American Journal of Respiratory and Critical Care Medicine

Rationale: To improve disease outcomes in idiopathic pulmonary fibrosis (IPF), it is essential to understand its early pathophysiology so that it can be targeted therapeutically.

Objectives: Perform three-dimensional assessment of the IPF lung microstructure using stereology and multiresolution computed tomography (CT) imaging.

Methods: Explanted lungs from patients with IPF (n = 8) and donor control subjects (n = 8) were inflated with air and frozen. CT scans were used to assess large airways. Unbiased, systematic uniform random samples (n = 8/lung) were scanned with microCT for stereological assessment of small airways (count number, and measure airway wall and lumen area) and parenchymal fibrosis (volume fraction of tissue, alveolar surface area, and septal wall thickness).

Measurements and Main Results: The total number of airways on clinical CT was greater in IPF lungs than control lungs (P < 0.01), owing to an increase in the wall (P < 0.05) and lumen area (P < 0.05) resulting in more visible airways with a lumen larger than 2 mm. In IPF tissue samples without microscopic fibrosis, assessed by the volume fraction of tissue using microCT, there was a reduction in the number of the terminal (P < 0.01) and transitional (P < 0.001) bronchioles, and an increase in terminal bronchiole wall area (P < 0.001) compared with control lungs. In IPF tissue samples with microscopic parenchymal fibrosis, terminal bronchioles had increased airway wall thickness (P < 0.05) and dilated airway lumens (P < 0.001) leading to honeycomb cyst formations.

Conclusions: This study has important implications for the current thinking on how the lung tissue is remodeled in IPF and highlights small airways as a potential target to modify IPF outcomes.

Scientific Knowledge on the Subject

To date, studies on the pathology of idiopathic pulmonary fibrosis (IPF) have primarily focused on the histopathological hallmarks of the disease: subpleural fibrosis, subepithelial fibroblast foci, and microscopic honeycombing. The recent use of ultra-high-resolution micro–computed tomography has greatly improved our understanding of the complex lung structure and how it is remodeled in IPF. However, there is limited information on the morphology of early IPF lesions, which are vital to understanding disease pathology.

What This Study Adds to the Field

Stereological assessment of the pathology of end-stage IPF lungs compared with age-matched control subjects using multiresolution three-dimensional imaging demonstrates that the smallest airways within the lung, the conducting terminal bronchioles and respiratory transitional bronchioles, are reduced in number, and the terminal bronchiolar walls are thickened, in regions of the lung that have no microscopic fibrosis. Furthermore, this study demonstrates that in regions of microscopic fibrosis in IPF lungs, the remaining terminal bronchioles have thicker airway walls and the airway lumen becomes distorted and dilated, leading to the formation of honeycomb cysts. The data presented in this study have important implications for the current thinking on how the lung tissue is remodeled in IPF and highlight small airways as a potential target to modify IPF outcomes.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease of unknown cause that primarily affects older adults. Despite two recently approved antifibrotic therapeutic agents (13), IPF remains a devastating disease with a median time of survival from diagnosis of only 3–5 years (4, 5). The histopathological hallmarks of the disease are subpleural fibrosis, subepithelial fibroblast foci, and microscopic honeycombing (69). To improve the disease outcomes in IPF, it is essential to understand the pathophysiology of the disease so that the early lesions can be targeted therapeutically. However, there is limited information on the morphology of early lesions within IPF lungs.

Multidetector computed tomography (MDCT) plays a central role in the clinical diagnosis of IPF because of its noninvasive nature (10). However, despite the advances in MDCT diagnosis, histopathological confirmation is still required in many cases to make a definitive diagnosis (4, 10). Recent studies using ultra–high-resolution microCT imaging have bridged the gap in resolution between MDCT and histopathology. These studies have provided new insights into the three-dimensional (3D) pathology of IPF, demonstrating 1) the nonconnectivity of fibroblastic foci (6), 2) the reduction of terminal bronchioles (last generation of conducting airways) in regions of lung tissue scored by a radiologist as minimal and established fibrosis on MDCT (11), and 3) the association between the reduction of terminal bronchioles and the formation of honeycomb cysts (12).

The goal of this study was to obtain new insights into the microscopic structural changes that occur within the IPF lung, by using stereology to assess the relationship of small airway remodeling to microscopic tissue fibrosis quantified on microCT by the volume fraction of tissue. Importantly, by using an unbiased, systematic uniform random (SUR) sampling design of whole lungs, we were able to assess the heterogeneity of disease lesions in lungs from patients with end-stage IPF compared with matched, donor-control lungs. The study demonstrates that a reduction in small airways and airway wall fibrosis in IPF can be found in regions of lung tissue without microscopic parenchymal fibrosis. However, when the surrounding tissue contains microscopic fibrosis, the small airways become dilated and distorted, leading to the formation of honeycomb cysts. These volumetric data have important implications for the current thinking on how the lung tissue is remodeled in IPF, and the potential of targeting small airway reduction to modify outcomes in patients with IPF. Some of the results in this study have been previously reported in the form of abstracts (13, 14).

Subjects

Single explanted lungs from eight patients with end-stage IPF treated with bilateral lung transplantation and eight unused donor control lungs, matched for age and sex, were obtained at the University of Pennsylvania Hospital with the approval of the University of Pennsylvania Hospital Institutional Review Board. Informed consent was obtained from the patients prior to lung transplantation or the donor’s next of kin. The study was conducted and approved by the University of British Columbia Research Ethics Board (#H07-0021). Subject demographics, pulmonary function, and MDCT data are provided in Table 1. The diagnosis of IPF was made through multidisciplinary discussions following current diagnostic guidelines (4). For this study, the MDCT scans and histopathology were reevaluated by independent radiologists (C.J.H. and D.M.) and a pathologist (T.V.C.); see Table E1 in the online supplement.

Table 1. Demographic and Quantitative MDCT Data for Study Subjects

 ControlIPF
Cases, n88
Age, yr60 ± 1061 ± 5
Sex, M/F7/15/3
Height, cm175 ± 5170 ± 8
Weight, kg89 ± 1478 ± 10
Smoking history, never/former4/3*2/6
Pack-years in former smokers15 ± 028 ± 16
Pulmonary function testing  
 FVC, L4.29 ± 0.502.08 ± 0.62
 FEV1, L3.33 ± 0.431.67 ± 0.50
 FEV1/FVC, %78 ± 280 ± 8
 DlCO, % predictedNA33 ± 11§
Specimen MDCT data  
 Total lung volume, ml3,340 (776)1,346 (799)
 Mean CT value, HU−876 (55)−540 (180)
 Percentage airspace volume, %88 (5)57 (17)
 Percentage tissue volume, %12 (5)43 (17)

Definition of abbreviations: CT = computed tomography; HU = Hounsfield units; IPF = idiopathic pulmonary fibrosis; MDCT = multidetector CT; NA = not applicable.

Data are shown as mean ± SD or median (interquartile range).

*Smoking history in one control subject is unknown.

Global Lung Function Initiative–estimated values are shown.

P < 0.001 versus control subjects.

§DlCO data in two patients are not available.

3D Imaging–based Stereology

The study workflow is shown in Figure 1, with procedures described in detail elsewhere (1517). Briefly, preoperative MDCT scans were performed for all patients with IPF before lung transplantation (parameters: 100–120 kV, 67–270 mA, 0.625–2.5 mm slice thickness).

Lung tissue samples were taken using a previously described SUR sampling method (1719). Briefly, lungs were inflated with air, frozen, and imaged with MDCT (parameters, 120 kV, 178 mA, 0.625 mm slice thickness). The specimen MDCT scans were used to obtain total lung volume, mean CT attenuation value, percentage tissue volume, percentage air space volume, total airway count, cross-sectional airway wall area, and airway lumen area (17, 20). The frozen lungs were sliced from apex to base, and eight SUR tissue samples per lung (23 × 23 mm) were taken.

MicroCT.

Tissue samples were maintained frozen (-30°C) using a cryo-stage during microCT (parameters: 50kV, 350μA, molybdenum target, 354ms exposure time, gain 24dB) as previously described(16, 17). Images were acquired at two isotropic resolutions: 13μm for airway analysis and 7μm for parenchymal analysis.

Stereology.

A detailed description of the stereology methods is provided in the online supplement (Figures E1–E9). All quantitative image analysis was performed in ImageJ (21). Terminal and transitional bronchioles were defined, counted, and segmented as previously described (22, 23). The centerlines of terminal bronchioles were used to generate exact cross-sectional images which enabled the computation of the following parameters: wall area, coefficient of variation of wall thickness, lumen area and roundness, branch roughness and curviness as previously described (17). Lung parenchyma was separated into aerated and nonaerated tissue (24), and point counts for each tissue type were used to determine the respective volume fractions. Line intercepts were used to calculate mean airspace chord length, alveolar surface density, alveolar surface area, and septal wall thickness as previously described (17, 19). After microCT scanning, tissue samples were formalin fixed and paraffin embedded. Tissue sections were stained using hematoxylin and eosin for histopathological diagnosis.

Statistical Analysis

A Shapiro-Wilk normality test was used to assess the distribution of the data. The demographic data were compared using a nonpaired t test and reported as mean ± SD. To compare all parameters between control and IPF cases, a nonparametric Mann-Whitney U test was used. For comparison between three groups, a likelihood ratio test was performed to compare the models with and without the grouping variable, and Tukey’s post hoc test for multiple comparisons. A linear mixed-effect model was used to investigate the difference between IPF and control samples, with the subject identifier as a random effect. All statistical analyses were performed using the statistical software R version 4.0.2 (25). A P value <0.05 was considered statistically significant.

Demographic and Quantitative MDCT Comparisons

Demographic, physiological features, and MDCT data for the patients with IPF and donor control subjects are shown in Table 1. The IPF cases had a reduced FEV1 and FVC but a preserved FEV1/FVC ratio compared to the calculated values derived from the demographic data of the control subjects (26). As shown in Table E1, three patients with IPF were assigned a radiologic pattern consistent with an alternative diagnosis, but all of the IPF cases had a pathological pattern of usual interstitial pneumonitis that supported a diagnosis of IPF. The quantitative analysis of the specimen MDCT scans showed that the IPF lungs had a smaller total lung volume (P < 0.01) and % airspace volume (P < 0.01), which coincided with a higher mean CT attenuation value (P < 0.01) and percentage tissue volume (P < 0.01) compared with control lungs (Table 1).

Comparison of Large and Small Airway Features between Control and IPF Lungs

Figures 2A and 2B are representative images from an IPF and a control lung for 1) a slice from the specimen MDCT scan showing the location of a SUR tissue sample taken from that slice (red circle), 2) the reconstructed MDCT airway tree, 3) a representative microCT slice for the SUR tissue sample taken, 4) the reconstructed airway tree from the microCT, and 5) a terminal bronchiole viewed in cross-section.

The total visible airway count derived from the specimen MDCT scans was greater in IPF lungs (561 ± 315) than in control lungs (196 ± 113; P < 0.01; Figure 2C). As shown in Figure 2D, the number of airways visible on the MDCT scans was increased in airway generations 7–17 in IPF lungs compared with control lungs (P < 0.05). Airway wall area (mm2) was greater in airway generations 10–13 in patients with IPF compared with control subjects (P < 0.05; Figure 2E). Airway lumen area (mm2) was greater in airway generations 12–13 in IPF lungs compared with control lungs (P < 0.05; Figure 2F). In IPF lungs, airways from generation 14–17 had airway wall thickening and lumen dilatation comparable to airways in generations 12–13, but no airways in generations 14–17 were visible in control lungs for statistical comparison.

From the analysis of small airways using microCT, it was found that the total number of both terminal (Figure 2G; P < 0.001) and transitional (Figure 2H; P < 0.001) bronchioles per lung were reduced in patients with IPF compared with control subjects. The wall area (Figure 2K; P < 0.001) and variance in the wall thickness (Figure 2L; P < 0.01) of terminal bronchioles was greater in IPF lungs than in control lungs. The lumen area of terminal bronchioles in IPF lungs was increased (Figure 2M; P < 0.01) and they were less round (Figure 2N; P < 0.05) compared with control lungs. As visualized in the 3D reconstruction of the microCT airway tree (Figure 2B), terminal bronchioles were distorted over their entire branch length with localized dilatations in IPF lungs, quantified by greater values for branch roughness (Figure 2J; P < 0.001) and curviness (Figure 2I; P < 0.05) compared with control lungs.

Comparison of Parenchymal Features between Control and IPF Lungs

The microCT images in Figure 3 show the heterogeneity of microscopic lung fibrosis in different lung tissue samples taken from an IPF lung (Figure 3A) compared with a control lung (Figure 3B). In IPF lungs, there was an increase in the volume fraction of total parenchymal tissue compared with control lungs (Figure 3C; P < 0.001). In the IPF lung parenchyma, 81% was aerated and 19% was nonaerated parenchyma, in contrast to control lungs, which contained no nonaerated parenchyma (Figure 3D; P < 0.001). In the regions of aerated parenchyma, the mean airspace chord length (Figure 3E) was not different between groups, but there was a substantial decrease in the alveolar surface area (Figure 3F; P < 0.001) in IPF lungs compared with control lungs. The reduction of alveolar surface area in IPF lungs was due to a reduction in the volume fraction of alveolar space (Figure 3G; P < 0.001), an increase in the volume fraction of septal tissue (Figure 3H; P < 0.001), and no change in the volume fraction of alveolar ducts (Figure 3I). The increase in the volume fraction of septal tissue was related to an increase in the average septal wall thickness in IPF lungs compared with control lungs (Figure 3J; P < 0.001). All stereological parameters assessed in the manuscript are provided by case for the IPF lungs in Table E2 and the control lungs in Table E3.

Association of Small Airway Remodeling with Microscopic Fibrosis

Figures 4A and 4B show the number of terminal and transitional bronchioles plotted against the volume fraction of tissue as measured on microCT for every control and IPF tissue sample. The upper limit for the volume fraction of tissue in controls (dashed line) was used to define a threshold for samples with and without microscopic tissue fibrosis. In IPF tissue samples with and without microscopic fibrosis, there was a reduction in the number of the terminal (Figure 4C; P < 0.01 and P < 0.05, respectively) and transitional (Figure 4D; P < 0.001 and P < 0.05, respectively) bronchioles compared with control samples. The wall area of terminal bronchioles (Figure 4E) was increased in IPF tissue samples with (P < 0.001) and without (P < 0.05) microscopic fibrosis compared with controls. IPF tissue samples with fibrosis also had a larger wall area compared with IPF tissue samples without fibrosis (P < 0.05). Terminal bronchiole branch roughness, which is an indicator of airway lumen distortion and dilatation (Figure 4F), was significantly increased in IPF tissue samples with fibrosis compared with IPF tissue samples without fibrosis (P < 0.001) and control samples (P < 0.001). These data indicate that in IPF lungs, the lumen of the terminal bronchioles only become dilated and the airway wall further thickened when the surrounding parenchyma becomes fibrotic. To visualize the relationship between microscopic parenchymal fibrosis and the dilation of terminal bronchioles, axial images of a microCT scan of IPF tissue samples without (Figure 4G) and with parenchymal fibrosis (Figure 4H) are provided. Compared with the IPF sample without fibrosis, the microCT image of the IPF sample with fibrosis shows typical features of honeycomb cysts (orange arrowheads, Figure 4H). The 3D renderings of the small airway structures overlaid onto the axial view of the microCT images demonstrate that in the samples with microscopic fibrosis, the honeycomb cysts are in fact part of the conducting airways, which are distorted and dilated by the surrounding parenchymal fibrosis (Figure 4J), compared with samples with no parenchymal fibrosis (Figure 4I).

Clinically, tissue fibrosis can be quantified by an increase in the mean CT attenuation value on MDCT. Figure 5 shows a positive correlation between the mean CT attenuation value for the regions within the IPF lung MDCT scans where tissue samples were taken, and the increase in terminal bronchiole wall area (Figure 5A; standardized β = 0.73; P < 0.001), lumen area (Figure 5B; standardized β = 0.38; P < 0.01) and the distortion of the lumen defined as branch roughness (Figure 5C; standardized β = 0.67; P < 0.001) measured using microCT. There was also a positive correlation between the regional mean CT attenuation values on the IPF lung MDCT scans and the volume fraction of tissue (Figure 5D; standardized β = 0.91; P < 0.001) as well as the septal wall thickness (Figure 5E; standardized β = 0.72; P < 0.001) but a negative correlation with alveolar surface density (Figure 5F; standardized β = −0.50; P < 0.001) measured using microCT.

This study demonstrates that the numbers of conducting terminal bronchioles and respiratory transitional bronchioles are significantly reduced and that the terminal bronchiole airway walls are significantly thickened in regions of the lung that have no microscopic fibrosis in patients with end-stage IPF compared with age-matched control subjects. Furthermore, this study demonstrates that in regions of microscopic fibrosis in IPF lungs, the remaining terminal bronchioles have thicker airway walls and the airway lumen becomes distorted and dilated, leading to the formation of honeycomb cysts. This study highlights that a reduction and fibrosis of small airways in IPF likely occurs early in the disease process as these features are present before microscopic parenchymal fibrosis occurs. This finding highlights the potential importance of the small airways for modification of disease outcomes in IPF.

An earlier report on end-stage IPF lungs, using microCT and a targeted sampling approach, demonstrated that the number of terminal bronchioles is reduced in regions of minimal or established fibrosis that were scored by a radiologist on clinical MDCT scans.

The current study, using a new cohort of IPF and control lungs, supports and extends the previous work, by using an unbiased uniform random sampling design (SURs) to quantify the total number of terminal and transitional bronchioles, airway dimensions, and volume fractions of parenchymal tissues per lung. Importantly, using this stereological analysis of the microanatomy (only visible using histology or microCT [7–13 μm]), these data show a reduction in the number of terminal and transitional bronchioles occurs in regions of no “microscopic” fibrosis. The current study also demonstrates that in regions of microscopic fibrosis in IPF lungs, the remaining terminal bronchioles have thicker airway walls and the airway lumen becomes distorted and dilated, forming honeycomb cysts. In support of our findings of thickened small airway walls in IPF, Figueira de Mello and colleagues have recently shown using histology on lung biopsies that small airways (<6 mm in diameter) have a greater wall area in patients with usual interstitial pneumonitis and nonspecific interstitial pneumonia compared with control subjects (27). In contrast to our data, these authors reported a narrower airway lumen area for these airways compared with donor control subjects. This discrepancy may be methodologic. An important aspect of studying the lung structure at any resolution and with any modality is the correct SUR sampling approach as detailed by the American Thoracic Society/European Respiratory Society guidelines to obtain unbiased, representative samples of the whole lung (18). Knudsen and colleagues have recently reviewed the advantages of 3D imaging for stereological studies and concluded that high-resolution microCT 3D imaging has significant advantages over two-dimensional (2D) histological studies (28). In the current study, the ability to visualize the entire branch length of the terminal and transitional bronchioles on microCT, and larger conducting airways using MDCT, revealed that conducting airways in the lungs of patients with IPF have marked heterogeneous lumen distortion, dilation, and wall thickening along their path length. This heterogeneity in the internal diameter of airways thus confounds the use of airway size to compare similar airway generations in 2D histological studies in IPF.

This study demonstrates that the conducting airway tree in end-stage IPF is remodeled from the seventh to the last generation (terminal bronchioles) with thickened walls and dilated lumens, resulting in an increased number of visible airways on MDCT compared with donor control subjects. These data confirm the previous findings of Verleden and colleagues, in a separate cohort of lungs, who reported increased numbers of airways from the 8th to the 14th generation on MDCT in IPF lungs (11). The authors hypothesized that the increase in the number of visible airways on MDCT was due to airway wall thickening. It is important to note that with the resolution of MDCT (∼0.5–1.0 mm), airways smaller than 2 mm are very difficult to quantify consistently. In this study, we quantified on MDCT that the conducting airway tree in IPF lungs is thickened and dilated along its path length, resulting in more visible airways due to a lumen larger than 2 mm. Furthermore, at the resolution of microCT (13 μm), we found that the smallest generation of conducting airways, the terminal bronchioles, were thickened and dilated but also reduced in number. Thus, if the resolution of MDCT was such that it could enable us to visualize all conducting airways, we would see that the absolute number of conducting airways must be reduced in IPF.

Irregular bronchial dilatation within or around areas of parenchymal abnormality has previously been described as traction bronchiectasis or bronchiolectasis, and this radiological term is used in the current IPF diagnosis guidelines (4, 2931). However, no measurements of force have been measured to demonstrate that traction induced by the surrounding fibrotic parenchyma is the mechanism for dilation and distortion of the conducting airways. In bronchiolitis obliterans syndrome and chronic obstructive pulmonary disease (COPD), the small airways have also been shown to be reduced, the remaining terminal bronchioles thickened, and the airway lumens narrowed and/or occluded (32, 33). In IPF lungs, we observed the opposite: the remaining terminal bronchioles have thickened walls, but the airway lumen is distorted and dilated when surrounded by fibrotic parenchymal tissue. These data indicate that traction by the fibrotic parenchyma may indeed be the mechanism for this alteration in airway structure in IPF. Future studies, with the use of precision-cut lung slices or other technologies, will be needed to determine if the traction of the fibrotic parenchymal tissue is indeed the mechanism for dilation of the conducting airways in IPF.

Importantly, the 3D visualization of the path length of terminal bronchioles demonstrated that in regions of microscopic fibrosis, thickened and dilated terminal bronchioles form honeycomb cysts. This novel finding may help to explain why honeycomb lesions on 2D histological sections are described as abnormally dilated airspaces with walls composed of fibrotic tissue, lined by an epithelium that shares characteristics with the airway epithelium (34, 35). In support of this finding, Club cells are the dominant airway epithelial cell present within the terminal bronchioles. Recent single-cell RNA sequencing data have identified two unique Club cell subpopulations expressing SCGB3A2 (a Club cell gene) or MUC5B (a known genetic risk factor of IPF) that are increased in the parenchymal tissue of patients with IPF and associated with extracellular matrix formation, or production of mucin and immune cell chemoattractants, respectively (3640). Future studies, combining single-cell sequencing with single-cell spatial imaging technologies, will be essential to understand the structural environment where Club cells reside, if their numbers are increased because of dilation of the terminal bronchiole lumen, and how they contribute to the pathogenesis of IPF. The anatomical data presented in this study, therefore, provide support to a previous hypothesis by Piciucchi and colleagues that proposed traction bronchiectasis of conducting airways and honeycombing is a continuum of airway disease throughout the airway tree, rather than separate pathological processes in IPF (41). Together, these observations may also explain why previous IPF studies show that traction bronchiectasis in the large conducting airways correlates with honeycombing on MDCT (42, 43).

It is well understood that the mechanical properties of the lung parenchyma and airways are interdependent (44). The lung parenchyma contains elastic fibers that act radially on the airways, whereas the airways contain longitudinal elastic fibers. When the lung is inflated, the transpulmonary pressure spreads uniformly in alveoli and parenchymal airways and acts on these fibers to increase the length and diameter of the airways. From the 3D analysis of terminal bronchioles in this study, we propose that parenchymal fibrosis results in the significant mechanical distention and dilatation of terminal bronchioles, resulting in traction bronchiolectasis and leading to a 320% increase in luminal cross-sectional area. Because the resistance of the tracheobronchial tree is a reflection of the combined resistances of all generations of airways arranged in series and parallel, the large increase in airway lumen size beyond seventh generation airways may contribute, together with increased elastic recoil, to the relatively preserved or often increased FEV1/FVC ratio due to a disproportionate reduction in FVC compared with FEV1 (33). In support of this hypothesis, the patients with end-stage IPF in this study had a comparable reduction of terminal bronchioles (81%) to that previously reported in patients with end-stage COPD (70%) (33); however, the patients with IPF had an FEV1/FVC ratio of 80%, versus the patients with end-stage COPD with an FEV1/FVC of 35%. Furthermore, the total cross-sectional area of all remaining terminal bronchioles was calculated per lung. In this study, control lungs had a total terminal bronchiole cross-sectional area of 2,299 mm2 per lung, compared with 1,310 mm2 per lung in IPF lungs and 934 mm2 per lung in end-stage COPD (recalculated from previously published data [33]). These data highlight the effect on FEV1 with terminal bronchiole reduction and dilatation in IPF, versus terminal bronchiole reduction and obstruction in COPD.

The application of stereology enabled the detailed assessment of both aerated and nonaerated parenchyma in IPF lungs. In regions of aerated parenchyma in IPF lungs, there was a substantial decrease in the alveolar surface area due to the reduction of alveolar air spaces and increased septal wall thickness, with no change in the alveolar duct space or mean airspace chord length. These findings are supported by a much earlier study using stereology on histological sections, which reported a decrease in alveolar surface area and thickening of septal walls in regions defined as “normal” in the lungs of patients with end-stage IPF compared with those from control subjects (45). Previous reports have shown that the mean CT attenuation of the whole lung is associated with the severity of pulmonary function impairment in patients with IPF (4648). Using image registration on the specimen MDCT, we found that the mean CT attenuation for the region of the tissue sample was strongly correlated with parenchymal fibrosis (volume fraction of parenchymal tissue) and terminal bronchiole wall thickening and lumen distortion quantified using microCT. In support of this finding, Miller and colleagues recently demonstrated that Pi10 (the average wall thickness of a hypothetical airway of 10 mm perimeter) was significantly greater in patients with interstitial lung abnormalities and in those with IPF (49). These studies highlight that radiologic features on MDCT have the potential to be used as an imaging biomarker for early changes in small airways and lung parenchyma. Lung texture analysis has been used to assess radiologic parenchyma features to predict disease outcomes in IPF (5052). In such studies, it will be important to validate whether existing quantitative MDCT biomarkers using texture analysis or parametric response mapping (53, 54) or novel biomarkers correlate with the microstructure of IPF lesions using microCT. In particular, for many of the radiological features used in the diagnosis of IPF such as ground-glass opacities and reticulation (believed to be active sites of tissue inflammation and remodeling), it is still unclear what tissue pathologies cause these radiological features. Quantitative studies combining MDCT, microCT, and histology will help determine the tissue pathologies associated with each of the radiological features used to diagnose different interstitial lung diseases. In the future, there is also the potential that the resolution of MDCT will improve to assess the small airways and parenchyma in vivo, so that individuals with underlying genetic risk factors and/or high-risk family histories may be screened early for disease.

Some limitations should be noted in the present study. First, the number of patients with IPF (n = 8) analyzed in this study is small. However, a number of the findings in this study are consistent with those in a previous independent cohort of nine patients with end-stage IPF (11). Second, although the availability of whole lungs enabled consistent inflation and unbiased SUR sampling of the lung for stereological assessment, such samples can only be obtained from patients with end-stage IPF after lung transplantation. Current biopsy strategies for sampling the peripheral lung or central airways in patients with mild IPF do not allow for such morphometric investigation. However, whole inflated lungs do provide the opportunity to assess the heterogeneous nature of disease pathology, with the assumption that fibrotic lesions represent advanced disease. An additional limitation of such a retrospective cohort study is that it does not allow investigation of disease progression, including whether patients with IPF were born with abnormal lung microstructures and a reduced number of airways. However, recent data on a cross-section cohort of control donor lungs with no smoking history or COPD have shown that the small airways are reduced with aging after the age of 25 years (55), which supports the notion that small airways can be reduced even further as part of the pathobiology of lung diseases such as COPD and IPF. Third, lung function data were not available for the donor control lungs and could only be imputed using the donor demographics. Fourth, only three control subjects had a smoking history, compared with six patients in the IPF group.

In conclusion, stereological assessment of IPF lung pathology using multi-resolution 3D imaging demonstrates that a reduction in small airway number and airway wall fibrosis in IPF likely occurs early in the disease process. Furthermore, terminal bronchioles undergo traction bronchiolectasis resulting in the formation of honeycomb cysts only in the presence of microscopic parenchymal fibrosis. The pathology data presented in this study have important implications for the current thinking on how the lung tissue is remodeled in IPF and highlights the potential of targeting small airway reduction to modify outcomes in patients with IPF.

The authors thank James Latham from the Radiology Department at St. Paul’s Hospital for performing multidetector computed tomography scans of whole lungs. They also thank Dr. Aaron Barlow from the Cellular Imaging and Biophysics Core for his help with microCT image acquisition, Darren Sutherland from the James Hogg Lung Registry for his help with sample preparation, and Chen Xi Yang for assisting in choosing the appropriate statistical methods, all from the University of British Columbia Centre for Heart Lung Innovation.

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Correspondence and requests for reprints should be addressed to Dragoş M. Vasilescu, Ph.D., B.Sc., UBC Centre for Heart Lung Innovation, St. Paul’s Hospital, Rm 166, 1081 Burrard Street, Vancouver, BC, V6Z 1Y6 Canada. E-mail: .

Supported by the MSD Life Science Foundation and the Public Interest Incorporated Foundation Fellowship (K.I.); by the Parker B. Francis Foundation Fellowship and a Canadian Lung Association grant (D.M.V.); by Canadian Institutes of Health Research and Michael Smith Foundation for Health Research New Investigator Awards (T.-L.H.); and, in part, through investigator-initiated grants from the British Columbia Lung Association and the Canadian Institutes of Health Research.

Author Contributions: K.I., T.-L.H., S.P., D.P., C.J.H., D.M., S.L., F.C., F.X., J.D.C., T.V.C., and D.M.V. contributed to data collection. K.I., T.-L.H., D.P., N.T., C.J.R., P.D.P., H.O.C., J.C.H., and D.M.V. contributed to study design, data interpretation, figures, and writing. All authors read and approved the manuscript.

This article has a related editorial.

This article has an online supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.202103-0585OC on August 3, 2021

Author disclosures are available with the text of this article at www.atsjournals.org.

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