Water Ppipe Ccondense (WPC) Rregulates the Eexpression of TLRs on Hhuman Mmacrophages via Rreactive Ooxygen Sspecies

Esmaeil Mortaz 1,2,3, 4, Gert Folkerts 2, Johan Garssen2 , Arda Kiani 1, Hamid Reza Jamaati 1, Marwan El-Sabbana 5, Ali A. Velayati 3 and Ian M. Adcock4

1Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran..2 Division of Pharmacology and Pathophysiology Utrecht Institute for Pharmaceutical Sciences, Faculty of Sciences, Utrecht University, Utrecht, The Netherlands, 3Department of Infectious Diseases, Mycobacteriology Research Center, NRITLD, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran and 4 Airways Disease Section, National Heart and Lung Institute, Imperial College London, London, UK and 5 Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut , Lebanon

Abstract:

Introduction: While water pipe smoking has become a global phenomenon, its potential health consequences are not well understood. Macrophages are considered important immune cells in the pathogenesis of COPD and induction of macrophage toll-like receptors (TLRs) is seen in COPD. Among the TLRs, TLR2, TLR4 and TLR9 play a critical role in innate immune sensing via the induction of proinflammatory cytokines.

Objectives: In this study we investigated the effect of water pipe condensate (WPC) on the expression of TLRs on these receptors.

Methods: Mainstream WSC was generated using a standardised laboratory protocol. U937 human macrophage cells were exposed to WPC for 16hrs before TLR-2 and TLR-4 were detected by flow cytometry (FACS) using anti-human TLR2, anti-human TLR4 or anti-human TLR9 antibodies or isotypes controls. Intracellular ROS levels were also measured by FACS using the redox-sensitive dye DCFH-DA (10M) and mRNA was determined using RT-qPCR.

Results: ROS induction by WPC was maximal (4-fold, p<0.05) at 30 min with 4mg/ml concentration of WPC. This was associated with an upregulation of TLR2 (5-fold increase, p0.05) and TLR4 (7.3-fold increase, p0.05) cell surface expression. Similarly, WPC induced TLR2 (4.1±0.2 versus 0.5±0.1, p<0.05) and TLR4 (5.3±0.8 versus 1.5±0.6, p<0.05) mRNA levels which was suppressed by pre-incubation with N-acetyl-L-cyteine (NAC, 10mM, p<0.05). In addition, WPC up-regulated the intracellular expression of TLR9 (6-fold increase, p0.05) but not its surface expression (1.4-fold increase, p=0.09).

Conclusions: WPC enhances the expression of TLRs in human macrophages via reactive oxygen specious. Further studies are required to determine the molecular and cellular effects of WPC on airway function.

Acknowledgements:

EM is supported by an ERS Long Term Fellowship (LTRF 2013–2052).