+1% Antibiotic-Antimycotic in 6 well tissue culture plates followed by incubation at 37uC in humidified 5% CO2 for 24 hours. The cells were then washed 3 times with 2 mL 37u C PBS (Gibco), followed by addition of 2 mL 37u C DMEM+10% FBS+1% Antibiotic-Antimycotic to each well. The transfection solution was created by combining 2.2 pmoles of either TLR2 Silencer Select siRNA s169 (ABI) or Silencer Select Negative Control #1 siRNA (ABI) to knock down TLR2 expression and serve as a negative control respectively, 8 mL INTERFERin (Polyplus) and 200 mL DMEM. After a 10 minute incubation period at room tempera- ture, 200 mL of the transfection solution was added to each well of a 6 well plate containing A549 cells, and 200 mL DMEM was added to untreated control wells. Plates were then incubated at 37uC, in humidified 5% CO2 for 48 hours. Tissue culture plates containing transfected A549 cells were washed 3 times with 2 mL Iscove’s medium, and M. abscessus 390S DmmpL4b was added to wells at a concentration of 16107 CFU/well, in 2 mL Is- cove’s+5% human serum. Control wells received only 2 mL Iscove’s medium+5% human serum. Plates were then incubated at 37uC, in humidified 5% CO2 for 8 hours. The wells were then washed three times with Iscove’s medium, prior to being lysed with 350 mL Qiagen RLT buffer (Qiagen). Lysates were placed in RNAse free tubes (VWR) and frozen 280uC for later RNA isolation. RNA was isolated using the RNeasy kit. The HbD2 primer/ probe sequences (see above), the ABI TLR2 gene expression assay Hs00152932_m1, and the ABI Human Eukaryotic 18 s rRNA Taqman Gene Expression Assay (Endogenous Control) were used for detection of HbD2, TLR2 and 18 s, respectively. qRTPCR of the RNA was performed as described above. A no template control was included to verify that amplification only took place in reactions containing RNA. Thermocycling conditions were as described above. Relative quantity was determined using the 22DDCT method [20], using the untreated control as the calibrator sample for HbD2 expression and the control treated with scrambled RNA as the control sample for TLR2 expression. Western blot analysis Transfected A549 cells were lysed with 300 mL RIPA buffer (Pierce). Lysates were placed in sterile tubes (VWR), and frozen 280uC for later analysis. Protein concentration was measured using the BCA protein Assay (Thermo). 50 mg protein was incubated in 16 Laemmli Sample Buffer (Biorad) at 95uC for 5 minutes, and separated by electrophoresis on a 10% Tris-HCL ReadyGel (Biorad) in Tris/Glycine/SDS Buffer (Biorad). The protein was then transferred to a 0.2 mm nitrocellulose mem- brane (Biorad) in 106 Tris/Glycine Buffer (Biorad)+20% methanol (Fisher), overnight at 4uC. The membrane was blocked with 5% nonfat dry milk in PBS-0.05% tween (PBS, Gibco; Tween-20, Sigma), for one hour at room temperature. Following 3 washes in PBS-0.05% tween, the membrane was cut such that the actin bands were on one half and the TLR2 bands on the other. The TLR2 membrane was incubated in 2 mg/mL Rabbit polyclonal antibody toTLR2 (Abcam), and the actin membrane in 0.5 mg/mL mouse monoclonal antibody to actin (Abcam), in 3% BSA- PBS-0.05% Tween (BSA, EM Science), for one hour at room temperature. Following 3 washes in PBS-0.05% Tween, the TLR2 membrane was incubated in 1:250,000 goat anti-rabbit IgG+HRP (Thermo), and the actin membrane in 1:250,000 goat anti-mouse IgG+HRP (Thermo), in 5% nonfat dry milk in PBS- 0.05% tween, for one hour at room temperature. After 3 washes in PBS-0.05% Tween, the membranes were blotted dry on bibulous paper before 1 minute of incubation with Supersignal West Dura Extended Duration Substrate (Thermo). The Glycopeptidolipid Prevents TLR2 Signaling membranes were dried with bibulous paper, and exposed to blue x-ray film (Phoenix). Results A549 alveolar epithelial cells generate an innate immune response to M. abscessus variants lacking GPL We have previously demonstrated that human macrophages recognize M. abscessus variants lacking GPL via TLR2, resulting in release of the proinflammatory cytokine TNFa. One class of M. abscessus surface molecules involved in this interaction are the phosphatidyl-myo-inositol mannosides which we have demonstrat- ed are ‘‘masked’’ in M. abscessus variants expressing GPL [9]. To determine whether the innate immune system of respiratory epithelial cells recognizes M. abscessus rough variants lacking GPL, we challenged A549 cells with the M. abscessus rough variants 390R and 390V, and the M. abscessus smooth variant 390S which expresses GPL [8,9]. HbD2, which is expressed by A549 cells in response to TLR stimulation [18], was assessed by real-time PCR. IL-1b stimulation without M. abscessus infection was included as one control because it stimulates HbD2 expression by a signaling pathway which is independent from the TLR signaling pathway [16]. MALP-2 stimulation without M. abscessus infection was Figure 1. A549 cells increase HbD2 gene expression in response to M. abscessus variants lacking GPL, but not the M. abscessus 390S variant expressing GPL. A549 cell monolayers were uninfected or challenged with M. abscessus variants 390R or 390S. In addition, some uninfected A549 cell monolayers were treated with IL1b or MALP-2 as controls for the ability of A549 cells to upregulate HbD2 gene expression. 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. * 390S versus 390R and 390V; P,0.05, t-test. doi:10.1371/journal.pone.0029148.g001 PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29148