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Schafer MJ, White TA, Iijima K, Haak AJ, Ligresti G, Atkinson EJ, Oberg AL,
Birch J, Salmonowicz H, Zhu Y, Mazula DL, Brooks RW, Fuhrmann-Stroissnigg
H, Pirtskhalava T, Prakash YS, Tchkonia T, Robbins PD, Aubry MC, Passos JF,
Kirkland JL, Tschumperlin DJ, Kita H, LeBrasseur NK.
Cellular senescence mediates fibrotic pulmonary disease.
Nature Communications 2017, 8: 14532.
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© The Authors 2017.
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DOI link to article:
https://doi.org/10.1038/ncomms14532
Date deposited:
13/04/2017
ARTICLE
Received 6 Apr 2016
| Accepted 9 Jan 2017 | Published 23 Feb 2017
Cellular senescence mediates fibrotic pulmonary
disease
Marissa J. Schafer
1,2
, Thomas A. White
1
, Koji Iijima
3
, Andrew J. Haak
4
, Giovanni Ligresti
4
, Elizabeth J. Atkinson
5
,
Ann L. Oberg
5
, Jodie Birch
6
, Hanna Salmonowicz
6
, Yi Zhu
1
, Daniel L. Mazula
1
, Robert W. Brooks
7
,
Heike Fuhrmann-Stroissnigg
7
, Tamar Pirtskhalava
1
, Y.S. Prakash
4,8
, Tamara Tchkonia
1
, Paul D. Robbins
7
,
Marie Christine Aubry
9
, Joa
˜
o F. Passos
6
, James L. Kirkland
1,4,10
, Daniel J. Tschumperlin
4
, Hirohito Kita
3
& Nathan K. LeBrasseur
1,2,4
Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by interstitial remodelling,
leading to compromised lung function. Cellular senescence markers are detectable within IPF
lung tissue and senescent cell deletion rejuvenates pulmonary health in aged mice. Whether
and how senescent cells regulate IPF or if their removal may be an efficacious intervention
strategy is unknown. Here we demonstrate elevated abundance of senescence biomarkers in
IPF lung, with p16 expression increasing with disease severity. We show that the secretome of
senescent fibroblasts, which are selectively killed by a senolytic cocktail, dasatinib plus
quercetin (DQ), is fibrogenic. Leveraging the bleomycin-injury IPF model, we demonstrate
that early-intervention suicide-gene-mediated senescent cell ablation improves pulmonary
function and physical health, although lung fibrosis is visibly unaltered. DQ treatment repli-
cates benefits of transgenic clearance. Thus, our findings establish that fibrotic lung disease is
mediated, in part, by senescent cells, which can be targeted to improve health and function.
DOI: 10.1038/ncomms14532
OPEN
1
Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Mayo Clinic 200 First Street Southwest, Rochester, Minnesota 55905, USA.
2
Department of Physical Medicine and Rehabilitation, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
3
Division of Allergic Diseases,
Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
4
Department of Physiology and Biomedical
Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
5
Division of Biomedical Statistics and Informatics, Department of Health
Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
6
Institute for Cell and Molecular Biosciences, Newcastle University
Institute for Ageing Newcastle upon Tyne NE4 5PL, UK.
7
Department of Metabolism and Aging, The Scripps Research Institute, Jupiter, Florida 33458, USA.
8
Department of Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
9
Department of Laboratory Medicine and Pathology,
Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
10
Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester,
Minnesota 55905, USA. Correspondence and requests for materials should be addressed to N.K.L. (email: lebrasseur.nathan@mayo.edu).
NATURE COMMUNICATIONS | 8:14532 | DOI: 10.1038/ncomms14532 | www.nature.com/naturecommunications 1
F
ibrosis and wound healing are fundamentally intertwined
processes, driven by a cascade of injury, inflammation,
fibroblast proliferation and migration, and matrix deposi-
tion and remodelling
1
. Older organisms display reduced ability
to heal wounds
2
and resolve fibrosis
3
, leading to tissue scarring
and irreparable organ damage. The origins of persistent injury
response and repair signalling underlying fibrotic tissue
destruction are poorly understood. This is particularly true of
idiopathic pulmonary fibrosis (IPF), a quintessential disease of
ageing with median diagnosis at 66 years and estimated survival
of 3–4 years
4
. IPF symptoms, including chronic shortness
of breath, cough, fatigue and weight loss, are progressive and
lead to a dramatic truncation of healthspan and lifespan. This is
due to destruction of lung parenchyma, which exhibits
characteristic honeycombing and fibroblastic foci patterns
1,5
.
Current IPF treatment regimens have limited efficacy
6,7
. Better
defining the mechanisms responsible for chronic activation
of profibrotic mechanisms and lung parenchymal destruction
is essential for devising more effective therapies.
Cellular senescence is an evolutionarily conserved state of
stable replicative arrest induced by pro-ageing stressors also
implicated in IPF pathogenesis, including telomere attrition,
oxidative stress, DNA damage and proteome instability. Damage
accumulation stimulates the activity of cyclin-dependent kinase
inhibitors p16
Ink4a
and/or p53-p21
Cip1/Waf1
, which antagonize
cyclin-dependent kinases to block cell cycle progression
8
.
Through secretion of the senescence-associated secretory
phenotype (SASP), a broad repertoire of cytokines, chemokines,
matrix remodelling proteases and growth factors, senescent cells
paracrinely promote proliferation and tissue deterioration
8
.
Conversely, senescence is autonomously anti-proliferative, may
be requisite for optimal cutaneous wound healing
9
and may
restrict pathological liver fibrosis
10
.
A growing body of evidence implicates accelerated mechanisms
of ageing, including cellular senescence, in IPF pathogenesis
11
.
Established senescence biomarkers, including p16, p21 and
senescence-associated b-galactosidase activity (SA-b-gal), have
been observed in both fibroblasts and epithelial cells in human
IPF lung tissue
12,13
, and human IPF cells show increased senescence
propensity ex vivo
14
. In mice, intratracheal instillation of the
chemotherapeutic agent bleomycin causes resolvable lung fibrosis
that recapitulates key features of human IPF
15
. Bleomycin lung
injury induces a molecular signature of senescence
16,17
and age-
dependent accumulation of senescent myofibroblasts may impede
fibrosis resolution following bleomycin exposure
3
. In contrast,
overexpression of an enzyme responsible for production of the
extracellular matrix component hyaluronan exacerbates bleomycin-
induced injury
18
, whereas its depletion appears to activate
senescence, challenging a negative role for senescence in
bleomycin-induced lung fibrosis
19
.
Very recently, Hashimoto et al. provided crucial evidence that
cellular senescence contributes to lung ageing and may be
targeted for functional improvement
20
. Using a novel suicide-
gene strategy, they discovered that elimination of naturally
occurring senescent cells restores lung compliance, structure and
elasticity in aged mice. An alternative approach to transgenic
senescent cell clearance is senolytics
21,22
, which may be a tractable
treatment option for humans. Administration of the senolytic
quercetin diminishes the proinflammatory phenotype of
bleomycin-induced senescence in fibroblasts in vitro
23
.
Similarly, delivery of rapamycin, a SASP inhibitor, attenuates
pulmonary fibrosis and myofibroblast activation in vivo
24
. The
culmination of prior reports suggests that senescent cells may
contribute to fibrotic lung disease; however, their mechanism of
action and whether they may be therapeutically targeted to
improve lung function remains untested.
Experimentation described herein explored the role of
cellular senescence in IPF pathology across the translational
continuum. We began by assessing human IPF and control
biospecimens and demonstrate that several senescence biomar-
kers accumulate in IPF lung, with p16 expression increasing
concordantly with disease severity. Using bleomycin-induced
lung injury as an IPF model, we show that, similar to human
IPF, murine lung fibrosis is characterized by accumulation
of p16- and SASP-positive fibroblasts and epithelial cells. We
hypothesized that SASP signalling is a mechanism by which
senescent cells exert negative effects, and our in vitro experiments
establish that the SASP of senescent fibroblasts is indeed
fibrogenic. Critically, senescent fibroblasts are selectively elimi-
nated through treatment with the senolytic drug cocktail,
dasatinib plus quercetin (DQ). Next, we tested the efficacy of
senescent cell deletion in improving bleomycin-induced lung
pathology in Ink-Attac mice, in which p16-positive cells are
deleted through suicide-gene activation. We show that senescent
cell clearance improves pulmonary function, body composition
and physical health when treatment is initiated at disease onset.
Notably, senolytic DQ treatment phenocopies the transgenic cell
clearance strategy. Thus, our results suggest that senescent cells,
through their SASP, wield potent effects on adjacent cells,
ultimately promoting functional lung deterioration. Our findings
provide important proof-of-concept evidence for targeting
senescent cells as a novel pharmacological approach for treatment
of human IPF.
Results
Senescence biomarkers accumulate in IPF lung. To explore
the hypothesis that senescent cells and the SASP regulate lung
fibrosis, we interrogated microarray and RNA sequencing
(RNAseq) data sets corresponding to independent IPF and
control human cohorts for differential expression of established
senescence genes. IPF subjects exhibited significant impairments
in lung function, as measured by forced vital capacity (FVC)
and diffusion capacity, and physical function, as measured by
the 12-item short form health survey physical component
score and 6 min walking distance, relative to control subjects
(Supplementary Tables 1 and 2). CDKN2A (p16) was significantly
upregulated within lung samples of individuals with IPF and
increased with disease severity (Fig. 1a). Correlation analyses
revealed that elevated pulmonary p16 expression assessed
via microarray was associated with reduced FVC, diffusion
capacity and 12-item short form health survey physical compo-
nent score (Supplementary Fig. 1).
To corroborate expression data, we investigated p16 cytospatial
distribution using immunohistochemistry in a subset of control
and IPF lung samples that were analysed by microarray.
We identified a rare population of p16-positive epithelial
cells in control lung samples (Fig. 1b). In IPF lung samples,
both epithelial cells and fibroblasts were p16 positive within
fibroblastic foci (Fig. 1c), the presumed leading edge of
IPF disease. In the honeycomb lung, reactive bronchiolar
epithelium and fibroblasts were equally positive for p16
(Fig. 1d). We next quantified an independent senescence
biomarker, telomere-associated foci (TAF), which are sites of
unresolved DNA damage within telomeres, demarcated by
gH2A.X and telomere immuno-fluorescence in situ hybridization
co-localization
25
. We observed a significant increase in both
the mean number of gH2A.X foci and the percentage of
TAF-positive cells in IPF samples, relative to controls
(Fig. 1e,f). Cumulatively, senescence biomarker results
demonstrate p16 expression increasing in register with disease
progression, accumulation of p16-positive fibroblasts and
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14532
2 NATURE COMMUNICATIONS | 8:14532 | DOI: 10.1038/ncomms14532 | www.nature.com/naturecommunications
epithelial cells within fibrotic foci and honeycomb lung, and
accretion of TAF in lung tissue of individuals with IPF.
Hypothesizing that senescent cells mediate IPF pathology
via their secretome, we assessed differential expression of
SASP components in human IPF transcriptome data sets,
focusing on growth and matrix remodelling factors, which play
essential roles in proliferation and tissue reorganization
26
.
Pregnancy-associated plasma protein A (PAPPA), which
mediates insulin-like growth factor (IGF) signalling by cleaving
IGF-binding proteins to liberate IGF
27
, was robustly upregulated,
as were IGFBP2 and 4, although to a lesser magnitude (Fig. 1a).
Expression of several matrix-remodelling proteins (MMPs)
strongly increased with disease severity and in concordance
with profibrotic factors, including collagen, type I, a1(COL1A1)
and vascular cell adhesion molecule 1 (VCAM1, Fig. 1a), the latter
of which is a potent mediator of fibroblast proliferation
28
.
Bleomycin injury induces cell senescence. We next established
experimental systems to further interrogate the cellular
identity and mechanisms by which senescent cells exert their
effects. Aerosolized intratracheal instillation of the chemother-
apeutic agent bleomycin induces lung fibrosis in mice and
recapitulates critical features of human IPF
15
. Using this
murine model, we fluorescence-activated cell sorted (FACS)
whole lungs isolated 14 days post bleomycin or phosphate-
buffered saline (PBS) exposure based on cell-surface marker
presentation (Fig. 2a–d) and conducted gene expression profiling
on populations of fibroblasts (PDGFRa
þ
, EPCAM
, CD31
and CD45
) (Fig. 2b,e), epithelial cells (EPCAM
þ
, PDGFRa
,
CD31
and CD45
) (Fig. 2c,f) and endothelial cells
(CD31
þ
, PDGFRa
, EPCAM
and CD45
) (Fig. 2d,g). We
observed significant upregulation of p16 within both fibroblasts
and epithelial cells but not endothelial cells (Fig. 2e–g). Transcript
CDKN2A
Control
–2
5 µm
–1 0
ab
c
d
f
e
12
RNAseq, LogFC
3456
Microarray,
Relative expression
–1 0 1
5
40
30
20
10
0
Control
IPF
*
*
Mean γ H2A.X foci
%TAF
4
3
2
1
0
IPF severity
TP53
IGFBP2
IGFBP4
PAPPA
PDGFA
PDGFB
VEGFA
ACTA2
COL1A1
MMP2
MMP3
MMP9
MMP10
MMP12
MMP13
VCAM1
TGF2
TGF3
Figure 1 | Biomarkers of cellular senescence in human IPF. (a) Transcriptional changes corresponding to senescence effectors (black), SASP growth
factors (dark grey) and SASP matrix remodelling (light grey) genes that were identified in independent RNAseq (control n ¼ 19, IPF n ¼ 20) and microarray
human lung IPF versus control data sets are shown. IPF samples analysed by microarray were severity classified by FVC as low (Z80%; n ¼ 17), moderate
(50–80%; n ¼ 60) or severe (o50%; n ¼ 16) and compared with control (n ¼ 64) (qo0.05 for both RNAseq and microarray). Human lung tissue sections
were IHC stained for p16 in b control and (c,d) IPF lung samples with (c) fibroblastic foci and (d) honeycomb lung depicted. p16-positive fibroblasts (stars)
and epithelial cells (arrows) are indicated ( 200 images). (e) Control (left panel) and IPF (right panel) lung sections were analysed for frequencies of
DNA damage foci (gH2A.X, green) and telomere immuno-fluorescence in situ hybridization (red) within alveolar compartments. Arrows indicate
gH2A.X foci co-localizing with telomeres (TAF) (scale bar, 5 mm), shown at higher magnification on the right (images are from maximum intensity
projection). (f) Mean number of gH2A.X foci (left) and percentage of cells containing at least one TAF (right) were determined through quantification
of Z-stack images with at least 100 cells per sample ( 100 images) (mean
±
s.e.m.; control n ¼ 10 (grey), IPF n ¼ 27 (red); t-test *Pr0.05).
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14532 ARTICLE
NATURE COMMUNICATIONS | 8:14532 | DOI: 10.1038/ncomms14532 | www.nature.com/naturecommunications 3
levels of proinflammatory and profibrotic SASP factors, monocyte
chemotactic protein 1 (Mcp1), plasminogen activator inhibitor 1
(Pai1), tumour necrosis factor-a (Tnfa), Mmp10, Mmp12, Col1a1
and transforming growth factor-b (Tgfb) were also increased in
fibroblasts (Fig. 2e) and epithelial cells (Fig. 2f). All three
cell populations exhibited upregulation of Mmp10 and Mmp12
(Fig. 2e–g). Thus, similar to human IPF, bleomycin-mediated
lung injury induces a senescent signature characterized
by increased transcriptional activation of p16 and SASP
components in fibroblasts and epithelial cells.
The secretome of senescent fibroblasts is profibrotic. To sub-
stantiate the SASP as a mediator of IPF pathology, we
devised an in vitro assay to test fibrotic activation. Exposure of
human IMR90 fibroblasts to 10 Gy irradiation induced
senescence observable after 21 days, which was confirmed by
staining for SA-b-gal (Fig. 3a) and expression profiling of
senescence effectors (p16 and p53) and SASP factors (MCP1 and
IL6) (Fig. 3b). Conditioned medium (CM) collected from senes-
cent cells (SASP-CM), relative to sham-irradiated control cells
(CCM), exhibited 3- to 4200-fold increases in the abundance of
established SASP proteins interleukin (IL)-1a, IL1b, IL6, IL10,
MCP1, PAI1, VCAM1, MMP2, MMP12 and TGFb (Fig. 3c).
To examine potential fibrogenic effects of the SASP, we
treated naive IMR90 fibroblasts with non-CM (NCM),
NCM þ 2ngml
1
TGFb (positive control), CCM or SASP-CM
and immunostained for a-smooth muscle actin (aSMA), an
indicator of fibrotic activation
29
. SASP-CM induced aSMA signal
intensity at a level comparable to TGFb treatment (Fig. 3d). Sixty-
two percent of fibroblasts treated with SASP-CM stained positive
for aSMA protein, whereas only 21% of cells treated with CCM
were aSMA positive. We next utilized traction force microscopy
30
to determine whether fibrotic activation translated to changes in
contractile behaviour. SASP-CM-treated fibroblast exhibited
significantly greater traction forces, relative to CCM-treated
fibroblasts (Fig. 3e). IMR90 cells treated with SASP-CM also
expressed higher levels of several fibrosis genes, including actin-a-2
(ACTA2,encodingaSMA), COL1A1, COL1A2 and fibronectin 1
(FN1), relative to NCM- and CCM-treated cells (Fig. 3f). Thus, our
results demonstrate that the secretome of senescent fibroblasts
robustly stimulates a fibrotic phenotype in healthy human
fibroblasts. Importantly, CM collected from irradiated bronchiolar
epithelial cells did not activate a fibrogenic response, as measured by
aSMA fibrotic activation of naive IMR90 cells (Supplementary
Fig. 2), suggesting that cell-type-specific SASP composition may
differentially affect pathological phenotypes within the lung.
(×1,000)
SSC-A
EPCAMEPCAMEPCAM
Fibroblasts (PDGFRα+)
Relative expression
Epithelial cells (EPCAM+)
Relative expression
Endothelial cells (CD31+)
Relative expression
250
ab
c
e
f
dg
2,500
PBS
Bleo
***
***
**
#
**
***
¥
*
**
***
**
*
*
*
*
*
*
*
#
#
**
**
1,500
500
15
10
5
0
2,500
1,500
500
15
10
5
0
2,500
1,500
500
15
10
5
0
p16
Mcp1
Pai1
Mmp10
Mmp12
Col1a1
Tnf
Tgf
p16
Mcp1
Pai1
Mmp10
Mmp12
Col1a1
Tnf
Tgf
p16
Mcp1
Pai1
Mmp10
Mmp12
Col1a1
Tnf
Tgf
10
5
10
4
10
3
10
2
10
2
10
3
10
4
10
5
0
0–304
10
2
10
3
CD31
10
4
10
5
0–105
10
2
10
3
CD31
10
4
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5
0–105
PDGFRα
M
–213
10
5
10
4
10
3
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2
0
M
–213
10
5
10
4
10
3
10
2
10
5
10
4
10
3
10
2
0
M
–213
200
150
100
50
(×1,000)
SSC-W
CD45
250
200
150
100
50
50
P1
P2
P3
100 150 200 250
(×1,000)FSC-A
50 100 150 200 250
(×1,000)SSC-H
50 100 150 200 250
(×1,000)FSC-A
Figure 2 | Bleomycin-induced senescence in murine lung cells. (a–d) Gating strategy for isolation of fibroblasts, epithelial and endothelial cells from the
lungs of mice 14 days post-aerosolized instillation of bleomycin (Bleo) or PBS. (a) Total single-cell suspensions (P1) were gated to exclude doublets (P2)
and CD45 þ cells (P3). (b) Fibroblasts (PDGFRa þ , EPCAM , CD31 and CD45 ), (c) epithelial cells (EPCAM þ , PDGFRa , CD31 and CD45 )
and (d) endothelial cells (CD31 þ , PDGFRa , EPCAM , CD45 ) were sorted from the P3 population. The expression of p16, SASP genes (Mcp1, Pai1,
Tnfa, Mmp10, Mmp12) and fibrotic genes (Col1a1 and Tgfb) were quantified by RT–PCR and are expressed relative to Hprt levels in sorted populations of
(e) fibroblasts, (f) epithelial cells and (g) endothelial cells (mean
±
s.e.m.; PBS n ¼ 8 (grey), Bleo n ¼ 6 (red); t-test, ***Po0.0005, **Po0.005, *Po0.05,
f
Pr0.07 and
#
Pr0.1.).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14532
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