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Effects of cigarette smoke extract and extracellular Hsp70 on TLR2 and TLR4 mRNA expression in THP-1 cells and monocyte-derived macrophages

Introduction: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease caused by long-term exposure to irritating gases or particulate matter, most often from cigarette smoke. Many systemic manifestations are displayed in patients with COPD. Elevated extracellular Hsp70 (eHsp70) level was found in COPD patients. eHsp70 may bind to Toll like receptors (TLR) 2 and 4 resulting in cytokine production and altered inflammatory response. The aim of this study was to explore the effects of cigarette smoke extract (CSE) and its combinations with eHsp70 on TLR2 and TLR4 mRNA expression in THP-1 cell line and monocyte-derived macrophages (MDMs). Material & methods: THP-1 cells and MDMs were treated with CSE (2.5% or 5%), low endotoxin recombinant human Hsp70 (rhHsp70 ; 1, 3 or 10 μg/ml), and their combinations for 24 h. RNA was isolated by Trizol/chlorophorm method, followed by cDNA synthesis and analysis of TLR2 and TLR4 mRNA expression by qPCR. Results: In THP-1 cells, 2.5% CSE alone and in combinations with 1 and 3 μg/ml rhHsp70 provoked a significant increase in TLR2 gene expression. In contrast, 5% CSE alone and in combinations with 1 and 3 μg/ml rhHsp70 as well as 10 μg/ml rhHsp70 decreased TLR2 gene expression in this cell line. Furthermore, 10 μg/ml rhHsp70 significantly reduced TLR4 mRNA expression, while 2.5% CSE and combinations of 2.5% CSE with 3 μg/ml rhHsp70 as well as 5% CSE with 3 μg/ml rhHsp70 resulted in increased TLR4 mRNA expression in THP-1 cells. In MDMs, TLR2 mRNA expression was increased after treatments with 3 and 10 μg/ml rhHsp70 and decreased after treatment with 2.5% and 5% CSE alone. Combinations of 5% CSE with rhHsp70 induced expression of TLR2 compared to both non-treated cells and CSE alone, while 2.5% CSE applied together with 3 μg/ml rhHsp70 increased TLR2 expression compared to 2.5% CSE in MDMs. 10 µg/ml rhHsp70 attenuated expression of TLR4 mRNA, and combination of 5% CSE with 3 µg/ml rhHsp70 increased its expression in MDMs. Conclusions: In both THP-1 cells and MDMs, TLR2 was more susceptible to CSE, whether applied alone or together with rhHsp70, but TLR2 and TLR4 expressions strongly depended on type of cells and concentrations of both CSE and rhHsp70. These results imply that rhHsp70 might modulate the inflammatory response to CSE in THP-1 cells and MDMs on the level of TLR2 and TLR4 gene expression, suggesting that eHsp70 might be implicated in systemic inflammation induced by cigarette smoke.

extracellular Hsp70, cigarette smoke extract, THP-1 cells, monocyte-derived macrophages

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