Gene expression profiling of familial and sporadic interstitial pneumonia

Abstract

Rationale: Idiopathic interstitial pneumonia (IIP) and its familial variants are progressive and largely untreatable disorders with poorly understood molecular mechanisms. Both the genetics and the histologic type of IIP play a role in the etiology and pathogenesis of interstitial lung disease, but transcriptional signatures of these subtypes are unknown.

Objectives: To evaluate gene expression in the lung tissue of patients with usual interstitial pneumonia or nonspecific interstitial pneumonia that was either familial or nonfamilial in origin, and to compare it with gene expression in normal lung parenchyma.

Methods: We profiled RNA from the lungs of 16 patients with sporadic IIP, 10 with familial IIP, and 9 normal control subjects on a whole human genome oligonucleotide microarray.

Results: Significant transcriptional differences exist in familial and sporadic IIPs. The genes distinguishing the genetic subtypes belong to the same functional categories as transcripts that distinguish IIP from normal samples. Relevant categories include chemokines and growth factors and their receptors, complement components, genes associated with cell proliferation and death, and genes in the Wnt pathway. The role of the chemokine CXCL12 in disease pathogenesis was confirmed in the murine bleomycin model of lung injury, with C57BL/6(CXCR4+/-) mice demonstrating significantly less collagen deposition than C57BL/6(CXCR4+/+) mice. Whereas substantial differences exist between familial and sporadic IIPs, we identified only minor gene expression changes between usual interstitial pneumonia and nonspecific interstitial pneumonia.

Conclusions: Taken together, our findings indicate that differences in gene expression profiles between familial and sporadic IIPs may provide clues to the etiology and pathogenesis of IIP.

Figure 1.

Hierarchical clustering of 35 samples based on 135 transcripts that best differentiated patients with pulmonary fibrosis from normal control subjects. Samples are color-coded according to disease inheritance status (orange = normal; blue = sporadic idiopathic interstitial pneumonia [IIP]; magenta = familial IIP) or histologic features (orange = normal; green = nonspecific interstitial pneumonia [NSIP]; red = usual interstitial pneumonia [UIP]); the asterisk (*) denotes one case of fibrotic NSIP. Average linkage clustering with euclidian distance metric was used.

Figure 2.

Venn diagram depicting the approach to identification of pulmonary fibrosis susceptibility genes on the basis of gene expression profiling. Genes (701) were identified as significantly differentially expressed (1% false discovery rate [FDR]) in three groups of patients (normal, sporadic, and familial IIP) by significance analysis of microarrays (SAM). Similarly, 676 genes were identified as significant at 1% FDR between sporadic and familial groups. Three hundred and thirty-two genes were common to the two analyses.

Figure 3.

Mean expression ratios in normal, sporadic IIP, and familial IIP groups of 62 genes with known function that best differentiate familial IIP from sporadic IIP. Genes are grouped according to their function (CC = coagulation cascade; nuclear factor [NF]-κB = regulation of I-κB/NF-κB cascade). Genes are labeled by gene symbol followed by familial/sporadic IIP fold change in parentheses.

Figure 4.

Venn diagram illustrating the approach taken to identify genes that differentiate UIP and NSIP. Genes (128) were identified as significantly differentially expressed (1% false discovery rate [FDR]) in three groups of patients (normal [N], UIP, and NSIP) by SAM. Similarly, 39 genes were identified as significant at a 1% FDR between UIP and NSIP groups. Thirty-three genes were in common to the two analyses.

Figure 5.

CXCL12 protein levels are significantly higher in (b) diseased lungs than in (a) normal control subjects because of the presence of a larger number of macrophages expressing CXCL12 in the parenchyma of subjects with pulmonary fibrosis.

Figure 6.

C57BL/6^CXCR4+/− mice develop less fibrosis than do wild-type control mice after bleomycin-induced lung injury. (a) Soluble collagen content in the right lungs of C57BL/6^CXCR4+/− and C57BL/6^CXCR4+/+ mice 14 d after administration of bleomycin or phosphate-buffered saline (PBS) was measured in a Sircol collagen assay (Biocolor, Newtownabbey, UK). *p < 0.05 by two-tailed Student t test. (b–e) Masson trichrome staining of left lung sections indicates dense and widespread fibrosis as revealed by collagen deposition in bleomycin-treated C57BL/6^CXCR4+/+ mice and much less fibrosis in C57BL/6^CXCR4+/− animals. No fibrosis is seen in animals that were administered PBS alone.

Figure 7.

Inflammation in whole lung lavage 14 d post-treatment with bleomycin or saline. (a) Significantly more cells in total (*p < 0.05 by two-tailed Student t test) were recovered from the bronchoalveolar lavage (BAL) fluid of C57BL/6^CXCR4+/− animals compared with wild-type control animals in response to bleomycin. Both murine strains were unresponsive to saline. (b) No differences in macrophage recruitment were observed between the two strains of mice. Significantly more lymphocytes (**p < 0.005 by two-tailed Student t test) were recruited to the lungs of C57BL/6^CXCR4+/− animals than C57BL/6^CXCR4+/+ control animals in response to bleomycin.