Mechanisms of induction of CCL20/MIP3-? in lung epithelial cells by Moraxella catarrhalis
The chemokine CCL20 is crucial for the development of the inflammatory response in the disease COPD. The alveolar epithelium is an important source of CCL20 after stimulation from particulate matter and bacteria. This process contributes to lymphocyte and dendritic cell activation, which is the link between innate and adaptive immunity, characteristic of chronic inflammation and chronic lung disease. Moraxella catarrhalis causes approximately 20% of COPD exacerbations; however, the cellular sources of the inflammatory mediators and the pathways by which it induces production of these mediators are as of yet unknown. This project examines the cytokine-inducing capacity of a wild-type Moraxella catarrhalis strain using the A549 lung epithelial cell line. Moraxella catarrhalis induces CCL20 expression at both mRNA and protein levels in A549 cells in a dose and time dependent manner as detected using RT-PCR and ELISA. TLR2 is the most important receptor for Moraxella catarrhalis in A549 cells, and THE TLR4 receptor plays a minor role in this recognition. This was demonstrated by TLR2- and TLR4-specific RNA interference and functional antibodies. The finding that A549 cells express few TLR4 receptors in their cell membrane and that HEK293 cells transfected with TLR2 or TLR4-MD2-CD14 respond equally to stimulation with Moraxella catarrhalis suggests that Moraxella catarrhalis can signal equally through TLR2 or TLR4, when available. To elucidate the intracellular signaling pathways that are activated after infection with Moraxella catarrhalis, dominant negative and siRNA specific forms of MyD88 and TRAF6 were used, showing a significant decrease in the expression of CCL20 by RT-PCR. Dual luciferase assays show increase activity of NF-?B luciferase when A549 cells were incubated with Moraxella catarrhalis, suggesting an important role of NF-?B in CCL20 expression. Moreover, the incubation of siRNA specific for MyD88 and TRAF6 in A549 cells decreased the activity of NF-?B luciferase, confirming the importance of these two adaptor molecules in NF-?B activation. We further investigated the role of NF?B using dominant negative mutant forms of IKK-? and IKK-?, which showed a dramatic decrease in the expression of CCL20. In addition, the use of NF?B inhibitors MG132 and PDTC showed a substantial decrease in CCL20 mRNA and protein level expression by RT-PCR and ELISA. Conclusions: Moraxella catarrhalis induces CCL20 expression primarily through TLR2 receptors, with a small role for TLR4 receptors. This is a MyD88-, TRAF6- and NF-?B-dependent pathway. We demonstrated the close relationship between innate and adaptive immunity and its importance for the development of chronic inflammation in the lung. Further research, directed towards targeted therapy to block specific intracellular signaling pathways (e.g.: CCL20/CCR6 axis), may therefore prove useful in the treatment of chronic lung disease.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:moraxella catarrhalis tlr4 tlr2 lung epithelial cells ccl20 mip3 ? a549
Date of Publication:01/01/2008