Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis
Lee et al. Respiratory Research
Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary fibrosis
Jong-Uk Lee 0
Hyun Sub Cheong
Eun-Young Shim 0
Da-Jeong Bae 0
Hun Soo Chang 0 1
Young Hoon Kim
Jong-Sook Park 1
Hyoung Doo Shin
Choon-Sik Park 0 1
0 Department of Interdisciplinary Program in Biomedical Science Major, Soonchunhyang Graduate School , Bucheon , Korea
1 Division of Allergy and Respiratory Medicine, Department of Internal Medicine , Soonchunhyang , University Bucheon Hospital , Bucheon , Korea
Background: Idiopathic pulmonary fibrosis (IPF) is characterized by the complex interaction of cells involved in chronic inflammation and fibrosis. Global gene expression of a homogenous cell population will identify novel candidate genes. Methods: Gene expression of fibroblasts derived from lung tissues (8 IPF and 4 controls) was profiled, and ontology and functional pathway were analyzed in the genes exhibiting >2 absolute fold changes with p-values < 0.05. CCL8 mRNA and protein levels were quantified using real-time PCR and ELISA. CCL8 localization was evaluated by immunofluorescence staining. Results: One hundred seventy eight genes differentially expressed and 15 genes exhibited >10-fold change. Among them, 13 were novel in relation with IPF. CCL8 expression was 22.8-fold higher in IPF fibroblasts. The levels of CCL8 mRNA and protein were 3 and 9-fold higher in 14 IPF fibroblasts than those in 10 control fibroblasts by real-time PCR and ELISA (p = 0.022 and p = 0.026, respectively). The CCL8 concentrations in BAL fluid was significantly higher in 86 patients with IPF than those in 41 controls, and other interstitial lung diseases including non-specific interstitial pneumonia (n = 22), hypersensitivity pneumonitis (n = 20) and sarcoidosis (n = 19) (p < 0.005, respectively). Cut-off values of 2.29 pg/mL and 0.43 pg/mL possessed 80.2 and 70.7% accuracy for the discrimination of IPF from NC and the other lung diseases, respectively. IPF subjects with CCL8 levels >28.61 pg/mL showed shorter survival compared to those with lower levels (p = 0.012). CCL8 was expressed by α-SMA-positive cells in the interstitium of IPF. Conclusions: Transcriptome analysis identified several novel IPF-related genes. Among them, CCL8 is a candidate molecule for the differential diagnosis and prediction of survival.
Gene expression; IPF; CCL8; Transcriptome
Idiopathic pulmonary fibrosis (IPF) is characterized by
alveolar epithelial cell hyperplasia and increased
myofibroblast with the interstitial deposition of extracellular
matrix (ECM) . The disease course is highly variable
due to interactions between chronic inflammatory and
fibrosis-related processes [2, 3]. Exploration of global
gene expression in lung tissues may facilitate the
identification of novel candidate genes to further explain the
complex mechanism and to predict the clinical courses
of IPF. In a human study, 164 differentially expressed
genes were demonstrated in IPF lung tissues . In this
study, fibrotic lungs showed changes in the expression
of genes involved in ECM formation and degradation. A
comparison of rapid-and slow-progressor patients
revealed 437 differentially expressed genes involved in
morphogenesis . In another study, integration of the
expression levels of 134 genes enabled the
discrimination of progressive and stable subjects . Almost
identical patterns of gene expression to those of stable
IPF were reported in cases of acute exacerbation .
These studies have demonstrated novel candidate genes
related with IPF.
The use of whole-lung tissues, however, may be a
limitation of transcriptomic studies because transcriptomic
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changes are cell-type-specific . The pathologic
characteristics of IPF include mixed features with normal
lungs, alveolar inflammation, interstitial fibrosis, and
honeycomb changes . Furthermore, the extent of
fibrosis and inflammation varies markedly during the
disease course. Accordingly, selective separation of
homogenous cells from diseased lungs would be optimal,
but is problematic. Among the various cell types present
in lung tissue, fibroblasts are easily obtained and
maintained, and the biologic properties of IPF fibroblasts
differ from those of normal lung fibroblasts [9, 10]. To
further investigate the molecular mechanisms of IPF
lungs, a global transcriptome analysis was conducted
using fibroblasts obtained from the lung tissues of 8
patients with IPF and normal lungs of 4 subjects with
localized lung lesions. The differential expression of
CCL8 was validated using an additional number of
fibroblasts and bronchoalveolar lavage (BAL) fluid samples
from normal controls (NC), patients with IPF and those
with other interstitial lung diseases including
nonspecific interstitial pneumonia (NSIP), hypersensitivity
pneumonitis (HP), and sarcoidosis.
Plasma, BAL fluids, and lung tissues of the subjects with
diffuse interstitial lung disease were obtained from a
biobank in Soonchunhyang University Bucheon Hospital
after the study protocol was approved by institutional
review board (IRB) in Korea National Institute for
Bioethics Policy (KoNIBP; P01-201408-BS-01-00). Control
BAL fluids were obtained from general population and
hospital personnel, and blood samples were obtained from
spouses, general population and hospital personnel after
approval by the hospital ethics committee (SCHBC
201508-025-005, schbc-biobank-2015-013). An informed
written consent to participate was obtained from each
subject. The diagnostic criteria for IPF, NSIP, HP and
sarcoidosis were based on the international consensus
statement [1, 11–14]. All subjects were examined by physicians
to obtain their medical history and underwent a chest
X-ray, pulmonary function tests, high-resolution chest
computed tomography (HRCT), and blood tests to
exclude collagen vascular diseases. None of the IPF patients
had any evidence of underlying collagen vascular diseases
through clinical manifestations or laboratory tests. IPF
was diagnosed by the presence of a UIP pattern in the
pathological specimen (surgical IPF) and/or by HRCT in
patients who were not subjected to surgical lung biopsy
(clinical IPF). Two pathologists examined each slide
independently after they were informed of the patients’ age,
sex, and HCRT results. The pathologic recognition of the
NSIP pattern included two major aspects: (1) recognition
of the characteristic histologic features and (2) exclusion
of other patterns of ILD as described in the ATS/ERS
2002 classification [14, 15], previous publication of ours
 and the modified version on the histologic definition
of the NSIP pattern .
HP was diagnosed by the presence of compatible
clinical manifestations with a non-necrotizing
granulomatous interstitial bronchiolocentric pneumonitis .
The diagnosis of sarcoidosis was made on the basis of
the compatible clinical pictures and histologic
demonstration of noncaseating granulomas [12, 13] The
diagnosis of HP and sarcoidosis needed exclusion of
other diseases capable of producing a similar histologic
picture: Biopsy tissues were subjected to special stains
(acid fast bacilli stain and Gömöri methenamine silver
stain) to rule out microorganisms and fungi. IPF patients
were evaluated using serial FVC and DLCO
measurements. The annual rate of FVC decline [dFVC(%/year)]
was calculated as follows: (last FVC - baseline FVC)/
baseline FVC/year. The normal controls had no
respiratory symptoms, as determined by a screening
questionnaire , had a predicted FEV1 and FVC > 80%, and
had normal chest radiogram results.
Lung fibroblasts were cultured from the surgical specimens
of 14 patients with IPF and normal lungs of 10 subjects
who underwent surgery to remove stage I or II lung cancer
as described in the previous publication . Briefly, lung
specimens were finely minced and placed into 150 cm2 cell
culture flasks with tissue culture media (TCM) consisting
of DMEM (Lonza Walkersville, Inc., Walkersville, MD,
USA), 10% fetal bovine serum (Thermo Fisher Scientific
Inc., Rockford, IL, USA), 2mmol/L glutamine, and 1%
Walkersville, Inc Cells were maintained at 37 °C in a 5% CO2
incubator and serially subcultured to yield a
morphologically homogeneous population of adherent
fibroblasts under the microscopy and α-smooth muscle actin
(SMA) immunohistochemical stain (Abcam, Cambridge,
MA, USA) until the fourth passage. The cells were then
stored at −170 °C. Fifth-passage fibroblasts (2.5 × 106) were
seeded in 1mL TCM in 10cm2 dishes. After reaching 90%
confluence, the fibroblasts were washed twice with PBS
(Thermo Fisher Scientific Inc.) and used for RNA. Total
RNA was extracted using TRI reagent (Ambion, Carlsbad,
CA, USA). The cell pellets were prepared in RIPA buffer
for immunoblot analysis, and protein concentrations were
measured using a BCA kit (Thermo Fisher Scientific Inc.).
Transcriptome microarray and analysis of gene ontology
and functional pathways
Total RNA was extracted from the fibroblasts and
converted to cDNA, which was amplified and purified using
an Illumina® Total Prep™ RNA Amplification Kit (Ambion,
Carlsbad, CA, USA). A transcriptome assay was
performed using a HumanHT-12 (BeadChip Illumina, San
Diego, CA, USA) containing sequences representing
~47,315 probes, which covered 27,455 curated and
putative genes. The quality and quantity of the extracted
RNA were examined by a RNA quantification reagent
(Ribogreen®, Invitrogen, Carlsbad, CA, USA). Fluorescence
was determined using a fluorometer (Victor 3, Perkin
Elmer, Boston, MA, USA). An IlluminaiScan scanner was
used to create images of the microarrays. Intensities of the
images were measured using GenomeStudio (v.2011.1,
Illumina, Inc., San Diego, CA, USA) with Gene Expression
Module (v1.0). The expression value of each gene was
determined by calculating differences by perfect match
intensity minus mismatch intensity of the probe pairs in
use. Genes showing detection call p-values <0.01 were
discarded to reduce the number of false positives, and the
remaining 15,020 genes were analyzed. Fold change of
gene expression was calculated as follows: a mean of
expression levels of IPF-fibroblasts divided by that of control
fibroblasts if the levels were higher in the IPF than the
controls. In cases of countertrend, fold change was
determined by dividing the mean value of control group by that
of the IPF group and presented as minus value. The
microarray data were analyzed using ScoreGenes software
package (http://compbio.cs.huji.ac.il/scoregenes/). The
general approach to analysis has been previously described
. Correction for multiple testing was performed by
calculating the false discovery rate, as previously described
. Genes were defined as being substantially changed if
they had a P-value of less than 0.05 by t-test, and a
threshold number of misclassifications (TNoM) score of 0 and a
t-test with a P value of less than 0.05 and absolute fold
change of greater than 2, as previously described . A
heat map of the differentially expressed genes was
constructed using the GenomeStudio software. Gene
ontology enrichment was performed by the Gene Ontology
(GO) database using the WebGestalt (http://www.webges
talt.org/), which was based on hypergeometric
distribution to show the overrepresented gene ontology
categories (p < 0.05). P-value was calculated using
BINOMDIST function on the basis of overrepresentation of gene
ontology categories when compared to all genes on the
chip. The online program Pathway-Express (Onto-Tools,
Wayne State University, Detroit, MI, USA,
http://vortex.cs.wayne.edu/Projects.html) was used to explore
biologically relevant pathways impacted by a list of
input genes. The gene expression data were deposited
in the NCBI Gene Expression Omnibus (series
accession number GSE71351; (http://www.ncbi.nlm.nih.gov/
geo/query/acc.cgi?acc=GSE71351). Gene ontology and
pathway predictions were performed using the Gene
Ontology database (http://www.webgestalt.org/) and
Pathway-Express software (Onto-Tools; http://vortex.cs.
RT-PCR and real-time PCR of CCL8 mRNA
Total RNA purified using TRI reagent was treated with
a Turbo DNA-Free™ Kit (Ambion). Total RNA (3 μg)
suspended in diethylpyrocarbonate-treated water was
heated at 65 °C for 5 min with 0.5 μg of
oligodeoxythymidine and 10 mM dNTPs, and then cooled on ice.
Amplification was performed for 30 cycles (5min at 94 °C,
30s at 94 °C, 30s at 60 °C, and 30s at 72 °C) with extension
at 72 °C for 7min. The following primer sequences were
used: CCL8: sense 5′-TGGAGAGCTACACAAGAATCA
CC-3′andantisense5′-TGGTCCAGATGCTTCATGGAA3′; β-actin: sense 5′-GGACTTCGAGCAAGAGATGG-3′
and antisense 5′-AGCACTGTGTTGGCGTACAG-3′.
PCR products were separated on a 1.0% agarose gel
containing ethidium bromide in Tris-borate EDTA buffer
at 100V for 40min and visualized under UV light. The
CCL8 band intensities were normalized to those of
β-actin. Real-time PCR was performed using the StepOneTM
Real-Time PCR System (Applied Biosystems, Foster city, CA,
USA). The PCR mixture (20μL) contained 1μg cDNA, 1μL
10pmol forward and reverse primers, and 10μL 2 × Power
SYBR Green PCR Master Mix (Applied Biosystems). The
reaction was carried out in a two-step procedure: denaturation
at 95 °C for 15s and 60 °C for 1min, and melting at 95 °C
for 15 s, 60 °C for 1min, and 95 °C for 15s. Data
were analyzed by the 2-ΔΔCT method , presented
as the relative fold change after normalization to
Determination of CCL8 protein levels
BAL was performed in the mostly involved segments of
IPF on HRCT without any immunosuppressive therapies
or in the right middle lobe of normal control subjects as
previously described [2, 23–25]. Total cell count was
done using a hemocytometer. Differential count of five
hundred cells was performed on slides of BAL cells
prepared by a cytocentrifuge and stained with Diff-Quik.
Cell pellets were separated from supernatants using
centrifugation (500G, 5 min), and the supernatants were
stored −80 °C. CCL8 protein concentrations in BAL
fluids and plasma were measured in normal controls
and IPF patients using an ELISA kit (Abnova, Taipei,
Taiwan) according to the manufacturer’s
recommendations. The lower limit of detection was 1.5pg/mL; values
below this limit were regarded as 0 pg/mL. The
coefficients of variance for inter- and intra-assays were less
than 15%. CCL8 protein concentrations were measured
in BAL fluids and plasma using an ELISA kit (Abnova,
Taipei, Taiwan), and normalized to total protein
Double immunofluorescence staining of CCL8 and
αsmooth muscle actin
Paraffin blocks of IPF and control lung tissues were cut
into 4-μm-thick slices, deparaffinized, rehydrated, and
stained using hematoxylin and eosin. The sections were
incubated in Fc receptor blocker (InnovexBiosciences,
Richmond, CA, USA) for 30 min, incubated in TBS with
5% BSA for 1 h to block non-specific binding, and then
incubated with monoclonal anti–human CCL8 antibody
(1:100, Origene, Rockville, MD, USA) or polyclonal
antihuman α-SMA antibody (1:200, Abcam, Cambridge,
MA, USA) in 5% BSA overnight at 4 °C. After washing
with 1 × TBS, the sections were incubated with
secondary antibodies: FITC-conjugated goat anti-rabbit
antibody (1:2000, Abcam) and PE-conjugated donkey
anti-mouse antibody (1:2000, Abcam). Nuclei were
counterstained with DAPI (Invitrogen, Carlsbad, CA,
USA). Confocal laser scanning was performed using a
microscope (LSM 510 META at 100× magnification)
coupled to a Photometrics Coolsnap HQ camera
(Photometrics, Tucson, AZ, USA), and images were generated
using the Zeiss LSM image browser.
The data were analyzed using SPSS v. 20.0. The
differences in gene expression between NC and IPF groups
were considered statistically significant when the
absolute fold-change in the mean value was >2 and the
pvalue was < 0.05 using t-tests and the nonparametric
TNoM scoring method . Comparisons of CCL8
concentrations between the study groups were performed
using the Kruskal-Wallis test and post hoc analysis
(Mann-Whitney U test). A receiver operating
characteristic (ROC) curve, AUC, and cut-off value were
calculated using MedCalc . Correlations between the
CCL8 levels and other parameters were analyzed by
Spearman’s correlation coefficient. The data were
presented as median values with 25 and 75% quartiles for
skewed variables, or as the means ± SEM for those with
a normal distribution. An optimal cutoff level of CCL8
was calculated using Cutoff Finder  and survival
rates were estimated by Kaplan-Meier’s method and
compared using log-rank test. Values of p < 0.05 were
considered statistical significance.
Clinical characteristics of the study groups
Lung fibroblasts were cultured from 14 IPF-lungs and
10 control lungs. Among them, 8 IPF-fibroblasts and 4
controls were used for the transcriptome study
(Additional file 1: Table S1). BAL samples were obtained from
patients with IPF (n = 86), NSIP (n = 22), hypersensitivity
pneumonitis (n = 20) and sarcoidosis (n = 19) (Table 1).
The patients with IPF had significantly higher values for
total cell count and numbers of macrophages,
neutrophils and eosinophils in the BAL fluid, and lower FVC
and DLCO values compared with those of NC (p < 0.05).
The IPF group was comprised of 34 surgical IPF and 52
clinical IPF patients. There were no significant
differences in the clinical and physiological parameters
between the two groups (Additional file 1: Table S2).
NSIP, hypersensitivity pneumonitis and sarcoidosis
groups also had significantly higher values for total cell
count and numbers of macrophages, neutrophils and
eosinophils in the BAL fluid, and lower FVC and DLCO
values compared with those of NC(p < 0.05).
Global gene expression profiling of the IPF and control
The cultured fibroblasts expressed α-SMA, but not
Ecadherin on Western blotting (Additional file 2:
Figure S1). The intensities of α-SMA were significantly
higher in the fibroblasts from IPF lungs (p = 0.011). The
expression levels of 15,020 genes were compared between
the two groups. Unsupervised hierarchical clustering was
done using the 15,020 genes in IPF fibroblasts and control
fibroblast (Additional file 3: Figure S2). Although no gene
passed the false discovery rate of less than 5% correction
for multiple testing, 178 genes showed different
expression levels according to the t-test and TNOM (p < 0.05
and fold changes > 2, Additional file 1: Table S3) (Fig. 1a).
The expressions of 109 genes were increased, while that of
69 genes decreased in the IPF group compared to the
control group. The top 15 genes showed 10-fold or greater
changes: 13 genes (PF4V1, MYOC, CCL8, ROR2, HBG2,
D4S234E, KCNJ2, RGS18, PITX1, EPB41L3, FGF7, LOC,
and POSTN) were increased, and 2 genes (FLJ25037 and
IGFBP2) were decreased (Table 2 and Fig. 1b). CCL8
expression was 22.8-fold higher in IPF-fibroblasts compared
Ontology and pathway analysis of the differentially
A gene ontology analysis of the 178 genes was
conducted. In comparison with those expected, the
observed gene numbers were significantly higher in a total
of 16 ontology categories (corrected p-values < 0.05;
Additional file 1: Table S4). A ratio of enrichment
of > 10 and a p-value < 0.001 was found in the following
categories: regulation of fibroblast growth factor
production (p = 4.6 × 10−5, ratio = 167.6) and cell adhesion
molecular binding (p = 0.0005, ratio = 19.2) (Fig. 1c). The
differentially expressed genes had a significant impact on
pathways. Among 109 up-regulated genes, 11genes
(BMP2, CCL26, CCL8, EPAS1, EPB41L3, FGF7, GAS1,
LEF1, PF4V1, PRKG2 andPTGS2) were mapped to 7
significant pathways (corrected gamma p-value < 0.05),
including Adherens junction, Melanoma, Cytokine-cytokine
Table 1 Clinical characteristics of the study subjects who underwent broncholaveolar lavage
Follow up duration (years)
BAL total cell count (×104/mL)
IPF: Idiopathic pulmonary fibrosis, NSIP: Nonspecific interstitial fibrosis, HP: Hypersensitivity pneumonitis
CS /ES/NS: current-smokers/ex-smokers/ never-smokers, ND: not determined, dFVC(%): annual decline rate of FVC
Difference in patient characteristics and pulmonary function test, shown as median (IQR), among the controls, IPF, NSIP, HP and sarcoidosis groups were
calculated with Kruskal-Wallis analysis of variance and Mann-Whitney U-test as post-hoc test
BAL cell numbers, shown as mean ± standard error of the mean, among the five groups were compared using one-way ANOVA analysis of variance with Tukey’s
honestly significant difference test as post-hoc test
Significances: Compared with control: *P <0.05, compared with IPF: †P < 0.05
receptor interaction, Pathways in cancer, Hedgehog
signaling pathway, Tight junction, and Long-term
depression (Additional file 1: Table S5 and Fig. 1d). Among 68
down-regulated genes, 5 genes (CADM1, ITGA10, LLGL2,
NLGN1 and PRKCB) were also mapped to 5 significant
pathways (Regulation of actin cytoskeleton, Tight junction,
Long-term potentiation, Cell adhesion molecules (CAMs),
MAPK signaling pathway). CCL8 was included in the
ontology categories of extracellular region, receptor
binding, heparin binding, G-protein-coupled receptor binding,
chemokine activity, carbohydrate derivative binding, and
glycosaminoglycan binding and the cytokine-cytokine
receptor interaction pathway.
Quantitation of CCL8 mRNA and protein levels in
Fibroblasts from 14 IPF lungs and 10 control lungs were
used. The CCL8 mRNA level normalized to that of
β-actin was 3-fold higher in the IPF fibroblasts than in
the control fibroblasts by RT-PCR (p = 0.0001; Fig. 2a
and b) and real-time PCR (p = 0.022; Fig. 2c). The CCL8
mRNA levels determined by the transcriptomic analysis
showed a strong correlation to those determined by
real-time PCR in the 12 subjects (r = 0.615, p = 0.033;
Fig. 2d). In addition, we measured the CCL8 protein
amount in supernatants and cell lysates of the cultured
fibroblasts (1 × 106) using CCL8 ELISA kit (Abnova, Taipei,
Taiwan). CCL8 levels were significantly higher in the
supernatants from the IPF- fibroblasts compared those from
the control-fibroblasts (p = 0.026) and cell lysates (p = 0.446)
(Fig. 2e, f ). The CCL8 protein levels were well correlated
with the CCL8 mRNA levels (r = 0.511, p = 0.011) (Fig. 2g).
CCL8 protein levels in BAL fluids
The CCL8 levels were significantly higher in the IPF
patients than those in the NC [6.01(2.75–17.16pg/mL)
vs. 0.90(0.00–2.07pg/mL), p = 7.62E-10], those in the
NSIP [0.12(0.00–7.47pg/mL), p = 0.00079], those in the
HP [1.52(0.00–7.37pg/mL), p = 0.0029] and those in the
sarcoidosis patients [0.00(0.00–1.9pg/mL), p = 0.000027]
(Fig. 3a). A ROC curve showed a clear difference
between the IPF patients and NC (AUC = 0.857, Fig. 3b),
and the other interstitial lung diseases groups (including
NSIP, HP and sarcoidosis, n = 61) (AUC = 0.750, Fig. 3c).
A cut-off value (2.29pg/mL) possessed 80.2% accuracy
with 86.0% specificity and 65.7% sensitivity between the
IPF patients and NC. A cut-off value (0.43pg/mL)
possessed 70.7% accuracy with 91.9% specificity and
57.4% sensitivity between the IPF patients and the other
interstitial lung diseases groups. The CCL8 levels were
analyzed in 69 subjects followed up for 1 to 8 years with
respect to the survival rate. When the subjects were divided
into two groups with a cut-off value of 28.61 pg/mL, the
survival rate was significantly lower in the group > 28.61
pg/mL compared with that in the group < 28.61 pg/mL
(hazard ratio = 3.93; CI: 1.25–12.39; p = 0.012; Fig. 3d). The
Fig. 1 Gene expression profiles of fibroblasts derived from the lung tissues of 8 patients with IPF and 4 controls. a A heat map of 178 genes
differentially expressed between the two groups (p-value <0.05 and absolute fold-change ratio >2 by t-test and TNoM). The maximum value (red)
of each gene was set to 3, the minimum value to −3, and the remaining values were linearly fitted in the range. b A heat map of top 15 genes
differentially expressed between the two groups (p-value < 0.05 by t-test and TNoM, absolute fold-change ratio >10). c The top 8 significantly
perturbed Gene Ontology nodes in the IPF patients versus the controls. Left, statistical significance of the perturbation as determined by a gene set test;
right, ratio of enrichment. The significance of differences between the dataset and the canonical pathway was measured as a ratio. Solid and open bars
represent upregulation and downregulation, respectively. d Biological pathway analysis of differentially expressed gene sets related to IPF (corrected
gamma p-value < 0.05). P-values and impact factors are plotted on the left and right axes, respectively
CCL8 protein concentrations showed a significant
correlation with neutrophil (p = 0.014, r = 0.297), while no
correlations with macrophage, lymphocytes and eosinophil
numbers in BAL fluid and physiological parameters
(Additional file 1: Table S6). There was no difference
of plasma CCL8 levels between 35 NC and 66 IPF
patients (p = 0.167; Additional file 4: Figure S3A), and no
correlation between CCL8 concentrations in plasma and
those in BAL fluids of 60 IPF patients (p = 0.169; Additional
file 4: Figure S3B).
Immunofluorescence staining for CCL8
To confirm CCL8 expression by myofibroblasts in IPF
lungs, CCL8 and α-SMA double immunofluorescence
staining was performed in 3 IPF and 3 control lung
tissues. In the control lungs, most probably smooth
muscle cells were stained for both α-SMA and CCL8. In
the IPF lungs, α-SMA was robustly expressed by
interstitial fibroblasts, most but not all of which expressed
CCL8 (Fig. 4, Additional file 5: Figure S4).
In our study, 178 genes were found to be differentially
expressed by the fibroblasts derived from IPF lungs.
Among the top 15 genes of them, only FGF7 and
POSTN have been previously reported as related with
IPF. The new genes identified in our study may provide
insight into the pathogenesis of IPF. Among the top
genes, CCL8 expression was >20-fold higher in the
IPFfibroblasts. CCL8 gene was reported to overexpress in
26 IPF lungs compared with 11 normal lungs (p = 0.099)
in the GDS1252 dataset, as well as in 6 sarcoidosis lung
1 PF4V1 63.21 ± 18.03 1.07 ± 0.27 0.048 0.039 59.2
3 CCL8 121.72 ± 45.48 5.33 ± 0.87 0.004 0.038 22.8
4 ROR2 70.83 ± 25.7 3.2 ± 2.7 0.048 0.034 22.1
5 HBG2 155.74 ± 48.34 8.07 ± 7.48 0.048 0.019 19.3
6 D4S234E 75.89 ± 21.97 4.04 ± 3.54 0.048 0.048 18.8
7 KCNJ2 111.29 ± 30.99 5.97 ± 2.12 0.004 0.011 18.6
8 RGS18 33.44 ± 12.08 1.99 ± 1.3 0.048 0.035 16.8
9 PITX1 592.66 ± 233.64 41.31 ± 14.57 0.048 0.05 14.3
Fig. 2 CCL8 mRNA and protein levels in lung tissue-derived fibroblasts from 14 IPF patients and 10 controls. (a) RT-PCR, (b) densitometry of the CCL8
RT-PCR band intensity normalized to that of β-actin, (c) real-time PCR, and (d) correlations of the CCL8 mRNA levels of 12 subjects determined by the
transcriptome chip with those by real-time PCR. e, f CCL8 protein level of Culture media and cell lysate, and (g) correlations of the CCL8 protein levels
and CCL8 mRNA levels of 24 subjects determined by the ELISA with those by real-time PCR. The data were presented as median values with 25 and
Fig. 3 CCL8 protein concentrations in BAL fluids and ROC curves. a CCL8 protein was detected in 25 of 41 normal controls, 80 of 86 IPF patients,
11 of 22 NSIP patients, 8 of 20 HP patients and 13 of 19 sarcoidosis patients. Open and closed circles indicate CCL8 protein levels detected
(>1.5pg/mL) and those below the lower limit of detection, respectively. The data were presented as median values with 25 and 75% quartiles.
b ROC curve of the CCL8 protein concentration between the two groups. A cut-off value of 2.17pg/mL had 80.2% accuracy, 86.0% specificity, and 65.7%
sensitivity for differentiating IPF patients from controls. c ROC curve of the CCL8 protein concentration between the other interstitial lung diseases groups.
A cut-off value 0.53pg/mL had 70.7% accuracy, 91.9% specificity, 57.4% sensitivity between the IPF patients and the other interstitial lung diseases group.
d A Kaplan-Meier plot of 69 subjects with IPF followed up for 1to 8 years. The percent survival rate was markedly lower in the group with a CCL8 level of
>28.61 pg/mL (red line) compared with that in the group with a CCL8 level of <28.61 pg/mL (black line) (hazard ratio = 3.93, CI: 1.25–12.39, p = 0.012)
compared with 6 normal lungs (p = 0.016) in the
GDS3580 dataset of NCBI GEO DataSet Browser
(https://www.ncbi.nlm.nih.gov/geo/). CCL8 was included
in the ontology categories of extracellular region, receptor
binding, heparin binding, G-protein-coupled receptor
binding, chemokine activity, carbohydrate derivative
binding, and glycosaminoglycan binding, which are essential
pathways in the pathogenesis of IPF. In validation, CCL8
levels in BAL fluids appeared useful as a candidate marker
for the differential diagnosis from NC and other chronic
interstitial lung diseases. Thus, we identified several novel
genes, and demonstrated the clinical relevance of CCL8 as
a candidate marker for the diagnosis and prognosis of IPF
patients for the first time to the best of our knowledge.
CCL8 is secreted into the peripheral circulation.
However, no correlation between CCL8 protein levels in BAL
fluids and peripheral bloods was observed. This may be
due to sources of CCL8 other than fibroblasts or
individual variance in the diffusion of CCL8 from IPF-fibroblasts
into the bloodstream. Among the CC chemokine ligand
family, CCL26 expression was increased in IPF patients
compared with controls (2.39-fold increase, p = 0.048) in
Fig. 4 Representative double immunofluorescence-stained images of IPF and control lung tissues. CCL8 and α-smooth muscle actin (α-SMA) were
stained using PE- (red) and FITC-conjugated antibodies (green), respectively. A proportion of interstitial fibroblasts (IT) and the peribronchial and
vascular area (VS) showed staining for both CCL8 and α-SMA (magnification, 200×)
our study. In other studies of IPF-patients, CCL2 are
present in metaplastic epithelial cells and vascular
endothelial cells  and CCL3, CCL4, and CCL7 expressions
are elevated in BAL fluids [29, 30]. The discrepancy
between our data and these reports may be due to the
presence of other sources of CCL2, CCL3, CCL4, and
CCL7 in the lung.
CCL8 activates various immune cells, including mast
cells, eosinophils, basophils, monocytes, T cells, and NK
cells . Recently, diverse functions of CCL8 have been
discovered in infection, immunity, and allergic
inflammation. CCL8 recruits gamma/delta T cells, which
preferentially express IL-17F and synergistically enhance
neutrophil chemotaxis in the presence of IL-8 .
CCL8 is induced by fibroblasts and endothelial cells
when co-stimulated with IL1-β and interferon (IFN)-γ.
IFN-γ has also a synergistic effect with activation of
TLR2, TLR3 or TLR4. The application of both IFN-γ and
dsRNA via TLRs resulted in the synergistic induction of
CCL8 expression . All of them are known mediators
involved in the development of IPF. Thus, CCL8 appears
to be related with innate immune response in the
development of IPF, but the exact role of CCL8 remains to be
solved in near future.
In the ontology analysis of the 178 genes, regulation of
fibroblast growth factor production, extracellular region,
positive regulation of the EMT, and positive regulation
of cell morphogenesis were the most relevant groups.
The fibroblasts used in our study expresses markers of
smooth muscle differentiation, such as α-SMA .
Lindahl GE and collaborators performed Gene ontology
analysis using 843 differentially expressed genes between
IPF and scleroderma fibroblasts and normal control
fibroblasts. Enriched functional groups represent 12 broad
categories as follows: anatomical structural development,
regulation of cell cycle, response to stress and wounding,
regulation of apoptosis, cell migration and smooth muscle
contraction in upregulation and inflammatory and
immune response, response to biotic stimulus, regulation of
apoptosis, regulation of cell migration, regulation of cell
proliferation, and regulation of I-kB/NF-kB cascade in
down regulation . The enriched functional groups are
in part compatible with our findings. Studies using human
whole lungs also showed elevated expression of genes
related to tissue remodeling/reorganization and ECM
formation/degradation [4, 5]. Interestingly, the expression of
CCL8 was positively correlated with those of IL-8,
IL-13RA2, and CCL2, ADAMTS1, ADAMTS8, MMP10,
MMP2, MMP3, TIMP2, ECM1, TGFBI, and CLEC3B, and
inversely correlated with FN1 (p < 0.05, respectively)
(Additional file 1: Tables S7 and S8). However, the
contents of the gene ontology are different between the
studies. In our study, the following 18 genes were included
in the extracellular region: PF4V1, NID1, PTHLH, CREG1,
TFPI, FBN2, RSPO3, TSKU, EPDR1, BMP2, CCL26,
POSTN, MYOC, CCL8, C1QTNF9B, CLEC18C, FGF7, and
TUBA4A. Among them, FGF7 , POSTN , TSKU
, and TFPI  have been suggested to be involved in
the pathogenesis of IPF. Two genes (PTGS2 and RGCC)
in our study were included in the fibroblast growth factor
production and EMT categories. Among them, PTGS2 is
known to be involved in the development of IPF .
These results indicate that more than half of the genes in
our study are newly identified.
Our study had the following limitation: the small
number of lung tissue samples available for microarray
analysis. Therefor we used the t-test and nonparametric
TNoM scoring method to compare the differences in
gene expression between NC and IPF groups because of
no gene passed of less than 5% correction for multiple
testing . The use of unadjusted P-values may be less
problematic than omitting informative genes in studies
aimed at identifying target genes responsible for
biological mechanisms . In addition, when the
differentially expressed genes in our study was compared with
those with Lindahl GE’s study using cultured fibroblasts
(n = 744, 10 controls and 3 IPF subjects) , those with
Sridhr S’s study using cultured fibroblasts (n = 1813, 4
controls and 10 IPF subjects; GSE44723), those with
Ronzani C’s study using (n = 3, 5 controls and 5 IPF
subjects; GES45686) and those with Emblom-Callahan’s
study using uncultured fibroblast (n = 1, 6 controls and
12 IPF subjects) . Only 3–10% of the differentially
expressed genes were overlapped between the studies as
seen in a Venn diagram in the Supplement (Additional
file 6: Figure S5). This discrepancy may be due to the
small numbers of fibroblasts in each study in addition to
the phenotypic changes of fibroblasts during the passage
Fibroblasts derived from IPF lungs have distinct
biological characteristics: a high percentage of apoptotic
cells, and increased collagen, fibronectin, gelatinase B,
TIMPs, β-FGF, and PDGF expression (i.e., a pro-fibrotic
secretory phenotype) , and a reduced capacity to
secrete anti-fibrotic molecules, such as prostaglandin E2
and hepatocyte growth factor . Interestingly, the
above-mentioned genes were not identified in our study.
Recently, a genomic expression in non-cultured
fibroblasts obtained from IPF-lungs demonstrated 1,813
significantly differentially expressed transcripts from those
of normal fibroblasts . When they were compared with
the 178 genes of our study, only 9 genes, including
ALDH3A2, CDC42EP3, IGFBP2, MOXD1, NBEAL2,
PITX1, POSTN, TMEM51, and UBE2K, were overlapped.
This may be due to biological differences between the
cultured fibroblasts used in our study and the uncultured
ones from IPF-lungs. However, we validated the CCL8
concentration in BAL fluid in 86 patients with IPF and
those in 41 controls and CCL8 protein expression using
immunohistochemical stain with antibodies to CCL8 and
the BAL fluids and lung tissues were obtained in
uncultured condition, so CCL8 protein may be generated per
se, not solely due to a phenotypic shift of the fibroblasts.
However, replication of the result is mandatory for useful
biomarkers. Another limitation was the use of control
fibroblasts from the resected cancer specimens. It cannot
be excluded that the gene expression in fibroblasts derived
from the lungs in which cancer has developed may be
different form fibroblasts derived from the normal lungs.
Epigenetic changes, such as CpG
miRNA, are widespread throughout the genome in
IPFlung tissues and may regulate the expression of important
genes [43–46]. Although fibroblasts have been studied
mainly at the steady-state RNA level, there is evidence for
the abnormal regulation of mRNA translation in the
fibroblasts . Furthermore, the surrounding environment
influences the gene expression of fibroblasts. Recently,
diseased ECM was reported to be the predominant driver
of pathological gene expression, and the expression of
ECM-sensitive genes is regulated primarily at the
translational level . Genes encoding IPF-associated ECM
proteins are targets for miR-29, which is downregulated in
fibroblasts grown on IPF-derived ECM. Other candidate
miRNAs are localized to the chromosome 14q32
microRNA cluster, many of which belong to the miR-154 family
. Thus, the differentially expressed genes in our study
should be analyzed together with global changes in CpG
methylation and miRNA expression in the future.
Global gene expression profiling of fibroblasts from lung
tissues from IPF patients was performed to identify
novel candidate genes. CCL8 was validated as a
candidate in BAL fluids and lung tissues samples. A total of
178 genes showed differential expression; among 15
genes showing ≥ 10-fold changes, 13 were newly
identified in relation with IPF. The CCL8 protein
concentrations in BAL fluids were significantly higher in patients
with IPF, and a cut-off value of 2.29pg/mL showed a
high degree of accuracy for diagnosis. The levels were
also higher compared to those in other interstitial lung
diseases including NSIP, HP and sarcoidosis with a
cut-off value (0.43 pg/mL) possessing a high degree of
accuracy for the discrimination from the other interstitial
lung diseases. The subjects with IPF with CCL8 levels
>28.61 pg/mL showed a reduced survival rate. In
conclusion, our transcriptome analysis identified new genes that
may be involved in the pathogenesis of IPF. Among them,
CCL8 may be useful as a candidate molecule for the
differential diagnosis of IPF and prediction of survival.
Additional file 1: Supplementary data. (DOC 379 kb)
Additional file 2: Figure S1. Expression of epithelial, mesenchyal and
myofibroblast markers of IPF fibroblasts and controls. Fibroblasts,
obtained from lung biopsies of 14 IPF and normal lung sections of
10 subjects with localized lung cancer, was characterized using
Western blot analysis for E-cadherin (epithelial marker), vimentin
(mesenchymal cell marker) and α-smooth muscle actin (myofibroblast
marker). The expression levels were normalized to β-actin as an
internal control protein. (TIF 8191 kb)
Additional file 3: Figure S2. A heat map of 15,020 genes in IPF
fibroblast and control fibroblast. A gradient scale ranging between green
(down-regulated) and red (up-regulated) was indicated. The maximum
value (red) of each gene was set to 3, the minimum value to −3, and the
remaining values were linearly fitted in the range. (TIF 18692 kb)
Additional file 4: Figure S3. Plasma CCL8 concentrations in the study
subjects and its correlation with CCL8 levels in BAL fluids. (A) CCL8
concentration in plasma from normal controls (n = 35) and IPF subjects
(n = 66), and, (B) correlation of the paired samples between plasma and
BAL fluids from IPF subjects (n = 60). The data were presented as median
values with 25 and 75% quartiles. (TIF 3385 kb)
Additional file 5: Figure S4. A blocking study using a recombinant
CCL8 protein in Immunofluorescence staining of CCL8 protein. The
mouse-anti human CCL8 antibody (1:100) were incubated with 10, 1,
0.1ng of a recombinant CCL8 (Origene, Rockville, MD, USA) for 2 h at
room temperature, then the mixtures were incubated for overnight with
the tissue sections of IPF lung tissues at 4 °C. The 2nd Ab (rabbit
antimouse-PE 1:2000) was incubated for 2 h and Confocal laser scanning was
performed using a microscope. As shown in the pictures, intensity of
CCL8 staining was decreased as the concentration of recombinant CCL8
protein increased. (TIF 8134 kb)
Additional file 6: Figure S5. Venn Diagram of the differentially
expressed genes by 4 studies using the cultured and uncultured
fibroblasts. The differentially expressed genes in our study (4 controls and
8 IPF-subjects, blue) were compared with those with Lindahl’s study
using cultured fibroblasts (10 controls and 3 IPF subjects, yellow) ,
those with Sridhr’s study using cultured fibroblasts (4 controls and 10 IPF
subjects, grey; GSE44723), those with Ronzani C’s study using cultured
fibroblasts (5 controls and 5 IPF subjects, green; GSE45686) and those with
Emblom-Callahan’s study using uncultured fibroblast (6 controls and 12
IPF subjects, red) . (TIF 7160 kb)
BAL: Bronchoalveolar lavage; ECM: Extracellular matrix; GO: Gene Ontology;
HP: Hypersensitivity pneumonitis; IFN: Interferon; IPF: Idiopathic pulmonary
fibrosis; NC: Normal controls; NSIP: Non-specific interstitial pneumonia;
ROC: Receiver operating characteristic
JU Lee and CS Park conceived and designed the experiments, EY Shim and
HS Chang performed the fibroblast culture and RNA isolation, HS Cheong
and HD Shin performed chip assay, DJ Bae performed ELISA, ST Uh, YH Kim,
and JS Park provided clinical samples, Bora Lee performed statistical analysis,
JU Lee, HS Cheong, and CS Park wrote the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
It was wrote at the materials and methods sections; as follows: The study protocol
was approved by institutional review board (IRB) in Korea National Institute for
Bioethics Policy (KoNIBP; P01-201408-BS-01-00) and the Soonchunhyang University
hospital ethics committee (SCHBC 2015-08-025-005).
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