Background {#Sec1}
==========

Idiopathic interstitial pneumonia (IIP) encompasses a group of diffuse parenchymal lung diseases characterized by interstitial involvement resulting from various patterns of inflammation and fibrosis of unknown cause. Based on histological features, IIP has been further classified into several subtypes, including idiopathic pulmonary fibrosis (IPF), which has the hallmark histopathologic feature described as usual interstitial pneumonia and nonspecific interstitial pneumonia (NSIP) \[[@CR1]--[@CR3]\]. The latest statement from American Thoracic Society (ATS) and European Respiratory Society (ERS) proposed the category "chronic fibrosing IIP" encompassing both IPF and NSIP \[[@CR3]\], because separation between these two diseases is difficult, with significant clinical, radiological, and pathological overlap between them \[[@CR4]\].

IPF is one of the most common and aggressive types of IIP and is characterized by alveolar epithelial damage that leads to inadequate tissue repair, collagen accumulation, and fibroblast proliferation, although the underlying molecular mechanisms remain unclear \[[@CR5]\]. Over the last decade, the few therapeutic options available have not been very effective and the outcome of IPF patients is poor \[[@CR6]\]. Pirfenidone is the first anti-fibrotic agent to be approved for IPF treatment, with its efficacy and tolerability supported by several clinical trials and surveillance \[[@CR7]--[@CR10]\]. Recently, nintedanib, a multiple tyrosine kinase inhibitor, also demonstrated clinical efficacy for IPF patients \[[@CR11]\]. However, these drugs only reduce the decline in forced vital capacity, without halting disease progression in all patients. Therefore, new diagnostic tools and therapeutic strategies, including molecular targeting drugs, are urgently needed.

Systematic analysis of the expression level of thousands of genes using microarray is an effective approach for identifying molecules that are altered in pulmonary fibrosis or after treatment with anti-fibrotic agents \[[@CR12]\]. Our group, as well as others, have performed high-throughput screens combined with gene expression analysis of lung diseases including cancers, and identified various potential targets for the development of new diagnostic tools and therapies \[[@CR13]--[@CR16]\]. However, few such analyses have been performed for IIP \[[@CR17]--[@CR21]\], and most of these studies have been performed in Caucasian populations with very limited data available from Japanese populations.

The present study aims to delineate the molecular mechanisms of pulmonary fibrosis and identify potential disease-specific biomarkers and/or therapeutic targets in the chronic fibrosing IIPs patients by using genome-wide microarray analysis followed by canonical pathway analysis.

Methods {#Sec2}
=======

Patients and clinical samples {#Sec3}
-----------------------------

Tissue samples were obtained by surgical lung biopsy from Japanese patients with newly diagnosed IIP at the Hiroshima University Hospital (Hiroshima, Japan) and who have never taken medication for IIPs before. All surgical lung specimens were immediately frozen and stored at −80 °C for later analysis. Each patient underwent physical examination, pulmonary function tests, high-resolution computed tomography, bronchoscopy, and bronchoalveolar lavage. IPF and NSIP were diagnosed according to the ATS/ERS criteria published in 2002 \[[@CR22]\]. Patients with evidence of collagen vascular disease, chronic hypersensitivity pneumonia, and other known causes of interstitial lung diseases (ILDs) were excluded. Control lung specimens for microarray analysis consisted of total RNA from three lungs (Caucasians aged 32--61 years; cause of death: sudden death) purchased from BD Biosciences Clontech (Lot Number 7080277; Palo Alto, CA, USA). Control tissues for immunohistochemistry were obtained from the healthy areas of lungs, removed locally, along with lung tumors. This study was approved by the Ethics Committee of Hiroshima University Hospital (IRB M33 and 326) and conducted in accordance with ethical standards established in the Helsinki Declaration of 1975. All participants provided written informed consent for the use of tissue specimens for the study and the publication of their individual data. Clinical characteristics of the 12 IIP patients are summarized in Table [1](#Tab1){ref-type="table"}.Table 1Clinical characteristics of idiopathic interstitial pneumonia casesVariablesMicroarray cohortIHC cohort(*n* = 12)(*n* = 10)Age (years)66.2 ± 2.163.1 ± 1.9Gender (male/female)\*9/37/3Smoking history (Smoker/non-smoker) \*9/37/3Pack-years\*23.8 ± 6.926.4 ± 7.8Disease category (IPF/ NSIP)7/55/5Pulmonary function test VC (% predicted)86.0 ± 6.288.9 ± 6.6 DLco (% predicted)57.2 ± 5.354.1 ± 5.5Values are expressed as mean ± SEM or as a number\**P* \< 0.05 (Mann-Whitney U or χ^2^ test between two groups)*IHC* immunohistochemical analysis, *IPF* idiopathic pulmonary fibrosis, *NSIP* nonspecific interstitial pneumonia, *VC* vital capacity, *DL* ~*CO*~ single-breath carbon monoxide diffusing capacity

RNA isolation and gene expression profiling {#Sec4}
-------------------------------------------

Gene expression profiles of frozen tissue specimens from 12 IIP patients, derived from the central part of the surgical lung biopsy, were analyzed by GP Biosciences Ltd. (Kanagawa, Japan). RNA quality was verified using the RNA6000 Nano Assay on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Illumina Human WG-6 v3 BeadArrays (Illumina Inc., San Diego, CA, USA) with about 48,000 transcripts were used according to the manufacturer's instructions. An Illumina TotalPrep RNA amplification kit (Ambion, Inc., Austin, TX, USA) was used to obtain biotin-labeled cRNA from 500 ng total RNA. As a control probe, normal human lung poly(A) RNA (BD Biosciences Clontech) was amplified under the same conditions. cRNA was synthesized overnight (18 h), labeled, and hybridized to the chip at 58 °C overnight. Hybridized arrays were labeled with streptavidin-Cy3 (PA43001; Amersham, Buckinghamshire, UK) and scanned with an Illumina BeadArray reader (Illumina Inc.). Scanned images were imported into BeadStudio v3 software (Illumina Inc.) for extraction, quality adjustment, and quantile normalization. Satisfactory quality was observed for all arrays and samples.

Functional and canonical pathway analyses {#Sec5}
-----------------------------------------

The microarray gene expression data was analyzed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood City, CA, USA) to determine whether genes associated with particular diseases, biological functions, or canonical signaling pathways were preferentially up- or downregulated in IIP patients relative to control subjects. Diseases and biological functions for which differential gene expression was observed were grouped into three categories: (1) diseases and disorders; (2) molecular and cellular functions; and (3) physiological system development and function.

Clustering analysis of microarray data {#Sec6}
--------------------------------------

To assess the difference and similarity in the gene expression profile between IPF and NSIP, a hierarchical clustering method was applied to genes and IIP subtypes. To obtain reproducible clusters for classifying the 12 IIP patients, 159 genes were selected for which valid data was obtained in 80% of the experiments, and whose expression ratios varied by standard deviations of \>3.0. Gene Cluster 3.0 and Java TreeView software developed by Eisen et al. were used to analyze the data \[[@CR23], [@CR24]\]. Before applying the clustering algorithm, the fluorescence ratio for each spot was log-transformed and the data for each sample was median-centered to remove experimental biases.

Immunohistochemical staining and morphometric analysis {#Sec7}
------------------------------------------------------

To evaluate the protein expression of two upregulated genes, desmoglein 3 (*DSG3*) and Krebs von den lungen-6 (*KL-6*)/Mucin 1 (*MUC1*), clinical tissue sections from 5 IPF patients, 5 NSIP patients, and 5 control lungs were stained using ENVISION+ Kit/horseradish peroxidase (HRP) (Dako Japan, Tokyo, Japan), as previously described \[[@CR25]\]. For antigen retrieval, slides were immersed in Target Retrieval Solution, Citrate pH 6 (Dako Japan) and boiled at 108 °C for 15 min in an autoclave. After blocking endogenous peroxidase activity with 0.03% H~2~O~2~ for 30 min, sections were incubated with mouse anti-human *DSG3* (Clone \#216519; R&D Systems, Minneapolis, MN, USA) and KL-6 antibodies, which were purified as previously described \[[@CR26]\]. The slides were then treated with HRP-labeled anti-mouse IgG secondary antibody followed by the addition of a chromogenic substrate. Sections were counterstained with hematoxylin. Image-Pro Plus 6.3 (Media Cybernetics. Inc. Rockville, MD, USA) was used for morphometric analysis to quantify the positively stained areas in the lung tissue, as previously described \[[@CR27]\].

Statistical analyses {#Sec8}
--------------------

Data were analyzed with SPSS for Windows, version 18.0 (SPSS Inc. Chicago, IL, USA) and are presented as mean ± SEM. Data for individual variables from the various groups were analyzed by the Kruskal-Wallis test followed by multiple comparisons using rank sums \[[@CR28]\]. Mean differences were considered statistically significant at *P* \< 0.05.

Results {#Sec9}
=======

Identification of genes up−/downregulated in IIP {#Sec10}
------------------------------------------------

Clinical characteristics of the 12 patients with chronic fibrosing IIP (IPF, *n* = 7; NSIP, *n* = 5) analyzed by microarray were shown in Table [1](#Tab1){ref-type="table"}. In total, 98 genes were upregulated while 1193 were downregulated in the lung tissue of IIP patients compared to control subjects, based on expression ratios that were \>20.0 or \<0.05 respectively, in at least 75% (i.e., 9 out of 12) of informative cases. The top 50 genes upregulated in IIP are listed in Table [2](#Tab2){ref-type="table"}. Some of the upregulated genes, namely *DSG3*, protocadherin gamma-A9 (*PCDHGA9*) and discoidin domain-containing receptor 1 (*DDR1*) are implicated in cell-cell interaction and/or adhesion; some, namely collagen type VII, alpha 1 (*COL7A1*), contactin-associated protein-like 3B (*CNTNAP3B*) and *MUC1* are encoding the extracellular matrix molecule or the molecules involved in cell-matrix interactions; and the others, namely *CDC25C* and growth factor independent protein 1B (*GFI1B*) are known to affect cell proliferation by affecting the progression of cell cycle or regulating transcription. Of these, DDR1 and KL-6/MUC1 have been previously reported as biomarkers for ILD \[[@CR29], [@CR30]\]. On the other hand, the top 50 genes downregulated in IIP are listed in Table [3](#Tab3){ref-type="table"}. Some of these, namely Defensin alpha 1 (*DEFA1*), *DEFA3* and Mucin 7 (*MUC7*) are known to play important roles in antimicrobial defense system in upper respiratory tract. Additionally, we also found that interleukin 10 (*IL-10*), which is known to be one of the inhibitor of Th1 cells was significantly downregulated.Table 2Top 50 genes upregulated in idiopathic interstitial pneumonia (IIP) patientsGene symbolGene nameFold change\
Fibrotic IIP/controlDSG3Desmoglein 3321.2KLRD1Killer cell lectin-like receptor subfamily D, member 1252.8OTUB1OTU domain, ubiquitin aldehyde binding 1246.7ZFP92ZFP92 zinc finger protein222.9NLGN4YNeuroligin 4, Y-linked217.2GLB1L3Galactosidase, beta 1-like 3214.8CDC25CCell division cycle 25C211.4CNTNAP3BContactin-associated protein-like 3B186.3SRGAP2SLIT-ROBO Rho GTPase-activating protein 2174.5NEK3NIMA-related kinase 3167.0SYT8Synaptotagmin VIII156.9TLR10Toll-like receptor 10145.6DSTDystonin140.3COL7A1Collagen type VII, alpha 1139.4UTYUbiquitously transcribed tetratricopeptide repeat-containing, Y-linked127.8CYorf15AChromosome Y open reading frame 15A121.8PCDHGA9Protocadherin gamma subfamily A, 9120.9OXTROxytocin receptor111.8AIM2Absent in melanoma 2107.5TMSB4YThymosin beta 4, Y-linked106.4YBEYybeY metallopeptidase104.4UBE2D2Ubiquitin-conjugating enzyme E2D 2102.7DDR1Discoidin domain receptor tyrosine kinase 199.2ZFYZinc finger protein, Y-linked98.0SLC17A9Solute carrier family 17, member 998.0EIF1AYEukaryotic translation initiation factor 1A, Y-linked96.8ANO9Anoctamin 991.6DDX3YDEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked88.7HBS1LHBS1-like (*S. cerevisiae*)88.6MSL3Male-specific lethal 3 homolog (*D. melanogaster*)85.2MUC1Mucin 1, cell surface-associated83.3PLCH2Phospholipase C, eta 282.5LPGAT1Lysophosphatidylglycerol acyltransferase 181.8DDX6DEAD (Asp-Glu-Ala-Asp) box helicase 680.4SDK2sidekick cell adhesion molecule 279.2C1QTNF1C1q and tumor necrosis factor related protein 178.8ZRANB2zinc finger, RAN-binding domain containing 278.6ZNF300zinc finger protein 30076.9DMBT1deleted in malignant brain tumors 176.7GFI1Bgrowth factor independent 1B transcription repressor75.1SLFN13schlafen family member 1372.1CDK5RAP3CDK5 regulatory subunit associated protein 370.8SLCO3A1solute carrier organic anion transporter family, member 3A170.5SNRNP70small nuclear ribonucleoprotein 70 kDa (U1)66.2IL18BPinterleukin 18 binding protein64.7PCNXL2pecanex-like 2 (Drosophila)64.6ZSCAN29zinc finger and SCAN domain containing 2963.7ESR2estrogen receptor 2 (ER beta)62.5FBXO41F-box protein 4161.7MRPL20mitochondrial ribosomal protein L2061.3 Table 3Top 50 known genes downregulated in IIP patientsGene symbolGene nameFold change\
Fibrotic IIP/controlPRB2Proline rich protein BstNI subfamily 28.35E-04DEFA3Defensin alpha 39.05E-04DEFA1Defensin alpha 11.09E-03PRB1Proline rich protein BstNI subfamily 11.21E-03HLA-DRB5Major histocompatibility complex, class II, DR beta 51.38E-03CYP1A2Cytochrome P450 family 1 subfamily A member 21.59E-03VNN2Vanin 21.73E-03MUC7Mucin 71.82E-03PRB3Proline rich protein BstNI subfamily 31.85E-03FPR2Formyl peptide receptor 21.88E-03WDR47WD repeat domain 471.90E-03HLA-CMajor histocompatibility complex, class I, C1.91E-03HIST1H2BDHistone cluster 1 H2B family member d1.94E-03CCRL1Chemokine (C-C motif) receptor-like 12.00E-03UMODUromodulin2.09E-03HIST1H1EHistone cluster 1 H1 family member e2.11E-03HIST1H2AEHistone cluster 1 H2A family member e2.19E-03FCARFc fragment of IgA receptor2.24E-03SLCO1A2Solute carrier organic anion transporter family member 1A22.26E-03RNASE3Ribonuclease A family member 32.27E-03WWP2WW domain containing E3 ubiquitin protein ligase 22.33E-03BPIFB2BPI fold containing family B member 22.34E-03LPOLactoperoxidase2.40E-03ITLN1Intelectin 12.42E-03NAIPNLR family apoptosis inhibitory protein2.42E-03CCDC85ACoiled-coil domain containing 85A2.46E-03MAP4Microtubule associated protein 42.50E-03PRH2Proline rich protein HaeIII subfamily 22.54E-03IL10Interleukin 102.59E-03CELA2AChymotrypsin like elastase family member 2A2.63E-03RGPD1RANBP2-like and GRIP domain containing 12.64E-03LAT2Linker for activation of T-cells family member 22.66E-03LRCH1Leucine rich repeats and calponin homology domain containing 12.67E-03HFEHemochromatosis2.79E-03GPM6AGlycoprotein M6A2.81E-03APOL1Apolipoprotein L12.84E-03FCGR3BFc fragment of IgG receptor IIIb2.89E-03S100A12S100 calcium binding protein A122.92E-03CCKCholecystokinin2.93E-03FCRL6Fc receptor like 62.94E-03OPRPNOpiorphin prepropeptide2.96E-03HMGCS23-hydroxy-3-methylglutaryl-CoA synthase 22.98E-03C8orf37chromosome 8 open reading frame 373.02E-03TCN1Transcobalamin 13.06E-03HIST1H4IHistone cluster 1 H4 family member i3.11E-03SLC22A12Solute carrier family 22 member 123.14E-03RGPD5RANBP2-like and GRIP domain containing 53.26E-03PRTGProtogenin3.32E-03C8BComplement C8 beta chain3.38E-03CLEC4MC-type lectin domain family 4 member M3.46E-03

Functional and canonical pathway analyses {#Sec11}
-----------------------------------------

As shown in Table [4](#Tab4){ref-type="table"}, IPA software revealed that the most highly-altered entry in IIP patients relative to control subjects for (1) diseases and disorders; (2) molecular and cellular functions; and (3) physiological system development and function was cancer, cellular movement, and cardiovascular system respectively. The top five canonical signaling pathways associated with the genes were antigen presentation pathway, cytotoxic T lymphocyte-mediated apoptosis of target cells, dendritic cell maturation, molecular mechanisms of cancer, and crosstalk between dendritic cells and natural killer cells. Thus, a number of genes and pathways related to cancer, cell death and survival, and cellular growth and proliferation were differentially expressed in IIP patients.Table 4Top 5 functional and canonical pathways from the Ingenuity Pathway AnalysisFunctional analysis/name*P* valueNumber of moleculesDiseases and disorders Cancer2.47E − 09--4.63E − 03770 Renal and urological disease3.66E − 08--3.83E − 0399 Inflammatory response6.96E − 08--4.28E − 03263 Immunological disease3.91E − 07--3.15E − 03219 Hematological disease4.54E − 07--4.41E − 03141Molecular and cellular functions Cellular movement5.28E − 11--4.43E − 03295 Cell death and survival2.11E − 07--4.72E − 03437 Cell morphology8.16E − 07--4.94E − 03105 Cellular growth and proliferation1.51E − 06--4.17E − 03423 Cellular development2.62E − 06--4.92E − 03372Physiological system development and function Cardiovascular system development and function1.54E − 08--4.83E − 03208 Organismal development3.30E − 08--4.83E − 03260 Hematological system development and function6.96E − 08--4.92E − 03258 Immune cell trafficking6.96E − 08--4.43E − 03268 Embryonic development8.16E − 07--3.34E − 03147Canonical pathway analysis/name*P* valueRatio Antigen presentation pathway1.89E − 0512/42 (0.286) Cytotoxic T lymphocyte-mediated apoptosis of target cells1.35E − 0412/88 (0.136) Dendritic cell maturation1.48E − 0428/211 (0.133) Molecular mechanisms of cancer2.27E − 0447/387 (0.121) Crosstalk between dendritic cells and natural killer cells2.7E − 0418/106 (0.170)

Clustering analysis of IPF and NSIP {#Sec12}
-----------------------------------

An unsupervised two-dimensional hierarchical clustering algorithm was used to analyze similarities among samples and genes by using data obtained from expression profiles of 12 patients with IIP (Fig. [1](#Fig1){ref-type="fig"}). After filtering using the criteria described in materials and methods, 159 genes remained. As shown in the dendrogram, three major groups---IPF1--4, IPF5--7 and NSIP1, and NSIP2--5---were distinguishable based on expression data, suggesting that the transcriptional profiles of IPF and NSIP were similar (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1The dendrogram (top) shows similarities between samples, with shorter branches indicating a higher degree of similarity. The heat map (bottom) shows the expression level of 159 genes in each case. Red: upregulated genes; green: downregulated genes. In the right panel, the names of 159 genes are listed

Validation of gene array data with immunohistochemistry {#Sec13}
-------------------------------------------------------

To validate the gene expression data at protein level, we selected two of the upregulated genes, *DSG3* and *KL-6/MUC1*, for immunohistochemical analysis. *DSG3* showed the highest upregulation in gene expression analysis, indicating its potential as a novel biomarker for IIPs. KL-6/MUC1 is well studied and is a clinically approved biomarker for IIPs \[[@CR26], [@CR30]--[@CR33]\]. Clinical characteristics of the 10 patients with IIPs included in the immunohistochemical analysis were similar to those of the patients included in the microarray analysis (Table [1](#Tab1){ref-type="table"}). As shown in Fig. [2a](#Fig2){ref-type="fig"}, DSG3 was mainly detected in the bronchiolar/alveolar epithelium and to a lesser extent in the fibrotic interstitium in IIP patients. The percentage of DSG3-positive areas in both IPF and NSIP lungs were significantly higher than those in control lungs (Fig. [2c](#Fig2){ref-type="fig"}). In agreement with earlier studies \[[@CR26], [@CR30], [@CR33]\], KL-6/MUC1 was expressed by type II pneumocytes in all the lung specimens (Fig. [2b](#Fig2){ref-type="fig"}). Furthermore, continuous KL-6/MUC1 staining was observed on the cell surface of regenerating type II pneumocytes in IIP patients, in contrast with normal lung tissue in which a discontinuous pattern was observed (Fig. [2b](#Fig2){ref-type="fig"}). The percentage of KL-6/MUC1-positive areas in both IPF and NSIP lung were significantly higher than that in control lungs (Fig. [2d](#Fig2){ref-type="fig"}).Fig. 2The expression of (**a**) DSG3 and (**b**) KL-6/MUC1 are strong in the lung affected by IPF or NSIP as compared to the controls. Morphometric analysis for (**c**) DSG3 and (**d**) KL-6/MUC1 confirmed that the rate of positively stained area is significantly high in the lung affected by IPF or NSIP as compared to the controls. \**P* \< 0.05; \*\**P* \< 0.01; \*\*\**P* \< 0.001

Discussion {#Sec14}
==========

A genome-wide gene expression analysis revealed that several genes were up- or downregulated in the lung tissue of Japanese IIP patients compared to control subjects. Among them, *DSG3* showed the highest upregulation in IIP lung as compared to control lung, and was considered to be a potential novel biomarker for IIPs. Subsequently, the function and pathway analysis demonstrated that genes and pathways related to cancer, cell death and survival, and cellular growth and proliferation were differentially expressed in Japanese IIP patients. These results suggest the possibility that several molecules involved in cancer cell growth can be novel biomarkers that can potentially be used for diagnosis or serve as therapeutic targets for IIPs.

In total, 98 genes were upregulated in the lung tissue of Japanese IIP patients compared to control subjects. Among the upregulated genes, *DSG3* showed the highest upregulation indicating its potential as a novel biomarker for IIPs. DSG3, a member of desmoglein family, is a calcium-binding transmembrane glycoprotein component of desmosomes in epithelial cells \[[@CR34]\]. Under normal conditions*,* DSG3 is expressed in oral mucosa and esophagus, but not in lungs \[[@CR34]\]. Therefore, DSG3 expression in IIP lung may be due to the differences in cell adhesion properties between normal pneumocytes and regenerative pneumocytes in IIP lung. Our study shows for the first time that DSG3 expression is significantly different between IPF or NSIP lungs and the control lungs, suggesting a novel biomarker for the diagnosis of IIP. Actually, it has been reported that DSG3 can be the useful biomarker for squamous cell lung cancer \[[@CR35]\]. As IIP and squamous cell lung cancer often occur simultaneously, both of these diseases may share the similar pathogenesis especially in the way cell adhesion properties are altered. In addition, some other molecules, which are known to be involved in cell-cell interaction and/or adhesion, were also shown to be upregulated. For example, KL-6/MUC1, which has been approved by Japan's Health Insurance Program as a diagnostic marker for ILDs since 1999 and is currently in wide clinical use in Japan was upregulated in IIP lungs. Given that the key pathologic features of IIPs are considered to be epithelial cell damage and abnormal regeneration \[[@CR36]\], we believe that our results from gene expression analysis are quite reasonable since these cell adhesion molecules are abundantly expressed in IIP tissues.

Other upregulated genes included transmembrane/secretory proteins such as DDR1, killer cell lectin-like receptor subfamily D member 1 (*KLRD1*) and toll-like receptor 10 (*TLR10*). These may also act as useful biomarkers since their cell surface localization makes them easily accessible to diagnostic methods and therapeutics. The biological and clinicopathologic significance of these candidate genes awaits validation through analysis of protein expression profiles in lung tissue obtained from IIP patients, as well as functional assays such as gene knockdown. Their potential as diagnostic markers in serum can be evaluated by enzyme-linked immunosorbent assay \[[@CR37]\], but a possible caveat is that gene expression in lung tissue and serum levels of the gene product are not always correlated. More recently, a mass spectrometry-based technique, multiple reaction monitoring, has proven to be a useful method for detecting proteins without specific antibodies \[[@CR38], [@CR39]\]. Thus, a high-throughput serum proteome analysis using this system combined with microarray gene expression profiling would be ideal for selecting candidate serum biomarkers.

We also demonstrated that lot of genes are downregulated in the lung tissue of Japanese IIP patients (Table [3](#Tab3){ref-type="table"}). Among them, *DEFA 1* and *3* are strongly downregulated; these genes are known to belong to human neutrophil peptides (HNPs) and the serum levels of HNPs have been reported to be elevated in patients with interstitial pneumonia associated with systemic sclerosis \[[@CR40]\] and also in those with acute exacerbation of IIPs \[[@CR41]\]. Our results would support the hypothesis that HNPs play important roles in the pathogenesis of IIPs. In addition, we also found that *IL-10*, which has been known as an inhibitor of cytokine production by Th1 cells is downregulated. As Th1 cytokine has been demonstrated to play important role in the progression of lung fibrosis \[[@CR42]\], we can speculate that the downregulation of IL-10 accompanied with increased production of Th1 cytokine may strongly promote the fibrotic change in the lung.

Interestingly, IPA analysis in our study revealed that IIP had a profile that was more similar to cancer than to inflammatory responses and immunological diseases. These findings were in marked contrast to those in patients with chronic hypersensitivity pneumonitis (CHP); several pathways related to inflammatory responses and immunological diseases were differentially expressed in patients with CHP \[[@CR43]\]. The canonical pathway analysis implicated dendritic cell maturation and molecular mechanisms of cancer pathways in IIP; indeed, the dendritic cell maturation signaling pathway is targeted by several molecular targeted agents mainly for chronic myelogenous leukemia such as nilotinib and dasatinib which inhibit Bcr-Abl tyrosine kinase activity \[[@CR44], [@CR45]\]. We speculate that these molecular targeted agents which interferes the dendritic cell maturation signaling pathway may also be beneficial for IIPs. Further study is required to determine whether these agents can, in fact, limit the progression of pulmonary fibrosis.

The differentially expressed genes and pathways in the patients with IIPs identified in the present study showed substantial overlap with those reported in the previous studies \[[@CR17]--[@CR19]\]. Selman M. et al. and Yang IV. et al. reported that genes encoding extracellular matrix molecules, cell surface molecules and cell adhesion molecules were highly expressed in IPF \[[@CR17], [@CR19]\]. In our study, *COL7A1*, which is involved in the extracellular matrix, *MUC1*, *KLRD1* and *TLR10*, which are the cell surface molecules, and *PCDHGA9* and *DSG3*, which are involved in cell-cell adhesion are upregulated in the patients with IIPs as compared to control. In addition, our results that functional pathways related to cellular growth and development are differentially expressed in the patients with IIPs as compared to control are similar to the results reported by Selman et al. \[[@CR17]\]. Based on these results, we can speculate that the genes and pathways differentially expressed in the patients with IIPs are not much different between Japanese and Caucasians.

The transcriptional profiles of IPF and NSIP were similar and only minor differences in gene expression were identified, consistent with the results of several previous investigations \[[@CR18], [@CR19]\]. It is still possible that differences exist and may have been detected if multiple samples from different lobes of the lung had been separately analyzed, since lung disease by nature has a patchy distribution and also because IPF and NSIP may coexist in the same lung \[[@CR46], [@CR47]\]. Moreover, fibrotic NSIP in some patients has a presentation similar to IPF. The classification of IPF and fibrotic NSIP as separate diseases has recently been challenged, and it has been suggested that they share a common clinical phenotype and pathogenesis \[[@CR48]\]. Importantly, patients with NSIP included in the present study were mostly consisted of fibrotic NSIP. The results presented here lend support to the reclassification of these IIP subtypes as a single clinical entity.

Although this study showed promising results, it has some limitations. First, control RNA for microarray analysis was derived from Caucasian subjects, because control RNA derived from Japanese subjects was not commercially available. Considering the ethnic differences in the relationship between genetic variants and the presence of IIP \[[@CR49], [@CR50]\], we cannot apply the results from the present study to Japanese patients with IIP without validation study. Second, the number of the subjects included in the immunohistochemical analysis is relatively small. We need further prospective studies with larger sample size in order to confirm the utility of DSG3 as the biomarker for IIPs.

Conclusions {#Sec15}
===========

To summarize, the genome-wide gene expression analysis of Japanese IIP patients revealed a set of upregulated genes including *DSG3*, a promising novel biomarker for IIPs. The differentially expressed genes between IIP patients and controls are implicated in cancer, cell death and survival, and cellular growth and proliferation. This dataset provides a resource for future studies investigating the molecular mechanisms underlying the development and progression of pulmonary fibrosis as well as a collection of molecules that can be targeted by novel therapeutics.

CNTNAP3B

:   Contactin-associated protein-like 3B

COL7A1

:   Collagen type VII, alpha 1

DDR1

:   Discoidin domain-containing receptor 1

DSG3

:   desmoglein 3

GFI1B

:   Growth factor independent protein 1B

HRP

:   Horseradish peroxidase

IIP

:   Idiopathic interstitial pneumonia

ILD

:   Interstitial lung disease

IPF

:   Idiopathic pulmonary fibrosis

KL-6

:   Krebs von den lungen-6

KLRD1

:   Killer cell lectin-like receptor subfamily D member 1

MUC1

:   Mucin 1

NSIP

:   Nonspecific interstitial pneumonia

PCDHGA9

:   protocadherin gamma-A9

TLR10

:   Toll-like receptor 10

UIP

:   Usual interstitial pneumonia

Not applicable.

Funding {#FPar1}
=======

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Availability of data and materials {#FPar2}
==================================

The gene expression data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE101286.

YH drafted and finalized the manuscript, performed a part of statistical analyses. NI conceived the study, conducted the gene expression profiling and pathway analysis, and drafted the manuscript. MT helped analyzing gene expression data. KY and KH performed immunohistochemical examination and serum measurements. HI, SO, HH, NH, MO, and NK participated in study design, coordination, and recruitment of the participants. KA and YO confirmed the pathological diagnosis of IIPs and helped immunohistochemical examination.

Ethics approval and consent to participate {#FPar3}
==========================================

This study was approved by the Ethics Committee of Hiroshima University Hospital (IRB M33 and 326) and conducted in accordance with the ethical standards established by the Helsinki Declaration of 1975. All participants presented their written informed consents for using their samples and publishing the data.

Consent for publication {#FPar4}
=======================

Not applicable.

Competing interests {#FPar5}
===================

All authors have no competing interest.

Publisher's Note {#FPar6}
================

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