Glycopeptidolipid Prevents TLR2 Signaling Figure 6. IL-8 release from BEAS 2B cells in response to M. abscessus variants lacking GPL is mediated by TLR2. (A) BEAS 2B bronchial epithelial cells received no bacteria or were challenged with M. abscessus variants 390R, 390S and 390V. 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. * 390S versus 390R and 390V; p,0.01, t-test. (B) BEAS 2B bronchial epithelial cells were preincubated with no antibody, antibody to TLR2 or isotype control antibody. Monolayers then received no bacteria or were challenged with the M. abscessus 390SDmmpL4b deletion mutant or the 390SDmmpL4b complemented 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 deletion mutant + anti-TLR2 antibody versus 390SDmmpL4b deletion mutant alone and versus 390SDmmpL4b deletion mutant + isotype antibody; p,0.01. * 390SDmmpL4b complemented mutant versus 390SDmmpL4b deletion mutant; p,0.01. doi:10.1371/journal.pone.0029148.g006 Engagement of TLRs on macrophages leads to gene expression of pro-inflammatory cytokines [23]. Ligand binding to TLRs present on respiratory epithelial cells leads to expression of the chemokine IL-8 involved in recruitment of circulating neutrophils to sites of infection/inflammation in the lung [24]. Stimulation of respiratory epithelial cells with the inflammatory cytokine IL-1b also leads to release of IL-8 through a mechanism that bypasses the TLR signaling [25]. In addition to IL-8, respiratory epithelial cells upregulate expression of the antimicrobial peptide HbD2 both in response to TLR engagement and IL1b stimulation [26]. Thus, there are two mechanisms by which respiratory epithelial cells are stimulated to express HbD2 and IL-8 in response to bacterial pathogens. In one mechanism, alveolar macrophages present in lung alveoli recognize bacteria via TLRs leading to expression of IL-1b. This in turn activates respiratory epithelial cells in the immediate vicinity to upregulate expression of HbD2 and release IL-8. This mechanism does not involve binding TLRs on respiratory epithelial cells [27]. In the second mechanism, direct engagement of TLRs at various levels in the respiratory tract by bacterial ligands results in a signaling cascade leading to HbD2 expression by respiratory epithelial cells [26] and release of IL-8. Thus, in upper portions of the respiratory tract where mucociliary clearance is operating and alveolar macrophages are absent, respiratory epithelial cells are the primary sentinels, sampling the airways for bacterial pathogens via TLRs on their surface. If bacteria are able to survive the upper airways and enter the alveoli, respiratory epithelial HbD2 expression and IL-8 release in response to direct TLR2 stimulation is augmented by IL-1b released from alveolar macrophages. In addition to inducing HbD2 by alveolar epithelial cells, IL-1b stimulates cell-mediated immune responses such as activation of B and T lymphocytes. This sequence of events is consistent with a vigorous host defense being mounted in the alveoli if upper airway defense mechanisms are breached. A549 cells are an alveolar epithelial tumor cell line used to study respiratory epithelial cellular responses [28]. Because freshly explanted human alveolar type II cells are difficult to obtain, A549 cells are a useful surrogate for the study of respiratory epithelial cell responses at the level of the alveolus [29]. Of relevance to our study is the fact that these cells have been used to study the interaction of mycobacteria with respiratory epithelium [30–34]. Also relevant to our study are previous reports indicating that A549 cells express TLR2, express HbD2 in response to IL1-b and TLR2 stimulation [16], and express HbD2 in response to M. tuberculosis infection [35]. In addition, evidence suggests that HbD2 possesses antimicrobial activity against M. tuberculosis when expressed by host cells coming into contact with this bacterium [36]. In addition to A549 cells, we examined the interaction of M. abscessus variants with bronchial epithelial BEAS 2B cells. This was done to assess responses to M. abscessus by a respiratory epithelial cell from a different site of origin (bronchus) than A459 cells. We also wanted to replicate our findings in a non-tumor cell line because genetic changes associated with tumorigenesis can alter cellular responses. We found that both cell lines responded similarly to M. abscessus variants. Our study demonstrates that M. abscessus GPL-expressing smooth variants are not recognized by TLRs present on respiratory epithelial cells. This is consistent with the hypothesis we have put forth which suggests that M. abscessus smooth variants are a colonizing phenotype by virtue of characteristics which include sliding motility, biofilm formation, and the ability to avoid detection by TLR2 [8,9,12,17]. These features enable M. abscessus smooth variants, which are likely the predominant phenotype found in the environment [37], to colonize ectatic airways and PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29148