Thomas M. Conlon

Cigarette smoke-induced iBALT mediates macrophage activation in a B cell-dependent manner in COPD

Chronic obstructive pulmonary disease (COPD) is characterized by a progressive decline in lung function, caused by exposure to exogenous particles, mainly cigarette smoke (CS). COPD is initiated and perpetuated by an abnormal CS-induced inflammatory response of the lungs, involving both innate and adaptive immunity. Specifically, B cells organized in iBALT structures and macrophages accumulate in the lungs and contribute to CS-induced emphysema, but the mechanisms thereof remain unclear. Here, we demonstrate that B cell-deficient mice are significantly protected against CS-induced emphysema. Chronic CS exposure led to an increased size and number of iBALT structures, and increased lung compliance and mean linear chord length in wild-type (WT) but not in B cell-deficient mice. The increased accumulation of lung resident macrophages around iBALT and in emphysematous alveolar areas in CS-exposed WT mice coincided with upregulated MMP12 expression. In vitro coculture experiments using B cells and macrophages demonstrated that B cell-derived IL-10 drives macrophage activation and MMP12 upregulation, which could be inhibited by an anti-IL-10 antibody. In summary, B cell function in iBALT formation seems necessary for macrophage activation and tissue destruction in CS-induced emphysema and possibly provides a new target for therapeutic intervention in COPD.

In Vivo Dark-Field Radiography for Early Diagnosis and Staging of Pulmonary Emphysema.

ObjectivesThe aim of this study was to evaluate the suitability of in vivo x-ray dark-field radiography for early-stage diagnosis of pulmonary emphysema in mice. Furthermore, we aimed to analyze how the dark-field signal correlates with morphological changes of lung architecture at distinct stages of emphysema. Materials and MethodsFemale 8- to 10-week-old C57Bl/6N mice were used throughout all experiments. Pulmonary emphysema was induced by orotracheal injection of porcine pancreatic elastase (80-U/kg body weight) (n = 30). Control mice (n = 11) received orotracheal injection of phosphate-buffered saline. To monitor the temporal patterns of emphysema development over time, the mice were imaged 7, 14, or 21 days after the application of elastase or phosphate-buffered saline. X-ray transmission and dark-field images were acquired with a prototype grating-based small-animal scanner. In vivo pulmonary function tests were performed before killing the animals. In addition, lungs were obtained for detailed histopathological analysis, including mean cord length (MCL) quantification as a parameter for the assessment of emphysema. Three blinded readers, all of them experienced radiologists and familiar with dark-field imaging, were asked to grade the severity of emphysema for both dark-field and transmission images. ResultsHistopathology and MCL quantification confirmed the introduction of different stages of emphysema, which could be clearly visualized and differentiated on the dark-field radiograms, whereas early stages were not detected on transmission images. The correlation between MCL and dark-field signal intensities (r = 0.85) was significantly higher than the correlation between MCL and transmission signal intensities (r = 0.37). The readers’ visual ratings for dark-field images correlated significantly better with MCL (r = 0.85) than visual ratings for transmission images (r = 0.36). Interreader agreement and the diagnostic accuracy of both quantitative and visual assessment were significantly higher for dark-field imaging than those for conventional transmission images. ConclusionsX-ray dark-field radiography can reliably visualize different stages of emphysema in vivo and demonstrates significantly higher diagnostic accuracy for early stages of emphysema than conventional attenuation-based radiography.

Improved In vivo Assessment of Pulmonary Fibrosis in Mice using X-Ray Dark-Field Radiography

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with a median life expectancy of 4–5 years after initial diagnosis. Early diagnosis and accurate monitoring of IPF are limited by a lack of sensitive imaging techniques that are able to visualize early fibrotic changes at the epithelial-mesenchymal interface. Here, we report a new x-ray imaging approach that directly visualizes the air-tissue interfaces in mice in vivo. This imaging method is based on the detection of small-angle x-ray scattering that occurs at the air-tissue interfaces in the lung. Small-angle scattering is detected with a Talbot-Lau interferometer, which provides the so-called x-ray dark-field signal. Using this imaging modality, we demonstrate-for the first time-the quantification of early pathogenic changes and their correlation with histological changes, as assessed by stereological morphometry. The presented radiography method is significantly more sensitive in detecting morphological changes compared with conventional x-ray imaging, and exhibits a significantly lower radiation dose than conventional x-ray CT. As a result of the improved imaging sensitivity, this new imaging modality could be used in future to reduce the number of animals required for pulmonary research studies.

Metabolomics screening identifies reduced L-carnitine to be associated with progressive emphysema.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, small airway remodelling and emphysema. Emphysema is the destruction of alveolar structures, leading to enlarged airspaces and reduced surface area impairing the ability for gaseous exchange. To further understand the pathological mechanisms underlying progressive emphysema, we used MS-based approaches to quantify the lung, bronchoalveolar lavage fluid (BALF) and serum metabolome during emphysema progression in the established murine porcine pancreatic elastase (PPE) model on days 28, 56 and 161, compared with PBS controls. Partial least squares (PLS) analysis revealed greater changes in the metabolome of lung followed by BALF rather than serum during emphysema progression. Furthermore, we demonstrate for the first time that emphysema progression is associated with a reduction in lung-specific L-carnitine, a metabolite critical for transporting long-chain fatty acids into the mitochondria for their subsequent β-oxidation. In vitro, stimulation of the alveolar epithelial type II (ATII)-like LA4 cell line with L-carnitine diminished apoptosis induced by both PPE and H2O2. Moreover, PPE-treated mice demonstrated impaired lung function compared with PBS-treated controls (lung compliance; 0.067±0.008 ml/cmH20 compared with 0.035±0.005 ml/cmH20, P<0.0001), which improved following supplementation with L-carnitine (0.051±0.006, P<0.01) and was associated with a reduction in apoptosis. In summary, our results provide a new insight into the role of L-carnitine and, importantly, suggest therapeutic avenues for COPD.

Inflammaging increases susceptibility to cigarette smoke-induced COPD

Chronic obstructive pulmonary disease (COPD) is related to an abnormal chronic inflammatory response of the lung to mainly cigarette smoke (CS) and the disease risk is increased in aged individuals. The source of this chronic inflammation is due to the repeated and progressive activation of immune cells. We hypothesize that in a chronic CS-induced mouse model, the predisposition to COPD pathogenesis in aged mice is characterized by an elevated immune response compared to young animals. We measured several characteristics of COPD in young and old mice (2 and 12 months of age) exposed to CS for 3 months. CS-exposed aged mice exhibited increased lung compliance (0.061 ± 0.008 vs. 0.055 ± 0.006 ml/cm H2O, p < 0.01), emphysema development (35.36 ± 0.71 vs. 25.31 ± 0.005 μm; p < 0.01) and airway remodeling (2.15 ± 0.37 vs. 1.09 ± 0.64 μm3/μm2; p < 0.01) compared to control animals, which was not seen in CS-exposed young mice. Quantification of lung tissue inflammation revealed a significantly greater volume of inducible bronchus-associated lymphoid tissue structures in aged mice after CS exposure (5.94 ± 2.89 vs. 2.37 ± 1.69 μm3/μm2; p < 0.01). Our results indicate that age-induced lung inflammation is further elevated after CS exposure in old mice, potentially via an age-induced change in immune cell susceptibility to CS thereby accelerating the pathophysiological hallmarks of COPD.

Impairment of Immunoproteasome Function by Cigarette Smoke and in Chronic Obstructive Pulmonary Disease

RATIONALE Patients with chronic obstructive pulmonary disease (COPD) and in particular smokers are more susceptible to respiratory infections contributing to acute exacerbations of disease. The immunoproteasome is a specialized type of proteasome destined to improve major histocompatibility complex (MHC) class I-mediated antigen presentation for the resolution of intracellular infections. OBJECTIVES To characterize immunoproteasome function in COPD and its regulation by cigarette smoke. METHODS Immunoproteasome expression and activity were determined in bronchoalveolar lavage (BAL) and lungs of human donors and patients with COPD or idiopathic pulmonary fibrosis (IPF), as well as in cigarette smoke-exposed mice. Smoke-mediated alterations of immunoproteasome activity and MHC I surface expression were analyzed in human blood-derived macrophages. Immunoproteasome-specific MHC I antigen presentation was evaluated in spleen and lung immune cells that had been smoke-exposed in vitro or in vivo. MEASUREMENTS AND MAIN RESULTS Immunoproteasome and MHC I mRNA expression was reduced in BAL cells of patients with COPD and in isolated alveolar macrophages of patients with COPD or IPF. Exposure of immune cells to cigarette smoke extract in vitro reduced immunoproteasome activity and impaired immunoproteasome-specific MHC I antigen presentation. In vivo, acute cigarette smoke exposure dynamically regulated immunoproteasome function and MHC I antigen presentation in mouse BAL cells. End-stage COPD lungs showed markedly impaired immunoproteasome activities. CONCLUSIONS We here show that the activity of the immunoproteasome is impaired by cigarette smoke resulting in reduced MHC I antigen presentation. Regulation of immunoproteasome function by cigarette smoke may thus alter adaptive immune responses and add to prolonged infections and exacerbations in COPD and IPF.

X-ray dark-field radiography facilitates the diagnosis of pulmonary fibrosis in a mouse model

The aim of this study was to evaluate whether diagnosing pulmonary fibrosis with projection radiography can be improved by using X-ray dark-field radiograms. Pulmonary X-ray transmission and dark-field images of C57Bl/6N mice, either treated with bleomycin to induce pulmonary fibrosis or PBS to serve as controls, were acquired with a prototype grating-based small-animal scanner. Two blinded readers, both experienced radiologists and familiar with dark-field imaging, had to assess dark-field and transmission images for the absence or presence of fibrosis. Furthermore readers were asked to grade their stage of diagnostic confidence. Histological evaluation of the lungs served as the standard of reference in this study. Both readers showed a notably higher diagnostic confidence when analyzing the dark-field radiographs (p < 0.001). Diagnostic accuracy improved significantly when evaluating the lungs in dark-field images alone (p = 0.02) or in combination with transmission images (p = 0.01) compared to sole analysis of absorption images. Interreader agreement improved from good when assessing only transmission images to excellent when analyzing dark-field images alone or in combination with transmission images. Adding dark-field images to conventional transmission images in a murine model of pulmonary fibrosis leads to an improved diagnosis of this disease on chest radiographs.

Cigarette smoke causes acute airway disease and exacerbates chronic obstructive lung disease in neonatal mice

Epidemiological evidence demonstrates a strong link between postnatal cigarette smoke (CS) exposure and increased respiratory morbidity in young children. However, how CS induces early onset airway disease in young children, and how it interacts with endogenous risk factors, remains poorly understood. We, therefore, exposed 10-day-old neonatal wild-type and β-epithelial sodium ion channel (β-ENaC)-transgenic mice with cystic fibrosis-like lung disease to CS for 4 days. Neonatal wild-type mice exposed to CS demonstrated increased numbers of macrophages and neutrophils in the bronchoalveolar lavage fluid (BALF), which was accompanied by increased levels of Mmp12 and Cxcl1 BALF from β-ENaC-transgenic mice contained greater numbers of macrophages, which did not increase following acute CS exposure; however, there was significant increase in airway neutrophilia compared with filtered air transgenic and CS-exposed wild-type controls. Interestingly, wild-type and β-ENaC-transgenic mice demonstrated epithelial airway and vascular remodeling following CS exposure. Morphometric analysis of lung sections revealed that CS exposure caused increased mucus accumulation in the airway lumen of neonatal β-ENaC-transgenic mice compared with wild-type controls, which was accompanied by an increase in the number of goblet cells and Muc5ac upregulation. We conclude that short-term CS exposure 1) induces acute airway disease with airway epithelial and vascular remodeling in neonatal wild-type mice; and 2) exacerbates airway inflammation, mucus hypersecretion, and mucus plugging in neonatal β-ENaC-transgenic mice with chronic lung disease. Our results in neonatal mice suggest that young children may be highly susceptible to develop airway disease in response to tobacco smoke exposure, and that adverse effects may be aggravated in children with underlying chronic lung diseases.

X-Ray Dark-field Imaging to Depict Acute Lung Inflammation in Mice

The aim of this study was to evaluate the feasibility of early stage imaging of acute lung inflammation in mice using grating-based X-ray dark-field imaging in vivo. Acute lung inflammation was induced in mice by orotracheal instillation of porcine pancreatic elastase. Control mice received orotracheal instillation of PBS. Mice were imaged immediately before and 1 day after the application of elastase or PBS to assess acute changes in pulmonary structure due to lung inflammation. Subsequently, 6 mice from each group were sacrificed and their lungs were lavaged and explanted for histological analysis. A further 7, 14 and 21 days later the remaining mice were imaged again. All images were acquired with a prototype grating-based small-animal scanner to generate dark-field and transmission radiographs. Lavage confirmed that mice in the experimental group had developed acute lung inflammation one day after administration of elastase. Acute lung inflammation was visible as a striking decrease in signal intensity of the pulmonary parenchyma on dark-field images at day 1. Quantitative analysis confirmed that dark-field signal intensity at day 1 was significantly lower than signal intensities measured at the remaining timepoints, confirming that acute lung inflammation can be depicted in vivo with dark-field radiography.

Cholesterol metabolism promotes B‐cell positioning during immune pathogenesis of chronic obstructive pulmonary disease

The development of chronic obstructive pulmonary disease (COPD) pathogenesis remains unclear, but emerging evidence supports a crucial role for inducible bronchus‐associated lymphoid tissue (iBALT) in disease progression. Mechanisms underlying iBALT generation, particularly during chronic CS exposure, remain to be defined. Oxysterol metabolism of cholesterol is crucial to immune cell localization in secondary lymphoid tissue. Here, we demonstrate that oxysterols also critically regulate iBALT generation and the immune pathogenesis of COPD. In both COPD patients and cigarette smoke (CS)‐exposed mice, we identified significantly upregulated CH25H and CYP7B1 expression in airway epithelial cells, regulating CS‐induced B‐cell migration and iBALT formation. Mice deficient in CH25H or the oxysterol receptor EBI2 exhibited decreased iBALT and subsequent CS‐induced emphysema. Further, inhibition of the oxysterol pathway using clotrimazole resolved iBALT formation and attenuated CS‐induced emphysema in vivo therapeutically. Collectively, our studies are the first to mechanistically interrogate oxysterol‐dependent iBALT formation in the pathogenesis of COPD, and identify a novel therapeutic target for the treatment of COPD and potentially other diseases driven by the generation of tertiary lymphoid organs.