Investigation of Fibroblast Markers in Idiopathic Pulmonary Fibrosis



Collins, Ashley Claire

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Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal form of interstitial lung disease (ILD) with no cure. The key effector cell of IPF is the fibroblast which is locked in dysregulated wound repair. To investigate the pathogenesis of this disease it is essential to be able to study this cell in isolation. However, despite its crucial role in the pathogenesis of IPF and all fibrotic disease, there is no isolation method for fibroblasts which does not involve long term in-vitro culture. This is due to the lack of an accepted surface marker for fibroblasts. To rectify this, we used genomic expression data and informatics tools to profile IPF fibroblasts and normal fibroblasts, isolated with minimal culture, to identify potential candidate markers. Using this approach, we ultimately identified three possible candidate markers; Brain Acid Soluble Protein 1 (BASP1), Brain Protein I3 (BRI3) and S100 Calcium Binding Protein P (S100P). Validation of gene expression was carried out using Q-PCR to compare the expression levels in lung cells including IPF derived macrophages and epithelial cells, to determine the uniqueness of these markers. Immunohistochemistry (IHC) of IPF lung tissue and normal lung tissue was performed to confirm protein expression and protein localization. Our data showed BASP1 is a fibroblast specific marker, however it is not disease specific due to its high expression in normal P0 fibroblasts. The absolute expression of BASP1 in fibroblasts is consistent in-vitro between IPF P0 fibroblasts and IPF P3 fibroblasts (fold difference of -1.1). BASP1 was present in fibrotic regions of IPF lung tissue and there was alpha-smooth muscle actin (ACTA2) co-localization. Taken together, these results indicate BASP1 is a fibroblast specific marker. BRI3 was highly expressed in IPF P0 fibroblasts and IPF P3 fibroblast (fold difference of 0.11), as confirmed by microarray analysis. Further, BRI3 expression was located in areas of fibrosis in IPF lung tissue and absent in normal lung tissue. BRI3 did not co-localize with ACTA indicating a subpopulation of fibroblasts that do not express ACTA2, a common fibroblast maker. There was a -15-fold difference between IPF P0 fibroblasts and IPF P3 fibroblasts indicating S100P may be specific to IPF fibroblasts as measured by cDNA microarray. The gene expression confirmed by Q-PCR of S100P showed high expression of IPF P0 fibroblasts compared to IPF P3 fibroblasts and this difference was statistically significant (p=0.005). In addition, the expression of S100P was significantly upregulated compared to macrophages (p=0.0105) and epithelial cells (p=0.0226). S100P was present in fibrotic areas not expressing ACTA2 of IPF lung tissue but was also present in normal lung tissue indicating S100P. Ultimately, we found that BASP1, BRI3, and S100P have potential as markers for fibroblasts. However due to their heterogeneous and patient dependent expression in the IPF lung tissue, further investigation into their expression in IPF and other fibrotic diseases is warranted in future studies.



IPF, Idiopathic pulmonary fibrosis, Fibroblast, Fibroblast marker