Glycopeptidolipid Prevents TLR2 Signaling Figure 4. TLR2 siRNA treatment decreases HbD2 gene expres- sion in A549 cells challenged with the M. abscessus 390S mmpL4b deletion mutant. A549 cells were transfected with scrambled RNA or TLR2 siRNA for 48 h with some cell monolayers then challenged with M. abscessus 390SDmmpL4b. After 8 h, HbD2 gene expression was quantified by real-time PCR. The results of real- time PCR are expressed as the relative change in HbD2 gene expression over that of the untreated, uninfected group and 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.g004 results indicate that M. abscessus variants lacking GPL either through spontaneous mutation, or targeted deletion of a gene critical for GPL formation, gain the ability to stimulate TLR2 in respiratory epithelial cells. These results are consistent with our previous findings which indicate that GPL masks underlying bioactive cell wall molecules capable of interacting with host cells [9]. Discussion In this study we demonstrate that a naturally occurring GPL- expressing smooth M. abscessus variant (390S) does not stimulate the innate immune response of respiratory epithelial cells, while rough variants lacking GPL (390R, 390V) stimulate respiratory epithelial cells through TLR2, resulting in gene expression of the downstream effector molecule HbD2 and release of IL-8. Furthermore, loss of M. abscessus 390S GPL through targeted deletion of the mmpL4b gene critical for GPL synthesis [13] converts the bacterium to a phenotype capable of stimulating respiratory epithelial TLR2. These results are in keeping with our prior studies using macrophages, which demonstrate that loss of GPL through either spontaneous mutation, physical removal, or targeted gene deletion converts M. abscessus to an immunostimu- latory phenotype capable of stimulating release of TNFa via Figure 5. Antibody to TLR2 decreases IL-8 release from A549 cells in response to the M. abscessus 390SDmmpL4b deletion mutant. A549 cell monolayers were preincubated with antibody to TLR2 or isotype control antibody and then received no bacteria or were challenged with the M. abscessus 390SDmmpL4b deletion mutant. Culture supernates were collected after 8 h and assayed by ELISA for IL- 8. Data are means 6 SEM of two experiments done in triplicate. * 390SDmmpL4b + anti-TLR2 antibody versus 390SDmmpL4b alone and 390SDmmpL4b + isotype antibody; p,0.01, t-test. doi:10.1371/journal.pone.0029148.g005 interaction with TLR2 [9,13]. In previous studies we have provided evidence that GPL masks underlying M. abscessus cell wall lipids which are known TLR2 ligands [9]. There has been increasing recognition of the role that respiratory epithelium plays in host innate immune responses to bacterial pathogens. The human innate immune response is the immediate response engendered by the host to a foreign antigen. In the lung, both alveolar macrophages, and respiratory epithelial cells are central to the innate immune response [15]. It is distinct from adaptive or cell-mediated immunity which takes time to develop and involves antigen presentation to T- lymphocytes, which orchestrate the subsequent host response. Because M. abscessus is primarily an opportunistic pathogen, it is probable that innate immune responses are important in preventing infection with this organism. The TLRs, which mediate the host innate immune response, are present on macrophages, dendritic cells and respiratory epithelial cells lining the lung. These receptors recognize pathogen-associated molecular patterns, which are conserved motifs expressed by microorganisms, but not by higher eukaryotes. In the case of mycobacteria, bacterial lipopeptides in the cell wall are recognized by host cell TLR2/TLR1 heterodi- mers [22,23]. We have previously demonstrated that M. abscessus expresses one type of these surface components, the phosphatidyl- myo-inositol mannosides [9], which stimulate the macrophage innate immune response via TLR2 signaling. PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29148