Elsevier

Cellular Immunology

Volume 325, March 2018, Pages 1-13
Cellular Immunology

Research paper
Transcriptomic evidence of immune activation in macroscopically normal-appearing and scarred lung tissues in idiopathic pulmonary fibrosis

https://doi.org/10.1016/j.cellimm.2018.01.002Get rights and content

Highlights

  • Macroscopically normal tissue in IPF patients is profoundly involved in the disease.

  • Immune activation is overt in normal-appearing and scarred tissue in IPF lungs.

  • Differences between normal-appearing and scarred tissue involve mostly epithelium.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease manifested by overtly scarred peripheral and basilar regions and more normal-appearing central lung areas. Lung tissues from macroscopically normal-appearing (IPFn) and scarred (IPFs) areas of explanted IPF lungs were analyzed by RNASeq and compared with healthy control (HC) lung tissues. There were profound transcriptomic changes in IPFn compared with HC tissues, which included elevated expression of numerous immune-, inflammation-, and extracellular matrix-related mRNAs, and these changes were similar to those observed with IPFs compared to HC. Comparing IPFn directly to IPFs, elevated expression of epithelial mucociliary mRNAs was observed in the IPFs tissues. Thus, despite the known geographic tissue heterogeneity in IPF, the entire lung is actively involved in the disease process, and demonstrates pronounced elevated expression of numerous immune-related genes. Differences between normal-appearing and scarred tissues may thus be driven by deranged epithelial homeostasis or possibly non-transcriptomic factors.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a distinct disorder within the broad group of diseases termed interstitial lung diseases (ILD), an inclusive group of lung disorders characterized by inflammation and/or fibrosis of the lung parenchyma [1], [2], [3]. As the most severe form of ILD, IPF causes substantial patient morbidity and mortality, has a median survival of approximately three years, and has limited proven efficacious therapies. Lung transplantation remains the only viable intervention in end-stage lung disease due to IPF.

As a fibrotic lung disease, IPF histologically demonstrates a pattern of lung injury termed usual interstitial pneumonia (UIP), which is characterized by dense regions of scarring, interspersed regions of relatively normal lung architecture, fibroblastic foci, patchy inflammatory cell infiltration, and honeycomb (cystic) change. The fibroblastic foci contain alpha-smooth muscle actin (α-SMA)-expressing myofibroblasts. These histologic observations support the commonly accepted paradigm that IPF is a disease of excess extracellular matrix (ECM) accumulation and dysregulated mesenchymal cell proliferation [4].

Despite intense research effort over the past several decades, the pathobiological mechanisms of IPF are not fully understood. As part of this effort, numerous transcriptomic profiling studies of lung tissues from patients with IPF have been performed using several approaches, including serial analysis of gene expression (SAGE) [5], microarray analysis [6], [7], [8], [9], [10], [11], [12], RNASeq [13], [14], [15], [16], and single-cell RNASeq [17]. These studies have revealed a wealth of phenomenological information with important mechanistic implications, stimulating and focusing research on several specific pathophysiological mechanisms of IPF, such as disturbances in expression of genes associated with extracellular matrix, inflammation and immunity, and pulmonary epithelia. Subsequent studies have more specifically focused on contributions from epithelial disturbances [17], including those affecting expression of surfactants, cilium-associated genes, and mucins, including MUC5B [11], [18]; matrix metalloproteinases, including MMP7 [8], [19]; and immune inflammation involving T cells, B cells, macrophages [20], [21], [22], [23], and numerous cytokines and chemokines [24]. The majority of molecular studies have focused on the most scarred areas of the lung since these areas are usually more accessible by standard surgical biopsy. However, the heavily scarred areas likely represent late stages of disease in which initial pathobiological mechanisms have dissipated.

IPF is characterized by scarring, but the IPF lung consistently demonstrates substantial geographic heterogeneity. Radiologic and gross pathology observations often show severe scarring of the lung in predominantly peripheral and basilar areas, whereas central and apical areas appear normal and seemingly unaffected. We recently analyzed and quantified histologic findings in macroscopically normal-appearing lung tissue in patients with IPF [25] and found that these areas exhibited patterns of lung injury termed organizing pneumonia ([OP], characterized by basophilic-staining deposits of ECM containing spindle-shaped fibroblasts or myofibroblasts), and nonspecific interstitial pneumonia ([NSIP], characterized by diffuse interstitial inflammation and/or fibrosis that occur in a spatially uniform pattern throughout the lung). Additionally, α-SMA-expressing myofibroblasts were present in the OP foci in the normal-appearing areas, similar to α-SMA-expressing myofibroblasts found in fibroblastic foci in scarred areas of UIP. Based on these findings, we concluded that lung injury is widespread throughout the IPF lung, extending beyond peripheral and basilar regions, and hypothesized a continuum of lung injury responses in IPF, with macroscopically normal-appearing areas representing earlier stages of disease and macroscopically scarred areas representing more advanced stages [25]. Transcriptomic profiles of macroscopically normal-appearing and scarred regions of IPF lungs have not been comparatively analyzed. Studying areas of the IPF lung that appear to represent earlier stages in the course of injury (macroscopically normal-appearing yet with microscopic evidence of lung injury) may allow for the detection of key mechanisms in disease pathogenesis.

In this study, we hypothesized that our recently reported histological abnormalities in macroscopically normal-appearing lung tissue in IPF [25] are accompanied by substantial transcriptomic disturbances. To challenge this hypothesis and thus gain a deeper understanding of the IPF disease process, we assessed transcriptomic profiles of macroscopically normal-appearing IPF lung tissues, and compared these profiles with transcriptomes of macroscopically scarred IPF lung tissues and healthy control lung tissues. It appears intuitive that macroscopically normal-appearing tissue represents earlier stages of disease, as opposed to densely scarred areas which represent later stages of disease. The scope of this study was to gain better insight into disease pathobiology in macroscopically normal-appearing (though microscopically abnormal [25]) areas, and such scope is consistent with the universal movement to understand and treat diseases at their earliest stages.

Section snippets

Patients and controls

This study was reviewed and approved by the University of Maryland Institutional Review Board, and all experiments were performed in accordance with relevant guidelines and regulations. Patients agreed to participate in the study and provided informed consent. From a group of 41 patients who had undergone lung transplantation in 2016 at the University of Maryland, we obtained lung explants from three patients with idiopathic pulmonary fibrosis (IPF). IPF was diagnosed based on standard

Characteristics of patients, controls, and lung tissues

From a group of 41 patients who had undergone lung transplantation in 2016 at the University of Maryland, we obtained lung explants from three patients who met established criteria for idiopathic pulmonary fibrosis (IPF) [2], [26]. Each of the three patients had idiopathic disease (no identifiable evidence of autoimmune or connective tissue disease, pulmonary drug toxicity, or significant environmental, occupational or avocational exposures), had chest computed tomography (CT) findings of a

Discussion

The primary goal of this study was to gain detailed insight into the pathobiology of IPF through comparative analyses of transcriptomes of lung tissues from macroscopically non-scarred, normal-appearing areas (IPFn) versus clearly diseased and overtly scarred areas (IPFs) of explanted IPF lungs. To our knowledge, this is the first investigation in which such comparisons have been performed. Previous transcriptomic profiling studies compared lung tissues from patients with IPF against tissues

Author contribution

IGL, NWT, APB, and SPA conceived the idea of the study. JRG, AS, JK, TJF, EJB, SMP, APB, and NWT identified and consented the participants, reviewed clinical, radiological, and pathology information, and contributed to the scientific message of the manuscript. IGL, NWT, and APB collected and processed explanted lungs and isolated lung tissues for subsequent analyses. IGL, AEW, and AC isolated RNA, performed quantitative reverse transcriptase-polymerase chain reactions, and performed primary

Disclosures

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or the U.S. Government.

Acknowledgments

This work was supported by the NIH R01HL126897 (SPA) and VA Merit Awards I01CX000101 (IGL) and I01BX002499 (SPA). The authors thank Dr. Paul Todd for his expert editorial support.

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