included as another control because it signals through the TLR2 signaling pathway [21]. Both IL-1b and MALP-2 stimulation resulted in substantial increases in HbD2 gene expression relative to untreated controls. Both M. abscessus rough variants lacking GPL also stimulated a significant increase in HbD2 gene expression over untreated control and M. abscessus 390S. In contrast, M. abscessus 390S did not increase HbD2 gene expression above untreated control (Figure 1). These results indicate that M. abscessus rough variants stimulate the innate immune response of respiratory epithelial cells. A M. abscessus GPL deletion mutant derived from the 390S smooth variant regains the ability to stimulate the A549 alveolar epithelial cell innate immune response We next sought to determine whether the previously characterized deletion mutant 390SDmmpL4b, which does not express GPL [13], gains the ability to stimulate the innate immune response of respiratory epithelial cells. When challenged with the 390SDmmpL4b variant, A549 cells responded with increased expression of HbD2 to a level comparable to that seen with the rough variant 390V (Figure 2A). M. abscessus 390V is the ideal comparator strain in this assay because it is a spontaneous mutant which arose on subculture of 390S, acquiring the rough phenotype and losing its ability to express significant quantities of GPL [8]. In addition, the complemented 390SDmmpL4b strain, which has regained the smooth phenotype and the ability to produce GPL [13], lacks the ability to stimulate A549 cell HbD2 gene expression (Figure 2B). Taken together, these results indicate that M. abscessus GPL expression interferes with activation of the respiratory epithelial cell innate immune response. Glycopeptidolipid Prevents TLR2 Signaling TLR2 siRNA treatment decreases expression of TLR2 in both uninfected and infected A549 alveolar epithelial cells In a prior study we demonstrated that human macrophage TLR2 mediates the macrophage innate immune response to M. abscessus rough variants [9]. As the first step in assessing the role of TLR2 in the respiratory epithelial response to M. abscessus, we examined the effect of TLR2 siRNA on TLR2 gene expression in uninfected A549 cells. TLR2 siRNA treatment resulted in a 76% reduction in TLR2 gene transcript relative to cells treated with scrambled RNA (Figure 3A). This was accompanied by a decrease in TLR2 protein as assessed by Western blotting (Figure 3B). When A549 cells were infected with M. abscessus 390SDmmpL4b, baseline expression of TLR2 increased. TLR2 siRNA treatment, however, resulted in decreased TLR2 gene expression, while treatment with scrambled RNA did not (Figure 3C). These results indicate that siRNA TLR2 treatment is able to reduce TLR2 gene expression in both uninfected and infected A549 cells. TLR2 siRNA treatment decreases expression of HbD2 gene transcript in A549 alveolar epithelial cells in response to challenge with the M. abscessus 390SDmmpL4b deletion mutant To determine whether the M. abscessus 390SDmmpL4b deletion mutant signals the innate immune response of A549 cells through TLR2, A549 cells were treated with scrambled RNA or TLR2 siRNA and then infected with this variant. Treatment with TLR2 siRNA, but not scrambled RNA, was associated with a significant reduction in HbD2 gene expression compared with cells receiving Figure 2. A M. abscessus 390SDmmpL4b deletion mutant lacking GPL has acquired the ability to stimulate HBD2 gene expression in A549 cells. (A) A549 cell monolayers were uninfected or challenged with M. abscessus variants 390V, 390S or 390SDmmpL4b, a deletion mutant lacking the mmpL4b gene which is a critical component of the GPL biosynthetic pathway. The results of real-time PCR are expressed as the relative fold increase in HbD2 gene expression over that of the untreated group and presented as mean +/2 SD of measurements from the same experiment performed in triplicate. * 390SDmmpL4b mutant vs 390S wild type; P,0.05, t-test. (B) A549 cell monolayers were uninfected or challenged with M. abscessus 390SDmmpL4b, or the complemented 390SDmmpL4b mutant. After 8 hours, HbD2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative fold increase in HbD2 gene expression over that of the untreated group and presented as mean +/2 SD of measurements from the same experiment performed in triplicate. * 390SDmmpL4b complemented versus 390SDmmpL4b mutant; P,0.05, t-test. doi:10.1371/journal.pone.0029148.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29148