Publicação
Macrophages in pulmonary fibrosis: characterization and fibroblasts transdifferentiation modulation
| Resumo: | Idiopathic pulmonary fibrosis (IPF) is the most common and lethal of interstitial lung diseases and affects around 3 million people worldwide. It is characterized by a progressive and irreversible loss of respiratory function due to lung architectural destruction. It is known that IPF comprises cycles of epithelial destruction, unbalanced formation of myofibroblasts and consequent excessive extracellular matrix deposition. Recent efforts have shifted the focus of uncovering the mechanisms of disease progression to understanding the role of inflammation, for instance pulmonary macrophages (MO) populations. In IPF, the roles of Alveolar (AMs) and Interstitial (IMs) macrophages described in literature are controversial. Thus, the main goal of this thesis was to contribute with new insights regarding whether AMs and IMs differentially modulate fibrosis progression through interactions with fibroblasts during the inflammatory phase. Bleomycin (BLM)-induced pulmonary fibrosis mouse model was used to study the proposed issues. First, MO characterization was performed by flow cytometry (FC), in the lung of untreated, BLM vehicle and BLM treated wild-type animals, at 3, 5, 7 and 9 days after its administration. We observed a progressive significant increase of IMs in treated animals. The expression of CD80, CD206 and MHCII was also assessed by FC and overall treated IMs present higher percentages of cells expressing these markers. Moreover, at day 7 after BLM administration, MO from untreated and treated mouse lungs cells were obtained by fluorescence activated cell sorting for further transcripts expression analysis of pro inflammatory and anti-inflammatory genes, by qRT-PCR. Treated IMs showed increase of several genes that can potentiate their action in promoting a pro-fibrotic micro-environment. Secondly, to answer whether AMs and IMs are differentially regulating fibrosis progression, a mouse lung fibroblasts cell line was stimulated with MO conditioned media of MO separated by fluorescence activated cell sorting at days 7 and 9 after BLM administration. After stimulation, apoptosis, proliferation, metabolic activity and transdifferentiation state were assessed. Overall, 7th day IMs conditioned media stimulate higher levels of fibroblast transdifferentiation. Taken together, the findings of this thesis indicate that, in the peak of inflammatory phase (day 7), IMs population is the one crucial for promoting lung fibrosis progression. |
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| Autores principais: | Ramos, Sófia Libório Passos |
| Assunto: | Alveolar and interstitial macrophages Fibroblast transdifferentiation Inflammatory phase Fase inflamatória Transdiferenciação de fibroblastos Macrófagos alveolares e intersticiais |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Idiopathic pulmonary fibrosis (IPF) is the most common and lethal of interstitial lung diseases and affects around 3 million people worldwide. It is characterized by a progressive and irreversible loss of respiratory function due to lung architectural destruction. It is known that IPF comprises cycles of epithelial destruction, unbalanced formation of myofibroblasts and consequent excessive extracellular matrix deposition. Recent efforts have shifted the focus of uncovering the mechanisms of disease progression to understanding the role of inflammation, for instance pulmonary macrophages (MO) populations. In IPF, the roles of Alveolar (AMs) and Interstitial (IMs) macrophages described in literature are controversial. Thus, the main goal of this thesis was to contribute with new insights regarding whether AMs and IMs differentially modulate fibrosis progression through interactions with fibroblasts during the inflammatory phase. Bleomycin (BLM)-induced pulmonary fibrosis mouse model was used to study the proposed issues. First, MO characterization was performed by flow cytometry (FC), in the lung of untreated, BLM vehicle and BLM treated wild-type animals, at 3, 5, 7 and 9 days after its administration. We observed a progressive significant increase of IMs in treated animals. The expression of CD80, CD206 and MHCII was also assessed by FC and overall treated IMs present higher percentages of cells expressing these markers. Moreover, at day 7 after BLM administration, MO from untreated and treated mouse lungs cells were obtained by fluorescence activated cell sorting for further transcripts expression analysis of pro inflammatory and anti-inflammatory genes, by qRT-PCR. Treated IMs showed increase of several genes that can potentiate their action in promoting a pro-fibrotic micro-environment. Secondly, to answer whether AMs and IMs are differentially regulating fibrosis progression, a mouse lung fibroblasts cell line was stimulated with MO conditioned media of MO separated by fluorescence activated cell sorting at days 7 and 9 after BLM administration. After stimulation, apoptosis, proliferation, metabolic activity and transdifferentiation state were assessed. Overall, 7th day IMs conditioned media stimulate higher levels of fibroblast transdifferentiation. Taken together, the findings of this thesis indicate that, in the peak of inflammatory phase (day 7), IMs population is the one crucial for promoting lung fibrosis progression. |
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