Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study

Abstract

Background: The observation that patients with idiopathic pulmonary fibrosis (IPF) can have higher than normal expiratory flow rates at low lung volumes led to the conclusion that the airways are spared in IPF. This study aimed to re-examine the hypothesis that airways are spared in IPF using a multiresolution imaging protocol that combines multidetector CT (MDCT), with micro-CT and histology.

Methods: This was a retrospective cohort study comparing explanted lungs from patients with severe IPF treated by lung transplantation with a cohort of unused donor (control) lungs. The donor control lungs had no known lung disease, comorbidities, or structural lung injury, and were deemed appropriate for transplantation on review of the clinical files. The diagnosis of IPF in the lungs from patients was established by a multidisciplinary consensus committee according to existing guidelines, and was confirmed by video-assisted thoracic surgical biopsy or by pathological examination of the contralateral lung. The control and IPF groups were matched for age, sex, height, and bodyweight. Samples of lung tissue were compared using the multiresolution imaging approach: a cascade of clinical MDCT, micro-CT, and histological imaging. We did two experiments: in experiment 1, all the lungs were randomly sampled; in experiment 2, samples were selected from regions of minimal and established fibrosis. The patients and donors were recruited from the Katholieke Universiteit Leuven (Leuven, Belgium) and the University of Pennsylvania Hospital (Philadelphia, PA, USA). The study took place at the Katholieke Universiteit Leuven, and the University of British Columbia (Vancouver, BC, Canada).

Findings: Between Oct 5, 2009, and July 22, 2016, explanted lungs from patients with severe IPF (n=11), were compared with a cohort of unused donor (control) lungs (n=10), providing 240 samples of lung tissue for comparison using the multiresolution imaging approach. The MDCT specimen scans show that the number of visible airways located between the ninth generation (control 69 [SD 22] versus patients with IPF 105 [33], p=0·0023) and 14th generation (control 9 [6] versus patients with IPF 49 [28], p<0·0001) of airway branching are increased in patients with IPF, which we show by micro-CT is due to thickening of their walls and distortion of their lumens. The micro-CT analysis showed that compared with healthy (control) lung anatomy (mean 5·6 terminal bronchioles per mL [SD 1·6]), minimal fibrosis in IPF tissue was associated with a 57% loss of the terminal bronchioles (mean 2·4 terminal bronchioles per mL [SD 1·0]; p<0·0001), the appearance of fibroblastic foci, and infiltration of the tissue by inflammatory immune cells capable of forming lymphoid follicles. Established fibrosis in IPF tissue had a similar reduction (66%) in the number of terminal bronchioles (mean 1·9 terminal bronchioles per mL [SD 1·4]; p<0·0001) and was dominated by increased airspace size, Ashcroft fibrosis score, and volume fractions of tissue and collagen.

Interpretation: Small airways disease is a feature of IPF, with significant loss of terminal bronchioles occuring within regions of minimal fibrosis. On the basis of these findings, we postulate that the small airways could become a potential therapeutic target in IPF.

Funding: Katholieke Universiteit Leuven, US National Institutes of Health, BC Lung Association, and Genentech.

Figure 1:

(A) Comparison of the distribution of mean ± SEM number of airways visible on the airway tree, reconstructed at the 800μM spatial resolution of the MDCT specimen scans at each branching generation shows an increase in the number of visible airway in IPF compared to the control lungs beyond generation 8 in experiment 1. (B). Shows this increase in number occurs primarily in airways ≤2 mm per generation show the greatest increase in visible airways occurs in the airways ≤2 mm in diameter. (C) MDCT specimen scan images of control lung (D and E) lungs affected by IPF where the circled areas represent regions of minimal (D) and established (E) fibrosis, selected by the radiologists who examined the preoperative MDCT scans. MicroCT scans of tissues taken from the circled areas were used for comparisons among control, minimal and established fibrotic regions (F, G, and H, respectively). § = <0∙0001, ¥ = 0∙0001, # = 0∙0023, ǂ = 0∙0027, V indicates vessels and * indicates airways.

Figure 2:

Representative cross-sectional microCT images of pre-terminal small airways cut at 90 degrees to the centreline (A-C) where the analysis showed an increase in wall area (D) and a reduction in the circularity of the lumen (E) in minimal and established fibrosis. The numbers of alveolar attachments tethered to the outer walls of pre-terminal bronchioles was significantly decreased in established fibrosis (F), and a trend toward a decline in lumen area that did not reach statistical significance (G) in minimal and established fibrosis compared to controls in experiment 2.

Figure 3:

Shows the number of terminal bronchioles and the mean linear intercept (Lm) in experiment 1. (A) Terminal bronchioles are sharply reduced in number per mL of tissue compared to controls in regions of minimal fibrosis, without further decline between regions of minimal and established fibrosis in IPF. (B) Lm increased in regions of established fibrosis in IPF due to the collapse of alveoli on alveolar ducts Values are expressed as per sample.

Figure 4:

The progression from control lung anatomy to minimal and established fibrosis is associated with an increase in Lm in experiment 2 (A). (B) The histology-based Ashcroft fibrosis score that ranges between 0 (normal) and 8 (dense fibrosis) was greater in established fibrosis than minimal fibrosis. (C) The volume fraction of the lung samples taken up by tissue. (D) The volume fraction of lung tissue taken up by collagen identified by picro sirius red staining used to identify collagen, in the parenchyma but not the airways. (E) Example of a fibroblastic focus, showing that the volume fraction was similarly increased in both minimal and established fibrosis. Values are expressed as per sample.

Figure 5:

Compares the volume fractions of infiltrating inflammatory immune cells in control lung tissue (red) to the volume fractions of the same cells in regions of minimal (blue) and established (green) fibrosis in experiment 2. This comparison shows (A) B-cell and CD4 lymphocyte infiltration increased in airways tissue and that the infiltration of macrophages, B-cells, CD4, CD8 and eosinophils are increased in the parenchyma in regions of minimal and established fibrosis compared to controls. These data show that the number airways containing tertiary lymphoid organs, as illustrated in (B), increased in both minimal and established regions of fibrosis compared to controls (C), but were not different from each other in the airways. Values are expressed as per sample.