Glycopeptidolipid Prevents TLR2 Signaling Figure 3. TLR2 siRNA treatment decreases TLR2 gene expression in uninfected and M. abscessus -infected A549 cells. As a first step in assessing the role of TLR2 in respiratory epithelial responses to M. abscessus, the effect of transfection of A549 cells with TLR2 siRNA on TLR2 gene expression was assessed. (A) Uninfected A549 cells were transfected with scrambled RNA or TLR2 siRNA. After 48 h TLR2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative difference in TLR2 gene expression using the A549 monolayers receiving scrambled RNA as the reference, with data presented as mean +/2 SD of measurements from the same experiment performed in triplicate. *TLR2 transfected cells versus cells receiving scrambled RNA P,0.05, t-test. (B) Western blotting of A549 cell extracts from (A) demonstrates decreased TLR2 in cells treated with TLR2 siRNA. (C) A549 cell monolayers were either untreated or transfected with RNA, and either uninfected or challenged with M. abscessus 390SDmmpL4b. TLR2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative difference in TLR2 gene expression using uninfected A549 monolayers receiving scrambled RNA as the reference, with data presented as mean +/2 SD of measurements from the same experiment performed in triplicate. * TLR2 siRNA + M. abscessus 390SDmmpL4b versus scrambled RNA + M. abscessus 390SDmmpL4b; P,0.05, t-test. doi:10.1371/journal.pone.0029148.g003 scrambled RNA (Figure 4). These results indicate that M. abscessus variants lacking GPL signal the respiratory epithelial cell response through TLR2. IL-8 release by A549 alveolar epithelial cells in response to the M. abscessus 390SDmmpL4b deletion mutant is mediated by TLR2 IL-8 has been used as a readout for TLR2 stimulation of respiratory epithelial cells. To replicate the HbD2 experiments using a different method, we assessed the response of A549 cells to M. abscessus 390SDmmpL4b by measuring IL-8 release. In addition, we assessed the role of TLR2 in mediating this response using a TLR2 blocking antibody which we have previously used to block the human macrophage TLR2 response to M. abscessus variants [9]. In this experiment 390SDmmpL4b stimulated release of substantial quantities of IL-8 which was blocked by preincubation with anti-TLR2 antibody, but not isotype control antibody (Figure 5). These results are consistent with the results obtained using HbD2 gene expression as a readout for stimulation of TLR2 by M. abscessus 390SDmmpL4b. GPL expression by M. abscessus 390S prevents TLR2 signaling in BEAS 2B cells as measured by IL-8 release To insure that our findings with A549 cells have relevance to other respiratory epithelial cells, we assessed the interaction of M. abscessus variants with BEAS 2B cells. These cells are a well- characterized SV40 immortalized cell line derived from normal human bronchial epithelium. They possess TLR2 receptors and release IL-8 in response to TLR2 stimulation [18,19]. In these experiments we measured IL-8 release in response to our M. abscessus rough variants 390R and 390V, and the smooth variant 390S which expresses GPL. Both M. abscessus rough variants lacking GPL stimulated a significant increase in IL-8 release compared to the untreated control and M. abscessus 390S (Figure 6A). Furthermore, M. abscessus 390SDmmpL4 regained the ability to stimulate IL-8 release via TLR2 (Figure 6B). These PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29148