Mechanisms of myeloid cell recruitment and biomarker potential in interstitial lung diseases

Interstitial lung diseases (ILDs) are fibrotic disorders with chronic inflammation and fibrinogenesis leading to lung scaring and lung function decline. Ultimately, progressive pulmonary fibrosis results in altered pulmonary physiology, abnormal gas exchange, and organ failure. ILDs include known causes and idiopathic causes, as it is the case of idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP). The most detrimental type of ILD is IPF in which anti-fibrotic drugs (nintedanib and pirfenidone) only decrease disease progression. For other ILD types, corticoid treatment helps to decrease exacerbation. Currently, clinical trials are evaluating the applicability of anti-fibrotic drugs for treating non-IPF ILDs. Therefore, mechanistic insights and in-depth cell characterization during tissue injury and remodeling in ILD are of great interest in the respiratory medical field. Circulating immune cell populations have been suggested to play a critical role in ILDs. For instance, mononuclear phagocytes are involved in the initiation, repair and regeneration of pulmonary fibrosis. Moreover, the close interaction between circulating and lung tissue-resident immune cells is critical to contribute to tissue homeostasis or lead to disease. However, precise myeloid phenotypes (e.g. myeloid-derived suppressor cells and monocytes) and their mechanisms of recruitment in ILDs have not yet been explored. In the first results chapter of this thesis, myeloid-derived suppressor cells (MDSC) abundance and function were investigated for the first time in IPF patients. For that, peripheral blood of 170 patients including IPF, non-IPF ILD, chronic obstructive pulmonary diseases (COPD) and controls were collected to characterize and quantify MDSC by flow cytometry. Circulating MDSC in IPF and non-IPF ILD were increased when compared with control. Moreover, cross sectional and longitudinal analysis of the abundance of MDSC inversely correlated with pulmonary function test in IPF only. IPF patients with high number of MDSC showed downregulation of co-stimulatory T cells signals quantified by qRT-PCR. Furthermore, MDSC were able to suppress lymphocytes CD4+ and CD8+ cells proliferation in vitro. Last, CD33 CD11b double positive cells, suggestive of MDSC, were found in neighboring fibrotic niches of the IPF lungs. Taking together, these results show that MDSC are potential biomarker for IPF and are suppressing T cell responses. In the second results chapter, we aimed at analyzing monocyte phenotype and recruitment from the blood to the lung tissue in ILD. Importantly, CX3CR1 expression on immune cells has been demonstrated to increase fibrosis features. For that, flow cytometry analysis of circulating monocytes was performed in 105 subjects (83 ILD, and 22 controls). Monocyte localization and abundance in the lung was assessed by immunofluorescence and flow cytometry analysis. For receptor-ligand function and transmigration pattern, monocytes were isolated from blood and cultured either alone or with endothelial cells. Here, we showed that classical monocytes (CM) were increased, while non-classical monocytes (NCM) were decreased in ILD: NSIP, hypersensitivity pneumonitis (HP) and connective tissue disease associated with ILD (CTD-ILD) compared with controls. Monocytes abundance positively correlated with lung function. Fractalkine levels, the ligand of CX3CR1, were higher in lung tissue than in plasma in ILD and also co-localized with bronchial ciliated cells. Fractalkine enhanced endothelial transmigration of NCM in ILD only. Flow cytometry and immunofluorescence staining showed increased NCM in ILD. NCM-derived cells in the ILD lungs co-stained with CX3CR1, M2-like and phagocytic markers. In summary, we show that epithelial-derived fractalkine drives the migration of NCM-CX3CR1 which provides an interstitial scavenger and phagocytic myeloid cells population in fibrotic ILD lungs

Mechanisms of myeloid cell recruitment and biomarker potential in interstitial lung diseases

Interstitial lung diseases (ILDs) are fibrotic disorders with chronic inflammation and fibrinogenesis leading to lung scaring and lung function decline. Ultimately, progressive pulmonary fibrosis results in altered pulmonary physiology, abnormal gas exchange, and organ failure. ILDs include known causes and idiopathic causes, as it is the case of idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP). The most detrimental type of ILD is IPF in which anti-fibrotic drugs (nintedanib and pirfenidone) only decrease disease progression. For other ILD types, corticoid treatment helps to decrease exacerbation. Currently, clinical trials are evaluating the applicability of anti-fibrotic drugs for treating non-IPF ILDs. Therefore, mechanistic insights and in-depth cell characterization during tissue injury and remodeling in ILD are of great interest in the respiratory medical field. Circulating immune cell populations have been suggested to play a critical role in ILDs. For instance, mononuclear phagocytes are involved in the initiation, repair and regeneration of pulmonary fibrosis. Moreover, the close interaction between circulating and lung tissue-resident immune cells is critical to contribute to tissue homeostasis or lead to disease. However, precise myeloid phenotypes (e.g. myeloid-derived suppressor cells and monocytes) and their mechanisms of recruitment in ILDs have not yet been explored. In the first results chapter of this thesis, myeloid-derived suppressor cells (MDSC) abundance and function were investigated for the first time in IPF patients. For that, peripheral blood of 170 patients including IPF, non-IPF ILD, chronic obstructive pulmonary diseases (COPD) and controls were collected to characterize and quantify MDSC by flow cytometry. Circulating MDSC in IPF and non-IPF ILD were increased when compared with control. Moreover, cross sectional and longitudinal analysis of the abundance of MDSC inversely correlated with pulmonary function test in IPF only. IPF patients with high number of MDSC showed downregulation of co-stimulatory T cells signals quantified by qRT-PCR. Furthermore, MDSC were able to suppress lymphocytes CD4+ and CD8+ cells proliferation in vitro. Last, CD33 CD11b double positive cells, suggestive of MDSC, were found in neighboring fibrotic niches of the IPF lungs. Taking together, these results show that MDSC are potential biomarker for IPF and are suppressing T cell responses. In the second results chapter, we aimed at analyzing monocyte phenotype and recruitment from the blood to the lung tissue in ILD. Importantly, CX3CR1 expression on immune cells has been demonstrated to increase fibrosis features. For that, flow cytometry analysis of circulating monocytes was performed in 105 subjects (83 ILD, and 22 controls). Monocyte localization and abundance in the lung was assessed by immunofluorescence and flow cytometry analysis. For receptor-ligand function and transmigration pattern, monocytes were isolated from blood and cultured either alone or with endothelial cells. Here, we showed that classical monocytes (CM) were increased, while non-classical monocytes (NCM) were decreased in ILD: NSIP, hypersensitivity pneumonitis (HP) and connective tissue disease associated with ILD (CTD-ILD) compared with controls. Monocytes abundance positively correlated with lung function. Fractalkine levels, the ligand of CX3CR1, were higher in lung tissue than in plasma in ILD and also co-localized with bronchial ciliated cells. Fractalkine enhanced endothelial transmigration of NCM in ILD only. Flow cytometry and immunofluorescence staining showed increased NCM in ILD. NCM-derived cells in the ILD lungs co-stained with CX3CR1, M2-like and phagocytic markers. In summary, we show that epithelial-derived fractalkine drives the migration of NCM-CX3CR1 which provides an interstitial scavenger and phagocytic myeloid cells population in fibrotic ILD lungs

Identification and quantification of the alveolar compartment by confocal laser endomicroscopy in patients with interstitial lung diseases

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Biofísica Médica e Fisiologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Doenças Intersticiais Pulmonares (DIP) é um termo que inclui mais de 200 doenças que afectam o parênquima pulmonar, partilhando manifestações clínicas, radiográficas e patológicas semelhantes. Este conjunto de doenças é bastante heterogéneo, apresentando cada tipo de DIP em diferente grau os elementos de inflamação e fibrose: enquanto a inflamação é reflectida pelo aumento de células inflamatórias e presença de nódulos ou edema, a fibrose reflecte-se pelas fibras adicionais de colagénio e elastina. Identificar o tipo de DIP de um doente é um processo difícil, sendo a Discussão Multidisciplinar o actual método de diagnóstico "gold standard": vários médicos especialistas compõem uma equipa multidisciplinar que vai ter em conta os dados clínicos, radiológicos e patológicos disponíveis para chegar a uma conclusão. Estes dados incluem imagens de tomografia computorizada de alta resolução (TCAR), a descrição da lavagem broncoalveolar e, quando possível, dados de biópsias. Apesar do esforço e competência da equipa multidisciplinar, 10% dos pacientes são categorizados como inclassificáveis devido a dados inadequados ou discrepância entre os dados existentes. A maior causa para DIP inclassificáveis é a ausência de dados histopatológicos associada aos riscos das biópsias cirúrgicas. É muito importante determinar a DIP específica de um doente, dadas as suas implicações no tratamento e gestão do mesmo. É particularmente crítica a distinção entre doentes com Fibrose Pulmonar Idiopática (FPI) e doentes sem FPI, dado que há terapias anti-fibróticas – como o Pirfenidone – indicadas para FPI que são extremamente dispendiosas, exigindo certeza no diagnóstico antes de serem prescritas. Além disso, o tratamento com agentes imunossupressores pode funcionar com o grupo dos não-FPI mas aumenta a morte e hospitalizações nos doentes com FPI. A discussão multidisciplinar pode beneficiar da informação adicional oferecida pelo Confocal Laser Endomicroscopy (CLE), uma técnica de imagiologia que torna possível visualizar os alvéolos pulmonares com resolução microscópica de forma minimamente invasiva, através de uma broncoscopia. O laser do CLE tem um comprimento de onda de 488 nm que permite observar a autofluorescência das fibras de elastina. Há evidências de que a quantidade de fibras de elastina é aumentada e a arquitectura destas fibras é alterada na presença de fibrose pulmonar, a qual está associada a algumas doenças intersticiais pulmonares incluindo a fibrose pulmonar idiopática. Até à data, os vídeos de Confocal Laser Endomicroscopy são, na maioria dos casos, analisados apenas visualmente, e pouca informação objectiva e consistente foi conseguida destes vídeos em doentes de DIP. No entanto, é possível obter informação mais relevante dos mesmos, convertendo-os em frames, pré-processando as imagens e extraindo atributos numéricos. Neste projecto, foram obtidas imagens dos alvéolos pulmonares de doentes de DIP através de CLE. O principal objectivo do projecto é melhorar a técnica de CLE e aumentar a sua usabilidade para que no futuro possa contribuir para facilitar a estratificação de doentes com DIP e eventualmente reduzir o número de biópsias pulmonares nestes doentes. Como mencionado, o instrumento de Confocal Laser Endomicroscopy emite uma luz laser azul de 488nm, a qual é reflectida no tecido e reorientada para o sistema de detecção pela mesma lente, passando por um pequeno orifício (pinhole). Isto permite que a luz focada seja recolhida e que feixes provenientes de planos fora de foco sejam excluídos, originando uma resolução microscópica que permite imagens ao nível celular. Quando o CLE é aplicado a imagem pulmonar, é possível observar as paredes alveolares pela autofluorescência natural presente nas fibras de elastina. No estudo clínico subjacente a este estudo, o protocolo de CLE foi aplicado a 20 pacientes, embora 8 tenham sido posteriormente excluídos da análise. Os vídeos de CLE obtidos sofreram duas selecções: uma com base na região onde uma biópsia (usada como referência) foi tirada e outra com base na qualidade técnica das imagens. Depois, os dados foram pré-processados: geraram-se imagens mosaico com um campo de visão alargado e, paralelamente converteram-se as sequências de vídeo em frames. A qualidade da imagem foi melhorada, filtrando o ruído electrónico para que posteriormente pudesse ser aplicada a análise de imagem. Esta análise extraiu valores numéricos que reflectem o estado do espaço alveolar, nomeadamente, variáveis de textura e medições relacionadas com as fibras de elastina. As imagens de CLE obtidas mostraram-se muito interessantes. A resolução é superior à tomografia computorizada de alta resolução e a tridimensionalidade acrescenta informação às biópsias. O facto de permitir feedback em tempo real e observar ao vivo os movimentos naturais da respiração contribui para a análise do estado do doente. A análise de textura feita às imagens serviu-se de um algoritmo de extracção de variáveis de Haralick a partir de uma Gray-Level Co-occurence Matrix (GLCM). Foram extraídas as variáveis de textura Momento Angular Secundário (Energia), Entropia, Momento de Diferença Inversa, Contraste, Variação e Correlação. O algoritmo de Ridge Detection (detecção de linhas) identificou a maior parte das fibras de elastina detectáveis por um observador humano e mediu o Número de Fibras, o seu Comprimento e Largura e o Número de Junções entre fibras, permitindo também calcular a Soma dos Comprimentos de todas as fibras. Estes algoritmos devolveram valores consistentes num processo mais eficiente comparado com um observador humano, conseguindo avaliar em poucos segundos múltiplas variáveis para todo o conjunto de dados. As medições relacionadas com as fibras de elastina pretendiam ajudar a identificar os doentes fibróticos. Era esperado que as fibras dos doentes fibróticos fossem mais largas, mas isso não se observou. Também se previa que este grupo de doentes apresentasse maior número de fibras e junções, mas não houve uma diferença significativa entre grupos. No entanto, quando o grupo fibrótico foi segregado, o número de fibras e junções parece separar a fibrose moderada da fibrose severa. Este resultado é interessante na medida em que sugere que a monitorização do número de fibras/junções com CLE pode potencialmente ser usado como medida de eficácia de medicação anti-fibrótica. Em relação às variáveis de textura, esperava-se que os doentes fibróticos apresentassem valores mais elevados de Entropia, Contraste e Variância e valores inferiores de Momento de Diferença Inversa, dado que o seu tecido pulmonar deveria corresponder a imagens mais complexas e heterogéneas com mais arestas presentes. No entanto, ainda não foi possível estabelecer diferenças significativas entre grupos. Apesar dos resultados com o conjunto de dados usado não ter demonstrado correlações fortes entre as conclusões do CLE e da TCAR/histopatologia, os valores das variáveis em si já contribuem para o estudo das DIP, nomeadamente da sua fisiologia. De facto, a amostra de doentes deste estudo era reduzida, mas com uma amostra maior, espera-se que algumas das varáveis se correlacionem com outras técnicas usadas no diagnóstico e permitam segregar os pacientes em grupos e eventualmente aplicar classificação de dados. Neste momento, é possível especular que algumas variáveis seriam melhores candidatas para um classificador, nomeadamente os Números de Fibras e Junções, a Soma dos Comprimentos das fibras e as variáveis de Haralick Entropia e Energia. O projecto apresentado nesta dissertação foi desenvolvido através de um estágio de 6 meses no departamento de Pneumologia no Academic Medical Center em Amsterdão, Países Baixos. No Academic Medical Center (AMC), fui acompanhada pelos estudantes de doutoramento Lizzy Wijmans - médica - e Paul Brinkman - engenheiro biomédico - e supervisionada pelo Dr. Jouke Annema, MD, PhD, Professor de endoscopia pulmonar. Este grupo de investigação do AMC está focado em técnicas inovadoras de imagiologia do sistema pulmonar e teve a oportunidade de reunir com a empresa MKT –que produz a tecnologia de Confocal Laser Endomicroscopy –, o que enriqueceu a discussão aqui apresentada. Do Departamento de Física da Faculdade de Ciências da Universidade de Lisboa, fui orientada pelo Prof. Nuno Matela.Interstitial Lung Diseases (ILD) is a heterogeneous group of more than 200 diseases which affect the lung parenchyma. To identify the type of ILD a patient suffers from is a difficult process, and 10% of the patients are categorized as unclassifiable, mostly due to the absence of histopathological data associated with the risks of lung biopsies. The patient specific diagnosis is important because of its implications to the patient treatment and management, being particularly relevant to identify lung fibrosis. The Confocal Laser Endomicroscopy (CLE) can add information to this process. CLE allows to image the lung tissue with a micrometer resolution in a minimally invasive way, through a bronchoscopy. The elastin fibers from the lung alveoli are visible with this technique due to their autofluorescence. Since there is evidence that the amount of elastin fibers increases, and their architecture is altered in lung fibrosis, CLE should be used to extract values reflecting this condition. Thus, the main goal of this project was to improve the CLE technique and increase its usability, by extracting numerical values from the images which would reflect the state of the alveolar space, particularly the elastin fibers. The ILD patients recruited for the study had their lung alveoli imaged with CLE. The CLE movies were selected, pre-processed – were converted into frames, had their image quality enhanced and some mosaics were obtained – and then analyzed. The ridge detection algorithm detected most fibers recognized by a human observer. It allowed the measurement of the Number of Detected Fibers, their Length and Width, the Number of Junctions between fibers and to calculate the Sum from all Fibers’ Lengths. The Gray-Level Co-occurrence Matrix allowed the extraction of the Haralick texture features: Angular Second Moment (Energy), Entropy, Inverse Difference Moment, Contrast, Variance and Correlation. These algorithms produced consistent and unbiased numerical features, in an efficient process which can analyze the entire data set in a few seconds. Regarding the fiber related measurements, it was expected for the fibrotic patients to have wider fibers and a higher number of fibers and junctions. In terms of texture variables, it was expected from the fibrotic patients to present higher values of Entropy, Contrast and Variance, and lower values of Inverse Difference Moment, given their lung tissue should correspond to more complex and heterogeneous images with more ridges present. Due to the small sample size, it was still not possible to stratify patients with this data set. Nevertheless, the measurements presented here already contribute to the study of ILD, helping to understand the disease physiology. It is hoped that in the future, these measurements will aid the diagnosis process specially in those cases when patients cannot undergo a surgical biopsy. Additionally, CLE could potentially be used as an anti-fibrotic medication efficiency measurement tool

From mouse to man and back : closing the correlation gap between imaging and histopathology for lung diseases

Lung diseases such as fibrosis, asthma, cystic fibrosis, infection and cancer are life-threatening conditions that slowly deteriorate quality of life and for which our diagnostic power is high, but our knowledge on etiology and/or effective treatment options still contains important gaps. In the context of day-to-day practice, clinical and preclinical studies, clinicians and basic researchers team up and continuously strive to increase insights into lung disease progression, diagnostic and treatment options. To unravel disease processes and to test novel therapeutic approaches, investigators typically rely on end-stage procedures such as serum analysis, cyto-/chemokine profiles and selective tissue histology from animal models. These techniques are useful but provide only a snapshot of disease processes that are essentially dynamic in time and space. Technology allowing evaluation of live animals repeatedly is indispensable to gain a better insight into the dynamics of lung disease progression and treatment effects. Computed tomography (CT) is a clinical diagnostic imaging technique that can have enormous benefits in a research context too. Yet, the implementation of imaging techniques in laboratories lags behind. In this review we want to showcase the integrated approaches and novel developments in imaging, lung functional testing and pathological techniques that are used to assess, diagnose, quantify and treat lung disease and that may be employed in research on patients and animals. Imaging approaches result in often novel anatomical and functional biomarkers, resulting in many advantages, such as better insight in disease progression and a reduction in the numbers of animals necessary. We here showcase integrated assessment of lung disease with imaging and histopathological technologies, applied to the example of lung fibrosis. Better integration of clinical and preclinical imaging technologies with pathology will ultimately result in improved clinical translation of (therapy) study results

Diseases of the Chest, Breast, Heart and Vessels 2019-2022

This open access book focuses on diagnostic and interventional imaging of the chest, breast, heart, and vessels. It consists of a remarkable collection of contributions authored by internationally respected experts, featuring the most recent diagnostic developments and technological advances with a highly didactical approach. The chapters are disease-oriented and cover all the relevant imaging modalities, including standard radiography, CT, nuclear medicine with PET, ultrasound and magnetic resonance imaging, as well as imaging-guided interventions. As such, it presents a comprehensive review of current knowledge on imaging of the heart and chest, as well as thoracic interventions and a selection of "hot topics". The book is intended for radiologists, however, it is also of interest to clinicians in oncology, cardiology, and pulmonology

Diseases of the Chest, Breast, Heart and Vessels 2019-2022

This open access book focuses on diagnostic and interventional imaging of the chest, breast, heart, and vessels. It consists of a remarkable collection of contributions authored by internationally respected experts, featuring the most recent diagnostic developments and technological advances with a highly didactical approach. The chapters are disease-oriented and cover all the relevant imaging modalities, including standard radiography, CT, nuclear medicine with PET, ultrasound and magnetic resonance imaging, as well as imaging-guided interventions. As such, it presents a comprehensive review of current knowledge on imaging of the heart and chest, as well as thoracic interventions and a selection of "hot topics". The book is intended for radiologists, however, it is also of interest to clinicians in oncology, cardiology, and pulmonology

Phenotyping Pulmonary Hypertension with CT and MR Imaging: Pulmonary Vessel and Right Ventricular Analysis

The thesis titled "Phenotyping Pulmonary Hypertension with CT and MR Imaging: Pulmonary Vessel and Right Ventricular Analysis" presents a body of work in clinical diagnostic and interventional radiology that aims to investigate the use of computed tomography (CT) imaging to analyse pulmonary blood vessels and magnetic resonance imaging (MRI) to evaluate the right ventricle in patients with pulmonary hypertension respectively, in addition to invasive interventional procedures such as right heart catheterisation. The work focuses on two subgroups of patients with pulmonary hypertension: those with chronic lung disease (CLD) and those with chronic thromboembolic pulmonary disease (CTEPH).’’ Pulmonary hypertension is a condition characterised by high blood pressure in the pulmonary arteries, which can lead to heart failure and other serious complications. Both CLD and CTEPH can cause or contribute to the development of pulmonary hypertension and both conditions have a direct impact on pulmonary blood vessels. The thesis aims to use CT and MRI to better understand the impact of these conditions on the pulmonary vessels and the right ventricle, and to identify potential biomarkers or other indicators that could be used to diagnose and manage pulmonary hypertension in these patients. The thesis discusses the results of the studies and the implications of these findings for the diagnosis and treatment of pulmonary hypertension in patients with CLD and CTEPH. It also describes the limitations and suggests potential directions for future research in this area. In the thesis, I aimed to investigate the utility of computed tomography (CT) imaging and magnetic resonance imaging (MRI) in the diagnosis and phenotyping of patients with PH due to CLD and CTEPH. My results show that CT pulmonary vessel analysis and cardiac MRI assessment of RV function can support the diagnosis and phenotyping of patients with PH due to CLD and CTEPH. Specifically, a lower volume of small pulmonary arteries on CT is associated with more severe PH and MRI has been shown to be an effective tool for assessing disease severity in PH in addition to assessment of therapy response. These imaging modalities can provide valuable information about the severity and morphological and functional changes in the right ventricle, as well as the presence and extent of underlying pulmonary vascular changes in CLD or CTEPH. Our findings suggest that CT and MRI can be valuable tools for the diagnosis and management of PH in these patient populations. Further research is needed to confirm and expand on these findings, and to identify potential biomarkers or other indicators that could be used to diagnose and manage PH in these patients

From Early Morphometrics to Machine Learning-What Future for Cardiovascular Imaging of the Pulmonary Circulation?

Imaging plays a cardinal role in the diagnosis and management of diseases of the pulmonary circulation. Behind the picture itself, every digital image contains a wealth of quantitative data, which are hardly analysed in current routine clinical practice and this is now being transformed by radiomics. Mathematical analyses of these data using novel techniques, such as vascular morphometry (including vascular tortuosity and vascular volumes), blood flow imaging (including quantitative lung perfusion and computational flow dynamics), and artificial intelligence, are opening a window on the complex pathophysiology and structure-function relationships of pulmonary vascular diseases. They have the potential to make dramatic alterations to how clinicians investigate the pulmonary circulation, with the consequences of more rapid diagnosis and a reduction in the need for invasive procedures in the future. Applied to multimodality imaging, they can provide new information to improve disease characterization and increase diagnostic accuracy. These new technologies may be used as sophisticated biomarkers for risk prediction modelling of prognosis and for optimising the long-term management of pulmonary circulatory diseases. These innovative techniques will require evaluation in clinical trials and may in themselves serve as successful surrogate end points in trials in the years to come

Frontiers of Targeted Therapy and Predictors of Treatment Response in Systemic Sclerosis

Systemic sclerosis (scleroderma) is an incurable connective tissue disease with an unclear etiology characterized by vasculopathy, dysregulation of the immune response, and progressive tissue fibrosis affecting the skin, lungs, digestive tract, heart, and kidneys. The collection of six original research manuscripts and five review articles included in this book highlights the most recent advances in this field and provides further elucidation of the pathogenesis, natural disease course, and treatment of this challenging disease