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Cell-based therapies hold great promise in re-establishing organ function for many diseases, including untreatable lung diseases such as idiopathic pulmonary fibrosis (IPF). However, many hurdles still remain, in part due to our lack of knowledge about the disease-driving mechanisms that may affect the cellular niche and thereby possibly hinder the function of any transplanted cells by imposing the disease phenotype onto the newly generated progeny. Recent findings have demonstrated increased ciliation of lung cells from IPF patients, but how this affects ciliated cell function and the airway milieu is not well-known. Here, we performed single-cell RNA sequencing on primary ciliated (FOXJ1

Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histologic correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, which ultimately lead to organ failure. The molecular composition of these lesions is unknown. In this sutdy, the protein composition of the lesions in explanted lungs from four end-stage bronchiolitis obliterans syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, and protein signatures for 14 obliterative bronchiolitis lesions were established. A set of 39 proteins, identified in >75% of lesions, included distinct structural proteins (collagen types IV and VI) and cellular components (actins, vimentin, and tryptase). Each respective lesion exhibited a unique composition of proteins (on average, n = 66 proteins), thereby mirroring the morphologic variation of the lesions. Antibody-based staining confirmed these mass spectrometry–based findings. The 14 analyzed obliterative bronchiolitis lesions showed variations in their protein content, but also common features. This study provides molecular and morphologic insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development, and wound healing processes.

Background: Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Methods: Primary distally-derived lung fibroblasts obtained from IPF patients and healthy individuals were cultured with LAD2 mast cells or stimulated with the mast cell proteases tryptase and chymase for 72 hours. Human lung fibroblasts and mast cells were cultured on traditional cell culture plastics or decellularized human lung tissue slices (lung scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and further evaluated for effects on epithelial cell migration. Results: Tryptase increased IL-6, VEGF and HGF release whereas co-culture with mast cells increased IL-6 in healthy lung fibroblasts. VEGF-C release was significantly increased in IPF fibroblasts and further induced by mast cells compared to healthy fibroblasts. Tryptase induced fibroblast migration but mast cells did not promote a myofibroblast phenotype such as altered αSMA levels. Culture in lung scaffolds increased the release of HGF and VEGF compared to culture on plastic plates. Migration of A549 epithelial cells was significantly reduced by IL-6, while HGF and conditioned media from scaffold cultures with mast cells and fibroblasts promoted migration. Conclusions: Mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in the pathogenesis of lung diseases.

Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Lung fibroblasts from IPF patients and healthy individuals were co-cultured with LAD2 mast cells or stimulated with the proteases tryptase and chymase. Human lung fibroblasts and mast cells were cultured on cell culture plastic plates or decellularized human lung tissue (scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and evaluated for effects on epithelial cell migration. Tryptase increased vascular endothelial growth factor (VEGF) release from fibroblasts, whereas co-culture with mast cells increased IL-6 and hepatocyte growth factor (HGF). Culture in scaffolds increased the release of VEGF compared to culture on plastic. Migration of epithelial cells was reduced by IL-6, while HGF and conditioned media from scaffold cultures promoted migration. In conclusion, mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells and tryptase in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in health and lung disease.

Contains fulltext : 169715.pdf (Publisher’s version ) (Open Access) Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by loss of lung function due to aberrant deposition of extracellular matrix (ECM) constituents. One such constituent is glycosaminoglycans (GAGs) consisting of unbranched polymers of repeating disaccharides. They are highly negatively charged which renders them the ability to interact with important mediators in the IPF pathogenesis. The abundance and localisation of GAGs in relation to tissue density is not known in IPF. The aim of this study was therefore to measure GAGs in lung samples from IPF patients and from control subjects. Methods: Lung tissue samples were collected from more or less dense areas in distal lungs from IPF patients (n=8) and from donor lungs (n=4). GAGs were isolated and quantified by HPLC after fluorochrome labelling. The total collagen content was quantified by the amount of hydroxyproline and fibrillar collagen was measured by picro-sirius red staining. Results: The total amount of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) was significantly higher in denser areas of IPF lungs compared to less dense areas (p ≤ 0.01 for HS, p ≤ 0.05 for CS/DS and p ≤ 0.01 for HA) and also compared to control lungs. HA was significantly increased in less fibrotic areas compared to control lungs. Moreover, the amount of GAGs in IPF tissue correlated with the volume fraction of picro-sirius red stain but not with the hydroxyproline content. Conclusions: Our results show that GAGs are quantatively elevated in fibrotic lesions characterized by high tissue density and increased levels of fibrillar collagen in IPF. This may create an altered ECM niche that promotes an active remodelling process.