Distinct intracellular signaling pathways control the synthesis of IL-8 and RANTES in TLR1/TLR2, TLR3 or NOD1 activated human airway epithelial cells
Introduction
The World Health Organization estimates that 210 million people suffer from chronic obstructive pulmonary disorder (COPD) and 300 million suffer from asthma worldwide. Inflammation is a central feature of these respiratory diseases. In the lung, inflammation must be resolved efficiently while preventing tissue damage that could impair gas exchange. The airways must also discriminate between the vast amount of non-threatening antigens and the much rarer pathogenic signals. Thus, the pulmonary epithelium not only plays a role as a physical barrier but also forms the first line of defense against infections by secreting cytokines and chemokines that will activate the innate immune defense.
However, in chronic inflammatory disorders like COPD, inflammation has been proposed to be the predominant mechanism of airflow limitation [1] and many inflammatory mediators have been found present in the sputum of COPD patients [2], [3]. Monocytes from patients with COPD showed enhanced chemotaxis towards GRO-α [4] and elevated IL-8 levels and increased neutrophil numbers are observed and negatively correlates with pulmonary function [5]. Asthma and COPD patients also suffer from episodes of exacerbations of their condition induced by further amplification of the inflammatory response triggered by various environmental factors, including bacterial and viral infections, which have a profound effect on patients' quality of life [6], [7]. During exacerbations, levels of the CC chemokine RANTES are markedly increased in airway epithelial cells and is accompanied by increased eosinophilia [8]. Thus understanding the molecular mechanisms involved in chemokines-initiated inflammation may provide valuable information for new clinical developments in chronic pulmonary diseases.
Human cells have evolved to recognize pathogens through receptors that bind different molecular patterns like lipids, carbohydrates, peptides and nucleic acids expressed by various microorganisms. Once activated, these pattern-recognition receptors (PRRs) trigger a network of intracellular signaling events leading to the production of inflammatory mediators. The two most studied PRR families are the TLR and Nucleotide-binding Oligomerization Domain (NOD)-like receptor (NLR) families. There are currently 12 known mammalian TLRs and more than 20 NLRs [9], [10].
Our understanding of TLR-mediated signaling has progressed rapidly in the last few years but many aspects remain unclear. Following dimerization, TLRs bind different adaptor molecules through their Toll/IL-1 receptor domain (TIR) [10]. The best characterized adaptor is MyD88 which was shown to, through the sequential recruitment of IL-1R-associated protein kinases (IRAKs) [11], [12], TNF-receptor-associated factor 6 (TRAF6) [13] and TGF-β Activated Kinase (TAK1) [14], [15], serve as the template for the activation of four major signaling pathways: the NFκB pathway and the three MAPK pathways, ERK1/ERK2, JNK and p38 MAPK. The TRIF adaptor, which can be recruited directly to TLR3, or indirectly via TRAM to TLR4, leads to the production of type I interferons via the activation of the IKK family members IKKε and TBK1 and phosphorylation of interferon response factors (IRFs) [16]. TRIF can also recruit TRAF6 [17] and in conjunction with Receptor-Interacting Protein 1 (RIP1) activate TAK1 and the downstream signaling pathways associated as outlined above [18]. The NOD receptors bind through their CARD domains to a protein kinase termed RIP2, (also called RICK or CARDIAK) [19], which will initiate downstream signaling via the recruitment of TAK1 and activation of the IKK complex [20], [21].
In this study we have investigated the PRR responsible for the production of IL-8 and RANTES by airway epithelial cells, and identify key protein kinases involved in their synthesis.
Section snippets
Materials
Human TLR1-9 Agonist kit, Human and Mouse NOD1/2 Agonist kit and SB203580 were obtained from Invivogen (San Diego, CA, USA). PD184352 was bought from USBiological (Swampscott, MA, USA). BIRB07896 and PS1145 were kindly provided by Professor Sir Philip Cohen (MRC PPU, University of Dundee, UK). All chemicals were bought from Fisher Scientific (Fair Lawn, NJ, USA). All enzymes were bought from Invitrogen (Carlsbad, CA, USA). Complete protease inhibitor cocktail tablets were from Roche (Mannheim,
RANTES and IL-8 are up regulated by similar TLR and NOD ligands in airway epithelial cells
When epithelial cells from a human bronchoalveolar carcinoma (A549) are stimulated with Flagelline to activate TLR5, three chemokines (namely RANTES, GRO-α and IL-8) were greatly up-regulated out of 36 common inflammatory mediators screened (Fig. 1A and B). GRO-α and IL-8 are two members of the same family of CXC chemokines that bind the CXCR2 receptor, whereas RANTES is a member of the CC chemokine family. In order to better understand which PRRs lead to an increase in synthesis of two
Discussion
In this manuscript we have shown that when three different receptor complexes acting through distinct adaptors are activated by their respective ligands they convey signals towards common signaling modules that differentially regulate the synthesis of IL-8 and RANTES (Fig. 6). This convergence mechanism to signaling module from multiple cellular receptors was also found in a systems biology study of RAW macrophages [33]. It is interesting to note there appears to be a correlation between signal
Conclusion
The distinction we made between pathways involved in the regulation of both IL-8 and RANTES (NFκB) or only IL-8 (MAPKs) gives rise to the possibility of designing more targeted clinical approaches based on the biological functions to be ablated. As RANTES and IL-8 are involved in recruiting and activating different immune cells, it may be beneficial in specific pathologies to only block the function of one family of chemokine. However, this may have consequences on other pathways as we have
Acknowledgements
We would like to thank Professor Sir Philip Cohen (MRC PPU, University of Dundee, UK) for the kind gift of BIRB0796 and PS-1145. We acknowledge the financial support of the Department of Medicine, McGill University and the McGill University Health Centre Research Institute (MUHC-RI). The Meakins-Christie Laboratories — MUHC-RI, are supported by a Centre grant from Les Fonds de la Recherche en Santé du Québec (FRSQ). SR would like to also acknowledge a salary award from FRSQ.
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These authors contributed equally to this work.