Induction of IL-8 but not IP-10 in response to Th2 stimulation.

<p>ppBECs were treated for 24-13 (10 ng/ml) and IL-4 (10 ng/ml) for 24 hours. Protein secretion in cell supernatant was measured by ELISA (R&D). Statistical analysis 1A n = 9 ANOVA P<0.05. 1B n = 15, Wilcoxon, p<0.05. NT-no treatment.</p

Mechanical Strain Causes Adaptive Change in Bronchial Fibroblasts Enhancing Profibrotic and Inflammatory Responses

<div><p>Asthma is characterized by periodic episodes of bronchoconstriction and reversible airway obstruction; these symptoms are attributable to a number of factors including increased mass and reactivity of bronchial smooth muscle and extracellular matrix (ECM) in asthmatic airways. Literature has suggested changes in cell responses and signaling can be elicited via modulation of mechanical stress acting upon them, potentially affecting the microenvironment of the cell. In this study, we hypothesized that mechanical strain directly affects the (myo)fibroblast phenotype in asthma. Therefore, we characterized responses of bronchial fibroblasts, from 6 normal and 11 asthmatic non-smoking volunteers, exposed to cyclical mechanical strain using flexible silastic membranes. Samples were analyzed for proteoglycans, α-smooth muscle actin (αSMA), collagens I and III, matrix metalloproteinase (MMP) 2 & 9 and interleukin-8 (IL-8) by qRT-PCR, Western blot, zymography and ELISA. Mechanical strain caused a decrease in αSMA mRNA but no change in either αSMA protein or proteoglycan expression. In contrast the inflammatory mediator IL-8, MMPs and interstitial collagens were increased at both the transcriptional and protein level. The results demonstrate an adaptive response of bronchial fibroblasts to mechanical strain, irrespective of donor. The adaptation involves cytoskeletal rearrangement, matrix remodelling and inflammatory cytokine release. These results suggest that mechanical strain could contribute to disease progression in asthma by promoting inflammation and remodelling responses.</p></div

Mechanical strain induces IL-8 expression.

<p>Fibroblasts from non-asthmatic (filled circles) or asthmatic (open circles) donors were cultured in the absence or presence of cyclical mechanical strain for 48 and 96h. A) IL-8 mRNA expression at 48h was assessed by RT-qPCR with the data normalized to GAPDH and displayed relative to the median of the unstrained non-asthmatic group using the ΔΔCT method. B) Release of IL-8 protein into the cell culture supernatants was assessed after 96 h by ELISA and normalised for cell number. The data were analysed using Wilcoxon’s signed rank test. Results shown represent data for fibroblasts from non-asthmatic (n = 6) or asthmatic (n = 11) donors for both the RT-qPCR and ELISA.</p

Comparison of the effect of cyclical mechanical strain on fibroblast proteoglycan expression.

<p>Fibroblasts derived from bronchial biopsies from non-asthmatic (n = 6, filled circles) or asthmatic donors (n = 11, open circles) were exposed to cyclical mechanical strain for 48h. RNA was extracted and RT-qPCR performed to measure expression of A) versican (VCN) and B) decorin (DCN). Data were normalized to GAPDH and displayed relative to the median of the non-asthmatic non-strain group using the ΔΔCT method. The data were analysed using Wilcoxon’s signed rank test. No differences were found between the treatments or subject groups.</p

The effect of cyclical mechanical strain on bronchial fibroblast morphology and cell number.

<p>A) Photomicrographs showing fibroblast morphology using phase contrast microscopy. For the strained cells, two views are shown demonstrating the differences in morphology in the centre or periphery of the culture well. B) Localization of F-actin stress fibres using FITC-conjugated phalloidin (green) and α smooth muscle actin (αSMA) using a FITC-conjugated mouse monoclonal anti αSMA antibody with 7AAD nuclear staining (red). Scale bar = 100μm. C) Cell numbers (mean+SD) for three individual fibroblast cultures (1 normal and 2 asthmatic donors). Cell number was determined by direct cell counting at 24, 48 or 96h. The difference between strained and non-strained cell numbers was tested for statistical significance using a paired-t test, and the differences between time points was tested for statistical significance using a one way ANOVA.</p

Mechanical strain induces protease expression.

<p>Fibroblasts derived from non-asthmatic (filled circles) or asthmatic (open circles) donors were subjected to mechanical strain (or left unstrained) for 96h. The resultant culture supernatants were analysed for A) the 72-kDa pro-enzyme form of MMP-2 (ProMMP-2) and B) the 92 kDa pro-enzyme MMP-9 (ProMMP-9) using gelatine zymography and densitometry. Data were normalized per 10,000 cells and displayed relative to the median of the unstrained non-asthmatic group. The data were analysed using Wilcoxon’s signed rank test. Results shown are from fibroblasts from non-asthmatic (n = 6) or asthmatic (n = 11) donors for ProMMP-2, and fibroblasts from non-asthmatic (n = 4) or asthmatic (n = 10) donors for ProMMP-9.</p

Ability of GSH, but not AA, to protect against CSE-induced cell death.

<p>PBEC obtained from non-asthmatic or asthmatic donors were treated for 24h with 20% CSE alone or in the presence of GSH (1mM) or AA (250nM). Changes in cell viability (a) and EA (b) were then evaluated with AxV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals of fold changes from CSE alone using PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) donors. <b>*</b> = p<0.05 according to Wilcoxon Signed Rank tests comparing GSH+CSE with CSE only. <b>∞</b> = p<0.05 according to a Mann Whitney U test comparing CSE+GSH treatment between PBECs from non-asthmatic and asthmatic donors.</p

Involvement of AIF in CSE-induced Apoptosis.

<p>PBECs were left untreated (A), or were treated for 24h with 20% CSE alone (B), or with a combination of CSE and GSH (1mM) (C) or AA (250nM) (D). Cells were then washed, fixed and stained with a primary antibody directed towards AIF, followed by a secondary FITC-conjugated antibody. The figure shows that in basal conditions, PBEC expressed AIF in their cytoplasm especially inside their mitochondria (A, insert); CSE treatment caused translocation of AIF from the mitochondria to inside the nuclei (B, green arrows and insert); GSH pre-treatment caused a significant decrease in the number of nuclear-AIF positive cells whereas AA was not so effective. Bar = 30μm. Data are representative of experiments performed with PBECs from 2 donors.</p

Effect of CSE on PBEC Viability and Apoptosis.

<p>PBEC obtained from non-asthmatic and asthmatic donors were treated for 24h with 20% CSE. Cell viability (a) and early apoptosis (b) were then evaluated with AnnexinV staining. The results are displayed as a box plot showing median, interquartile range and 5–95% confidence intervals using PBECs from 10 non-asthmatic (○) and 10 asthmatic (Δ) volunteers. * represents significance (p<0.05) according to Wilcoxon Signed Rank tests comparing treatment with untreated control. ∞ represents significance (p = 0.003) according to a Mann Whitney U test comparing CSE treatment between PBECs from non-asthmatic and asthmatic donors.</p

Mechanical strain enhances the production of interstitial collagens.

<p>Expression of A) collagen I (COL1A1) and B) collagen III (COL3A1) mRNA was determined using RT-qPCR after exposing fibroblasts from non-asthmatic (filled circles) or asthmatic (open circles) donors to cyclic strain or no strain for 48h. Data were normalized to GAPDH and expressed relative to the median value of the non-strain non-asthmatic group using the ΔΔCT method. The expression of collagen in cell culture supernatants C) was measured at 96h using the Sircol dye binding method. The data were analysed using Wilcoxon’s signed rank test. The results for the RT-qPCR represent data for bronchial fibroblasts from non-asthmatic (n = 6) or asthmatic (n = 11) subjects, and for the soluble collagen assay data for fibroblasts from non-asthmatic (n = 5) or asthmatic (n = 9) donors. The group sizes for the soluble collagen experiment were reduced to 5 for non-asthmatic and 9 for the asthmatic groups respectively, as a result of inadequate samples to perform analysis.</p