Pulmonary fibrosis is a common consequence of lung inflammation, leading to organ dysfunction and significant morbidity and mortality. Macrophages, through their diverse functions associated with polarisation status, play a role in lung homeostasis and alternatively activated (M2) macrophages have been associated with lung fibrosis. Prolyl hydroxylases (PHDs) are the main oxygen sensors and regulators of hypoxia inducible factors (HIFs). The PHD/HIF pathway is known to play a role in tissue inflammation and fibrosis, but their role in macrophage polarisation is not fully understood. Aim To study the role of the PHD/HIF pathway in macrophage polarisation and lung fibrosis, and specifically in Idiopathic Pulmonary Fibrosis (IPF). Hypothesis It was hypothesised that pan hydroxylase inhibition alters macrophage polarisation and modulates lung inflammation and fibrosis. Methods A combination of pharmacological (pan hydroxylase inhibitors DMOG and FG41) and genetic (HIF and PHD-null) tools were used to manipulate the PHD/HIF pathway. The bleomycin induced lung fibrosis model was used to define the effect of pan hydroxylase inhibition during the early, inflammatory or the late, fibrotic phase of this model. Murine bone marrow derived macrophages (BMDM), human monocyte derived macrophages and alveolar macrophages obtained from patients with lung fibrosis were used to study the effect of pan hydroxylase inhibition on macrophage polarisation. Bronchoalveolar lavage fluid (BALF) from patients was used to define the association between lung CCL18, an M2 associated chemokine, and disease progression in IPF. Results DMOG therapy during the early phase of the bleomycin model significantly reduced lung fibrosis at day 24. In contrast, late phase pan hydroxylase inhibition enhanced lung fibrosis at day 24. In both instances there was evidence of enhanced alveolar macrophage M2-like polarisation following pan hydroxylase inhibition. Reduced fibrosis after early pan hydroxylase inhibition was not a consequence of reduced acute lung inflammation or direct inhibition of collagen synthesis. In BMDM, pan hydroxylase inhibition resulted in an ‘augmented M2-like’ macrophage. Using LysM-Cre HIF-1α, HIF-2α and PHD-3 KO mice as well as chetomin, a potent inhibitor of HIF-1α and HIF-2α mediated gene expression, the HIF-dependent and HIF-independent polarisation markers were defined. PHD-3 deficiency was not sufficient to enhance M2 skewing. In contrast to murine BMDM, in human monocyte derived macrophages and alveolar macrophages from healthy volunteers and patients with interstitial lung disease including IPF, pan hydroxylase inhibition did not augment M2 polarisation and indeed significantly inhibited macrophage CCL18 expression. CCL18 studies in clinical BALF samples confirmed that CCL18 was elevated in the lungs of patients with IPF and other ILDs compared to controls. However, baseline BALF CCL18 concentrations did not correlate with disease severity or with disease progression, suggesting this is not a useful biomarker in IPF. Further, a unique study of serial BAL in IPF patients showed no association between 12-month change in CCL18 and disease progression over the same period. Indeed CCL18 concentrations mostly fell over 12 months in patients that did progress, strongly suggesting that CCL18 does not play a major pathogenic role in IPF. Concluding, it was shown that in both BMDM and murine lung pan hydroxylase inhibition promoted an ‘augmented M2-like’ polarisation. Pharmacological pan hydroxylase inhibition during the late fibrotic phase of injury enhanced fibrosis but it is not known if there was a causal association between M2 macrophages and lung fibrosis. Similarly, the functional relevance of finding enhanced M2 polarisation observed during early DMOG therapy, which subsequently resulted in attenuated fibrosis, is not known. In human macrophages, pan hydroxylase inhibition unexpectedly attenuated CCL18 production, a chemokine associated with an M2-like phenotype in man whilst other M2 markers were unchanged. However, there was no evidence to support a pathogenic role for CCL18 in IPF, and therefore there is little potential for using pan hydroxylase inhibition to target CCL18 and treat IPF.

Introdução: Pneumopatias que cursam com hipóxia crônica, como a doença pulmonar obstrutiva crônica, podem ser acompanhadas por alterações cognitivas secundárias a queda prolongada da saturação de oxigênio. Objetivo: 1) Averiguar se existem alterações cognitivas nos pacientes com fibrose pulmonar idiopática; 2) Caso estejam presentes, avaliar o seu perfil neuropsicológico e 3) Verificar se há correlação entre a gravidade da doença e desempenho cognitivo. Métodos: Vinte e um pacientes com diagnóstico de fibrose pulmonar idiopática foram selecionados do Ambulatório de Doenças do Interstício Pulmonar da Divisão de Pneumologia do Hospital das Clínicas da USP e foram submetidos a exame neurológico convencional, testes laboratoriais, neuroimagem, avaliação neuropsicológica abrangente e prova de função pulmonar. O grupo controle consistiu de vinte indivíduos não dementes clinicamente saudáveis equiparados por sexo, idade, escolaridade e nível sócio-econômico com o grupo anterior. Resultados: Os resultados do Mini-exame do estado mental, fluência verbal (categoria frutas), Teste de memória de figuras-aprendizado, Teste de dígito-símbolo e o Teste de trilhas parte B apresentaram diferença entre os dois grupos (p < 0,05). Conclusões: O estudo mostrou que os pacientes com fibrose pulmonar idiopática apresentam desempenho pior que o grupo controle em testes de funções executivas e velocidade de processamento mental, o que vai ao encontro com a hipótese de uma encefalopatia do tipo subcortical, semelhante àquela encontrada em pacientes com outras doenças pulmonares que cursam com hipóxia. É necessária a confirmação desses resultados com testes de tempo de reação mais precisos / Background: Lung disease with hypoxaemia as chronic pulmonary obstructive disease may be associated with cognitive impairment caused by continuous fall in oxygen saturation. Objective: 1) to identity if there is cognitive impairment in patients with idiopathic pulmonary fibrosis; 2) if present, to evaluate the neuropsychological profile of these alterations and 3) to verify if there is correlation of disease severity and cognitive performance. Methods: Twenty-one patients with idiopathic pulmonary fibrosis were selected from the Interstitial Lung disease Out-patient Clinic and were submitted throughout conventional neurological exam, blood lab, neuroimaging, broad neuropsychological and lung function evaluation. The control group consisted of twenty non-demented healthy subjects paired by gender, age, schooling and socio-economic level. Results: The Mini-mental state examination, Semantic verbal fluency (fruit category), learning item of the Brief Cognitive Battery, Digit symbol test and Trail making part B results were different between both groups (p < 0.05). Conclusions: This study showed that patients with IPF performed worse in tests of executive functions and mental speed, which is congruent with the hypotheses of encephalopathy of subcortical type similar to the one found in patients with other lung hypoxaemic disease. These results should be confirmed with more accurate reaction time tests

A fibrose pulmonar é uma via comum de várias doenças agudas e crônicas do interstício pulmonar que pode resultar na cicatrização anormal do pulmão. Há acúmulo excessivo das proteínas da matriz extracelular levando a desestruturação das paredes alveolares, e consequente perda das trocas gasosas pelos pulmões. As células NKT são grande fonte de citocinas e podem ser cruciais na polarização de macrófagos para o fenótipo M2. O projeto tem a hipótese de que as células NKT podem influenciar o desenvolvimento da fibrose pulmonar via modulação de macrófagos. Para isso, animais selvagens e knockout para células NKT invariante (Ja18-/-) foram submetidos ao protocolo de indução de fibrose pulmonar pela bleomicina. Os resultados indicam que o grupo Ja18-/- assim como os grupos experimentais que receberam agonistas para células NKT apresentaram uma proteção contra a fibrose pulmonar uma vez que houve menor síntese de hidroxiprolina, deposição de colágeno, citocinas pró-fibróticas e a manutenção de macrófagos M1 no tecido pulmonar. / Pulmonary fibrosis is a common pathway of various acute and chronic interstitial lung diseases that may result in abnormal healing of the lung. There is excessive accumulation of extracellular matrix proteins, leading to disruption of the alveolar walls and the consequent loss of gas exchange through the lungs. NKT cells are a big source of cytokines and may be crucial in the polarization of macrophages to the M2 phenotype. This project has hypothesized that NKT cells can influence the development of pulmonary fibrosis through modulation of macrophages. For this, wild and knockout invariant NKT cells (Ja18-/-) mice were subjected to the protocol of bleomycin induced pulmonary fibrosis. The results indicate that the group Ja18-/- as well as the experimental groups receiving agonists for NKT cells showed protection against lung fibrosis since there was less synthesis of hydroxyproline, collagen deposition, pro-fibrotic cytokines and maintenance of macrophages M1 in lung tissue.

Made available in DSpace on 2015-12-06T23:47:21Z (GMT). No. of bitstreams: 0 / Objetivo: Analisar as diferenças histopatológicas entre os lobos pulmonares submetidos a biópsia cirúrgica de pacientes com fibrose pulmonar idiopática. Métodos: Foram incluídos 16 pacientes com resultados de biópsias concordantes de dois sítios pulmonares, o lobo médio ou o segmento lingular. Técnicas semiquantitativas foram utilizadas na avaliação histológica pulmonar, aplicando-se um escore com base nas alterações encontradas no padrão histológico de pneumonia intersticial usual (PIU). Resultados: Encontrou-se maior incidência de foco fibroblástico em lobo inferior esquerdo e maior volume médio dos fragmentos pulmonares originários do lobo médio. Nenhuma diferença significante, que viesse a mudar o estadiamento da doença, e independentemente do local de origem da biópsia, foi encontrada. Conclusão: Não há dados que fundamentem a rejeição ao uso dos lobos médios e segmentos lingulares na investigação histológica de doenças intersticiais, em especial a fibrose pulmonar idiopática.. / Objective: To analyze the histopathology differences between the pulmonary lobes submitted to surgical biopsy in patients with idiopathic pulmonary fibrosis. Methods: Sixteen patients with usual interstitial pneumonia and concordant biopsies of two distinct lobes (the middle lobe or the lingula) were included in this study. Semiquantitative techniques were used in the histological evaluation of fragments of different lobes of the lung. A score was applied for the evaluation of usual interstitial pneumonia. Results: A greater incidence of fibroblastic foci was found in the left inferior lobe, and a greater mean volume of pulmonary fragments was obtained from the middle lobe. No significant difference was found in variables related to disease staging, no matter what was the pulmonary lobe. Conclusion: There is no reason to despise the middle lobe and the lingula in the histopathological investigation of interstitial diseases of the lung, especially the idiopathic pulmonary fibrosis. / BV UNIFESP: Teses e dissertações

A fibrose pulmonar é uma via comum de várias doenças agudas e crônicas do interstício pulmonar que pode resultar na cicatrização anormal do pulmão. Há acúmulo excessivo das proteínas da matriz extracelular levando a desestruturação das paredes alveolares, e consequente perda das trocas gasosas pelos pulmões. As células NKT são grande fonte de citocinas e podem ser cruciais na polarização de macrófagos para o fenótipo M2. O projeto tem a hipótese de que as células NKT podem influenciar o desenvolvimento da fibrose pulmonar via modulação de macrófagos. Para isso, animais selvagens e knockout para células NKT invariante (Ja18-/-) foram submetidos ao protocolo de indução de fibrose pulmonar pela bleomicina. Os resultados indicam que o grupo Ja18-/- assim como os grupos experimentais que receberam agonistas para células NKT apresentaram uma proteção contra a fibrose pulmonar uma vez que houve menor síntese de hidroxiprolina, deposição de colágeno, citocinas pró-fibróticas e a manutenção de macrófagos M1 no tecido pulmonar. / Pulmonary fibrosis is a common pathway of various acute and chronic interstitial lung diseases that may result in abnormal healing of the lung. There is excessive accumulation of extracellular matrix proteins, leading to disruption of the alveolar walls and the consequent loss of gas exchange through the lungs. NKT cells are a big source of cytokines and may be crucial in the polarization of macrophages to the M2 phenotype. This project has hypothesized that NKT cells can influence the development of pulmonary fibrosis through modulation of macrophages. For this, wild and knockout invariant NKT cells (Ja18-/-) mice were subjected to the protocol of bleomycin induced pulmonary fibrosis. The results indicate that the group Ja18-/- as well as the experimental groups receiving agonists for NKT cells showed protection against lung fibrosis since there was less synthesis of hydroxyproline, collagen deposition, pro-fibrotic cytokines and maintenance of macrophages M1 in lung tissue.

Introdução: Pneumopatias que cursam com hipóxia crônica, como a doença pulmonar obstrutiva crônica, podem ser acompanhadas por alterações cognitivas secundárias a queda prolongada da saturação de oxigênio. Objetivo: 1) Averiguar se existem alterações cognitivas nos pacientes com fibrose pulmonar idiopática; 2) Caso estejam presentes, avaliar o seu perfil neuropsicológico e 3) Verificar se há correlação entre a gravidade da doença e desempenho cognitivo. Métodos: Vinte e um pacientes com diagnóstico de fibrose pulmonar idiopática foram selecionados do Ambulatório de Doenças do Interstício Pulmonar da Divisão de Pneumologia do Hospital das Clínicas da USP e foram submetidos a exame neurológico convencional, testes laboratoriais, neuroimagem, avaliação neuropsicológica abrangente e prova de função pulmonar. O grupo controle consistiu de vinte indivíduos não dementes clinicamente saudáveis equiparados por sexo, idade, escolaridade e nível sócio-econômico com o grupo anterior. Resultados: Os resultados do Mini-exame do estado mental, fluência verbal (categoria frutas), Teste de memória de figuras-aprendizado, Teste de dígito-símbolo e o Teste de trilhas parte B apresentaram diferença entre os dois grupos (p < 0,05). Conclusões: O estudo mostrou que os pacientes com fibrose pulmonar idiopática apresentam desempenho pior que o grupo controle em testes de funções executivas e velocidade de processamento mental, o que vai ao encontro com a hipótese de uma encefalopatia do tipo subcortical, semelhante àquela encontrada em pacientes com outras doenças pulmonares que cursam com hipóxia. É necessária a confirmação desses resultados com testes de tempo de reação mais precisos / Background: Lung disease with hypoxaemia as chronic pulmonary obstructive disease may be associated with cognitive impairment caused by continuous fall in oxygen saturation. Objective: 1) to identity if there is cognitive impairment in patients with idiopathic pulmonary fibrosis; 2) if present, to evaluate the neuropsychological profile of these alterations and 3) to verify if there is correlation of disease severity and cognitive performance. Methods: Twenty-one patients with idiopathic pulmonary fibrosis were selected from the Interstitial Lung disease Out-patient Clinic and were submitted throughout conventional neurological exam, blood lab, neuroimaging, broad neuropsychological and lung function evaluation. The control group consisted of twenty non-demented healthy subjects paired by gender, age, schooling and socio-economic level. Results: The Mini-mental state examination, Semantic verbal fluency (fruit category), learning item of the Brief Cognitive Battery, Digit symbol test and Trail making part B results were different between both groups (p < 0.05). Conclusions: This study showed that patients with IPF performed worse in tests of executive functions and mental speed, which is congruent with the hypotheses of encephalopathy of subcortical type similar to the one found in patients with other lung hypoxaemic disease. These results should be confirmed with more accurate reaction time tests

La radiothérapie thoracique peut induire le développement de pneumopathies aiguës et de fibroses. La dysfonction du système vasculaire participe au développement de lésions radiques. Dans l'intestin, un KO endothélial de PAI-1 protège les souris de la fibrose radique. Le premier objectif de ce projet est d'explorer le rôle de PAI-1 dans l'apparition de la fibrose radique pulmonaire. L'irradiation thoracique de souris à 17 Gy altère sévèrement le parenchyme pulmonaire et l'analyse histologique révèle que l'invalidation de PAI-1 aggrave les lésions à 2 et 13 semaines. Cette invalidation ne protège donc pas les animaux des dommages radiques pulmonaires. L'organisation en parallèle du poumon permet d'envisager une tolérance à de fortes doses par fraction sur des petits volumes. Des irradiations en conditions stéréotaxiques ont donc été réalisées chez la souris. Les analyses histologiques montrent une déstructuration alvéolaire et un fort infiltrat inflammatoire au niveau de la zone cible. Un œdème est observable dans l'ensemble du poumon ipsilatéral deux semaines après irradiation. Le poumon ipsilatéral est également affecté par des altérations de structure, tel un épaississement des septa alvéolaires. Ces bouleversements se traduisent également au niveau transcriptomique. A la vue de l'ensemble de ces altérations, un test à l'effort a été réalisé pour évaluer l'impact potentiel sur la fonction pulmonaire. Les résultats mettent en évidence une diminution des performances des animaux. Les analyses sont à approfondir mais elles démontrent l'importance de s'intéresser aux tissus sains situés hors du volume cible mais recevant des fractions variables de la dose délivrée. / Radiation-induced endothelial dysfunction is known to participate to the development of normal tissue damage. PAI- is implicated in the phenotypic changes of irradiated endothelial cells and KOendo mice are protected from radiation damage to the gut. Whole thorax of PAI-1 KOendo and floxed mice were exposed to 17 Gy. Histological analyzes showed that PAI-1 KOendo induces a worsening of injuries at 2 and 13 weeks. Consequently, contrary to the gut no protection from radiation-induced lung damage is observed in PAI-1 KOendo mice. Our second aim was to study the effects of a single high dose stereotactic irradiation on pulmonary tissues. Histological analyzes and scanner imaging show important injuries on the targeted volume. An ipsilateral edema can also be observed 2 weeks after irradiation. Ipsilateral lung is moreover importantly damaged. A thickening of alveolar septa is notably observable. A transcriptomic analysis show important similarities between tissues from the ipsilateral lung and the focal lesion. As really highly damages have been observed in both scanner and histological analyzes, we decided to perform forced physical activity test on treadmill. A drastic decrease of maximal distance traveled has been observed from two weeks. These experiments highlighted a deficiency in respiratory function and all of these results show the importance of non-targeted irradiated pulmonary volume in the development of radiation-induced fibrosis. Effect of an endothelium-specific deletion of HIF-1α has been investigated in this model of stereotactic irradiation. Only few differences have been observed between KOendo and control mice. Experiments are still ongoing.

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<p>This PhD thesis focuses on the mechanical activation of TGFβ in the context of pulmonary fibrosis. Mechanical TGFβ activation occurs by physical force of breathing and signals to the nucleus via phospho‐Smad2. This activation occurs in presence of strong pan‐serine protease and matrix metalloproteinase inhibition. The augmented expression of latent TGFβ in lung tissue also lead to TGFβ activity following tissue stretch. Tissue biopsies from pulmonary fibrosis patients exhibited the same mechanical TGFβ activation and subsequent accumulation of phospho‐Smad2 as was seen in animal models. In rodent models and human control tissue, TGFβ was not released in detectable quantities, nor was there any significant upregulation of phospho‐Smad2. These data show that mechanical TGFβ activation is a relevant and limited to the context of a fibrotic disease process. Non‐invasive investigation of lung fibrosis was evaluated for correlation to classical assessments. We found that non‐invasive lung function parameters measured by a rodent ventilator, and small animal CT imaging correlated significantly with histomorphometic Ashcroft scoring. Exercise testing and quantification of the maximal oxygen consumption rate was a valuable indicator of overall rodent lung health but did not correlated significantly with Ashcroft scoring. Non‐invasive investigation tools evaluated here represent important advances in the quality of interpretation of preclinical lung fibrosis trials. Finally, collagen turnover was investigated by measurement of pyridinolines and serum collagen metabolic peptides. A novel method was developed and tested to detect pyridinolines in facile procedure. We found that deoxypyridinolines, but not pyridinolines, were significantly increased in the serum of lung fibrosis patients with respect to healthy controls. Furthermore, collagen type 1 telopeptide, a collagen breakdown product, was significantly increased in lung fibrosis patient serum. These data intriguingly indicate that under stable lung fibrosis conditions, more collagen appears to be breaking down into the serum then is synthesized.</p> / Doctor of Philosophy (PhD)