Am J Physiol Lung Cell Mol Physiol 307: L681–L691, 2014.

First published September 26, 2014; doi:10.1152/ajplung.00014.2014. Review

CALL FOR PAPERS Biomarkers in Lung Diseases: from Pathogenesis to Prediction

to New Therapies

Molecular biomarkers in idiopathic pulmonary fibrosis

Brett Ley,1 Kevin K. Brown,2 and Harold R. Collard1
1
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Francisco,
San Francisco, California; and 2Department of Medicine, National Jewish Health and the University of Colorado,
Denver, Colorado
Submitted 17 January 2014; accepted in final form 12 August 2014

Ley B, Brown KK, Collard HR. Molecular biomarkers in idiopathic pulmonary

fibrosis. Am J Physiol Lung Cell Mol Physiol 307: L681–L691, 2014. First
published September 26, 2014; doi:10.1152/ajplung.00014.2014.—Molecular bio-
markers are highly desired in idiopathic pulmonary fibrosis (IPF), where they hold
the potential to elucidate underlying disease mechanisms, accelerated drug devel-
opment, and advance clinical management. Currently, there are no molecular
biomarkers in widespread clinical use for IPF, and the search for potential markers
remains in its infancy. Proposed core mechanisms in the pathogenesis of IPF for
which candidate markers have been offered include alveolar epithelial cell dys-
function, immune dysregulation, and fibrogenesis. Useful markers reflect important
pathological pathways, are practically and accurately measured, have undergone
extensive validation, and are an improvement upon the current approach for their
intended use. The successful development of useful molecular biomarkers is a
central challenge for the future of translational research in IPF and will require
collaborative efforts among those parties invested in advancing the care of patients
with IPF.
biomarker; diagnosis; prediction; pulmonary fibrosis

IDIOPATHIC PULMONARY FIBROSIS (IPF) is a chronic fibrotic lung
disease of unknown cause that generally affects adults over the
age of 50 years (75). On surgical lung biopsy, it is characterized
by the underlying histopathological pattern of usual interstitial
pneumonia (UIP) in the absence of secondary causes or associa-
tions (i.e., idiopathic UIP). IPF has a poor prognosis with an
average survival of 3–5 years, but there is appreciable heteroge-
neity among individual patients in disease course. At present,
there are no FDA-approved medications for IPF, leaving limited
therapeutic options for patients with progressive disease.
In categorizing patients with IPF, clinicians and researchers
currently rely on clinical data — history and physical exam,
radiology, pulmonary function tests, and histopathological pat-
tern — that do not directly reflect the underlying pathobiologi-
cal mechanisms of the disease. We are unable, therefore, to
adequately subphenotype patients with IPF who may have
quantitatively or qualitatively different biological mechanisms
responsible for their underlying disease. This is a major limi-
tation to both clinical care and research in the field.
In this article, we explore the field of molecular biomarkers,
which we use to refer to any objectively quantifiable substance at
the cellular level or smaller (e.g., protein) that can be measured in
Address for reprint requests and other correspondence: H. R. Collard, 505
Parnassus Ave., Box 0111, San Francisco, CA 94143 (e-mail: hal.collard
@ucsf.edu).
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biological tissue or fluids from patients with IPF. At their core,
molecular biomarkers should reflect and inform the underlying
biological mechanisms involved in a disease. This article aims to
provide a framework for thinking about molecular biomarker
development for IPF, using three core mechanistic themes rele-
vant to IPF to review the steps necessary to the development of
molecular biomarkers and to describe how the current crop of
molecular biomarkers perform.
DEVELOPMENT OF MOLECULAR BIOMARKERS FOR IPF
In theory, molecular biomarkers may be used in IPF in several
ways. These include identifying patients at risk for developing IPF
(predisposition biomarkers); making the diagnosis of IPF (diag-
nostic or screening biomarkers); determining a patient’s baseline
prognosis, staging disease severity, and monitoring for progres-
sion (prognostic biomarkers); and identifying target engagement
with a specific mechanistic response or predicting response or
toxicity to therapy, either by identifying a specific subgroup most
likely to respond to a therapy (therapeutic, predictive, or compan-
ion biomarker) or by substituting for a clinically meaningful
outcome/end point (surrogate biomarker/end point) (10, 23). Cur-
rently, there are no molecular biomarkers in widespread clinical
use for IPF for any of these uses.
There are two fundamentally different approaches to bio-
marker discovery, which both have value. In the hypothesis-
based approach, a candidate marker is selected a priori on the
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L682 MOLECULAR BIOMARKERS IN IPF

basis of preexisting rationale/evidence. This is the approach
that has been taken in the majority of biomarker studies in IPF
to date. This approach has the advantage of a strong biological
rationale and preliminary data, but it suffers from a lack of
efficiency in the discovery process. The unbiased or hypothe-
sis-free approach utilizes systems biology methods (e.g.,
genomic, transcriptomic, proteomic, and integratomic) to
screen a large number of candidate markers for their associa-
tion with the disease or outcome of interest, greatly increasing
the efficiency and scope of the discovery process but increasing
the risk of false discovery.
Most fundamentally, molecular biomarkers should reflect
the presence and activity of relevant pathobiological processes
or mechanisms. In addition, biomarker measurement should be
simple, technically accurate, and broadly reproducible and
have acceptable risk. Therefore, measurement from easily
obtainable body fluids or tissues is preferred [i.e., blood/serum,
urine, exhaled breath condensates ⬎ bronchoalveolar lavage
fluid (BALF) ⬎ transbronchial biopsy ⬎ surgical lung biopsy].
Molecular biomarkers require broad validation across sexes,
ages, ethnicities, and disease severity to assure generalizability
(13, 53). Practically, validation of the biomarker begins with a
derivation (or discovery) cohort of appropriate size and rele-
vance to its proposed role, in which reference ranges, thresh-
olds, inter- and intrasubject variability, and predictive perfor-
mance for the biomarker’s intended use are measured with the
appropriate statistical tests. Although techniques to reduce
optimism (e.g., cross-validation, bootstrap resampling) can be
used for “internal validation,” true validation (external valida-
tion) involves replicating results in one or more separate
cohorts (ideally prospective and multicentered).
If considered for use in clinical practice, molecular biomark-
ers should improve upon current clinical practice compared
with non-biomarker-driven strategies and their use should
ultimately lead to improved patient outcomes. For example, a
novel prognostic biomarker should add statistical performance
to well-established prognostic variables (e.g., pulmonary func-
tion tests), and its measurement should lead to alterations in
clinical management (e.g., change of therapy, referral for lung
transplantation). If molecular biomarkers do not have all of
these clinical characteristics, they still may add research value
by providing insights into disease biology. Figure 1 outlines
the ideal qualities of molecular biomarkers in IPF.
CANDIDATE MOLECULAR BIOMARKERS IN IPF
Over the past 15 years, the pathogenic paradigm for IPF
has shifted from one of uncontrolled inflammation toward

Fig. 1. Ideal qualities of molecular biomarkers for idiopathic pulmonary fibrosis. BAL, bronchoalveolar lavage; IPF, idiopathic pulmonary fibrosis; ILD,
interstitial lung disease.

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MOLECULAR BIOMARKERS IN IPF
one of alveolar epithelial cell dysfunction, immune dysregu-
lation, and fibroproliferation/fibrogenesis/matrix remodel-
ing (39, 85). In this paradigm, alveolar epithelial stress and
dysfunction results in activation of profibrotic signaling
pathways, fibroblast proliferation, and exuberant extracellu-
lar matrix deposition (39, 85). The resulting tissue destruc-
tion and architectural distortion eventually lead to organ
dysfunction (e.g., restricted ventilation, hypoxemia), dis-
ability (shortness of breath and exercise limitation), and
death from respiratory failure. In the remainder of this
article, we use these proposed core mechanistic pathways to
discuss and contextualize candidate biomarkers (Fig. 2).
Table 1 demonstrates the level of evidence supporting a
clinical role for each candidate biomarker.
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L683
Core Pathway 1: Alveolar Epithelial Cell Dysfunction
Surfactant proteins. Surfactant proteins are synthesized
and secreted by type II alveolar epithelial cells (AECs).
Their functions include facilitating surfactant function and
transport and innate host defense. Quantitative or qualitative
abnormalities in surfactant proteins are hypothesized to
increase alveolar epithelial endoplasmic reticulum (ER)
stress and trigger the unfolded protein response (UPR) (94).
Both mutations in genes encoding surfactant proteins and
the level of surfactant proteins in blood and the extracellular
lining fluid of the lung in patients have been proposed as
potential molecular biomarkers of IPF.
MUTATIONS IN GENES ENCODING SURFACTANT PROTEINS. Muta-
tions in surfactant protein genes, especially A2 and C, have

Infection (viral), microaspiration, inhalational (tobacco)

SPC ER stress
SPA2 MUC5B
Alveolar
TERT/TERC Epithelial cell TOLLIP
UPR macrophage
Short telomeres dysfunction ELMOD2
TLR3
Apoptosis

Th2 Lymphocytes
ECM
SPA, SPD, Epithelial to Collagen
CXCL13
KL6/MUC1, mesenchymal
TGFβ CCL18
α-defensins, transition
cCK18, MMP7, Fibrogenesis OPN
Immune dysregulation
MMP1, OPN YKL40
Fibroblasts and
myofibroblasts TLR3?
OPN
Periostin
CD28
Anti-
CD4 CD25
OPN CD4 CD25 HSP70
Periostin IgG
CD45
CD34
CCL18 CD4+
OPN FOXP3 T cell
Coll1 Sema7a FOXP3 Treg
Leukocytes Fibrocyte YKL40
Blood proteins Fibronectin Sema7a
SPA, SPD,
KL6/MUC1,
α-defensins,
cCK18, YKL40, Genomic Blood cells
Anti-HSP70 IgG, SPC TERT/TERC CD28+ CD4+ T cells
CXCL13, MMP7, TOLLIP SPA2 Tregs
MMP1, OPN, TLR3 Telomere length Semaphorin 7a+ T regs
Periostin MUC5B ELMOD2 Fibrocytes

Fig. 2. Core mechanisms and candidate molecular biomarkers for idiopathic pulmonary fibrosis (see text and Table 1). Pictured is the alveolar space (top, light blue),
epithelial cell layer (tan), interstitial space (white), and capillary (bottom, red). Injury to the alveolar epithelium from a variety of inciting factors (dark blue box) leads
to epithelial cell dysfunction, fibrogenesis/extracellular matrix (ECM) deposition, and immune dysregulation. Proposed molecular biomarkers of these processes include
blood proteins, genomic markers, and blood cells (see light green boxes). cCK18, cleaved cytokeratin 18; CXCL, C-X-C chemokine ligand; ELMOD2, ELMO
containing domain 2 gene mutations; EMT, epithelial-to-mesenchymal transition; ER, endoplasmic reticulum; HSP, heat shock protein; MMP, matrix metalloproteinase;
KL6/MUC1, Krebs von den Lungen 6/mucin 1; MUC5B, mucin 5B promoter polymorphism; OPN, osteopontin; SPA, surfactant protein A; SPA2, surfactant protein
A2 gene mutations; SPC, surfactant protein C gene mutations; SPD, surfactant protein D; TERT/TERC, telomerase gene mutations; TGF, transforming growth factor;
TLR3, Toll-like receptor 3 gene mutation; TOLLIP, Toll-interacting protein gene mutations; Treg, regulatory T cell; UPR, unfolded protein response.

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Table 1. Candidate molecular biomarkers for idiopathic and D (SPA and SPD), have been evaluated as diagnostic and
pulmonary fibrosis and the strength of evidence supporting prognostic molecular biomarkers in IPF. Because surfactant
their clinical role proteins are synthesized and secreted by type II AECs, their
levels in extracellular fluids may reflect alterations in produc-
Biomarker Predisposition Diagnosis Prognosis Therapeutic
tion and secretion by abnormal type II AECs, increased per-
Genomic meability of the epithelial-endothelial barrier, and/or type II
SPC ⫾ AEC injury leading to nonsecretory release. Gene expression
SPA2 ⫾
MUC5B ⫾ ⫹
profiling in IPF lungs demonstrates overexpression of the
TERT/TERC ⫾ SPA1 gene in some patients with progressive disease compared
Telomere length ⫾ ⫾ with those with relatively stable disease, lending support to
ELMOD2 ⫾ altered surfactant production in patients with progressive IPF
TOLLIP ⫾ ⫾ (11). Both SPA and SPD levels are decreased in BALF of
TLR3 ⫺ ⫾
Blood proteins patients with IPF compared with healthy controls but are
SPA ⫾ ⫾ ⫺ similar to other interstitial lung diseases (ILDs) including
SPD ⫾ ⫾ ⫺ nonspecific interstitial pneumonia (NSIP) (8, 34). Conversely,
KL6/MUC1 ⫾ ⫾ ⫺ serum levels of both SPA and SPD are elevated in IPF
␣-Defensins ⫾
cCK18 ⫾
compared with healthy controls (28, 34, 62). In small studies,
CCL18 ⫾ serum levels of SPA and/or SPD were significantly higher in
YKL40 ⫾ subjects with IPF compared with other ILDs, but with insuf-
Anti-HSP70 IgG ⫾ ficient evidence to establish them as diagnostic markers (28,
CXCL13 ⫾ 34, 62). Serum SPD levels have been reported to be signifi-
MMP7 ⫾ ⫹
MMP1 ⫾ ⫾ cantly elevated in acute exacerbations of IPF (AE-IPF) com-
OPN ⫾ pared with patients with stable IPF, suggesting that type II
Periostin ⫾ AECs may play a direct or indirect role in the pathobiology of
Blood cells AE-IPF (18). Prognostically, higher serum levels of both SPA
CD28% CD4⫹
T cells ⫾
and SPD have been associated with reduced survival in IPF
Tregs ⫾ independent of clinical variables (8, 28, 38, 92). However, in a
Semaphorin 7a⫹ recent prediction model study including 118 IPF patients,
Tregs ⫾ neither SPA nor SPD significantly improved discrimination of
Fibrocytes ⫾ survival times when added to a clinical model [age, forced vital
Key: No molecular or cellular biomarkers have a proven clinical role in IPF, capacity (FVC), lung CO-diffusing capacity (DLCO), and
but the strength of evidence supporting a potential role is denoted as follows: ⫹, change in FVC] (89). In two prospective treatment trials of
Consistent or strong evidence supporting potential role; ⫾, conflicting or pirfenidone, longitudinal changes in SPA and SPD levels were
inadequate evidence to support potential role; ⫺, consistent or strong evidence
against a potential role; blank, no evidence. SPC, surfactant protein C gene not significantly different between treatment and placebo
mutations; SPA2, surfactant protein A2 gene mutations; MUC5B, mucin 5B groups (7, 93). In sum, serum levels of SPA and SPD may be
promoter polymorphism; TERT/TERC, telomerase gene mutations; ELMOD2, markers of abnormal function, injury, apoptosis, or prolifera-
ELMO containing domain 2 gene mutations; TOLLIP, Toll-interacting protein tion of type II AECs, but their measurement has yet to be
gene mutations; TLR3, toll-like receptor 3 gene mutation; SPA, surfactant
protein A; SPD, surfactant protein D; KL6/MUC1, Krebs von den Lungen
proven clinically useful in making the diagnosis or predicting
6/mucin 1; cCK18, cleaved cytokeratin 18; HSP, heat shock protein; CXCL, prognosis in patients with IPF. Whether serial measurements
C-X-C motif chemokine; MMP, matrix metalloproteinase; OPN, osteopontin; can reflect disease activity over time is unknown.
Treg, regulatory T cell. KL6/MUC1. Krebs von den Lungen-6 (KL6)/mucin 1
(MUC1) is a large membrane-bound glycoprotein in the mucin
family expressed on the extracellular surface of type II AECs
provided important insights into the pathogenesis of IPF (4, 14, and bronchiolar epithelial cells in the lung, as well as glandular
15, 17, 45– 47, 50, 52, 57, 59, 66, 86, 94, 95, 97, 99, 101, 103). epithelial cells in other tissues including pancreas and breast
For example, some mutations have been shown to be associ- (35). Originally investigated as a potential tumor marker for
ated with the development of pulmonary fibrosis and with the adenocarcinomas, it has since been studied extensively as a
induction of chronic ER stress and the UPR in AECs (14, 15, diagnostic and prognostic biomarker in ILDs. Expression of
45, 50, 57). In animal models, a second profibrotic stimulus KL6 is increased in affected lung and regenerating type II
(e.g., viral infection, bleomycin) may be necessary for the AECs in a variety of ILDs including IPF (64). In vitro, KL6 has
development of fibrosis, suggesting that IPF may involve a also demonstrated chemotactic, proproliferative, and antiapo-
“two-hit” pathobiology, similar to that proposed for some ptotic effects on lung fibroblasts, suggesting a potential patho-
malignancies (15, 45). Because IPF is an uncommon condition genic role in pulmonary fibrosis (30, 63).
in the general population, and surfactant protein mutations are Serum levels of KL6 are significantly elevated in IPF and
rarely associated with sporadic IPF, the most likely role for the other ILDs compared with controls without ILD (34, 62).
use of surfactant protein mutations as molecular biomarkers in Elevated serum levels, especially greater than 1,000 U/ml, are
clinical practice would be identifying at-risk individuals within significantly associated with reduced survival in many ILDs,
families with pulmonary fibrosis known to have specific sur- including IPF (82, 107, 108). However, in the largest study of
factant protein mutations. IPF patients (n ⫽ 118), KL6 level at baseline demonstrated
SURFACTANT PROTEIN LEVELS. Surfactant protein levels mea- poor discrimination for survival time (C-statistic 0.58) and did
surable in BALF and blood, specifically surfactant proteins A not improve the prediction of survival beyond clinical predic-

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MOLECULAR BIOMARKERS IN IPF
tors (age, FVC, DLCO, and change in FVC) (89). In patients
with AE-IPF, serum KL6 was elevated compared with patients
with stable IPF, supporting a role for type II AEC injury in
AE-IPF (18). However, when evaluated longitudinally in pro-
spective treatment trials, changes in serum KL6 levels did not
track with treatment response (7, 93). In sum, KL6 may be a
marker of type II AEC injury and may indicate worse survival
among patients with ILDs, but it may not be specific to the
pathogenesis of IPF or useful for diagnosing or prognosticating
in IPF beyond readily available clinical information. Whether
serial changes in KL6 prove useful for monitoring disease
progression and/or diagnosing acute exacerbation in IPF de-
serves further study.
MUC5B. Genome-wide linkage scanning involving 82 fam-
ilies with familial pulmonary fibrosis (FPF) followed by fine
mapping of a region of interest on chromosome 11 in 575
subjects with pulmonary fibrosis (83 FPF, 492 sporadic IPF)
and 322 controls, identified a common single nucleotide poly-
morphism (SNP) in the putative promoter region of the mucin
5B (MUC5B) gene (rs35705950) that was highly associated
with disease (83). The association has been confirmed in
independent cohorts from the US and Europe (12, 109). In
total, the minor allele occurred in 34% of FPF, 34 – 42% of
sporadic IPF, and 9 –11% of control subjects. In the European
study, no association was found with systemic sclerosis-asso-
ciated ILD, suggesting potential specificity for IPF (12). In a
population-based study, there was an allele-dose-dependent
association with an increased prevalence of interstitial lung
abnormalities on chest imaging by computerized tomography
in individuals without clinically recognized ILD (i.e., subclin-
ical ILD), lending further epidemiological support for these
genetic polymorphisms in MUC5B as a risk factor for the
development of pulmonary fibrosis (32).
In addition to its association with an increased risk of
developing IPF, the MUC5B promoter polymorphism appears
to have prognostic value. In two independent IPF cohorts, one
drawn from clinical practice (n ⫽ 148) and one drawn from a
large clinical trial (n ⫽ 438), the presence of the MUC5B
mutation (measured by two different assays) was associated
with decreased mortality compared with the wild-type SNP,
independent of clinical factors (sex, FVC, and DLCO), and
when included in a clinical model significantly improved the
prediction of mortality (C-statistic increased from 0.68 – 0.69
to 0.71– 0.73) (70). It is unclear how to reconcile MUC5B’s
association with increased risk of developing IPF and yet a
better prognosis among those with established IPF. (70). The
mechanism by which the MUC5B promoter polymorphism
could cause or contribute to IPF is unknown. MUC5B is
overexpressed in the lung of subjects with IPF regardless of
allele status, as well as in unaffected carriers compared with
unaffected noncarriers, potentially implicating a direct role of
the MUC5B protein in the pathogenesis of IPF (83). Elucidat-
ing a mechanistic role for MUC5B in IPF could lead to
important advancements in our understanding of disease risk
and prognosis in a large proportion of familial and sporadic
cases.
Telomeres. Telomeres, the noncoding repetitive nucleotide
sequence at the ends of DNA that protect against degradation,
shorten with age (5). After telomeres reach a critical length,
activation of a p53-dependent checkpoint leads to apoptosis or
cellular senescence. Therefore, short telomeres, potentially
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L685
leading to AEC senescence and apoptosis, provide an attractive
pathogenic link between aging and the development of IPF (19,
76). In 2007, heterozygous mutations in telomerase genes
(TERT and TERC) were reported in 8 –15% of families with
IPF and rarely in sporadic cases of IPF (6, 98). Although
telomerase mutations are rare in sporadic cases of IPF, shorter
telomeres are common in sporadic IPF compared with age-
matched controls (2). In fact, nearly one-quarter of patients
with IPF have peripheral blood leukocyte telomere lengths in
the ⬍10th percentile (21). Short telomeres have also been
associated with risk of developing chronic obstructive pulmo-
nary disease (COPD), another aging-related lung disease and
thus may not be specific to the pathogenesis of IPF (80).
Recently, peripheral blood leukocyte telomere length has been
shown to be predictive of transplant-free survival in three
separate cohorts of patients with IPF (total n ⫽ 342), indepen-
dent of other prognostic factors (age, sex, FVC, and DLCO)
(90). Pending further validation and delineation of normal and
threshold values, peripheral blood telomere length holds prom-
ise as a mechanistic biomarker that may help predict predis-
position to disease and disease prognosis.
cCK18. In exploring the putative role of ER stress-UPR-
induced AEC apoptosis in the pathogenesis of IPF, a marker of
the activity of this process would be highly desirable. Cyto-
keratin 18 is a cytoskeletal protein found in AECs (and other
epithelial cells). During epithelial cell apoptosis, cytokeratin 18
is cleaved by caspases yielding a fragment, cleaved cytokeratin
18 (cCK18), which is detectable by a specific antibody. In a
recent study, cCK18 was detected by immunohistochemistry in
AECs of IPF lungs but not in normal lung tissue (16). In vitro,
cCK18 and mediators of the UPR increased following induc-
tion of ER stress in type II AECs. cCK18 was significantly
elevated in the serum of IPF patients compared with normal
controls and patients with hypersensitivity pneumonitis (HP)
and NSIP, distinguishing IPF from HP/NSIP with moderate
accuracy (AUROC 0.76). However, cCK18 level at baseline
was not associated with disease severity or outcomes in IPF.
Therefore, cCK18 may be mechanistically informative for the
ER stress-UPR-AEC apoptosis in IPF, with elevated levels
suggesting higher degrees of AEC apoptosis in IPF compared
with other ILDs. Although serum cCK18 levels at baseline do
not appear to be useful for determining prognosis, serial levels
have not been evaluated for measuring ongoing disease activity
and progression. Further study will be necessary to determine
whether cCK18 serum levels could serve as an efficacy marker
for drugs intended to target ER stress-UPR-AEC apoptotic
pathways.
Core Pathway 2: Immune Dysregulation
Innate and adaptive immunity contribute to tissue injury and
repair including fibrogenesis in many tissues (24). The contri-
bution of the immune system to tissue injury and fibrosis in IPF
is controversial. Clinically, IPF patients do not respond to
traditional immunosuppressive therapies. In fact, treatment of
IPF with prednisone and azathioprine was associated with
increased mortality and hospitalizations in a recent random-
ized-controlled trial (33). In addition, on histopathology, cel-
lular infiltration by inflammatory cells is not conspicuous in
IPF, consisting of minimal patchy lymphocytic and plasma cell
infiltrates with occasional lymphoid aggregates in areas of
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L686 MOLECULAR BIOMARKERS IN IPF
dense fibrosis (51, 75). A more nuanced look at immunity,
however, suggests that there are important aspects of the IPF
disease pathobiology that may be influenced by components of
the immune system.
Innate immunity. TLR3. Toll-like receptors (TLRs) are com-
ponents of the innate immune system that are critical for
response to infection and tissue injury. The pathogenic role of
a functional SNP in the TLR3 gene (L412F) was recently
investigated in IPF (61). Through a series of in vitro studies,
activation of TLR3 in IPF lung fibroblasts with the L412F
mutation demonstrated reduced cytokine responses and dys-
regulated fibroproliferation compared with wild-type (61).
Also, TLR3null mice had increased collagen and profibrotic
cytokine production compared with wild type. In two indepen-
dent cohorts of IPF totaling 308 subjects, this mutation was
associated with increased mortality independent of clinical
parameters (age, FVC, and DLCO), and in a clinical trial cohort
it was associated with accelerated physiological progression
(61). The TLR3 SNP was common (32– 43% were heterozy-
gous, 6.5–11% were homozygous) and may be a factor leading
to increased progression in established IPF.
ELMOD2. Genome-wide linkage scanning of six multiplex
FPF families from southeastern Finland, an area with cluster-
ing of FPF due to common ancestry, identified mutations in
ELMOD2 (ELMO domain containing 2) that was associated
with decreased ELMOD2 mRNA expression in the lung (31).
In the lung, ELMOD2 is expressed in macrophages and type II
AECs. In vitro, AEC cell lines overexpressing ELMOD2
activated gene expression pathways involved in response to
viral infections, and ELMOD2 inhibition blunted antiviral
cytokine expression after TLR3 activation, thus supporting an
antiviral response role for ELMOD2 (74).
TOLLIP. In a large genome-wide association study of IPF and
control subjects, SNPs in the TOLLIP gene (Toll-interacting
protein) were associated with development of IPF and de-
creased expression of TOLLIP protein (60). Counterintuitively,
one of the minor alleles was protective against the development
of IPF but associated with increased mortality among estab-
lished IPF subjects. TOLLIP is an adapter protein with ubiq-
uitin-binding domains that modulates TLR, interleukin-1, and
TGF-␤ signaling through receptor trafficking and degradation
(110).
␣-DEFENSINS. The ␣-defensins are small antimicrobial pro-
teins secreted by neutrophils and epithelial cells that are in-
volved in multiple functions of innate immunity (41). In a
small study, plasma protein levels were elevated in IPF and
correlated with measures of disease severity (56). More re-
cently, global gene expression profiling demonstrated ␣-de-
fensins 3 and 4 to be significantly upregulated in the blood and
lung tissue of AE-IPF compared with stable IPF (41). In lung
tissue from AE-IPF, ␣-defensin proteins were localized to type
II AECs in the setting of widespread evidence of AEC apo-
ptosis and proliferation. Peripheral blood gene expression pro-
filing of 130 patients with IPF demonstrated that ␣-defensins 3
and 4 were among 13 genes whose expression levels distin-
guished severe from mild IPF as defined by impairment in
DLCO (106). Interestingly, ␣-defensins are activated by MMP7,
which is also highly expressed in type II AECs in IPF lungs
and may be important in disease pathogenesis (see section on
MMPs below).
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Alveolar macrophage activation. CCL18. CC chemokine li-
gand 18 (CCL18), also known as PARC (for pulmonary and
activation-regulated chemokine), is a chemokine protein pro-
duced by alveolar macrophages that stimulates collagen pro-
duction in pulmonary fibroblasts (40, 71, 72). In fibrotic ILDs
including IPF, there are increased numbers of CCL18-positive
macrophages (71). CCL18 gene expression is increased in
alveolar macrophages derived from BALF, and this expression
correlates with impairment in pulmonary function (71, 72). In
vitro, upregulation of CCL18 in alveolar macrophages occurs
with Th2 cytokine stimulation (e.g., IL-13) and coculture with
fibroblasts via a ␤2-integrin/scavenger receptor pathway (71).
In turn, CCL18 derived from alveolar macrophages induces
fibroblast production of collagen, which appears independent
of TGF-␤ signaling pathways (49, 71). These studies suggest a
positive feedback loop for collagen production in which Th2-
activated alveolar macrophages [so called alternatively acti-
vated (M2) macrophages] produce CCL18, CCL18 stimulates
collagen production by pulmonary fibroblasts, and fibroblast-
derived collagen stimulates further CCL18 production by al-
veolar macrophages (71). In 72 patients with IPF, serum
CCL18 concentration at baseline predicted subsequent physi-
ological progression and survival independent of other clinical
parameters (73). Therefore, CCL18 may be a mediator and
marker of a fibrogenic pathway common to IPF and other
fibrotic ILDs, and its serum level may prove to be a useful
prognostic biomarker.
YKL40. YKL40 is a chitinase-like protein expressed in many
cell types that is involved in tissue response to injury and is
dysregulated in many acute and chronic inflammatory condi-
tions including COPD and asthma (48). Mechanisms by which
it may be involved in tissue fibrosis include mediating Th2
cell-IL-13 signaling pathways, inflammatory cell and fibroblast
survival and proliferation, and alternative alveolar macrophage
activation (48). YKL40 expression localizes to bronchiolar
epithelial cells and alveolar macrophages adjacent to fibrotic
areas in IPF, and protein levels are elevated in the BALF and
serum of patients with IPF compared with healthy controls (25,
42). Elevated serum concentrations correlate with worse gas
exchange (25), and in a small single-center study both BALF
and serum levels were associated with survival in IPF (42).
Adaptive immunity. ANTI-HSP70 ANTIBODIES. In a study inves-
tigating the role of antigen-specific autoimmunity in IPF, IgG
autoantibodies to heat shock protein (HSP)70 were identified in
the serum of 25% of patients with IPF compared with 3% of
healthy controls (37). HSP70 protein, antibody-antigen com-
plexes, and complement were common in IPF lung tissue.
HSP70 protein has been shown to induce CD4 T cells from IPF
patients to proliferate and produce profibrotic cytokines (IL-4),
and anti-HSP70 antibodies isolated from IPF patients have
been shown to activate monocytes and induce IL-8 production.
In a small, single-center study, anti-HSP70 IgG positivity in
IPF patients was associated with physiological progression and
worse 1-year survival.
CXCL13. The importance of B cells in the pathogenesis of IPF
is unknown, but a potential role is suggested by the identifi-
cation of lymphoid aggregates in IPF lungs along with evi-
dence for antibody dysregulation in some IPF patients (37, 51,
111). C-X-C motif chemokine 13 (CXCL13) is a chemokine
important for homing CXCR5-expressing B lymphocytes into
lymphoid aggregates (100). In a recent study of 95 subjects

MOLECULAR BIOMARKERS IN IPF
with IPF, CXCL13 was significantly elevated in blood and
lung tissue compared with controls (healthy subjects and sub-
jects with COPD), and CXCL13 expression localized to lym-
phoid aggregates in the lung (100). Both baseline levels of
CXCL13 and increases in CXCL13 over time were associated
with reduced survival, independent of baseline demographic
and physiological parameters. These findings require external
validation but suggest a potential role for CXCL13 as prog-
nostic biomarker in IPF, and the study authors theorize that
CXCL13 measurement could identify IPF patients who may be
responsive to therapies that target B cells.
T CELL SUBSETS. T cells are the predominant mononuclear cell
type in IPF lungs, and their density in IPF lung tissue has been
associated with disease severity and poor survival (69). CD4⫹
T cells express CD28, a costimulatory protein whose expres-
sion may be lost after repetitive cycles of antigen-driven
stimulation and T cell proliferation. CD4⫹ CD28null cells are
therefore considered a marker of chronic adaptive immune
activation (27). In a study involving 89 patients with IPF, the
proportion of CD28⫹ CD4 T cells in the peripheral blood
(CD28%) was low compared with healthy controls (27). Also,
low CD28% was correlated with low DLCO and reduced sur-
vival. In a subgroup of patients with serial measurements,
longitudinal changes in CD28% correlated with longitudinal
changes in FVC (27). Clustering of peripheral blood mononu-
clear cell gene expression profiles in IPF patients identified a
subgroup with downregulated genes involved in the “costimu-
latory signal during T cell activation” pathway including
CD28, ICOS, LCK, and ITK (29). Quantitative expression
levels of these four genes correlated with the percentage of
peripheral blood CD4⫹CD28⫹ T cells, and in two small
cohorts, improved prediction of transplant-free survival when
added to clinical predictors (29). These findings were con-
firmed in a validation cohort and were not explained by
immunosuppression use.
Regulatory T cells (Tregs) play an important role in modu-
lation of the adaptive immune response. In a small study, Tregs
(CD4⫹CD25⫹FOXP3⫹) were reduced in the BALF and
blood of patients with IPF compared with healthy controls and
other ILDs, and Treg suppressor function, not number, was
correlated with impairment in pulmonary function (43). Sema-
phorin 7a (Sema 7a) is a membrane protein that modulates
lymphocyte function. Its expression on Tregs is increased in
the lung and peripheral blood of patients with IPF, and periph-
eral blood Sema 7a⫹ Tregs are elevated in patients with
rapidly progressive disease (78). Sema 7a⫹ Tregs have re-
duced expression of regulatory cytokines (e.g., IL-10) and
were able to induce fibrosis in a TGF-␤-overexpression mouse
model.
Core Pathway 3: Fibroproliferation, Fibrogenesis,
and Extracellular Matrix Remodeling
MMPs. MMPs are a large family of zinc-containing metal-
lopeptidases that degrade multiple components of ECM, acti-
vate and degrade biological mediators (e.g., growth factors,
chemokines, and cell surface receptors), and facilitate cell
migration (68). There are 23 known MMPs expressed from 24
gene loci, and most are secreted (68). MMPs are critically
important in the homeostasis of the ECM, expressed at low
levels in healthy tissue and upregulated in wound healing (68).
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L687
MMPs are some of the most highly expressed genes in IPF
lungs (87, 111). In IPF lungs, MMPs are primarily produced by
activated type II AECs, but a few are produced by fibroblasts
within fibroblast foci (39). MMP1 degrades type 1 and 3
collagen and is expressed by type II AECs. MMP1 is increased
in lung tissue and BALF from patients with IPF, and serum
levels of MMP1 are higher in IPF compared with HP, sarcoid-
osis, and COPD (81). MMP7 is also produced by activated type
II AECs and upregulated in IPF (67, 87, 111). It has broad
substrate activity including multiple basement membrane and
ECM components, signaling molecules, and receptors, sug-
gesting a potential role in mediating an array of biological
processes (68). MMP7 colocalizes in IPF AECs with osteo-
pontin (OPN), a proinflammatory and profibrotic cytokine (see
next section on OPN below), and MMP7 expression/activation
is induced by OPN, which in turn is cleaved and activated by
MMP7 (1, 67). Similar to MMP1, serum protein levels of
MMP7 are higher in IPF compared with HP, sarcoidosis, and
COPD (81). In one study, the combination of serum MMP1
and MMP7 levels was able to distinguish IPF from HP with
96% sensitivity and 87% specificity (81). Serum MMP7 levels
also correlated with impairment in pulmonary function (FVC
and DLCO) (81). In a candidate serum protein screening ap-
proach in IPF patients, MMP7 concentration was identified as
significantly associated with survival and progression-free and
transplant-free survival in the discovery cohort (n ⫽ 140) and
transplant-free survival in the validation cohort (n ⫽ 101) (79).
MMP7 was further included in a prognostic model along with
clinical parameters (sex, FVC, and DLCO) derived from the
discovery cohort, demonstrating a good to excellent discrimi-
native performance for survival in the validation cohort (C-
statistic 0.73– 0.84) (89). However, model calibration was not
presented. In a large clinical trial cohort of IPF subjects (n ⫽
438), MMP7 was an independent predictor of survival in a
model including clinical parameters (sex, FVC, DLCO) and
MUC5B genotype (P ⫽ 0.04) (70). Change in serum MMP7
level is currently being used as a primary efficacy outcome in
a phase 2 trial of inhaled carbon monoxide therapy for IPF
(clinicaltrials.gov NCT01214187).
Several other MMPs are of interest in the pathobiology
of IPF. MMP3 expression is increased in IPF lungs, and
MMP3null mice are protected from bleomycin-induced lung
injury, suggesting a potential pathogenic role (104). In vitro,
MMP3 also cleaves and activates OPN (1), and exposure of
lung epithelial cells to MMP3 results in ␤-catenin signaling
activation via cleavage of E-cadherin and subsequent activa-
tion of epithelial-to-mesenchymal transition (104). MMPs may
play a role in fibrocyte migration into lung tissue. Human
fibrocytes strongly express matrix metalloproteinases (MMPs)
2, 7, 8, and 9, where MMP8 specifically mediates migration
through collagen 1 and MMP2 and 9 may facilitate migration
through basement membrane components (26). MMP9 is ex-
pressed in phenotypically altered (Thy-1 negative) fibroblasts
within fibroblast foci in response to TGF-␤1 stimulation (77),
and its activated form is increased in the BALF of patients
with rapidly progressive compared with slowly progressive
disease (84).
OPN. OPN is a secreted phosphorylated glycoprotein (also
called secreted phosphoprotein 1 or SPP1) that functions as a
mediator of inflammation and wound healing in many organs
and may play a role in multiple TGF-␤-mediated processes
Review
L688 MOLECULAR BIOMARKERS IN IPF
(22, 102). Its expression is increased in IPF lungs and localizes
to AECs and macrophages (67, 91, 105, 111). In vitro, OPN
promotes migration and proliferation of fibroblasts, and its
expression is increased by bleomycin-induced lung injury in
mice (67, 91). OPN induces upregulation of MMP7 in AECs
and colocalizes with MMP7 in AECs of IPF lungs (67). OPN
is elevated in the BALF and plasma of IPF patients compared
with healthy controls, but plasma levels are not significantly
different from other ILDs (36, 67). In a mix of ILD including
IPF, plasma OPN levels correlated with oxygenation but not
pulmonary function parameters (36). OPN gene expression is
increased in the lung tissue of IPF patients with progressive
disease compared with those with stable disease (11).
Periostin. Periostin is an ECM and intracellular protein
(so-called matricellular) that promotes ECM deposition, mes-
enchymal cell proliferation, and wound closure and contributes
to fibrosis in multiple organs (44, 58). In mice, periostin is
upregulated after bleomycin-induced lung injury, and blocking
periostin improves survival and limits collagen deposition.
Periostinnull mice are protected from bleomycin-induced fibro-
sis. Periostin is increased in lung tissue of IPF patients and
localizes to fibroblasts, especially to fibroblasts in fibroblast
foci (58, 65). Serum periostin levels are elevated in IPF and
correlate with physiological progression (58, 65). In asthma,
periostin is secreted by bronchial epithelial cells in response to
IL-13 and can serve as a marker of IL-13 expression and
treatment response to IL-13 antagonists (20, 88).
Circulating fibrocytes. Circulating fibrocytes are a subpop-
ulation of leukocytes that express hematopoietic (CD45,
CD34) and mesenchymal (Coll 1, fibronectin) markers (9).
Fibrocytes are mesenchymal progenitor cells believed to be
capable of producing ECM and differentiating into fibroblasts
and myofibroblasts (9). In murine pulmonary fibrosis models,
circulating fibrocytes contributed to the lung fibroblast popu-
lation (96) and impaired recruitment of fibrocytes protected
against fibrosis (55). Fibrocytes are detected in IPF lungs but
not healthy control lungs and are recruited by CXCL12 ex-
pression by AECs (3). In one study, circulating fibrocytes were
higher in IPF patients compared with normal controls and were
elevated in AE-IPF compared with stable IPF (54). In three
patients who recovered from AE-IPF, circulating fibrocyte per-
centage returned to preexacerbation levels in recovery. Circulat-
ing fibrocyte percent did not correlate with pulmonary func-
tion, exercise capacity, or extent of fibrosis on high-resolution
computed tomography; however, ⬎5% circulating fibrocytes
was associated with worse survival (54).
CONCLUSIONS AND FUTURE DIRECTIONS
Major advances in the understanding the pathobiology of
IPF have occurred over the past decade, leading to the recog-
nition of numerous potential molecular biomarkers, but the
field of molecular biomarkers for IPF remains in its infancy.
Only two prognostic biomarkers (MUC5B and MMP7) appear
promising enough at this time to consider for translation into
clinical practice. Currently, there is insufficient evidence to
support any biomarkers for other clinical roles. Mechanisti-
cally informative molecular biomarkers that are practical, ac-
curate, validated, and clinically useful are needed to move the
field forward. Characterization of potential molecular biomark-
ers for IPF should continue to take advantage of the wealth of
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data generated by systems biology research, which provides an
unbiased approach to identifying and validating candidate
biomarkers and mechanistic pathways.
IPF, like many other clinically defined diseases, is a biolog-
ically heterogenous disease, involving multiple complex and
interactive mechanisms, the relative importance of which may
vary among individual patients. Molecular biomarkers could
someday soon lead to diagnostic reclassification and/or sub-
phenotyping of IPF patients on the basis of an individual
patient’s relevant biology, directly informing treatment op-
tions. We believe that the development of clinically useful
molecular biomarkers for IPF is the central challenge to trans-
lational research in the field over the next decade and that
meeting this challenge will require collaborative efforts among
those parties invested in advancing the care of patients with
IPF.
ACKNOWLEDGMENTS
The authors wish to thank Dean Sheppard for thoughtful review of the
manuscript.
DISCLOSURES
H. R. Collard is a consultant to companies interested in the development of
biomarkers in IPF and is partially funded by an NIH grant focused on
biomarker development in IPF.
AUTHOR CONTRIBUTIONS
B.L., K.K.B., and H.R.C. conception and design of research; B.L., K.K.B.,
and H.R.C. prepared figures; B.L., K.K.B., and H.R.C. drafted manuscript;
B.L., K.K.B., and H.R.C. edited and revised manuscript; B.L., K.K.B., and
H.R.C. approved final version of manuscript.
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