Relationship between gene expression and lung function in Idiopathic Interstitial Pneumonias
- Mark P. Steele1,
- Leah G. Luna2,
- Christopher D. Coldren3,
- Elissa Murphy3,
- Corinne E. Hennessy3,
- David Heinz3,
- Christopher M. Evans3,
- Steve Groshong3, 4,
- Carlyne Cool3, 4,
- Gregory P. Cosgrove3, 4,
- Kevin K. Brown3, 4,
- Tasha E. Fingerlin2, 5,
- Marvin I. Schwarz3,
- David A. Schwartz2, 3, 4 and
- Ivana V. Yang2, 3, 6Email author
© Steele et al. 2015
Received: 15 June 2015
Accepted: 16 October 2015
Published: 26 October 2015
Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs.
Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples.
We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.
There is substantial clinical heterogeneity in the clinical, radiologic, and histopathologic features within each subtype of idiopathic interstitial pneumonias (IIPs). For instance, all forms of IIP have a somewhat unpredictable prognosis and many but not all patients can progress to end stage lung disease [1–3]. While subtypes of IIPs differ in clinical, radiographic, and histopathologic presentation [1–3], the type of IIP often cannot be determined and many of the subtypes of IIP have overlapping clinical and laboratory features indicating that the current definitions remain too broad.
Idiopathic pulmonary fibrosis (IPF), by far the most common form of IIP, is histopathologically defined by the presence of the prototypical form of pulmonary fibrosis, usual interstitial pneumonia (UIP), a fibrosing interstitial pneumonia characterized by a pattern of heterogeneous, subpleural regions of fibrotic, and remodeled lung that often results in death within 2–3 years of diagnosis . Other IIPs, such as respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), are more cellular, occur earlier in life, and have a considerably lower mortality . By contrast, idiopathic nonspecific interstitial pneumonia (iNSIP), a pattern of IIP that is more likely a syndrome than a disease, is most commonly characterized by interstitial fibrosis but in a more uniform pattern than UIP, and carries a better prognosis than IPF/UIP [6, 7]. These differences within the subtype and overlaps among subtypes create a distinct challenge to achieve an accurate diagnosis for an individual patient with this potentially life-threatening diagnosis.
The molecular mechanisms that account for the extreme heterogeneity in clinical presentation, radiologic patterns, histopathologic variation, and disease progression are largely unknown. We hypothesize that biological heterogeneity in IIPs will be reflected by gene expression patterns in lung tissue from patients with IIP, and gene expression patterns will change as a function of disease activity, progression, and severity. To test this hypothesis, we measured gene expression in lung tissue of patients with IIP, and correlated gene expression patterns with diffusing capacity of the lung for carbon monoxide (DLCO) and forced vital capacity (FVC).
Demographic characteristics of the Lung Tissue Research Consortium (LTRC) cohort
Subject demographics and clinical characteristics of the derivation (LTRC) cohort by IIP subcategory
P value (IIP vs control)
P value (IIP subtypes)
Age - mean (std dev)
Gender – % male
Race - % Caucasian
21 (42 %)
3 (3 %)
0 (0 %)
1 (6 %)
0 (0 %)
1 (9 %)
7 (14 %)
96 (55 %)
70 (59 %)
11 (65 %)
6 (46 %)
6 (55 %)
20 (40 %)
58 (35 %)
41 (34 %)
4 (24 %)
7 (54 %)
4 (36 %)
2 (4 %)
10 (7 %)
8 (7 %)
1 (6 %)
0 (0 %)
0 (0 %)
Pack years - mean (std dev)b
St George’s score - mean (std dev)
Pre-BD FVC, %predicted - mean (std dev)
DLCO, %predicted - mean (std dev)
Identification of genes associated with the IIPs clinical subtype
To establish whether there are significant differences in expression in clinical subtypes of IIP, we first identified molecular profiles associated with clinically defined subtypes of IIP. For this initial analysis, we used an ANCOVA model that incorporates clinical subtype with at least 10 individuals per group (including controls), age, gender and smoking status as factors. Venn diagrams in Additional file 1: Figure S1 illustrate overlap of differentially expressed mRNAs amongst IIP categories versus controls using the 5 % FDR criterion alone or combined 5 % FDR and 2-fold change criteria. While the statistical interpretation of intersection-union testing among these groups is compromised by differences in the power of individual contrasts and by the common comparator group, a large proportion of the differentially expressed mRNA transcripts exhibit differences between control lung and two or more of the IIP subgroups. However, we also observed genes unique to each IIP subtype, especially in the IPF/UIP group which has the most differentially expressed transcripts overall as well as the most unique transcripts. This result is likely a combination of IPF/UIP being the largest group of IIPs examined in our study and the fact that IPF/UIP has the most remodeled lung of all IIPs. Examination of 29 genes in common to all IIPs using the 5 % FDR and 2 fold change criteria reveals genes involved in inflammation (IL1RL1, IL1R2, IL18R1, IL8RAP), extracellular matrix (ITGA10, FCN3), Wnt signaling (SFRP2), coagulation (alpha-1 antichymotrypsin or SERPINA3) and host defense (DEFA3) (Additional file 1: Table S1). Interestingly, defensin DEFA3 is one of the genes we identified as differentially expressed in peripheral blood of severe vs mild IPF/UIP categorized by differences in the DLCO . We used the high degree of overlap among IIPs and biological relevance of genes identified in common to all IIPs as a rationale for inclusion of all IIP subtypes in the analysis of lung function variables as opposed to focusing only on IPF/UIP; however, we adjust for IIP subtype in all further analyses.
Identification of genes associated with DLCO
We next sought to identify genes whose expression is associated with the DLCO measurement. For this analysis, we used an ANOVA model that incorporates categories of disease based on predicted DLCO (see below), IIP subtype and smoking status, or an ANCOVA model that incorporates the % predicted DLCO measurement, IIP subtype and smoking status as factors. Although no significant difference in smoking status exist among IIPs due to high variability in each IIP subtype, differences in smoking histories in these individuals may influence gene expression and we therefore included them in the model. We did not include age and gender because they are accounted for in the calculation of % predicted lung function variables. By categorical analysis, 91 unique transcripts differentiate mild disease (DLCO ≥65 %; n = 33) from severe disease (DLCO ≤35 %; n = 40) at 5 % FDR (Additional file 1: Table S2). When DLCO is treated as a continuous variable (135 subjects with DLCO measurement available), 706 genes correlate with changes in % predicted DLCO at 5 % FDR (Additional file 1: Table S3). Six hundred fourteen genes not identified in the categorical analysis were found to be associated with changes in DLCO in the continuous analysis.
Identification of genes associated with FVC
We also identified gene expression changes associated with the FVC measurement, in an analogous manner to the analysis of DLCO. By categorical analysis, 681 genes differentiate mild disease (FVC ≥75 %; n = 50) from severe disease (FVC ≤40 %; n = 40) when categorized by % predicted FVC at 5 % FDR (Additional file 1: Table S4). Two thousand four hundred sixty seven genes correlate with changes in % predicted FVC in 164 subjects with available FVC data at 5 % FDR (Additional file 1: Table S5) with 1794 of these transcripts not overlapping with categorical analysis.
Transcriptional changes in common to decline in DLCO and FVC
Differentially expressed genes (5 % FDR) in severe vs mild disease as characterized both by a decline in % predicted DLCO and FVC
p-value (Severe vs Mild DLCO)
Severe/Mild Fold Change (DLCO)
p-value (Severe vs Mild FVC)
Severe/Mild Fold Change (FVC)
Validation of gene expression changes in an independent cohort
Validation of gene expression changes from the LTRC IIP cohort in an independent cohort of IIPs (NJH cohort)
Localization of gene expression changes
Results of our investigation of global gene expression changes in lungs of patients with IIPs demonstrate that, at the molecular level, there are more commonalities than differences among different subtypes of IIPs. On the other hand, the extent of disease, as characterized by lower measures of FVC and DLCO, is associated with marked changes in expression of novel and established genes and pathways involved in IIPs.
Our findings that lung tissue from IPF, iNSIP, RB-ILD, and uncharacterized fibrosis share many common expression patterns supports the concept that, despite differences in clinical presentation, these diseases may be related etiologically and pathogenically. Three publications [17–19] indicate that IPF/UIP and iNSIP, presumed distinct clinical-pathologic processes, may be related etiologically and pathogenically. Two of these studies [17, 19] have used transcriptional profiles of affected lung tissue to create IIP molecular signatures, and their findings suggest that dissimilar histological patterns may be related biologically.
In addition to some differences in expression among different IIP subtypes, we identified more pronounced changes in gene expression associated with more advanced disease. This conclusion is supported by previous reports [20, 21]. Selman et al. identified 437 differentially expressed genes in rapid compared to slow progressors, including overexpression of genes involved in morphogenesis, oxidative stress, migration/proliferation, and genes from fibroblasts/smooth muscle cells . Our group also demonstrated differential expression (p <0.05 and >5 fold change) of 191 transcripts in individuals with progressive IPF (based on changes in DLCO and FVC over 12 months) compared to slow progressors . While only less than 10 % of the genes in Table 2 (AGER, C11orf9, NFIL3, PSAT1, RTKN2, and SERPINE2) were identified by these earlier studies, additional overlap was observed when we considered gene families (for example GADD45A in the present study and GADD45B in our earlier publication), suggesting that the present study was successful at confirming published observations but also identifying novel gene transcripts. Two potential explanations for limited overlap are the fact that technologies and genome annotations have changed (our earlier study used the Serial analysis of gene expression [SAGE] as opposed to microarrays) and sample size (our current study has considerably larger sample size than previous publications).
Among the gene transcripts most strongly associated with disease severity are rhotekin 2 (RTKN2), selecting E (SELE), and peptidase inhibitor 15 (PI15). Rhotekin 2 is a member of a family of proteins containing a Rho-binding domain that are target peptides for the Rho-GTPases and are important in lymphocyte development and function ; however rhotekin 2 is ubiquitously expressed . Genetic variants in rhotekin 2 have been associated with rheumatoid arthritis (RA) and activation of the NF-κB pathway in Japanese . These known roles for rhotekin 2, the observation of decreased expression of rhotekin 2 in more severe disease, both in our earlier publication  and in the current study, and localization of expression to the lung epithelium in the current study, point to this gene as a candidate that warrants further functional investigation in IIPs. Selectin E is a member of the selectin family of cell adhesion molecules and plays a central role in adhesion of leukocytes to the endothelium . Its expression inhibits bleomycin-induced lung fibrosis  but the soluble form of the protein product is increased in serum of patients with pulmonary fibrosis . Finally, PI15 is a trypsin inhibitor that is developmentally regulated in the lung mesenchyme  but otherwise is not well characterized; its role in development is consistent with recapitulation of developmental pathways in lung injury that is a hallmark of IIPs .
One of the strengths of the present study is the use of two independent cohorts of IIP for derivation and validation. However, we were only able to validate the most pronounced transcriptional changes identified in the derivation cohort. One possible explanation for this is that overall the NJH cohort has milder disease, an observation we have previously made . We also considered the possibility that lack of RB-ILD cases in the replication cohort contributed to limited validation but ruled this out by showing that exclusion of RB-ILD cases from the derivation cohort did not substantially influence the results. A notable weaknesses of the present study in that, analogous to earlier publications, we have used whole lung tissue with the mixture of cells. Immunohistochemistry analysis of rhotekin 2, one of the genes with strongest correlation with disease severity in our study, demonstrates that reduced expression may be a combination of reduced expression in specific cells and loss of healthy airway epithelia that are replaced by honeycomb cysts. Further future studies will be needed to determine which of the genes identified by our analysis are differentially regulated in specific cell populations. Another weakness of our study is cross-sectional design in which we identify gene expression changes associated with a decline in lung function across a cohort of different individuals. A study of longitudinal design with measurements of gene expression and lung function over time in same individuals will be needed to validate these findings. However, the finding that rhotekin 2 expression was associated with more rapid disease progression in our earlier study  supports the notion that genes identified in our study have direct relevance to disease progression.
In summary, we identified commonalities and differences in gene among different subtypes of IIP. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIP. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.
Subjects and tissue samples
All human tissue was collected with appropriate ethical review for the protection of human subjects. Written informed consent was obtained for all subjects’ participation in research by the Lung Tissue Research Consortium and National Jewish Health ILD Research Program. The current study only used de-identified information and was determined to be non-human subject research by both National Jewish Health and Colorado Multiple Institution IRBs. ATS/ERS guidelines were followed for diagnosis of IIP subtypes. The LTRC IIP cohort was used to derive gene expression signatures. NJH IIP cohort was used to validate gene expression signatures. The control tissue cohort was split to provide control lung expression profiles for both derivation and validation stages.
Lung tissue specimens from lower (n = 121), upper (n = 31), and middle/lingula (n = 15) lobes from subjects with IIP (119 IPF, 17 iNSIP, 13 uncharacterized fibrosis, 11 RB-ILD, 4 DIP, and 3 COP) were obtained from the LTRC. The LTRC is a resource created by the NHLBI to provide human lung tissues and DNA to qualified investigators for use in research. The program enrolls donor subjects who are anticipating lung surgery, collects blood and extensive phenotypic data from the prospective donors, and then processes their surgical waste tissues for research use. Most donor subjects have fibrotic interstitial lung disease or COPD. Clinical data include clinical and pathological diagnoses, chest CT images, pulmonary function tests (spirometry, DLCO, and ABG), exposure (including cigarette smoking history) and symptom questionnaires (including Borg dyspnea scale), and family history of lung disease.
The NJH ILD cohort consists of 131 patients with biopsy-proven IIP (111 IPF/UIP, 12 iNSIP, and 8 uncharacterized fibrosis) that were clinically evaluated by investigators at National Jewish Health. All subjects in this cohort have undergone a standardized evaluation designed to provide a specific diagnosis. The evaluation included a standardized history focused on the presence of current or previous systemic disease; medications; tobacco and recreational drug use; familial lung disease; avocational, occupational, environmental, and accidental exposures. Additional testing includes serologic evaluation for evidence of systemic disease, chest radiography, pulmonary physiology (including lung volumes by body plethysmography, spirometry before and after inhaled bronchodilator, and diffusing capacity), pressure volume curves, and gas exchange with exercise (formal six-minute walk testing and/or cardiopulmonary exercise testing). Video assisted thorascopic (VAT) or open surgical lung biopsy was performed as clinically indicated. The diagnosis of IIP was established using the criteria defined in the ATS/ERS consensus statement [1, 2].
Control, non-diseased lung tissue from lower (n = 86), and middle (n = 4) lobes was obtained from International Institute for Advancement of Medicine, formerly Tissue Transformation Technologies (Ediston, NJ). All individuals had suffered brain death and were evaluated for organ transplantation before research consent. Informed consent was obtained at the time of transplant evaluation. All specimens failed regional lung selection criteria for transplantation. Subjects had to demonstrate no evidence of active infection or chest radiographic abnormalities, mechanical ventilation <48 h, PaO2/FiO2 ratio >200, and no past medical history of underlying lung disease or systemic disease that involves the lungs (e.g., rheumatoid arthritis). Lung samples were procured within 34 h after brain death (mean, 16.2 h; range, 4.5–33.25 h). The control cohort was divided to proportionally provide the same percentage controls to LTRC and NJH IIP cohorts; 50 controls were used with the LTRC cohort and 40 with the NJH cohort.
Microarray data generation
Total RNA including small RNA species was isolated from approximately 100 mg of snap-frozen lung tissue using the mirVana kit (AB/Ambion, Austin TX). RNA purity and concentration were determined by spectrophotometry, and RNA integrity was determined using the Bioanalyzer (Agilent, Santa Clara, CA). mRNA microarray target labeling was conducted using 300 ng of total RNA and the Message Amp II kit (AB/Ambion, Austin TX), hybridized to the Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) and processed according to the manufacturer’s instructions. All microarray data met the quality control criteria established by the Tumor Analysis Best Practices Working Group  and are available in the Gene Expression Omnibus repository as GSE31962.
Microarray data analysis
Expression data from 217 mRNA arrays (LTRC cohort; 167 IIPs and 50 controls) were analyzed using ANOVA implemented in Partek (St Louis, MO). Intensity data were imported, log2-transformed, and quantile normalized using RMA , and expression levels were summarized on a transcript level using the mean value of all probesets mapping to a transcript. Non-expressed and invariant transcripts were removed using a median variance filter, corrected by a Benjamini-Hochberg false discovery rate (FDR) of 0.10 , resulting in a final dataset of 11950 transcript measurements across 217 samples. Differential expression of individual transcripts was identified using an ANOVA (for categorical analysis) or ANCOVA (for continuous analysis) model incorporating lung function variable (%predicted FVC or DLCO), the final clinical diagnosis of each subject and smoking status. We included age and smoking status in the model as there are significant differences in these variables between IIP and control groups. We considered the impact of several technical variables including array batch, RNA preservative, RNA quality (RIN) and anatomic location of the lung biopsy; minimal expression changes were associated with these variables and we therefore did not include them in the final model. NJH cohort mRNA expression profiles were collected and processed in the same manner as the LTRC cohort data, with the exception of the final filtering step; in this case, 11950 transcripts from the LTRC dataset were retained in the dataset. Pathway analysis was performed using the Ingenuity Pathway Analysis (IPA) database and software (www.ingenuity.com) and the Fisher Exact Test to determine significant enrichments. We used the Network Analyst tool to generate the protein-protein interactome. NetworkAnalyst uses a comprehensive high-quality protein-protein interaction (PPI) database based on InnateDB . The database contains manually curated protein interaction data from published literature as well as experimental data from several PPI databases including IntAct, MINT, DIP, BIND, and BioGRID. The database currently contains 14755 proteins and 145955 interactions for human, and 5657 proteins and 14491 interactions for mouse.
Primers for mRNA expression were designed using Primer-BLAST and are listed in Additional file 1: Table S8. RNA was normalized to a concentration of 100 ng/μL and reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit. Each 20-μL PCR contained 15 ng cDNA, 0.5 μM final concentration of forward and reverse primers and 1× final concentration of the Power SYBR Green master mix. Real-time PCR was performed on an Applied Biosystems Viia 7 instrument using the following profile: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Dissociation curves were collected at the end of each run. ΔCT values were calculated relative to GAPDH, and ΔΔCT values were calculated by comparison among different groups of samples.
Standard immunohistochemical staining protocols were followed. Briefly, histological sections of normal and IPF lung were deparaffinized, blocked with hydrogen peroxide, followed by antigen retrieval in citrate buffer, and non-immune serum block. RTKN2 anti-rabbit antibody (Sigma, St. Louis MO; product number HPA037946) was added to histological sections at 1:200 final dilution and incubated overnight. Secondary antibody staining was performed with 3, 3′-diaminobenzidine (DAB) using the ImmPRESS kit (Vector Laboratories, Burlingame CA) and RTKN2 was visualized using the peroxidase substrate (ImmPact DAB kit). The sections were counterstained with hematoxylin. The primary antibody was replaced by non-immune serum for negative control slides.
This study was funded by the National Heart Lung and Blood Institute (HL095393, HL097163, HL099571, HL101715, and HL121572). The funding agency had no role in design, in the collection, analysis, and interpretation of data; in the writing of the manuscript; nor in the decision to submit the manuscript for publication.
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