International audienceAbstractThe activity of cysteine cathepsin B increased markedly in lung homogenates and in bronchoalveolar lavage fluids (BALF) of the mouse model of bleomycin-induced lung fibrosis after 14 days of challenge. In contrast the level of the cysteine cathepsin inhibitor cystatin C was unaffected in BALF of wild-type and cathepsin B-deficient mice. Therefore, murine cystatin C is not a reliable marker of fibrosis during bleomycin-induced lung fibrosis. Current data are in sharp contrast to previous analysis carried on human BALF from patients with idiopathic pulmonary fibrosis, for which the level of cathepsin B remained unchanged while cystatin C was significantly increased

Couillin, Isabelle

Gombault, Aurélie

Kasabova, Mariana

Lalmanach, Gilles

Lecaille, Fabien

Marchand-Adam, Sylvain

Reinheckel, Thomas

Villeret, Bérengère

PubMed

Mariana Kasabova

Bérengère Villeret

Aurélie Gombault

Fabien Lecaille

Thomas Reinheckel

Sylvain Marchand-Adam

Isabelle Couillin

Gilles Lalmanach

Crossref

Discordance in cathepsin B and cystatin C expressions in bronchoalveolar fluids between murine bleomycin-induced fibrosis and human idiopathic fibrosis

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LETTER TO THE EDITOR Open Access
Discordance in cathepsin B and cystatin C
expressions in bronchoalveolar fluids
between murine bleomycin-induced
fibrosis and human idiopathic fibrosis
Mariana Kasabova1,5, Bérengère Villeret2, Aurélie Gombault2, Fabien Lecaille1, Thomas Reinheckel3,
Sylvain Marchand-Adam1,4, Isabelle Couillin2 and Gilles Lalmanach1*
Abstract
The activity of cysteine cathepsin B increased markedly in lung homogenates and in bronchoalveolar lavage fluids
(BALF) of the mouse model of bleomycin-induced lung fibrosis after 14 days of challenge. In contrast the level of
the cysteine cathepsin inhibitor cystatin C was unaffected in BALF of wild-type and cathepsin B-deficient mice.
Therefore, murine cystatin C is not a reliable marker of fibrosis during bleomycin-induced lung fibrosis. Current data
are in sharp contrast to previous analysis carried on human BALF from patients with idiopathic pulmonary fibrosis,
for which the level of cathepsin B remained unchanged while cystatin C was significantly increased.
Keywords: Bronchoalveolar lavage fluid, Bleomycin, Cathepsins, Cystatins, Fibrosis, Lung, Proteases
Findings
Cathepsin B, cystatin C and idiopathic pulmonary fibrosis:
A quick focus
Idiopathic pulmonary fibrosis (IPF) is a chronic irrever-
sible lung disease of unknown etiology characterized by
an important deposition of extracellular matrix (ECM)
components in the interstitial space and alveoli [1]. Most
of in vivo studies relied on the murine model of
bleomycin (BLM)-induced lung fibrosis [2]. Besides
irradiation, fluorescein isothiocyanate (FITC) or silica
model, the bleomycin model is the best characterized
model with common characteristics of human IPF and is
considered to be clinically relevant. Moreover, the
experimental time frame is short and reproducible with
the development of fibrosis occurring by day 14 (D14) as
seen both biochemically and histologically [2]. However,
fibrotic mechanisms for IPF and bleomycin-induced lung
damage may be different, since experimental lung
fibrosis is induced by a single dose of bleomycin applied
via the intratracheal route and the role of inflammation
is crucial, while human IPF is the result of repeated and
diverse aggressions throughout the patient’s life and
inflammatory episodes are of secondary importance.
Moreover in the murine model, fibrosis is reversible as
opposed to IPF that is a progressive and irreversible
process leading to respiratory failure.
Cysteine cathepsins (11 members in humans) are
proteases participating actively in ECM remodeling and
in fibrotic disorders [3]. Cathepsin B (CatB) may
contribute to lung myofibrogenesis by triggering the
TGF-β1-driven Smad 2–3 pathway [4]. Conversely,
pharmacological inhibition and genetic silencing of CatB
diminished α-SMA expression, delayed fibroblast differ-
entiation and led to an accumulation of intracellular
pro-TGF-β1. In addition CatB drives activation of hep-
atic stellate cells, and participates in liver fibrogenesis.
Recently, a conclusive trial (phase I) for the treatment of
hepatic fibrosis (VBY-376, a CatB inhibitor from Virobay,
Menlo Park, Ca, USA) supported the notion that the use
of CatB inhibitors could be appropriate for therapy of lung
fibrosis.
* Correspondence: gilles.lalmanach@univ-tours.fr
1INSERM, UMR 1100/Centre d’Etude des Pathologies Respiratoires (CEPR),
Université François Rabelais, Faculté de Médecine, 10 Boulevard Tonnellé,
F-37032 Tours, cedex, France
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kasabova et al. Respiratory Research  (2016) 17:118 
DOI 10.1186/s12931-016-0432-6
The proteolytic activity of cysteine cathepsins is regu-
lated by their natural endogenous inhibitors belonging
to the cystatin family [5, 6]. Among this family, cystatin
C is the most potent inhibitor of cysteine cathepsins.
Cystatin C is synthesized and secreted by an extensive
variety of human cells with a widespread distribution in
body fluids and tissues [7]. A significant increase of
immunoreactive cystatin C was found in human bron-
choalveolar lavage fluids (BALF) from IPF patients,
raising the question of its use as a potential marker of
IPF [8]. On the other hand, mouse and human cystatin
C share 71 % of identity on amino acid level implying
highly similar structural and functional properties of the
homologues. Mouse cystatin C is expressed by all tissues
with a relative content very similar to that of human
tissues and has a widespread distribution in body fluids
[9]. In terms of regulation cystatin C is rather seen as a
housekeeping gene, because previous work could not
a)
b)
0 
100 
200 
300 
400 
500 
600 
700 
800 
WT NaCl WT BLM CatB-/- 
NaCl 
CatB-/- 
BLM 
WT NaCl WT BLM CatB-/- 
NaCl 
CatB-/- 
BLM  
P
ep
tis
ad
e 
ac
tiv
ity
 (
%
) 
Z-FR-AMC 
CA-074 
**
** **
**
**
**
0 
50 
100 
150 
200 
250 
300 
P
ep
tid
as
e 
ac
tiv
ity
 (
%
) 
Z-FR-AMC 
CA-074 
WT NaCl    WT BLM    CatB-/- 
NaCl 
CatB-/- 
BLM 
CatB-/- 
BLM 
CatB-/- 
NaCl 
WT BLM    WT NaCl    
** ** **
**
D1 D14
D1 D14
**
Fig. 1 Peptidase activity of cysteine cathepsins after bleomycin administration to wild type and cathepsin B-deficient mice. Mice (C57BL/6 strain)
were challenged with BLM (7.5 mg/kg for day 1; 3 mg/kg for day 14) or saline solution by intranasal instillation and sacrificed after 1 and 14 days
[19]. Lung homogenates and BALF were prepared as described earlier by Gasse et al. ([19]) and Kasabova et al. ([8]). a Lung homogenates and
(b) BALF were incubated in the activity buffer (0.1 M sodium acetate pH 5.5, 2 mM DTT, 2 mM EDTA, 0.01 % Brij35) for 5 min. at 30 °C. The overall
cathepsin activity was measured toward Z-FR-AMC (50 μM, λexc = 350 nm and λem = 460 nm) at day 1 (D1) and day 14 (D14) (Gemini spectrofluorimeter,
Molecular Devices). White bars: endopeptidase cathepsin activity. Grey bars: control experiments. Samples were preincubated in the presence of CA-074
(10 μM) for 30 min prior to measuring the peptidase activity. Data are expressed as mean values ± SEM. **p< 0.05 (Kruskall and Wallis non-parametric test)
(WT NaCl, n= 5; WT BLM, n= 6; CatB-/- NaCl, n= 5; CatB-/- BLM, n= 8)
Kasabova et al. Respiratory Research  (2016) 17:118 Page 2 of 5
establish its transcriptional regulation in response to
various stimuli (e.g. bacterial and viral infections, stress,
cytokines, growth factors) [10].
Increase of CatB activity in both lung homogenates and
bronchoalveolar lavage fluids
BALF and lung homogenates from wild type (C57BL/6
strain) and CatB-deficient (CatB-/-) mice [11] were
collected at D1 or D14 post-BLM treatment. To our
knowledge consequences of the genetic deletion of CatB
in bleomycin-induced fibrosis have not been reported or
studied elsewhere. In this study, CatB-/- mice served pri-
marily as control for assessment of the CatB-dependent
peptidase activity. Samples were handled as described
previously [8]. At D1, the overall cathepsin activity in
lung homogenates remained unchanged for BLM- and
saline-treated WT mice (Fig. 1a). Pre-incubation with
CA-074, a selective CatB inhibitor [12], showed that
CatB is the prevailing active cathepsin. Consistently, the
peptidase activity decreased dramatically (~85 %) in
lungs from saline- and BLM-treated CatB-/- mice. At
D14, BLM instillation resulted in a 3-fold increase of the
cathepsin activity in WT mice. Addition of CA-074 indi-
cated that this activity is mostly CatB-dependent. Again
cathepsin activity in BLM-treated CatB-/- mice was
markedly decreased (~80 %), confirming that BLM
administration to WT mice induces an overexpression
of CatB at D14 (Fig. 1a). Results correlate with a recent
article uncovering an increase of lung cathepsins after
BLM administration [13]. In this elegant study, the use
of an optical probe revealed a specific and maximal
labeling (D14) of cathepsins at sites of fibrotic lesions
correlated with the extent of disease burden [13].
Cysteine cathepsin activity was next considered in
BALF. At D1 post-BLM treatment, a ~2-fold increase of
the activity was observed in BLM-treated WT mice
compared to saline control. Pre-incubation with CA-074
demonstrated that active CatB, in contrast to the measure-
ments in lung homogenates, is not the major cathepsin
found in BALF at D1. The equivalent overall cathepsin ac-
tivity in BALF from CatB-/- saline control and BLM-treated
CatB-/- mice corroborated this statement (Fig. 1b). A 4-fold
increase of cathepsin activity was assessed for WT mice at
D14 post-BLM treatment and ~50 % of the peptidase
activity related to CatB contrary to that observed at D1.
Moreover, unlike lung homogenates, the cathepsin activity
increased ~6-fold in CatB-/- mice after BLM challenge
(Fig. 1b), supporting functional redundancy and/or the
establishment of compensatory mechanisms between
cysteine cathepsins [14, 15]. Present results differ from
those observed in human BALF where no significant differ-
ence in cathepsin activity, including CatB, was observed
between non-fibrotic and IPF patients [8]. A key point is
that a similar amount of macrophages, which are the
primary source of cysteine cathepsins in BALF, was found
for both groups of patients [8]. Conversely in the murine
BLM-induced lung fibrosis activated macrophages (with a
M2/M2-like phenotype) are predominant in the immune
infiltrate and BALF at D14, in association with the
presence of markers of the Th2 profibrotic response [13].
Nevertheless recruited macrophages have intermediate
proinflammatory and profibrotic phenotypes [16] and in
overall the changes in the levels of key cytokines and che-
mokines upon bleomycin-induced fibrosis were consistent
with those observed in human IPF [17].
The concentration of cystatin C is not affected by BLM
challenge
We reported earlier a significant increase of immunore-
active cystatin C, in human BALF from IPF patients rais-
ing the question of its potential use as a new biomarker
[8]. The increase of cystatin C level was significant for
each of three IPF severity grades (stages I, II, II). Cystatin
C has long been validated besides creatinine as a serum
a)
b)
0 
1000 
2000 
3000 
4000 
5000 
6000 
WT NaCl  WT BLM CatB-/- 
NaCl 
CatB-/- 
BLM  
C
ys
ta
tin
 C
 (p
g/
m
l) 
D1 
D14 
0 
200 
400 
600 
800 
1000 
1200 
1400 
1600 
WT NaCl WT BLM CatB-/- NaCl CatB-/- BLM  
C
ys
ta
tin
 C
 (p
g/
m
l) 
D1 
D14 
Fig. 2 Cystatin C immunoassay. Dosage of immunoreactive cystatin C
was performed after BLM (or saline solution) administration to wild-type
and CatB-deficient mice (see legend to Fig. 1). The concentration of
cystatin C was determined using the mouse/rat ELISA Quantikine kit
according to the manufacturer’s instructions (R&D Systems Inc,
Minneapolis, MN, USA). a Lung homogenates and (b) BALF. Data are
expressed as mean values ± SEM (WT NaCl, n= 5; WT BLM, n= 6;
CatB-/- NaCl, n= 5; CatB-/- BLM, n= 8)
Kasabova et al. Respiratory Research  (2016) 17:118 Page 3 of 5
biomarker of glomerular filtration rate, but we did not
measure a statistically confident variation in alveolar
concentration of cystatin C between patients with a low
(<60 ml/min) or a high (>60 ml/min) glomerular
clearance (p > 0.1) and no co-morbidity in conjunction
with cystatin C level was observed [8]. Here, cystatin C
concentration remained unchanged both in lung extracts
(Fig. 2a) and BALF (Fig. 2b) for BLM-treated and control
mice at D1 and D14. The same level of BALF cystatin C
was also measured for saline- and BLM-treated CatB-/-
mice. Conversely to that observed in human BALF from
IPF patients, cystatin C level is unaffected at D14 follow-
ing BLM challenge, supporting clearly that murine cysta-
tin C cannot be embraced as a marker in BLM-induced
lung fibrosis.
Of interest bronchopulmonary dysplasia (BPD) is
characterized by impaired alveolar development and
widespread bronchial disease, and consecutive fibrotic
changes are observed. Results in accordance with the
present study were reported using a non-human primate
model of BPD: mRNA and protein levels of CatB were
significantly increased in the lung tissue of baboons with
BPD. In contrast, both mRNA and protein levels of
cystatin C remained unchanged in lung tissue lysates
and BALF [18]. Although the rodent model of BLM-
induced fibrosis is of mandatory concern to decipher
proteolytic mechanisms of fibrogenesis, the present data
confirm and point out that transposition of the results
to human IPF should be done with caution.
Abbreviations
AMC: 7-Amino-4-methylcoumarin; BALF: Bronchoalveolar lavage fluid, BLM,
Bleomycin; CA-074: N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-
isoleucyl-L-proline; Cat: Cathepsin; ECM: Extracellular matrix; IPF: Idiopathic
pulmonary fibrosis; WT: Wild type; Z: Benzyloxycarbonyl
Acknowledgements
Not applicable.
Funding
This work was supported by la Région Centre-Val de Loire (FibroCat project;
number: 201000049823). We acknowledge the Institut National de la Santé
et de la Recherche Médicale (INSERM) for institutional fundings. MK held a
doctoral fellowship from MENRT (Ministère de l’Education Nationale, de la
Recherche et de la Technologie, France). Funding agencies had no role in
the design of the study, in the analysis and interpretation of data, and in
writing the manuscript.
Availability of data and material
All data generated or analysed during this study are included in this
published article.
Authors’ contributions
MK, IC and GL planned and designed research; MK, BV and AG performed
experiments; FL and SMA contributed analytic tools; TR provided genetically
engineered mice; MK, FL, IC and GL analyzed data; AG, FL, TR, SMA and IC
revised the paper; MK and GL wrote the paper; All authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
All animal experiments were examined and approved under the number
D-45-234-6 by the Ethic committee for animal experimentation of CNRS and
University of Orléans and complied with the French Government’s ethical
and animal experiment regulations.
Author details
1INSERM, UMR 1100/Centre d’Etude des Pathologies Respiratoires (CEPR),
Université François Rabelais, Faculté de Médecine, 10 Boulevard Tonnellé,
F-37032 Tours, cedex, France. 2CNRS, UMR-INEM 7355, Immunologie et
Neurogénétique Experimentales et Moléculaires, Université d’Orléans,
Orléans, France. 3Institute of Molecular Medicine and Cell Research, Medical
Faculty, Albert-Ludwigs-University Freiburg, Freiburg, Germany. 4Centre
Hospitalier Régional Universitaire Tours, Service de Pneumologie, Tours,
France. 5Present address: Laboratoire de Biologie des Tumeurs et du
Développement (LBTD), Université de Liège, Liège, Belgium.
Received: 9 April 2016 Accepted: 17 September 2016
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Kasabova et al. Respiratory Research  (2016) 17:118 Page 5 of 5


The activity of cysteine cathepsin B increased markedly in lung homogenates and in bronchoalveolar lavage fluids (BALF) of the mouse model of bleomycin-induced lung fibrosis after 14 days of challenge. In contrast the level of the cysteine cathepsin inhibitor cystatin C was unaffected in BALF of wild-type and cathepsin B-deficient mice. Therefore, murine cystatin C is not a reliable marker of fibrosis during bleomycin-induced lung fibrosis. Current data are in sharp contrast to previous analysis carried on human BALF from patients with idiopathic pulmonary fibrosis, for which the level of cathepsin B remained unchanged while cystatin C was significantly increased

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Discordance in cathepsin B and cystatin C expressions in bronchoalveolar fluids between murine bleomycin-induced fibrosis and human idiopathic fibrosis

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