Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis

Abstract

Rationale: Many of the interstitial lung diseases represent a diagnostic and therapeutic challenge because their clinical and even histologic features are often nonspecific. Likewise, the transcriptional signatures of most of them are unknown.

Objective: To compare the gene expression patterns from patients with idiopathic pulmonary fibrosis (IPF) hypersensitivity pneumonitis (HP), and nonspecific interstitial pneumonia (NSIP) using custom oligonucleotide microarrays.

Methods: We profiled lung biopsies from 15 patients with IPF, 12 with HP, and eight with NSIP. Labeled complementary ribonucleic acid was hybridized to a custom Affymetrix oligonucleotide DNA microarray using standard Affymetrix protocols. The custom array, Hu03, contained 59,619 probe sets representing an estimated 46,000 gene clusters.

Results: We identified statistically significant gene expression signatures that characterize HP and IPF. The HP gene expression signature was enriched for genes that are functionally associated with inflammation, T-cell activation, and immune responses, whereas the IPF signature was characterized by the expression of tissue remodeling, epithelial, and myofibroblast genes. We then compared these gene expression signatures to classify NSIP, a histologic pattern that is often difficult to differentiate consistently from HP and IPF. Two cases exhibited an IPF-like gene expression, another one could be more properly classified as HP, whereas others did not resemble HP or IPF, suggesting that they may represent idiopathic NSIP.

Conclusions: Our results underscore the value of gene expression signatures to classify the interstitial lung diseases and to understand pathogenic mechanisms, and suggest new ways to improve the diagnosis and treatment of patients with these diseases.

Figure 1.

Hypersensitiviy pneumonitis (HP) and idiopathic pulmonary fibrosis (IPF) can be distinguished using gene expression profiling. (A) Overabundance analysis of the actual and expected number of genes that significantly distinguish whole lung gene expression between patients with IPF or HP and healthy subjects. The x axis denotes the number of genes (probes) in log scale, and the y axis shows the p value (threshold number of misclassifications [TNoM]) in log scale. Actual number of genes is significantly more abundant than would be expected (pink line). (B) Leave-One-Out Cross-Validation (LOOCV) classification graph. The x axis denotes the TNoM p value in log scale, and the y axis denotes the error rate in percentages. The pink arrow is the highest p value that gets the lowest error rate. The orange arrow is the lowest p value that gets the lowest error rate. FDR = false-discovery rate.

Figure 2.

The distribution of differentially expressed genes among functional categories. (A) Genes that had locus-link information and were significantly increased in IPF compared with HP. (B) Genes that were increased in HP compared with IPF. Note the large percentage of genes that belong to the distinct functional annotations. All enrichments are statistically significant (Fisher's exact score p value < 0.05).

Figure 3.

Immunolocalization of insulinlike growth factor binding protein 4 (IGFBP-4), N-cadherin, and matrix metalloproteinase 1 (MMP-1) in IPF and normal lungs. (A) MMP-1 immunoreactive protein was found in alveolar epithelial cells. It can be noticed that the enzyme is virtually absent in fibroblastic foci (original magnification, 40×). (B) IGFBP-4 immunoreactive bronchiolar basal epithelial cells in IPF lungs (40×). (C) N-cadherin immunoreactive protein was observed in alveolar epithelial cells usually covering fibroblastic foci (40×). (D) Normal lungs were usually negative for the three molecules as exemplified for N-cadherin (40×). (E) A negative control omitting the primary (in this case, IGFBP-4) antibody (40×). Results illustrate the analysis of six IPF and five control lungs.

Figure 4.

ELISA for IGFBP-4 protein in bronchoalveolar lavage (BAL) fluid samples. The protein was not detected in BAL from healthy individuals (controls). An increased concentration of IGFBP-4 was detected in BAL obtained from patients with IPF compared with patients with HP. The data represent the mean ± SD. For the IPF samples, p < 0.01.

Figure 5.

Infogram and classification score. (A) Infogram of 1,058 genes that made the least classification errors by LOOCV. The expression levels for each gene were normalized to the geometric mean of all the samples for each gene. Increased genes are shown in progressively brighter shades of yellow, and decreased genes are shown in progressively darker shades of blue. Genes shown in gray are not different between the groups. The genes were ranked according to their significance level. (B) Classification score of every sample in the dataset. Samples below the zero line were considered as usual interstitial pneumonia. Samples above the separation line were classified as HP. Note that only one HP sample was misclassified. NSIP = nonspecific interstitial pneumonia.