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Pathology of Idiopathic Pulmonary Fibrosis Assessed by a Combination of Microcomputed Tomography, Histology, and Immunohistochemistry

Open ArchivePublished:September 10, 2020DOI:https://doi.org/10.1016/j.ajpath.2020.09.001
      Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease with the histology of usual interstitial pneumonia (UIP). Although the pathologist's visual inspection is central in histologic assessments, three-dimensional microcomputed tomography (microCT) assessment may complement the pathologist's scoring. We examined associations between the histopathologic features of UIP and IPF in explanted lungs and quantitative microCT measurements, including alveolar surface density, total lung volume taken up by tissue (%), and terminal bronchiolar number. Sixty frozen samples from 10 air-inflated explanted lungs with severe IPF and 36 samples from 6 donor control lungs were scanned with microCT and processed for histologic analysis. An experienced pathologist scored three major UIP criteria (patchy fibrosis, honeycomb, and fibroblastic foci), five additional pathologic changes, and immunohistochemical staining for CD68-, CD4-, CD8-, and CD79a-positive cells, graded on a 0 to 3+ scale. The alveolar surface density and terminal bronchiolar number decreased and the tissue percentage increased in lungs with IPF compared with controls. In lungs with IPF, lower alveolar surface density and higher tissue percentage were correlated with greater scores of patchy fibrosis, fibroblastic foci, honeycomb, CD79a-positive cells, and lymphoid follicles. A decreased number of terminal bronchioles was correlated with honeycomb score but not with the other scores. The three-dimensional microCT measurements reflect the pathological UIP and IPF criteria and suggest that the reduction in the terminal bronchioles may be associated with honeycomb cyst formation.
      Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic disease characterized by a rapid decline in lung function and poor prognosis.
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      ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis
      An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.
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      ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis
      An official ATS/ERS/JRS/ALAT clinical practice guideline.
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      An official ATS/ERS/JRS/ALAT clinical practice guideline.
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      The aim of this study was to extend the understanding of the pathology of lungs with UIP and IPF by investigating the relationship between the experienced pathologist's scorings of histologic features, such as honeycomb cysts, and microCT measurements of alveolar surface density, tissue percentage, Lm, and the number of the terminal bronchioles.

      Materials and Methods

      Study Protocol

      A diagnosis of IPF was based on the current American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin American Thoracic Association guidelines that include dominant airway-centered changes as an exclusion criterion.
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      • Lynch D.A.
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      • Swigris J.J.
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      • Ancochea J.
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      • Costabel U.
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      • Hansell D.M.
      • Johkoh T.
      • Kim D.S.
      • King Jr., T.E.
      • Kondoh Y.
      • Myers J.
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      • Nicholson A.G.
      • Richeldi L.
      • Selman M.
      • Dudden R.F.
      • Griss B.S.
      • Protzko S.L.
      • Schunemann H.J.
      ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis
      An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.
      ,
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      • Myers J.L.
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      • Behr J.
      • Cottin V.
      • Danoff S.K.
      • Morell F.
      • Flaherty K.R.
      • Wells A.
      • Martinez F.J.
      • Azuma A.
      • Bice T.J.
      • Bouros D.
      • Brown K.K.
      • Collard H.R.
      • Duggal A.
      • Galvin L.
      • Inoue Y.
      • Jenkins R.G.
      • Johkoh T.
      • Kazerooni E.A.
      • Kitaichi M.
      • Knight S.L.
      • Mansour G.
      • Nicholson A.G.
      • Pipavath S.N.J.
      • Buendia-Roldan I.
      • Selman M.
      • Travis W.D.
      • Walsh S.
      • Wilson K.C.
      ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis
      An official ATS/ERS/JRS/ALAT clinical practice guideline.
      The major features of this protocol have been described in detail elsewhere.
      • McDonough J.E.
      • Kaminski N.
      • Thienpont B.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      Gene correlation network analysis to identify regulatory factors in idiopathic pulmonary fibrosis.
      ,
      • Verleden S.E.
      • Tanabe N.
      • McDonough J.E.
      • Vasilescu D.M.
      • Xu F.
      • Wuyts W.A.
      • Piloni D.
      • De Sadeleer L.
      • Willems S.
      • Mai C.
      • Hostens J.
      • Cooper J.D.
      • Verbeken E.K.
      • Verschakelen J.
      • Galban C.J.
      • Van Raemdonck D.E.
      • Colby T.V.
      • Decramer M.
      • Verleden G.M.
      • Kaminski N.
      • Hackett T.L.
      • Vanaudenaerde B.M.
      • Hogg J.C.
      Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.
      ,
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      • Hogg J.C.
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      • Wuyts W.A.
      • Kaminski N.
      Transcriptional regulatory model of fibrosis progression in the human lung.
      Briefly intact lung specimens donated by patients with severe IPF treated by lung transplantation and unused donor lungs that served as controls were inflated with air and frozen solid with liquid nitrogen vapor. The specimen was kept frozen while cutting it into 2-cm-thick transverse slices. Two lung tissue samples were obtained from the upper, middle, and lower part of the lung (n = 6 per lung) to compare samples with different severity of the disease.
      • McDonough J.E.
      • Kaminski N.
      • Thienpont B.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      Gene correlation network analysis to identify regulatory factors in idiopathic pulmonary fibrosis.
      ,
      • Verleden S.E.
      • Tanabe N.
      • McDonough J.E.
      • Vasilescu D.M.
      • Xu F.
      • Wuyts W.A.
      • Piloni D.
      • De Sadeleer L.
      • Willems S.
      • Mai C.
      • Hostens J.
      • Cooper J.D.
      • Verbeken E.K.
      • Verschakelen J.
      • Galban C.J.
      • Van Raemdonck D.E.
      • Colby T.V.
      • Decramer M.
      • Verleden G.M.
      • Kaminski N.
      • Hackett T.L.
      • Vanaudenaerde B.M.
      • Hogg J.C.
      Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.
      ,
      • McDonough J.E.
      • Ahangari F.
      • Li Q.
      • Jain S.
      • Verleden S.E.
      • Herazo-Maya J.
      • Vukmirovic M.
      • DeIuliis G.
      • Tzouvelekis A.
      • Tanabe N.
      • Chu F.
      • Yan X.
      • Verschakelen J.
      • Homer R.J.
      • Manatakis D.V.
      • Zhang J.
      • Ding J.
      • Maes K.
      • De Sadeleer L.
      • Vos R.
      • Neyrinck A.
      • Benos P.V.
      • Bar-Joseph Z.
      • Tantin D.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      • Kaminski N.
      Transcriptional regulatory model of fibrosis progression in the human lung.
      Informed consent was obtained directly from the patient or from the next of kin of the donors who served as controls under conditions approved by the ethical (S52174) and biosafety (MS20101571) committees at the Katholieke Universiteit Leuven and accepted by all the other participating institutions.

      microCT-Based Morphometric Quantification

      The tissue samples were kept frozen while scanned at 9.98-μm voxel resolution with a SkyScan 1172 scanner (Kontich, Belgium).
      • Verleden S.E.
      • Tanabe N.
      • McDonough J.E.
      • Vasilescu D.M.
      • Xu F.
      • Wuyts W.A.
      • Piloni D.
      • De Sadeleer L.
      • Willems S.
      • Mai C.
      • Hostens J.
      • Cooper J.D.
      • Verbeken E.K.
      • Verschakelen J.
      • Galban C.J.
      • Van Raemdonck D.E.
      • Colby T.V.
      • Decramer M.
      • Verleden G.M.
      • Kaminski N.
      • Hackett T.L.
      • Vanaudenaerde B.M.
      • Hogg J.C.
      Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.
      As previously described,
      • McDonough J.E.
      • Kaminski N.
      • Thienpont B.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      Gene correlation network analysis to identify regulatory factors in idiopathic pulmonary fibrosis.
      • McDonough J.E.
      • Ahangari F.
      • Li Q.
      • Jain S.
      • Verleden S.E.
      • Herazo-Maya J.
      • Vukmirovic M.
      • DeIuliis G.
      • Tzouvelekis A.
      • Tanabe N.
      • Chu F.
      • Yan X.
      • Verschakelen J.
      • Homer R.J.
      • Manatakis D.V.
      • Zhang J.
      • Ding J.
      • Maes K.
      • De Sadeleer L.
      • Vos R.
      • Neyrinck A.
      • Benos P.V.
      • Bar-Joseph Z.
      • Tantin D.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      • Kaminski N.
      Transcriptional regulatory model of fibrosis progression in the human lung.
      image thresholding was applied to separate tissue and airspaces. The tissue segmentation was used to compute tissue percentage and alveolar surface density (defined as alveolar surface area per volume of lung). The airspace segmentation was used to compute the mean airspace size (Lm) by measuring and averaging interalveolar wall distances.
      • Verleden S.E.
      • Tanabe N.
      • McDonough J.E.
      • Vasilescu D.M.
      • Xu F.
      • Wuyts W.A.
      • Piloni D.
      • De Sadeleer L.
      • Willems S.
      • Mai C.
      • Hostens J.
      • Cooper J.D.
      • Verbeken E.K.
      • Verschakelen J.
      • Galban C.J.
      • Van Raemdonck D.E.
      • Colby T.V.
      • Decramer M.
      • Verleden G.M.
      • Kaminski N.
      • Hackett T.L.
      • Vanaudenaerde B.M.
      • Hogg J.C.
      Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.
      The terminal bronchioles were defined as the last generation of conducting bronchioles and counted manually in the microCT scans of each sample. The number of terminal bronchioles per milliliter of lung was calculated by dividing the number per sample by the sample volume.
      • McDonough J.E.
      • Yuan R.
      • Suzuki M.
      • Seyednejad N.
      • Elliott W.M.
      • Sanchez P.G.
      • Wright A.C.
      • Gefter W.B.
      • Litzky L.
      • Coxson H.O.
      • Pare P.D.
      • Sin D.D.
      • Pierce R.A.
      • Woods J.C.
      • McWilliams A.M.
      • Mayo J.R.
      • Lam S.C.
      • Cooper J.D.
      • Hogg J.C.
      Small-airway obstruction and emphysema in chronic obstructive pulmonary disease.
      ,
      • Vasilescu D.M.
      • Phillion A.B.
      • Tanabe N.
      • Kinose D.
      • Paige D.F.
      • Kantrowitz J.J.
      • Liu G.
      • Liu H.
      • Fishbane N.
      • Verleden S.E.
      • Vanaudenaerde B.M.
      • Lenburg M.E.
      • Stevenson C.S.
      • Spira A.
      • Cooper J.D.
      • Hackett T.L.
      • Hogg J.C.
      Non-destructive cryo micro CT imaging enables structural and molecular analysis of human lung tissue.

      Histologic Analysis

      The pathologist's scoring was performed in the present study using histologic sections obtained in the previous IPF study.
      • Verleden S.E.
      • Tanabe N.
      • McDonough J.E.
      • Vasilescu D.M.
      • Xu F.
      • Wuyts W.A.
      • Piloni D.
      • De Sadeleer L.
      • Willems S.
      • Mai C.
      • Hostens J.
      • Cooper J.D.
      • Verbeken E.K.
      • Verschakelen J.
      • Galban C.J.
      • Van Raemdonck D.E.
      • Colby T.V.
      • Decramer M.
      • Verleden G.M.
      • Kaminski N.
      • Hackett T.L.
      • Vanaudenaerde B.M.
      • Hogg J.C.
      Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.
      After microCT, portions of the frozen samples were fixed in alcohol-based formalin at −20°C overnight, warmed to room temperature, and then processed into paraffin blocks from which histologic sections were cut and stained with hematoxylin and eosin and Movat Pentachrome stains. These histologic sections were examined by an experienced pulmonary pathologist (T.V.C.), who scored eight pathological features that consisted of the three major UIP criteria, including patchy fibrosis, honeycomb cyst formation, and fibroblastic foci, as well as five additional pathologic changes, including emphysema, degree of inflammation, hyaline membrane formation, lymphoid follicles, and respiratory bronchiolitis, all on a 0 to 3+ scale (Figure 1). In addition, other portions of the frozen samples were briefly warmed to −1°C, vacuum embedded in the OCT compound (Sakura Finetek, Torrance, CA), immediately returned to −80°C, and cut into serial frozen sections (8-μm thick) for immunohistochemistry. These sections were stained with primary antibodies for CD68 (M0876; 1:200 dilution; Dako Cytomation, Carpinteria, CA), CD4 (M7310; 1:200 dilution; Dako Cytomation), CD8 (M7103; 1:400 dilution; Dako Cytomation), and CD79a (M7050; 1:200 dilution; Dako Cytomation) as previously reported.
      • Verleden S.E.
      • Tanabe N.
      • McDonough J.E.
      • Vasilescu D.M.
      • Xu F.
      • Wuyts W.A.
      • Piloni D.
      • De Sadeleer L.
      • Willems S.
      • Mai C.
      • Hostens J.
      • Cooper J.D.
      • Verbeken E.K.
      • Verschakelen J.
      • Galban C.J.
      • Van Raemdonck D.E.
      • Colby T.V.
      • Decramer M.
      • Verleden G.M.
      • Kaminski N.
      • Hackett T.L.
      • Vanaudenaerde B.M.
      • Hogg J.C.
      Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.
      These sections were also scored by the same pathologist on a 0 to 3+ scale.
      Figure thumbnail gr1
      Figure 1Examples of pathological scores on idiopathic pulmonary fibrosis (IPF) tissue section (hematoxylin and eosin staining). A: Mild patchy fibrosis (score = 1) without honeycomb cysts formation (score = 0) or emphysema (score = 0). B: Severe patchy fibrosis (score = 3) without honeycomb cysts formation (score = 0) or emphysema (score = 0). C: Mild patchy fibrosis (score = 1) and emphysema (score = 1) without honeycomb cysts formation (score = 0). D: Severe patchy fibrosis (score = 3) and honeycomb (score = 1) without emphysema (score = 0). Scale bars = 2 mm.

      Statistical Analysis

      Data are expressed as means ± SD. Statistical analysis was performed with the R statistical program version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org). The Spearman correlation test and U-test were used for correlation tests and group comparisons, respectively. Multiple comparisons were performed with Wilcoxon tests with Holm correction.

      Results

      There is no significant difference in age, sex, height, or weight between the patients with IPF and the controls (Table 1). Table 2 summarizes microCT and histologic scores. Alveolar surface density and the number of terminal bronchioles per milliliter of lung were lower, whereas tissue percentage and Lm were higher in patients with IPF compared with controls. In addition, CD68-, CD4-, CD8-, and CD79a-positive cells were greater in patients with IPF than controls. The significant difference between the patients with IPF and controls was also present when comparing microCT indexes in the upper, middle, and lower regions separately (Supplemental Figure S1).
      Table 1Demographic Characteristics of Study Participants
      CharacteristicControlIPF
      Age, y58 ± 1057 ± 5
      Height, cm175 ± 6173 ± 7
      Weight, kg80 ± 1573 ± 10
      Sex, n M:F6:010:0
      Smoking history, n former:never2:410:0
      FEV1, % predictedNA61 ± 15
      FVC, % predictedNA59 ± 20
      DLCO, % predictedNA28 ± 8
      Data are expressed as means ± SD unless otherwise indicated. n = 6 control; n = 10 IPF.
      DLCO, diffusing capacity for carbon monoxide; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; IPF, idiopathic pulmonary fibrosis; NA, not applicable.
      Table2Comparisons of microCT and Histologic Findings between the Control and IPF Groups
      VariableControlIPF
      Tissue cores, n3660
      microCT
       Alveolar surface density, mm2/mm315.5 ± 2.08.9 ± 3.6∗
       Tissue, %28.4 ± 3.750.5 ± 14.2∗
       Lm, μm360 ± 53529 ± 298∗
       Terminal bronchioles in dung, n/mL4.1 ± 1.61.8 ± 1.3∗
      Histologic scoring
       Patchy fibrosisNA1.3 ± 1.0
       Fibroblastic fociNA0.8 ± 0.8
       HoneycombNA0.2 ± 0.5
       CD680.7 ± 0.51.6 ± 0.6∗
       CD40.4 ± 0.61.6 ± 0.7∗
       CD81.1 ± 0.41.6 ± 0.6∗
       CD79a0.1 ± 0.31.2 ± 0.8∗
       Lymphoid follicle0 ± 00.8 ± 0.8∗
      Data are expressed as means ± SD unless otherwise indicated. All scores range from 0 to 3.
      P < 0.005.
      Lm, the mean linear intercept; IPF, idiopathic pulmonary fibrosis; microCT, microcomputed tomography; NA, not available; tissue, %, total lung volume taken up by tissue.
      Figure 2, A and B gives examples of histologic regions with and without honeycomb formation (score 0 and 1, respectively) that were registered to the microCTs. The alveolar surface density and number of terminal bronchioles were lower in the honeycomb regions (score ≥ 1) than in the nonhoneycomb regions (score = 0) (Figure 2, C and D). This finding was visualized (Figure 2E) and a video that shows a microCT stack of the same sample (Figure 2, B and E) (Supplemental Video S1). The video shows that many branches of the small airway tree (pink) were located in the normal-appearing regions but not in the honeycomb region and that the conducting airway leading into the honeycomb region was directly connected to those severely distorted airspaces (orange). Furthermore, the decreases in the alveolar surface density and number of terminal bronchioles in the honeycomb region were also confirmed in a subanalysis that included nonemphysematous IPF samples (histologic emphysema score = 0) and controls (Figure 3).
      Figure thumbnail gr2
      Figure 2Comparisons of microcomputed tomographic (microCT) measures between regions with and without honeycomb cysts formation in idiopathic pulmonary fibrosis (IPF) samples (hematoxylin and eosin staining). A: Patchy fibrosis (score = 2) without honeycomb cysts formation (score = 0). B: Patchy fibrosis (score = 2) with honeycomb cysts formation (score = 1). Arrow indicates honeycomb region. The histologic sections were matched with microCT images. C and D: The alveolar surface density and number of terminal bronchioles per milliliter of lung volume on microCT were decreased in honeycomb regions compared with nonhoneycomb regions. E: Three-dimensional rendering of the small airway tree (pink) overlaid onto microCT images from the same stack as used in B (). The small airway was connected with airspace in the honeycomb region (orange). n = 12 honeycomb regions; n = 47 nonhoneycomb regions. ∗P < 0.05 versus nonhoneycomb regions. Original magnification, ×1 (A, B, and E).
      Figure thumbnail gr3
      Figure 3Comparisons of alveolar surface density and number of terminal bronchioles between nonemphysematous regions with and without honeycomb cysts formation. The alveolar surface density (A) and number of terminal bronchioles (B) were compared between control, nonemphysematous idiopathic pulmonary fibrosis (IPF) samples with and without honeycomb regions. The absence of emphysema was determined based on the histologic emphysema score of 0. n = 36 control; n = 10 nonemphysematous idiopathic pulmonary fibrosis (IPF) samples with honeycomb regions; n = 23 nonemphysematous idiopathic pulmonary fibrosis (IPF) samples without honeycomb regions. ∗P < 0.05 versus controls; P < 0.05 versus nonemphysematous IPF samples without honeycomb regions.
      Table 3 shows Spearman correlation coefficients between microCT indexes and pathologic scores in IPF samples (n = 59). Decreased alveolar surface density and increased tissue percentage and Lm on microCT were correlated with the histologic scores of patchy fibrosis, fibroblastic foci, and honeycomb. In contrast, a decreased number of the terminal bronchioles was correlated with an increased score of honeycomb but not with patchy fibrosis and fibroblastic foci.
      Table 3Spearman Correlation Coefficients between Microcomputed Tomography Indexes and Pathological Scores in Idiopathic Pulmonary Fibrosis Samples
      VariableAlveolar surface density, mm2/mm3Tissue, %LmTerminal bronchioles, n/mL
      Major criteria for UIP
       Patchy fibrosis−0.66∗0.69∗0.41∗−0.02
       Fibroblastic foci−0.52∗0.58∗0.39∗0.05
       Honeycomb−0.53∗0.38∗0.40∗−0.34∗
      Other scores
       Emphysema0.01−0.56∗0.19−0.09
       Inflammation−0.250.31∗∗−0.030.09
       Hyaline membrane0.010.06−0.130.00
       Respiratory bronchiolitis0.18−0.17−0.140.00
      P < 0.05, ∗∗P < 0.005.
      Lm, mean linear intercept; tissue, %, total lung volume taken up by tissue; UIP, usual interstitial pneumonia.
      Table 4 shows Spearman correlation coefficients between microCT indexes and scores of lymphoid cells in IPF samples (n = 60). Increased tissue percentage was correlated with increased scores for CD68-, CD4-, CD8-, and CD79a-positive cells and lymphoid follicles, whereas Lm and the number of terminal bronchioles were not associated with any of the scores of immune cells and lymphoid follicles.
      Table 4Spearman Correlation Coefficients between Microcomputed Tomography Indexes and Scores of Infiltrated Inflammatory Immune Cells in Idiopathic Pulmonary Fibrosis Samples
      VariableAlveolar surface density, mm2/mm3Tissue, %LmTerminal bronchioles, n/mL
      CD68−0.200.49∗0.020.20
      CD4−0.110.30∗∗0.090.19
      CD8−0.170.41∗0.050.09
      CD79a−0.44∗0.53∗0.190.03
      Lymphoid follicle−0.41∗0.44∗0.14−0.06
      P < 0.05, ∗∗<P < 0.005.
      Lm, mean linear intercept; tissue, %, total lung volume taken up by tissue.

      Discussion

      This study compared standard histopathologic criteria of UIP and IPF and quantitative morphologic measures obtained from microCT. The microCT findings of decreased alveolar surface density and increased tissue percentage were positively associated with the pathologist's scoring of patchy fibrosis, fibroblastic foci, honeycomb formation, infiltration of CD79a-positive lymphocytes, and lymphoid follicle formation. Furthermore, a combination of histologic assessment and the 3D microCT information revealed that a reduction in the number of terminal bronchioles was associated with honeycomb formation but not with patchy fibrosis or fibroblastic foci. These findings indicate that 3D morphometric assessment via microCT can be used to complement the pathologist's visual inspection by showing the pathological relationship between the peripheral airways and parenchyma in lungs with IPF.
      From a histopathologic perspective, lungs with UIP and IPF characterized by spatially heterogeneous fibrosis with fibroblastic foci and honeycomb lesion,
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      and are in line with the present microCT findings that indicate a direct communication between the small airway tree and honeycomb regions in lungs with IPF.
      Together with previous findings that the polymorphism in the promoter region of the MUC5B gene, which regulates mucin production from bronchiolar epithelium, is associated with the pathogenesis of IPF
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      and the honeycomb regions are lined with bronchiolar-like epithelium,
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      The idiopathic pulmonary fibrosis honeycomb cyst contains a mucocilary pseudostratified epithelium.
      we speculate that the terminal bronchiole remodeling might be involved in the honeycomb formation.
      This study used a single histologic section for each tissue sample. Although microscopic pathologic findings could vary throughout the tissue sample, it is speculated that within-sample variation is smaller than the intersample variation because the cylindrical tissue samples used in this study are relatively small (20-mm high and 14-mm in diameter). Moreover, the close correlation between the pathologist's score of patchy fibrosis and the tissue percentage on microCT that was obtained from the entire microCT stack suggests that the single histologic section is sufficiently representative of the pathologic findings of the sample core.
      Extensive research indicates that a transition of fibroblasts into synthetic myofibroblast and subsequent deposition of collagen play an important role in the progressive fibrotic process after repeated injuries in IPF
      • Scotton C.J.
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      Molecular targets in pulmonary fibrosis: the myofibroblast in focus.
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      • Anstrom K.J.
      • et al.
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      ; however, the role of inflammatory immune cells is not established. Histologic studies have found infiltration of inflammatory immune cells, such as B-cell aggregates,
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      • Donahoe M.
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      • Duncan S.R.
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      • Janossy G.
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      Immunohistological analysis of lung tissue from patients with cryptogenic fibrosing alveolitis suggesting local expression of immune hypersensitivity.
      whereas clinical trials using immunosuppressive therapy have consistently failed to find the effectiveness in patients with IPF.
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      A recent study by Verleden et al
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      • Xu F.
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      • Willems S.
      • Mai C.
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      found that the CD79a-positive cell infiltration and lymphoid follicle formation are present even in minimal fibrotic regions of lungs with IPF. The present finding extends this observation by finding an association of lymphoid follicle formation score with increased tissue percentage and decreased alveolar surface density and further supports the notion that the persistent adaptive immune response contributes to a fibrotic remodeling process in IPF.
      Moreover, an increase in CD68-positive cells was associated with the increased tissue percentage in lungs with IPF. This finding is consistent with the hypothesis that macrophages are mainly involved in the pathogenesis of IPF
      • Zhang L.
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      • Maher T.M.
      • Lloyd C.M.
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      The transferrin receptor CD71 delineates functionally distinct airway macrophage subsets during idiopathic pulmonary fibrosis.
      but could also in part reflect the smoking history in all patients. Macrophages are subcategorized into functional phenotypes, such as M1 and M2, and play various roles in the lung, including host defense to external insults and wound healing after injury.
      • Song E.
      • Ouyang N.
      • Horbelt M.
      • Antus B.
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      Influence of alternatively and classically activated macrophages on fibrogenic activities of human fibroblasts.
      Therefore, in addition to staining with CD68 antibody, different approach, such as gene expression profiling and flowcytometry, should be integrated in a future study to explore the pathogenic roles of each macrophage phenotype in IPF.
      There are limitations to the present study worth noting. First, all cases with IPF were from former smokers. Because smoking is a major cause of emphysema that is closely associated with the loss of the terminal bronchioles,
      • McDonough J.E.
      • Yuan R.
      • Suzuki M.
      • Seyednejad N.
      • Elliott W.M.
      • Sanchez P.G.
      • Wright A.C.
      • Gefter W.B.
      • Litzky L.
      • Coxson H.O.
      • Pare P.D.
      • Sin D.D.
      • Pierce R.A.
      • Woods J.C.
      • McWilliams A.M.
      • Mayo J.R.
      • Lam S.C.
      • Cooper J.D.
      • Hogg J.C.
      Small-airway obstruction and emphysema in chronic obstructive pulmonary disease.
      ,
      • Koo H.K.
      • Vasilescu D.M.
      • Booth S.
      • Hsieh A.
      • Katsamenis O.L.
      • Fishbane N.
      • Elliott W.M.
      • Kirby M.
      • Lackie P.
      • Sinclair I.
      • Warner J.A.
      • Cooper J.D.
      • Coxson H.O.
      • Pare P.D.
      • Hogg J.C.
      • Hackett T.L.
      Small airways disease in mild and moderate chronic obstructive pulmonary disease: a cross-sectional study.
      ,
      • Tanabe N.
      • Vasilescu D.M.
      • Kirby M.
      • Coxson H.O.
      • Verleden S.E.
      • Vanaudenaerde B.M.
      • Kinose D.
      • Nakano Y.
      • Pare P.D.
      • Hogg J.C.
      Analysis of airway pathology in COPD using a combination of computed tomography, micro-computed tomography and histology.
      the present finding of a reduced number of terminal bronchioles in IPF might have been affected by smoking. However, no correlation was found between the pathologist's score for emphysema and the number of terminal bronchioles, suggesting that the influence of smoking-related emphysematous destruction is minimal in this study. Second, the sample number is small, and all cases of IPF were very severe and required lung transplantation. To broaden insights into disease phenotypes, it would be beneficial if the design of future studies would include lung specimen from indiiduals with different stages of IPF as well as different smoking status (ie, both smokers and nonsmokers). Third, the static cross-sectional nature of the study limits causal inferences. Therefore, the present study was not able to test whether patchy fibrosis and honeycomb cyst formation induce infiltration of immune cells in lungs with IPF or if specific immune cells induce a fibrotic process in lungs with IPF.
      In conclusion, this is the first study, to our knowledge, to find that quantitative morphometric microCT measurements of alveolar surface density, tissue percentage, and Lm are closely associated with the general histopathologic scoring of patchy fibrosis, fibroblastic foci, and honeycomb lesions in IPF. These data suggest that microCT measurements provide a reliable structural assessment of lungs with IPF, especially since because established major criteria of UIP and IPF are associated with a reduced alveolar surface area, which potentially impairs diffusion capacity in patients with IPF. Furthermore, the 3D microCT evaluation revealed that honeycomb formation, but not patchy fibrosis, is associated with a greater reduction in the number of terminal bronchioles in IPF. Volumetric microCT-based quantification of the lung structure complements histologic assessment of cellular composition of lungs with IPF, and by combining these methods with subsequent gene expression analysis or single-cell sequencing, it may be possible to identify a novel therapeutic target for this devastating lung disease.

      Acknowledgments

      We thank Fanny Chu and Jingwen Pan (Center for Heart Lung Innovation, University of British Columbia) for their assistance in histologic preparation and staining.

      Supplemental Data

      • Supplemental Figure S1

        Comparisons of microcomputed tomography indexes in the upper, middle, and lower regions between lungs with idiopathic pulmonary fibrosis (IPF) and control lungs. Alveolar surface density (A), tissue percentage (B), mean linear intercept (Lm) (C), number of the terminal bronchioles per milliliter of lung in the upper, middle, and lower regions (D) were separately compared between control lungs and lungs with IPF. ∗P < 0.05 versus control.

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