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Rationale: The molecular mechanisms underlying acute exacerbations of idiopathic pulmonary fibrosis (IPF) are poorly understood. We studied the global gene expression signature of acute exacerbations of IPF.
Objectives: To understand the gene expression patterns of acute exacerbations of IPF.
Methods: RNA was extracted from 23 stable IPF lungs, 8 IPF lungs with acute exacerbation (IPF-AEx), and 15 control lungs and used for hybridization on Agilent gene expression microarrays. Functional analysis of genes was performed with Spotfire and Genomica. Gene validations for MMP1, MMP7, AGER, DEFA1–3, COL1A2, and CCNA2 were performed by real-time quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry and in situ terminal deoxynucleotidyltransferase dUTP nick end-labeling assays were performed on the same tissues used for the microarray. ELISA for α-defensins was performed on plasma from control subjects, patients with stable IPF, and patients with IPF-AEx.
Measurements and Main Results: Gene expression patterns in IPF-AEx and IPF samples were similar for the genes that distinguish IPF from control lungs. Five hundred and seventy-nine genes were differentially expressed (false discovery rate < 5%) between stable IPF and IPF-AEx. Functional analysis of these genes did not indicate any evidence of an infectious or overwhelming inflammatory etiology. CCNA2 and α-defensins were among the most up-regulated genes. CCNA2 and α-defensin protein levels were also higher and localized to the epithelium of IPF-AEx, where widespread apoptosis was also detected. α-Defensin protein levels were increased in the peripheral blood of patients with IPF-AEx.
Conclusions: Our results indicate that IPF-AEx is characterized by enhanced epithelial injury and proliferation, as reflected by increases in CCNA2 and α-defensins and apoptosis of epithelium. The concomitant increase in α-defensins in the peripheral blood and lungs may suggest their use as biomarkers for this disorder.
The mechanisms of acute exacerbation of idiopathic pulmonary fibrosis (IPF-AEx), a syndrome characterized by new development of pulmonary infiltrates, deterioration of lung function and hypoxemia, are unknown.
This analysis of genome-scale gene expression patterns in lungs of patients with IPF-AEx identifies epithelial injury and proliferation as the key molecular genetic events in IPF-AEx, and suggests plasma defensins as new biomarkers. Therapeutic strategies that protect the epithelium should be evaluated in this syndrome.
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease with a median survival of 2.5–3 years (1), and is largely unaffected by currently available medical therapies (2). Although most patients experience a gradual disease course characterized by steady worsening of symptoms, lung function, and gas exchange, some experience rapid deteriorations that are of unknown etiology. These deteriorations have been defined as acute exacerbations of IPF (IPF-AEx) (3–10). The pathological hallmark of IPF-AEx is diffuse alveolar damage superimposed on the usual interstitial pneumonia pattern characteristic in IPF (7). IPF-AEx can occur at any time during the disease course, and the risk of an exacerbation does not appear to be linked to the level of pulmonary function derangement, age, or smoking history (11). Little is known about the pathogenesis of IPF-AEx. Along with histopathology of diffuse alveolar damage, there is evidence of loss of alveolar epithelial cell integrity (12). It has been suggested that IPF-AEx may represent a response to a clinically occult infection (4, 13) but direct evidence of an association with infections is still missing (9).
Gene expression profiling was previously applied to stable sporadic (14–20) and familial IPF (21). To generate new hypotheses regarding the molecular events that underlie IPF-AEx and to identify new potential biomarkers for this syndrome, we analyzed global gene expression patterns in the lungs of patients undergoing IPF-AEx and compared them with stable IPF and control lungs. Some of the results have been previously reported in the form of an abstract (22).
See the online supplement for details on methods.
Lung tissue samples for microarray analysis were obtained through the University of Pittsburgh Health Sciences Tissue Bank (Pittsburgh, PA) as previously described (15) (and see the online supplement). Those included 23 lungs from patients with IPF, 8 lungs from patients with acute exacerbation of IPF (IPF-AEx; obtained from explanted lungs or via the warm autopsy protocol) (23), and normal lung histology samples from control subjects. Plasma samples of patients with stable IPF (n = 10), patients with IPF-AEx (n = 16), and healthy control subjects (n = 12) were obtained from Asan Medical Center (Seoul, South Korea). The diagnosis of IPF was based on the American Thoracic Society and European Respiratory Society definition (24). The definition of IPF-AEx was based on criteria provided by Collard and colleagues (3) or Akira and colleagues (25). All cases were reviewed by expert pulmonologists and pathologists. Detailed clinical information about the subjects with IPF-AEx is provided (see Tables E1 and E2 in the online supplement).
The mean forced vital capacity (expressed as a percentage of the normal expected value) (FVC%) and diffusing capacity for carbon monoxide (expressed as a percentage of the normal expected value) (DlCO%) of patients with stable IPF and patients with IPF-AEx are provided in Tables 1A and 1B. All studies were approved by the Institutional Review Boards at the University of Pittsburgh and Asan Medical Center.
Total RNA extracted from snap-frozen lung tissue was used as template for the generation of labeled cRNA that was hybridized to Agilent 4 × 4 4k whole human genome microarrays and scanned with an Agilent scanner (Agilent Technologies, Santa Clara, CA) as recommended and previously described by us (16). The complete data set is available in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE10667).
The same RNA samples used for microarray experiments were used to run real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on TaqMan system (Applied Biosystems, Foster City, CA). PCR was performed with TaqMan universal PCR master mix (Applied Biosystems) for the following genes: CCNA2, DEFA1–3, AGER, COL1A2, MMP1, MMP7, and GUSB. The results were analyzed by the ΔΔCt method and (GUSB, encoding β-glucuronidase) was used as a housekeeping gene. Fold change was calculated by taking the average over all the control samples as the baseline.
OCT-embedded sections of normal and IPF-AEx samples were used for fluorescence immunohistochemistry. Rabbit polyclonal antibody against cyclin A2 (CCNA2; Abcam, Cambridge, MA), prosurfactant protein C (Abcam), and mouse monoclonal antibodies for cytokeratin (Vector Laboratories, Burlingame, CA), vimentin (Vector Laboratories), Ki-67 (Abcam), and α-defensins (Hycult Biotechnology, Uden, The Netherlands) were used as described in the online supplement. Each antigen–antibody complex was labeled with biotinylated antibody against mouse or rabbit IgG, and visualized with fluorescein green or Texas red (Vector Laboratories). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO).
Plasma concentrations of α-defensins were determined with an ELISA kit for α-defensins (DEFA1–3) (Hycult Biotechnology).
Total protein was denatured by adding Laemmli sample buffer (Bio-Rad, Hercules, CA) 2-mercaptoethanol and boiling. Fifteen micrograms of total protein was used in the immunoblotting process.
Formalin-fixed, paraffin-embedded tissue samples were used for the terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay, done with an in situ cell death detection kit (Roche Applied Sciences, Indianapolis, IN). After proteinase digestion the sections were incubated in a mixture containing terminal deoxynucleotidyltransferase and fluorescein isothiocyanate–labeled dUTP. The TUNEL conjugates were labeled with alkaline phosphatase, visualized with Vector red, and counterstained with hematoxylin. The samples were observed under a light microscope.
Array images were processed according to the Agilent Feature Extraction protocol (26). All arrays were cyclic-LOESS normalized, using the Bioconductor package as previously described (27). For statistical analysis we applied significance analysis of microarrays (SAM) (28). A q value of 5, which corresponds to a 5% false discovery rate, was used as a cutoff of statistical significance in microarray data. Data visualization and clustering were performed with Genomica (28), Scoregene (29), and Spotfire DecisionSite 9 (TIBCO, Palo Alto, CA). For qRT-PCR, the Student t test was used and significance was defined as P < 0.05.
When compared with control samples, the global gene expression patterns of IPF-AEx are almost identical to those of stable IPF (Figure 1A). To better characterize this similarity we looked at genes that characterized IPF lungs in previous studies (Figure 1B) and compared their expression in IPF-AEx with that in stable IPF (Figure 1C). Impressively, all the highlighted genes were not significantly different between IPF and IPF-AEx (Figure 1C; see also Table 2). These results indicate that compared with control samples, IPF-AEx exhibits a fibrosis signature that is identical to that of stable IPF.
To confirm this observation we performed qRT-PCR for MMP1, MMP7, COL1A2, and AGER, which are among the genes that consistently distinguish patients with IPF from control subjects (16, 20) (Figure 2). As expected, COL1A2, MMP1, and MMP7 were significantly higher and AGER was significantly lower in IPF and IPF-AEx compared with control samples, but there was no significant difference in their expression between IPF and IPF-AEx.
To identify the subtler gene expression changes that distinguish IPF-AEx from stable IPF, and to focus on clusters of genes that seemed differentially expressed in IPF and IPF-AEx (orange quadrangles; Figure 1A), we performed a direct comparison of IPF-AEx and stable IPF. We identified 579 significantly differentially expressed genes (q < 5) (Figure 3A). Among them were genes related to stress response such as heat shock proteins and α-defensins and mitosis-related genes including histones and CCNA2 (Figure 3B and Table 3).
Impressively, the gene expression signature of IPF-AEx did not exhibit an increase in inflammatory response compared with stable IPF. Genes known to be associated with the general inflammatory response, adaptive or innate immunity, were not significantly enriched in genes that characterize IPF-AEx. Similarly, only 2 (the α-defensins DEFA3 and DEFA4) of the 100 genes on the array that belong to gene ontology (GO) annotations associated with response to viral infection were significantly changed (see Figure E1 in the online supplement).
When compared with stable IPF, CCNA2 was one of the top 20 up-regulated genes, with a q value of 0 and a 2.27-fold increase (Table 3). Considering that this gene is a regulator of the cell cycle we chose to validate and localize its expression. qRT-PCR confirmed the microarray data (Figures 3B and 3C), and Western blots indicated an increase in CCNA2 protein (Figure 3D). To localize CCNA2 overexpression we performed double-fluorescence labeling for CCNA2 with either cytokeratin or vimentin. The double labeling demonstrated coexpression of CCNA2 with cytokeratin (Figures 4A–4C), but not with vimentin (Figures 4D–4F), indicating that the increase in CCNA2 was localized to epithelial cells and not fibroblasts. Confocal microscopy revealed localization of CCNA2 in the alveolar epithelium as well as the basal portion of the bronchial epithelium (Figures 4G and 4H). To determine whether increases in CCNA2 were associated with cellular proliferation, we double-labeled IPF-AEx tissues with CCNA2 and Ki-67. CCNA2 and Ki-67 colocalized to the pulmonary epithelium (Figure 4I), suggesting accelerated epithelial cell proliferation, potentially as a compensatory response of the injured epithelium.
To determine whether increased epithelial proliferation was associated with epithelial cell death in IPF-AEx, we studied apoptosis in IPF-AEx tissues by in situ TUNEL assay. We observed significant and widespread positive epithelial TUNEL staining in IPF-AEx tissues (Figure 4K). This pattern was consistent with previous observations in IPF (30). In addition, positive TUNEL stains were also observed in the hyaline membranes typical of diffuse alveolar damage, a pathological hallmark of IPF-AEx (Figure 4J). In control lungs, rare TUNEL-positive structures were predominantly observed in apoptotic bodies engulfed by alveolar macrophages (Figure 4L).
Gene expression of α-defensins (DEFA3 and DEFA4) was significantly increased in IPF-AEx lungs compared with stable IPF in the microarray data (Figure 3B), and the increase was confirmed by qRT-PCR (Figure 5A). To determine whether α-defensins may serve as peripheral blood markers for IPF-AEx, we analyzed their levels in the plasma of patients with IPF-AEx from Asan Medical Center. Plasma defensin concentrations were significantly higher in patients with IPF-AEx compared with control subjects (P = 0.0007) or patients with stable IPF (P = 0.025) (Figure 5B). To determine the cellular origins of α-defensins in IPF-AEx, we performed double labeling on IPF-AEx lung sections with antibodies against α-defensins and against the alveolar type II cell marker surfactant protein C (SFTPC). These experiments identified alveolar type II cells as the source of α-defensins in IPF-AEx (Figures 5C–5E).
In this study, we used gene expression microarrays to characterize acute exacerbations of IPF. Compared with control samples, IPF and IPF-AEx lungs exhibited similar gene expression signatures. However, on direct comparison of IPF and IPF-AEx we identified differentially expressed genes and chose to focus our validation on CCNA2 and α-defensins. CCNA2, a general regulator of the cell cycle, was among the most up-regulated genes in IPF-AEx. Increased CCNA2 protein expression was localized to proliferating epithelial cells but not to mesenchymal cells. TUNEL staining was also positive and localized to the epithelium in IPF-AEx. Gene expression levels of α-defensins were up-regulated in IPF-AEx and their protein expression was localized to the alveolar epithelium in IPF-AEx. Plasma α-defensin concentrations were higher in patients with IPF-AEx compared with those with stable IPF or control subjects. Taken together, these results indicate the central role of the pulmonary epithelium in IPF-AEx and suggest a potential role for α-defensins as peripheral blood biomarkers in IPF-AEx.
One impressive feature of our results is the relative similarity of the gene expression patterns that distinguish IPF or IPF-AEx from control lungs. We have previously reported the up-regulation of matrix metalloproteinase-7 (MMP7), matrix metalloproteinase-1 (MMP1), collagens I and III, and osteopontin (14–21), as well as down-regulation of caveolin-1 (31) and advanced glycosylation end products-specific receptor (AGER) (32), in IPF. All these genes behaved similarly in stable IPF and IPF-AEx, as did the majority of all other genes that distinguished IPF from control samples. We did not detect any dramatic shift in gene expression that would indicate a new process or a dramatic shift in lung cellular phenotype or content. Although we found increases in some genes associated with response to stress (HMOX1 and HSP1A1), we did not find any changes in known inflammation-related genes, such as IL-1, IL-6, tumor necrosis factor-α, or NF-κB target genes in the comparison of IPF and IPF-AEx. Interestingly, other genes increased in acute lung injury such as AGER, a known marker of generalized inflammation (33), were not increased in IPF-AEx lungs. In fact, AGER was significantly decreased in IPF-AEx compared with control samples, potentially reflecting loss of type I alveolar epithelial cells (16, 32). Taken together, these results do not support an overwhelming lung inflammatory response as a potential mechanism for acute exacerbation. We also did not find any gene expression patterns indicative of a response of the lung to viral or bacterial infections, a mechanism observed in animals (34) but not yet confirmed in human IPF-AEx (9, 35). Although our results do not rule out an occult viral infection or a previous viral infection as the triggering mechanism for IPF-AEx, neither do they support a role for an active infection during the last phase of the syndrome.
Naturally, our analysis is limited by our dependence on tissue harvested at explant or warm autopsy. It is entirely possible that by the time the patients experienced the final deterioration all evidence of response to an infection or infected tissue was destroyed. In this context the finding of increased α-defensin levels and the evidence of epithelial injury may be interpreted as remnants of an infectious process that triggered the acute lung injury but was cleared by the time the lungs were harvested. Although we cannot disprove this interpretation, we do think that the lack of expression of viral response genes reduces the likelihood of an active infection. A definite answer regarding the role of infections will require sampling earlier at presentation and longitudinal studies of the same patient, a task impossible with lung tissue but attainable with bronchial lavage or peripheral blood samples.
One of the remarkable features of our study is the localization of increased CCNA2 expression to the alveolar epithelium, rather than to fibroblasts or myofibroblastic foci. CCNA2 is the main A-type cyclin present in somatic cells (36) and a mediator of the cell cycle. The overexpression and localization of CCNA2 to epithelial cells but not to mesenchymal cells suggests that IPF-AEx is probably an extension of the epithelial injury and dysregulation that characterizes IPF (37) and definitely is not a result of uncontrolled fibroblast proliferation. The fact that the majority of CCNA2-expressing cells were also positive for Ki-67, a proliferation marker, suggests that CCNA2 expression represents a proliferative response of the epithelium. In light of the positive TUNEL staining in the epithelium and hyaline membranes, it is tempting to hypothesize that this enhanced proliferation represents a failed compensatory response to injury, localizing the pathogenesis of IPF-AEx to the epithelium.
One use of lung gene expression data is in the identification of differentially expressed genes that encode secreted proteins. Such secreted proteins may be detected in the alveolar fluid or peripheral blood and thus be useful as potential surrogate markers for disease activity (16). Previous studies suggested that peripheral blood IL-8, KL-6, and most recently circulating fibrocytes may be increased in IPF-AEx (38–40). In our study the genes encoding α-defensins were significantly increased in IPF-AEx lungs compared with stable IPF or control samples, and their protein expression was increased in the plasma of patients with IPF-AEx. α-Defensins are innate immunity antimicrobial peptides abundant in neutrophil granules and mucosal surfaces (41, 42). α-Defensins affect various immune functions. α-Defensins are involved in activation of the classical complement pathway (43, 44). In vitro α-defensins induce the production of heat shock proteins and type I collagens in human lung fibroblasts (45), and stimulate cytokine production of bronchial epithelial cells (46). Elevation of α-defensins has been described in pulmonary alveolar proteinosis (47), α1-antitrypsin deficiency (48), acute respiratory distress (49), and chronic lung allograft rejection (50) and in patients with IPF but not in the context of acute exacerbation (51). In this context, it is important to note that we observed α-defensin expression in surfactant protein C–expressing cells in IPF-AEx lungs—a finding that suggests that the plasma increases in α-defensins may be indicative of the lung microenvironment in IPF-AEx and again highlights the central role of the epithelium in IPF-AEx.
In summary, this is the first study of lung gene expression patterns in IPF-AEx lungs. Gene expression patterns indicate that IPF-AEx represents an extension of the molecular process that underlies IPF and not a new process. Although expression patterns that distinguish stable IPF and IPF-AEx lungs from normal lung are similar, we have identified genes that are differentially expressed in a direct comparison of IPF and IPF-AEx lungs. The increased expression of CCNA2 and α-defensins is localized to the epithelium of IPF-AEx lungs, where widespread proliferation and apoptosis are detected, suggesting that the central molecular events in IPF-AEx are localized to the alveolar epithelium. Taken together, our results indicate the central role of alveolar epithelial injury in IPF-AEx and thus support the study of agents that protect the epithelium as therapeutic measures in this devastating syndrome. The identification of increases in plasma concentrations of proteins originating from the pulmonary epithelium in patients with IPF-AEx suggests their use as tools for evaluating patients with IPF during the course of the disease.
The authors thank Lara Chensny and Dr. Simon Watkins for support of the study, as well as the patient members of the Simmons Center ILD support group.
Supported by NIH grants HL073745, HL0894932, and HL095397 and by the Dorothy P. and Richard P. Simmons Endowed Chair for Interstitial Lung Disease.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200810-1596OC on April 16, 2009
Conflict of Interest Statement: K.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.F.G. is an inventor on a patent application of the use of peripheral blood proteins as biomarkers. K.O.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.J.R. is an inventor on a patent application of the use of peripheral blood proteins as biomarkers. Y.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.A.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.W.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.S.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.K. is a primary investigator on two industry investigator initiated grants, one from Biogen Idec for $674,000 and the other from Centocor for $250,000. N.K. is an inventor on a patent application for the use of peripheral blood proteins as biomarkers.