Gene expression profiling of idiopathic interstitial pneumonias (IIPs): identification of potential diagnostic markers and therapeutic targets

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) [1‐3]. The latest statement from American Thoracic Society (ATS) and European Respiratory Society (ERS) proposed the category “chronic fibrosing IIP” encompassing both IPF and NSIP [3], because separation between these two diseases is difficult, with significant clinical, radiological, and pathological overlap between them [4].

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 [5]. Over the last decade, the few therapeutic options available have not been very effective and the outcome of IPF patients is poor [6]. 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 [7‐10]. Recently, nintedanib, a multiple tyrosine kinase inhibitor, also demonstrated clinical efficacy for IPF patients [11]. 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 [12]. 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 [13‐16]. However, few such analyses have been performed for IIP [17‐21], 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.

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 [34]. Under normal conditions, DSG3 is expressed in oral mucosa and esophagus, but not in lungs [34]. 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 [35]. 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 [36], 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 [37], 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 [38, 39]. 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). 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 [40] and also in those with acute exacerbation of IIPs [41]. 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 [42], 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 [43]. 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 [44, 45]. 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 [17‐19]. 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 [17, 19]. 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. [17]. 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 [18, 19]. 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 [46, 47]. 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 [48]. 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 [49, 50], 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.