lung diseases. Chest. 148:1011–1018. https://doi.org/10.1378/ Mosteiro, L., C. Pantoja, N. Alcazar, R.M. Marión, D. Chondronasiou, M. chest.14-3078 Rovira, P.J. Fernandez-Marcos, M. Muñoz-Martin, C. Blanco-Aparicio, J. Somogyi, V., N. Chaudhuri, S.E. Torrisi, N. Kahn, V. Müller, and M. Kreuter. Pastor, et al. 2016. Tissue damage and senescence provide critical sig- 2019. The therapy of idiopathic pulmonary fibrosis: what is next? Eur. nals for cellular reprogramming in vivo. Science. 354. aaf4445. https:// Respir. Rev. 28. 190021. https://doi.org/10.1183/16000617.0021-2019 doi.org/10.1126/science.aaf4445 Spagnolo, P., T.M. Maher, and L. Richeldi. 2015. Idiopathic pulmonary fi- Muñoz-Lorente, M.A., P. Martı́nez, Á. Tejera, K. Whittemore, A.C. Moisés- brosis: Recent advances on pharmacological therapy. Pharmacol. Ther. Silva, F. Bosch, and M.A. Blasco. 2018. AAV9-mediated telomerase ac- 152:18–27. https://doi.org/10.1016/j.pharmthera.2015.04.005 tivation does not accelerate tumorigenesis in the context of oncogenic Stanley, S.E., J.J.L. Chen, J.D. Podlevsky, J.K. Alder, N.N. Hansel, R.A. Mathias, K-Ras-induced lung cancer. PLoS Genet. 14. e1007562. https://doi.org/10 X. Qi, N.M. Rafaels, R.A. Wise, E.K. Silverman, et al. 2015. Telomerase .1371/journal.pgen.1007562 mutations in smokers with severe emphysema. J. Clin. Invest. 125: Olovnikov, A.M.. 1973. A theory of marginotomy. The incomplete copying of 563–570. https://doi.org/10.1172/JCI78554 template margin in enzymic synthesis of polynucleotides and biological Sun, L., M.C. Louie, K.M. Vannella, C.A. Wilke, A.M. LeVine, B.B. Moore, and significance of the phenomenon. J. Theor. Biol. 41:181–190. https://doi T.P. Shanley. 2011. New concepts of IL-10-induced lung fibrosis: fi- .org/10.1016/0022-5193(73)90198-7 brocyte recruitment and M2 activation in a CCL2/CCR2 axis. Am. Piñeiro-Hermida et al. Journal of Cell Biology 19 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 J. Physiol. Lung Cell. Mol. Physiol. 300:L341–L353. https://doi.org/10.1152/ Tsang, A.R., H.D.M. Wyatt, N.S.Y. Ting, and T.L. Beattie. 2012. hTERT mu- ajplung.00122.2010 tations associated with idiopathic pulmonary fibrosis affect telomerase Takai, H., A. Smogorzewska, and T. de Lange. 2003. DNA damage foci at activity, telomere length, and cell growth by distinct mechanisms. Aging dysfunctional telomeres. Curr. Biol. 13:1549–1556. https://doi.org/10 Cell. 11:482–490. https://doi.org/10.1111/j.1474-9726.2012.00810.x .1016/S0960-9822(03)00542-6 Watson, J.D.. 1972. Origin of concatemeric T7 DNA. Nat. New Biol. 239:197–201. Tompkins, D.H., V. Besnard, A.W. Lange, S.E. Wert, A.R. Keiser, A.N. Smith, https://doi.org/10.1038/newbio239197a0 R. Lang, and J.A. Whitsett. 2009. Sox2 is required for maintenance and Wong, C.K., C.A. Smith, K. Sakamoto, N. Kaminski, J.L. Koff, and D.R. Gold- differentiation of bronchiolar Clara, ciliated, and goblet cells. PLoS One. stein. 2017. Aging Impairs Alveolar Macrophage Phagocytosis and In- 4. e8248. https://doi.org/10.1371/journal.pone.0008248 creases Influenza-Induced Mortality in Mice. J. Immunol. 199: Tsakiri, K.D., J.T. Cronkhite, P.J. Kuan, C. Xing, G. Raghu, J.C. Weissler, R.L. 1060–1068. https://doi.org/10.4049/jimmunol.1700397 Rosenblatt, J.W. Shay, and C.K. Garcia. 2007. Adult-onset pulmonary Zhang, L., Y. Wang, G. Wu, W. Xiong, W. Gu, and C.Y. Wang. 2018. Macro- fibrosis caused by mutations in telomerase. Proc. Natl. Acad. Sci. USA. phages: friend or foe in idiopathic pulmonary fibrosis? Respir. Res. 19: 104:7552–7557. https://doi.org/10.1073/pnas.0701009104 170. https://doi.org/10.1186/s12931-018-0864-2 Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Piñeiro-Hermida et al. Journal of Cell Biology 20 of 20 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120 Supplemental material Downloaded from http://rupress.org/jcb/article-pdf/219/10/e202002120/1048391/jcb_202002120.pdf by guest on 30 April 2021 Figure S1. Physiological aging leads to impaired surfactant activity in wild-type mice, and this is anticipated in telomerase-deficient mice. Interfacial film stability and WB of hydrophobic surfactant proteins from Tert+/+ and G3 Tert−/− mice with increasing weeks of age. (A) Surface tension obtained after hitting five-times-compressed surfactant membranes to test their stability. (B) SP-B and SP-C bands obtained by performing WB analysis in reducing con- ditions, loading the same amount of PC for each sample. Densitometry analysis was performed using as reference the most intense band among the samples tested within each run. White bars represent Tert+/+ mice. Gray bars represent G3 Tert−/− mice. Data are expressed as mean ± SEM (n = 4–6 animals per group). *, P < 0.05; **, P < 0.01 between different ages for each condition using one-way ANOVA with post hoc Dunn–Sidak multiple comparison test or between different conditions within the same age following a Shapiro–Wilk normality test, two-sample unpaired t test, or Mann–Whitney test when appropriate. γ, surface tension; A.D.U., arbitrary densitometry units. Figure S2. Telomerase gene therapy prevents age-related worsening in surfactant activity in wild-type and telomerase-deficient mice. Membrane stability and WB of hydrophobic surfactant proteins from 71–72-wk-old Tert+/+ and G3 Tert−/− mice treated with AAV9-empty or AAV9-Tert. (A) Surface tension obtained after hitting five-times-compressed surfactant membranes to test their stability. (B) SP-B and SP-C bands obtained by performing WB analysis in reducing conditions, loading the same amount of PC for each sample. Densitometry analysis was performed using as reference the most intense band among the samples tested within each run. White bars represent Tert+/+ mice. Gray bars represent G3 Tert−/− mice. Data are expressed as mean ± SEM (n = 3–5 animals per group). *, P < 0.05 between different conditions following a Shapiro–Wilk normality test, two-sample unpaired t test, or Mann–Whitney test when appropriate. γ, surface tension; A.D.U., arbitrary densitometry units. Piñeiro-Hermida et al. Journal of Cell Biology S1 Telomerase treatment prevents lung fibrosis https://doi.org/10.1083/jcb.202002120