Immunofluorescence-guided segmentation of three-dimensional features in micro-computed tomography datasets of human lung tissue.

Micro-computed tomography (µCT) provides non-destructive three-dimensional (3D) imaging of soft tissue microstructures. Specific features in µCT images can be identified using correlated two-dimensional (2D) histology images allowing manual segmentation. However, this is very time-consuming and requires specialist knowledge of the tissue and imaging modalities involved. Using a custom-designed µCT system optimized for imaging unstained formalin-fixed paraffin-embedded soft tissues, we imaged human lung tissue at isotropic voxel sizes less than 10 µm. Tissue sections were stained with haematoxylin and eosin or cytokeratin 18 in columnar airway epithelial cells using immunofluorescence (IF), as an exemplar of this workflow. Novel utilization of tissue autofluorescence allowed automatic alignment of 2D microscopy images to the 3D µCT data using scripted co-registration and automated image warping algorithms. Warped IF images, which were accurately aligned with the µCT datasets, allowed 3D segmentation of immunoreactive tissue microstructures in the human lung. Blood vessels were segmented semi-automatically using the co-registered µCT datasets. Correlating 2D IF and 3D µCT data enables accurate identification, localization and segmentation of features in fixed soft lung tissue. Our novel correlative imaging workflow provides faster and more automated 3D segmentation of µCT datasets. This is applicable to the huge range of formalin-fixed paraffin-embedded tissues held in biobanks and archives.

X-ray Micro-Computed Tomography for Nondestructive Three-Dimensional (3D) X-ray Histology.

Historically, micro-computed tomography (µCT) has been considered unsuitable for histologic analysis of unstained formalin-fixed, paraffin-embedded soft tissue biopsy specimens because of a lack of image contrast between the tissue and the paraffin. However, we recently demonstrated that µCT can successfully resolve microstructural detail in routinely prepared tissue specimens. Herein, we illustrate how µCT imaging of standard formalin-fixed, paraffin-embedded biopsy specimens can be seamlessly integrated into conventional histology workflows, enabling nondestructive three-dimensional (3D) X-ray histology, the use and benefits of which we showcase for the exemplar of human lung biopsy specimens. This technology advancement was achieved through manufacturing a first-of-kind µCT scanner for X-ray histology and developing optimized imaging protocols, which do not require any additional sample preparation. 3D X-ray histology allows for nondestructive 3D imaging of tissue microstructure, resolving structural connectivity and heterogeneity of complex tissue networks, such as the vascular network or the respiratory tract. We also demonstrate that 3D X-ray histology can yield consistent and reproducible image quality, enabling quantitative assessment of a tissue's 3D microstructures, which is inaccessible to conventional two-dimensional histology. Being nondestructive, the technique does not interfere with histology workflows, permitting subsequent tissue characterization by means of conventional light microscopy-based histology, immunohistochemistry, and immunofluorescence. 3D X-ray histology can be readily applied to a plethora of archival materials, yielding unprecedented opportunities in diagnosis and research of disease.

Correlative 3D Imaging and Microfluidic Modelling of Human Pulmonary Lymphatics using Immunohistochemistry and High-resolution µCT.

Lung lymphatics maintain fluid homoeostasis by providing a drainage system that returns fluid, cells and metabolites to the circulatory system. The 3D structure of the human pulmonary lymphatic network is essential to lung function, but it is poorly characterised. Image-based 3D mathematical modelling of pulmonary lymphatic microfluidics has been limited by the lack of accurate and representative image geometries. This is due to the microstructural similarity of the lymphatics to the blood vessel network, the lack of lymphatic-specific biomarkers, the technical limitations associated with image resolution in 3D, and sectioning artefacts present in 2D techniques. We present a method that combines lymphatic specific (D240 antibody) immunohistochemistry (IHC), optimised high-resolution X-ray microfocus computed tomography (µCT) and finite-element mathematical modelling to assess the function of human peripheral lung tissue. The initial results identify lymphatic heterogeneity within and between lung tissue. Lymphatic vessel volume fraction and fractal dimension significantly decreases away from the lung pleural surface (p < 0.001, n = 25 and p < 0.01, n = 20, respectively). Microfluidic modelling successfully shows that in lung tissue the fluid derived from the blood vessels drains through the interstitium into the lymphatic vessel network and this drainage is different in the subpleural space compared to the intralobular space. When comparing lung tissue from health and disease, human pulmonary lymphatics were significantly different across five morphometric measures used in this study (p ≤ 0.0001). This proof of principle study establishes a new engineering technology and workflow for further studies of pulmonary lymphatics and demonstrates for the first time the combination of correlative µCT and IHC to enable 3D mathematical modelling of human lung microfluidics at micrometre resolution.

Small airways disease in mild and moderate chronic obstructive pulmonary disease: a cross-sectional study.

BACKGROUND: The concept that small conducting airways less than 2 mm in diameter become the major site of airflow obstruction in chronic obstructive pulmonary disease (COPD) is well established in the scientific literature, and the last generation of small conducting airways, terminal bronchioles, are known to be destroyed in patients with very severe COPD. We aimed to determine whether destruction of the terminal and transitional bronchioles (the first generation of respiratory airways) occurs before, or in parallel with, emphysematous tissue destruction. METHODS: In this cross-sectional analysis, we applied a novel multiresolution CT imaging protocol to tissue samples obtained using a systematic uniform sampling method to obtain representative unbiased samples of the whole lung or lobe of smokers with normal lung function (controls) and patients with mild COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1), moderate COPD (GOLD 2), or very severe COPD (GOLD 4). Patients with GOLD 1 or GOLD 2 COPD and smokers with normal lung function had undergone lobectomy and pneumonectomy, and patients with GOLD 4 COPD had undergone lung transplantation. Lung tissue samples were used for stereological assessment of the number and morphology of terminal and transitional bronchioles, airspace size (mean linear intercept), and alveolar surface area. FINDINGS: Of the 34 patients included in this study, ten were controls (smokers with normal lung function), ten patients had GOLD 1 COPD, eight had GOLD 2 COPD, and six had GOLD 4 COPD with centrilobular emphysema. The 34 lung specimens provided 262 lung samples. Compared with control smokers, the number of terminal bronchioles decreased by 40% in patients with GOLD 1 COPD (p=0·014) and 43% in patients with GOLD 2 COPD (p=0·036), the number of transitional bronchioles decreased by 56% in patients with GOLD 1 COPD (p=0·0001) and 59% in patients with GOLD 2 COPD (p=0·0001), and alveolar surface area decreased by 33% in patients with GOLD 1 COPD (p=0·019) and 45% in patients with GOLD 2 COPD (p=0·0021). These pathological changes were found to correlate with lung function decline. We also showed significant loss of terminal and transitional bronchioles in lung samples from patients with GOLD 1 or GOLD 2 COPD that had a normal alveolar surface area. Remaining small airways were found to have thickened walls and narrowed lumens, which become more obstructed with increasing COPD GOLD stage. INTERPRETATION: These data show that small airways disease is a pathological feature in mild and moderate COPD. Importantly, this study emphasises that early intervention for disease modification might be required by patients with mild or moderate COPD. FUNDING: Canadian Institutes of Health Research.

Resolvin E1, resolvin D1 and resolvin D2 inhibit constriction of rat thoracic aorta and human pulmonary artery induced by the thromboxane mimetic U46619.

BACKGROUND AND PURPOSE: The ω-6 fatty acid-derived lipid mediators such as prostanoids, thromboxane and leukotrienes have well-established roles in regulating both inflammation and smooth muscle contractility. Resolvins are derived from ω-3 fatty acids and have important roles in promoting the resolution of inflammation, but their activity on smooth muscle contractility is unknown. We investigated whether resolvin E1 (RvE1), resolvin D1 (RvD1) and resolvin D2 (RvD2) can modulate contractions of isolated segments of rat thoracic aorta (RTA) or human pulmonary artery (HPA) induced by the α1 -adrenoceptor agonist phenylephrine or the stable thromboxane A2 mimetic U46619. EXPERIMENTAL APPROACH: Contractile responses in RTA and HPA were measured using wire myography. Receptor expression was investigated by immunohistochemistry. KEY RESULTS: Constriction of RTA segments by U46619, but not by phenylephrine, was significantly inhibited by pretreatment for 1 or 24 h with 10-100 nM RvE1, RvD1 or RvD2. The inhibitory effect of RvE1 was partially blocked by a chemerin receptor antagonist (CCX832). RvE1 at only 1-10 nM also significantly inhibited U46619-induced constriction of HPA segments, and the chemerin receptor, GPR32 and FPR2/ALX were identified in HPA smooth muscle. CONCLUSION AND IMPLICATIONS: These data suggest that resolvins or their mimetics may prove useful novel therapeutics in diseases such as pulmonary arterial hypertension, which are characterized by increased thromboxane contractile activity.

Manning the Barricades: Lung Fibroblasts and CD4+ T Cells as the Last Line of Defense against Bacterial Invasion?

Fibroblasts are a major structural cell in the human lung, being responsible for the production of extracellular matrix components that provide the intricate structure necessary for correct lung function. Generally located in the submucosa, fibroblasts do not usually directly interact with the commensal microbes that we now know are resident in the airways. However, during situations in which alveolar macrophages and epithelial cells are impaired, for example, during severe viral infections leading to pneumonia, bacteria can invade the lung mesenchyme. In these circumstances, fibroblasts may represent another immunological barrier to bacterial invasion, not just as innate immune effectors, but also by interacting with migrating and tissue-resident adaptive immune cell populations, such as CD4+ T cells. The cytokines produced by CD4+ T helper cells are integral in directing appropriate innate and adaptive immune responses against bacteria, but the nature of fibroblast-CD4+ cell interaction, unlike the CD8+ T-cell interaction, is not clearly established. Here, we review the responses of lung fibroblasts to bacteria and discuss emerging data indicating a key role for these cells in directly presenting bacterial antigens to CD4+ T cells.

The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.

Human Lung Fibroblasts Present Bacterial Antigens to Autologous Lung Th Cells.

Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4+ Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4+ T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4+ T cells isolated from the lung were predominantly (mean 97.5%) CD45RO+ memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4+ T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4+ T cells.

Three-dimensional characterization of fibroblast foci in idiopathic pulmonary fibrosis.

In idiopathic pulmonary fibrosis (IPF), the fibroblast focus is a key histological feature representing active fibroproliferation. On standard 2D pathologic examination, fibroblast foci are considered small, distinct lesions, although they have been proposed to form a highly interconnected reticulum as the leading edge of a "wave" of fibrosis. Here, we characterized fibroblast focus morphology and interrelationships in 3D using an integrated micro-CT and histological methodology. In 3D, fibroblast foci were morphologically complex structures, with large variations in shape and volume (range, 1.3 × 104 to 9.9 × 107 µm3). Within each tissue sample numerous multiform foci were present, ranging from a minimum of 0.9 per mm3 of lung tissue to a maximum of 11.1 per mm3 of lung tissue. Each focus was an independent structure, and no interconnections were observed. Together, our data indicate that in 3D fibroblast foci form a constellation of heterogeneous structures with large variations in shape and volume, suggesting previously unrecognized plasticity. No evidence of interconnectivity was identified, consistent with the concept that foci represent discrete sites of lung injury and repair.

Three dimensional imaging of paraffin embedded human lung tissue samples by micro-computed tomography.

BACKGROUND: Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. METHODS: FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. RESULTS: The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15 mm x 7 mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. CONCLUSION: We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.

Overcoming barriers to influenza vaccination.

Healthcare professionals face an ongoing challenge in overcoming barriers to influenza vaccination. This article explores the external and internal influences related to the uptake of flu vaccination with regard to both healthcare workers and their patients. The article discusses the ways in which nurses can help patients eligible for free flu vaccinations to make informed choices.

Interleukin-13, but not indomethacin, increases cysteinyl-leukotriene synthesis in human lung macrophages.

Aspirin-exacerbated respiratory disease (AERD) is associated with constitutively elevated synthesis of bronchoconstrictor cysteinyl-leukotrienes, associated with increased expression of leukotriene (LT)C(4) synthase and Th2 cytokines and airway eosinophilia. We examined whether interleukin-13 can increase LTC(4) synthase gene transcription and cysteinyl-leukotriene synthesis in macrophages isolated from resected human lung tissue and whether an NSAID (indomethacin) can trigger further cysteinyl-leukotriene synthesis in these cells. Overnight culture of human lung macrophages with IL-13 (10 ng/mL) increased spontaneous and ionophore-stimulated production of cysteinyl-leukotrienes by 42% (P = 0.02) and 52% (P = 0.005), respectively, as quantified by enzyme immunoassays, but PCR gene transcription assays did not demonstrate an effect on LTC4S mRNA. The addition of indomethacin (100 µM) did not modulate cysteinyl-leukotriene production in either IL-13-treated or untreated macrophages. We conclude that while IL-13 enhances cysteinyl-leukotriene synthesis in human lung macrophages, it does not replicate the enhanced LTC(4) synthase expression observed in the AERD lung nor confer sensitivity to NSAIDs.