Reduced proliferation and increased TSLP expression by lung fibroblasts from COPD patients
Chronic obstructive pulmonary disease (COPD) is identified with partially reversible airflow limitation, chronic bronchitis, small airway remodelling, and alveolar destruction. COPD is also progressive in nature. TSLP (Thymic stromal lymphopoietin), an Interleukin (IL)-7 like cytokine expressed by structural cells, is a determinant of inflammation. We aimed to characterize human lung fibroblasts (HLF) from human donors with COPD as well as patients without COPD (non-COPD), comparing proliferation and TSLP release. Statins can exert anti-proliferative and anti-inflammatory effects, and their use has been linked to improved lung health, thus we also examined the effect of statins on proliferation and cytokine release by lung fibroblasts from COPD and non-COPD donors. Primary HLF cultures from three COPD diagnosed and non- COPD donors were used. Proliferation was measured using laser scanning cytometry (LSC) counting of H33248-stained cells with 5% fetal bovine along with the addition of simvastatin (0.1µM and 0.5µM). TNFα (tumor necrosis factor α) and/or IL-1β (interleukin 1β) (10ng/mL), and various concentrations of cigarette smoke extract (CSE) were used to stimulate cells. TSLP release, mRNA abundance and transcriptional activity were measured by ELISA, Real Time Polymerase Chain Recation (RT- PCR) and luciferase assay, respectively. RT-PCR was also utilized to profile TNFα and Interleukin 1β receptors. We also ascertained the effect of various stimuli on receptor-mediated signaling pathways using Western blotting. Impact of simvastatin (1-10µM) on TSLP release was determined by ELISA. During exponential growth phase, HLF from COPD donors proliferated 46.7% slower than from non-COPD. Simvastatin (0.5µM) inhibited proliferation, as indicated by 53% (P<0.01) and 48% (P< 0.001) fewer COPD and non-COPD donor HLFs at Day 6 culture. At baseline COPD HLFs make approximately 2-fold (P<0.05) more TSLP/cell compared to non-COPD HLFs. TNFα and/or IL-1β (10ng/ml, 48h) induced approximately 1.8 to 2.3-fold (P<0.05) more TSLP release in COPD HLFs. Interestingly, simvastatin had no impact on basal TSLP release, but in the presence of TNFα (5µM, 72h), TSLP release was actually increased approximately 2.19-fold (COPD) and 1.8-fold (non-COPD). TSLP mRNA levels were maximum at 6h in both COPD and non-COPD HLFs and relative TSLP mRNA was approximately 15-fold higher in COPD HLFs compared to non-COPD HLFs (P<0.01). This correlated with human TSLP promoter luciferase reporter assays that showed baseline transcription in COPD HLFs is markedly (approximately 17-fold) higher than non-COPD HLFs. In TNFα-stimulated cultures, TSLP luciferase activity was approximately 10-fold higher in COPD HLFs compared to HLFs from non-COPD. There was no difference in mRNA abundance of receptor subunits for TNFα or IL-1β between patient groups. Phosphorylation of mitogen activated protein kinases, ERK1/2 and p38MAPK, were also comparatively higher in COPD HLFs when stimulated with TNFα suggesting increased response via TNFα receptors. Additionally, in TNFα-stimulated cultures, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional activity was approximately 2.6-fold higher in COPD HLFs as compared to HLFs from non-COPD donors. Collectively our data show that proliferation of HLFs from COPD subjects is lower and can be further reduced by simvastatin. COPD HLFs exhibit increased basal and cytokine-stimulated TSLP mRNA expression, transcription and release, suggesting these cells carry stable intrinsic differences in regulatory mechanisms. Differences in TSLP levels are not due to differential abundance of TNFα and IL-1β receptors although the intracellular responses mediated by TNF α receptor is higher in COPD HLFs. Simvastatin augments TNFα-induced TSLP release in COPD and non-COPD HLFs. These data indicate further investigation of the role of TSLP and its response to therapy, in COPD is warranted.