bial peptide known to be upregulated in respiratory epithelial cells by TLR signaling [16]. In this study we demonstrate the utility of measuring HbD2 gene expression, as well as IL-8 release, as readouts of respiratory epithelial cell responses to M. abscessus. Using these assays, we demonstrate that respiratory epithelial cell TLRs do not recognize the colonizing phenotype of M. abscessus which expresses GPL, and that loss of GPL through targeted deletion of the mmpLb4 gene converts M. abscessus to a phenotype which is recognized by toll-like receptor 2 (TLR2) on respiratory epithelial cells. Methods Bacteria The isogenic M. abscessus 390S (smooth colony morphotype expressing GPL), 390R and 390V (rough colony morphotype lacking GPL expression) variants have been previously character- ized [8,9,12,17]. The mmpL4b gene deletion mutant derived from the 390S variant, and the mmpL4b complemented mutant have been described in a recent publication [13]. Bacteria were maintained as titrated frozen stocks stored at 270u C with intermittent passage for 3 days on Middlebrook 7H11 agar plates supplemented with Middlebrook OADC (BD), followed by flash freezing. To prepare single-cell frozen bacterial stocks for experiments, lawns of the different bacterial strains were plated on Middlebrook 7H11 OADC agar plates and incubated at 37u C. After 3 days, bacteria were harvested and placed into sterile Eppendorf tubes containing 1.0 mL sterile PBS and three glass beads. Tubes were pulse- vortexed 50 times, after which residual aggregates of bacteria were allowed to settle for 20 min. The top 500 mL of bacterial supernate was removed and the supernates from two to four tubes were pooled in 50 mL conical tubes. Bacteria were then sonicated on high power in a sonicating waterbath for 20 s to break apart any residual bacterial aggregates. The bacterial suspension was aliquoted into multiple Eppendorf tubes, which were then flash frozen and stored at 270u C. Individual tubes were thawed and titrated to determine CFU for a particular frozen stock. Infection of A549 cells for measurement of HBD2 gene expression and IL-8 levels The A549 cell line is a well characterized Type II alveolar epithelial tumor cell line which was obtained from the American Type Culture Collection (Rockville, MD). A549 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma) with 10% Fetal Bovine Serum (FBS)(Invitrogen) and 1% Antibiotic-Antimycotic (Gibco) at 37uC in 5% CO2. All experi- ments were performed prior to the twenty fifth cell passage. A549 cells were plated at 1.56105 cells/mL, in a volume of 3 mL of DMEM+10% FBS+1% Antibiotic-Antimycotic in 6-well tissue culture treated plates (Greiner) and incubated for 72 h at 37uC, in humidified 5% CO2. The cells were then washed 3 times with 3 mL Iscove’s Modified Dulbecco’s Medium (with sodium bicarbonate; without L glutamine: Sigma), and the media was replaced with 2 mL Iscoves+5% human serum (Human AB Serum, male—SeraCare). Wells were infected with M. abscessus variants at a concentration of 16107 CFU/well. Uninfected control wells were left untreated or in some cases received either 100 ng/mL MALP-2 (TLR6/TLR2 ligand; Imgenex), or 20 ng/ mL recombinant human interleukin-1b (IL1ß) (PeproTech). The plates were then incubated at 37uC, in humidified 5% CO2 for 8 hr. The wells were washed 3 times with 3 mL Iscove’s medium, and 350 mL Qiagen RLT buffer (Qiagen) was added to the wells to lyse the cells. Lysates were placed in RNAse free tubes (VWR) and frozen 280uC for later RNA isolation. Glycopeptidolipid Prevents TLR2 Signaling For experiments measuring A549 IL-8 release, cells were plated at a concentration of 56105 cells/mL using the same medium in 24-well tissue culture treated plates (Greiner), and incubated for 24 h at 37uC, in humidified 5% CO2. The cells were then washed 3 times with 0.5 mL Iscoves medium and the media replaced with Iscoves medium alone. Wells then received medium alone, anti- TLR2 antibody (10 mg/mL)(Ebioscience 16902483) or IgG1 isotype control antibody (10 mg/mL)(Ebioscience 16902483). After a 1 h incubation, wells received no bacteria, or M. abscessus variants at a concentration of 2.56106 CFU/well. After an additional 8 hours the supernates were collected, filtered with 0.2 um centrifugal filters (VWR 82031-358), and frozen at 280uC. Supernates were analyzed per manufacturer’s protocol with the BD Human IL-8 ELISA set (BD555244). Infection of BEAS 2B cells for measurement of IL-8 levels The BEAS 2B cell line, a well-characterized SV40 immortalized cell line derived from normal human bronchial epithelium, was obtained from the ATCC. These cells contain TLR1 through TLR6 and respond to the TLR2 agonist Pam 3 Cys with expression of IL-8 [18,19]. BEAS2b cells were plated at a concentration of 26105 cells per well in 0.5 mL BEBM+BEGM kit (without gentamicin-amphotericin B) (Lonza cc3171 and cc3170), in 24-well tissue culture plates coated with 0.01 mg/mL fibronectin, 0.3 mg/ mL bovine collagen type 1 and 0.01 mg/mL BSA in BEBM (Sigma F1141; Advanced Biomatrix 5005-B; Sigma A9418), and incubated at 37uC, 5% CO2 for 24 hours. The wells were rinsed 3 times with BEBM and refilled with 0.5 mL BEBM. Wells then received medium alone, anti-TLR2 antibody (10 mg/mL)(Ebioscience 16902483) or IgG1 isotype control antibody (10 mg/mL)(E- bioscience 16902483). Wells were then incubated at 37uC, 5% CO2 for 1 hour. After 1 h incubation, wells received no bacteria, or M. abscessus variants at a concentration of 2.56106 CFU/well. After an additional 8 hours, the supernates were collected and filtered with 0.2 um centrifugal filters (VWR 82031-358) and frozen at 280uC. Supernates were analyzed per manufacturers protocol with the BD Human IL-8 ELISA set (BD555244). Real-time PCR assessment of HBD2 gene expression RNA from lysates of A549 cells receiving various treatments was isolated using the RNeasy kit (Qiagen). For HbD2, the primer/ probe sequences and final reaction concentrations were based on previous reports, and were as follows: forward: 59-GAGGAGGC- CAAGAAGCTGC-39 (300 nM); reverse: 59-CGCACGTCTCT- GATGAGGG-39 (300 nM); probe: 59-FAM-TGGCTGATGCG- GATTCAGAAAGGG-TAMRA-39 (250 nM) [16]. The ABI Human Eukaryotic 18S rRNA TaqmanH Gene Expression Assay (Endogenous Control) was used per the manufacturer’s instruction for detection of 18S rRNA. qRTPCR of the RNA was performed using the ABI TaqmanH One Step RT-PCR Master Mix Reagents kit, per manufacturer’s instruction. A no template control was included to verify that amplification only took place in reactions containing RNA. Thermocycling conditions began with reverse transcription, consisting of one cycle at 48uC for 30 minutes, followed by one cycle at 95uC for 10 minutes. PCR consisted of 40 cycles at 95uC for 15 seconds, and 60uC for 60 seconds. Relative quantity was determined using the 22DDCT method [20], using the untreated control as the calibrator sample. Assessment of the effect of siRNA TLR2 knockdown on HBD2 expression in response to M. abscessus Transfection of A549 cells was performed by plating at a concentration of 46105 cells per well in 4 mL DMEM+10% FBS PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29148