Comprehensive gene expression profiling identifies distinct and overlapping transcriptional profiles in non-specific interstitial pneumonia and idiopathic pulmonary fibrosis

Matthew J Cecchini, Karishma Hosein, Christopher J Howlett, Mariamma Joseph, Marco Mura, Matthew J Cecchini, Karishma Hosein, Christopher J Howlett, Mariamma Joseph, Marco Mura

Abstract

Background: The clinical-radiographic distinction between idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) is challenging. We sought to investigate the gene expression profiles of IPF and NSIP vs. normal controls.

Methods: Gene expression from explanted lungs of patients with IPF (n = 22), NSIP (n = 10) and from normal controls (n = 11) was assessed. Microarray analysis included Significance Analysis of Microarray (SAM), Ingenuity Pathway, Gene-Set Enrichment and unsupervised hierarchical clustering analyses. Immunohistochemistry and serology of proteins of interest were conducted.

Results: NSIP cases were significantly enriched for genes related to mechanisms of immune reaction, such as T-cell response and recruitment of leukocytes into the lung compartment. In IPF, in contrast, these involved senescence, epithelial-to-mesenchymal transition, myofibroblast differentiation and collagen deposition. Unlike the IPF group, NSIP cases exhibited a strikingly homogenous gene signature. Clustering analysis identified a subgroup of IPF patients with intermediate and ambiguous expression of SAM-selected genes, with the interesting upregulation of both NSIP-specific and senescence-related genes. Immunohistochemistry for p16, a senescence marker, on fibroblasts differentiated most IPF cases from NSIP. Serial serum levels of periostin, a senescence effector, predicted clinical progression in a cohort of patients with IPF.

Conclusions: Comprehensive gene expression profiling in explanted lungs identifies distinct transcriptional profiles and differentially expressed genes in IPF and NSIP, supporting the notion of NSIP as a standalone condition. Potential gene and protein markers to discriminate IPF from NSIP were identified, with a prominent role of senescence in IPF. The finding of a subgroup of IPF patients with transcriptional features of both NSIP and senescence raises the hypothesis that "senescent" NSIP may represent a risk factor to develop superimposed IPF.

Keywords: Idiopathic pulmonary fibrosis; Microarray; Non-specific interstitial pneumonia; Usual interstitial pneumonia.

Conflict of interest statement

Ethics approval and consent to participate

The study was approved by the Human Tissue Committees and Research Ethics Boards of the University Health Network (protocol n.11–0932) and Western University (n.105214).

Consent for publication

No personal identifiers of patients are included in the study.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Outline of microarray analysis. SAM = Significance Analysis of Microarray. RIN = RNA integrity number
Fig. 2
Fig. 2
Gene expression levels determined by oligonucleotide microarray in the IPF (blue), NSIP (green) and normal control (red) groups. Examples of genes upregulated in both IPF and NSIP: a. Matrix Metalloproteinase 7 (MMP-7). b Osteopontin (also known as Secreted Phosphoprotein 1). Examples of genes specifically upregulated in IPF: c Insulin Growth Factor Binding Protein 5 (IGFBP-5). d Smooth muscle alpha-2 actin (ACTA-2). Examples of genes upregulated in both NSIP and normal controls: e Long Pentraxin 3 (PTX-3). f Indoleamine 2–3-dioxygenase-1 (IDO-1). Examples of genes specifically upregulated in NSIP: g Interferon-induced protein 44-like (IFI-44). H. LDL receptor related protein 2 (LRP-2)
Fig. 3
Fig. 3
Heat map representing differentially expressed genes (fold change ≥1.50 and q valuea Hierarchic clustering based on 146 differentially expressed genes (IPF vs. NSIP groups) in IPF (n = 22), NSIP (n = 10) and IPF-NSIP (n = 5) groups. Each row corresponds to an individual sample, and each column corresponds to an individual gene. Each square on the matrix represents the expression level of an individual gene in each sample, with red and green indicating gene expression levels above or below, respectively, compared with each other. While an IPF and a NSIP cluster are identifiable, an “unpaired” group of 7 IPF subjects and 2 NSIP patients showed intermediate expression and no clear clustering
Fig. 4
Fig. 4
Immunohistochemistry studies. a-f Lower power (4X) and high power (20X) photomicrographs of hematoxylin and eosin stained sections of representative IPF (A,D), NSIP (B,E) cases and normal control lung tissue (C,F). g-l high power photomicrographs (20X) of immunohistochemistry for p53 and p16 of representative IPF (G,J), NSIP (H,K) cases and normal control lung tissue (I,L). Inserts show high power magnification. Scale bar for A-C = 500 μm, D-L = 100 μm

References

    1. American Thoracic Society, European Respiratory Society American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society. Am J Respir Crit Care Med. 2002;165:277–304. doi: 10.1164/ajrccm.165.2.ats01.
    1. Latsi PI, du Bois RM, Nicholson AG, Colby TV, Bisirtzoglou D, Nikolakopoulou A, Veeraraghavan S, Hansell DM, Wells AU. Fibrotic idiopathic interstitial pneumonia: the prognostic value of longitudinal functional trends. Am J Respir Crit Care Med. 2003;168:531–537. doi: 10.1164/rccm.200210-1245OC.
    1. Christie JD, Edwards LB, Kucheryavaya AY, Benden C, Dobbels F, Kirk R, et al. The registry of the international society for heart and lung transplantation: twenty-eighth adult lung and heart-lung transplant report-2011. J Heart Lung Transplant. 2011;30:1104–1122. doi: 10.1016/j.healun.2011.08.004.
    1. King TE, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet. 2011;378:1949–1961. doi: 10.1016/S0140-6736(11)60052-4.
    1. MacDonald SL, Rubens MB, Hansell DM, Copley SJ, Desai SR, du Bois RM, et al. Nonspecific interstitial pneumonia and usual interstitial pneumonia: comparative appearances at and diagnostic accuracy of thin-section CT. Radiology. 2001;221:600–605. doi: 10.1148/radiol.2213010158.
    1. Idiopathic Pulmonary Fibrosis Clinical Research Network. Raghu G, Anstrom KJ, King TE, Lasky JA, Martinez FJ. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med. 2012;366:1968–1977. doi: 10.1056/NEJMoa1113354.
    1. King TE, Bradford WZ, Castro-Bernardini S, Fagan EA, Glaspole I, Glassberg MK, et al. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med. 2014;370:2083–2092. doi: 10.1056/NEJMoa1402582.
    1. Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U, et al. INPULSIS trial investigators. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med. 2014;370:2071–2082. doi: 10.1056/NEJMoa1402584.
    1. Flaherty KR, Travis WD, Colby TV, Toews GB, Kazerooni EA, Gross BH, et al. Histopathologic variability in usual and nonspecific interstitial pneumonias. Am J Respir Crit Care Med. 2001;164:1722–1727. doi: 10.1164/ajrccm.164.9.2103074.
    1. Flaherty KR, King TE, Raghu G, Lynch JP, Colby TV, Travis WD, et al. Idiopathic interstitial pneumonia: what is the effect of a multidisciplinary approach to diagnosis? Am J Respir Crit Care Med. 2004;170:904–910. doi: 10.1164/rccm.200402-147OC.
    1. Belloli EA, Beckford R, Hadley R, Flaherty KR. Idiopathic non-specific interstitial pneumonia. Respirology. 2016;21:259–268. doi: 10.1111/resp.12674.
    1. Sverzellati N, Lynch DA, Hansell DM, Johkoh T, King TE, Travis WD. American Thoracic Society-European Respiratory Society classification of the idiopathic interstitial pneumonias: advances in knowledge since 2002. Radiographics. 2015;35:1849–1871. doi: 10.1148/rg.2015140334.
    1. Nicholson AG, Addis BJ, Bharucha H, Clelland CA, Corrin B, Gibbs AR, et al. Inter-observer variation between pathologists in diffuse parenchymal lung disease. Thorax. 2004;59:500–505. doi: 10.1136/thx.2003.011734.
    1. Harada T, Watanabe K, Nabeshima K, Hamasaki M, Iwasaki H. Prognostic significance of fibroblastic foci in usual interstitial pneumonia and non-specific interstitial pneumonia. Respirology. 2013;18:278–283. doi: 10.1111/j.1440-1843.2012.02272.x.
    1. Rosas IO, Kaminski N. When it comes to genes--IPF or NSIP, familial or sporadic--they’re all the same. Am J Respir Crit Care Med. 2007;175:5–6. doi: 10.1164/rccm.200610-1415ED.
    1. Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183:788–824. doi: 10.1164/rccm.2009-040GL.
    1. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci. 2005;102:15545–15550. doi: 10.1073/pnas.0506580102.
    1. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Consortium GO. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25:25–29. doi: 10.1038/75556.
    1. Kim KS, Seu YB, Baek S-H, Kim MJ, Kim KJ, Kim JH, Kim J-R. Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism. Mol Biol Cell. 2007;18:4543–4552. doi: 10.1091/mbc.e07-03-0280.
    1. Kim SY, Diggans J, Pankratz D, Huang J, Pagan M, Sindy N, et al. Classification of usual interstitial pneumonia in patients with interstitial lung disease: assessment of a machine learning approach using high-dimensional transcriptional data. Lancet Respir Med. 2015;3:473–482. doi: 10.1016/S2213-2600(15)00140-X.
    1. Selman M, Pardo A, Barrera L, Estrada A, Watson SR, Wilson K, et al. Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis. Am J Respir Crit Care Med. 2006;173:188–198. doi: 10.1164/rccm.200504-644OC.
    1. Yang IV, Burch LH, Steele MP, Savov JD, Hollingsworth JW, McElvania-Tekippe E, et al. Gene expression profiling of familial and sporadic interstitial pneumonia. Am J Respir Crit Care Med. 2007;175:45–54. doi: 10.1164/rccm.200601-062OC.
    1. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-γ: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75:163–189. doi: 10.1189/jlb.0603252.
    1. Pollard KM, Cauvi DM, Toomey CB, Morris KV, Kono DH. Interferon-γ and systemic autoimmunity. Discov Med. 2013;16:123–131.
    1. Rossios C, Pavlidis S, Gibeon D, Mumby S, Durham A, Ojo O, et al. Impaired innate immune gene profiling in airway smooth muscle cells from chronic cough patients. Biosci Rep. 2017;37:BSR20171090. doi: 10.1042/BSR20171090.
    1. Suber T, Mallampalli RK. An emerging role for megalin as a regulator of protein leak in acute lung injury. Am J Respir Cell Mol Biol. 2017;57:504–505. doi: 10.1165/rcmb.2017-0224ED.
    1. Pardo A, Gibson K, Cisneros J, Richards TJ, Yang Y, Becerril C, et al. Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. PLoS Med. 2005;2:e251. doi: 10.1371/journal.pmed.0020251.
    1. DePianto DJ, Chandriani S, Abbas AR, Jia G, N’Diaye EN, Caplazi P, et al. Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis. Thorax. 2015;70:48–56. doi: 10.1136/thoraxjnl-2013-204596.
    1. Selman M, Pardo A, Barrera L, Estrada A, Watson SR, Wilson K, Aziz N, Kaminski N, Zlotnik A. Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis. Am J Respir Crit Care Med. 2006;173:188–198. doi: 10.1164/rccm.200504-644OC.
    1. Huh JW, Kim DS, Oh Y-M, Shim TS, Lim CM, Do Lee S, et al. Is metalloproteinase-7 specific for idiopathic pulmonary fibrosis? Chest. 2008;133:1101–1106. doi: 10.1378/chest.07-2116.
    1. Kojima H, Kunimoto H, Inoue T, Nakajima K. The STAT3-IGFBP5 axis is critical for IL-6/gp130-induced premature senescence in human fibroblasts. Cell Cycle. 2012;11:730–739. doi: 10.4161/cc.11.4.19172.
    1. Schafer MJ, White TA, Iijima K, Haak AJ, Ligresti G, Atkinson EJ, et al. Cellular senescence mediates fibrotic pulmonary disease. Nat Commun. 2017;8:14532. doi: 10.1038/ncomms14532.
    1. Álvarez D, Cárdenes N, Sellarés J, Bueno M, Corey C, Hanumanthu VS, et al. IPF lung fibroblasts have a senescent phenotype. Am J Physiol Lung Cell Mol Physiol. 2017;313:L1164–L1173. doi: 10.1152/ajplung.00220.2017.
    1. Mailleux AA, Crestani B. Licence to kill senescent cells in idiopathic pulmonary fibrosis? Eur Respir J. 2017;50(2). 10.1183/13993003.01360-2017.
    1. Lehmann M, Korfei M, Mutze K, Klee S, Skronska-Wasek W, Alsafadi HN, et al. Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo. Eur Respir J. 2017;50
    1. van Deursen JM. The role of senescent cells in ageing. Nature. 2014;509:439–446. doi: 10.1038/nature13193.
    1. Coppé JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol. 2010;5:99–118. doi: 10.1146/annurev-pathol-121808-102144.
    1. Naik PK, Bozyk PD, Bentley JK, Popova AP, Birch CM, Wilke CA, et al. Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2012;303(12):L1046–L1056. doi: 10.1152/ajplung.00139.2012.
    1. Ohta S, Okamoto M, Fujimoto K, Sakamoto N, Takahashi K, Yamamoto H, Kushima H, Ishii H, Akasaka K, Ono J, Kamei A, Azuma Y, Matsumoto H, Yamaguchi Y, Aihara M, Johkoh T, Kawaguchi A, Ichiki M, Sagara H, Kadota J-I, Hanaoka M, Hayashi S, Kohno S, Hoshino T, Izuhara K. Consortium for development of diagnostics for pulmonary fibrosis patients (CoDD-PF). The usefulness of monomeric periostin as a biomarker for idiopathic pulmonary fibrosis. PLoS One. 2017;12:e0174547. doi: 10.1371/journal.pone.0174547.
    1. Lee HY, Lee KS, Jeong YJ, Hwang JH, Kim HJ, Chung MP, Han J. High-resolution CT findings in fibrotic idiopathic interstitial pneumonias with little honeycombing: serial changes and prognostic implications. AJR Am J Roentgenol. 2012;199:982–989. doi: 10.2214/AJR.11.8192.

Source: PubMed

Sponzoři a spolupracovníci

Zdravotní podmínky

Drogové intervence

3
Předplatit