A549 cells increase HβD2 gene expression in response to <i>M. abscessus</i> variants lacking GPL, but not the <i>M. abscessus</i> 390S variant expressing GPL.

<p>A549 cell monolayers were uninfected or challenged with <i>M. abscessus</i> variants 390R or 390S. In addition, some uninfected A549 cell monolayers were treated with IL1β or MALP-2 as controls for the ability of A549 cells to upregulate HβD2 gene expression. After 8 hours, HβD2 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 +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> 390S versus 390R and 390V; <i>P</i><0.05, <i>t</i>-test.</p

Antibody to TLR2 decreases IL-8 release from A549 cells in response to the <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant.

<p>A549 cell monolayers were preincubated with antibody to TLR2 or isotype control antibody and then received no bacteria or were challenged with the <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant. Culture supernates were collected after 8 h and assayed by ELISA for IL-8. Data are means ± SEM of two experiments done in triplicate. * 390SΔ<i>mmpL4b</i> + anti-TLR2 antibody versus 390SΔ<i>mmpL4b</i> alone and 390SΔ<i>mmpL4b</i> + isotype antibody; p<0.01, <i>t</i>-test.</p

TLR2 siRNA treatment decreases HβD2 gene expression in A549 cells challenged with the <i>M. abscessus</i> 390S <i>mmpL4b</i> deletion mutant.

<p>A549 cells were transfected with scrambled RNA or TLR2 siRNA for 48 h with some cell monolayers then challenged with <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>. After 8 h, HβD2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative change in HβD2 gene expression over that of the untreated, uninfected group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> TLR2 siRNA <b>+</b><i>M. abscessus</i> 390SΔ<i>mmpL4b</i> versus scrambled RNA + <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>; <i>P</i><0.05, <i>t</i>-test.</p

IL-8 release from BEAS 2B cells in response to <i>M. abscessus</i> variants lacking GPL is mediated by TLR2.

<p>(A) BEAS 2B bronchial epithelial cells received no bacteria or were challenged with <i>M. abscessus</i> variants 390R, 390S and 390V. Culture supernates were collected after 8 h and assayed by ELISA for IL-8. Data are means ± SEM of two experiments done in triplicate. * 390S versus 390R and 390V; p<0.01, <i>t</i>-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 <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant or the 390SΔ<i>mmpL4b</i> complemented mutant. Culture supernates were collected after 8 h and assayed by ELISA for IL-8. Data are means ± SEM of two experiments done in triplicate. * 390SΔ<i>mmpL4b</i> deletion mutant + anti-TLR2 antibody versus 390SΔ<i>mmpL4b</i> deletion mutant alone and versus 390SΔ<i>mmpL4b</i> deletion mutant + isotype antibody; p<0.01. * 390SΔ<i>mmpL4b</i> complemented mutant versus 390SΔ<i>mmpL4b</i> deletion mutant; p<0.01.</p

A <i>M. abscessus</i> 390SΔ<i>mmpL4b</i> deletion mutant lacking GPL has acquired the ability to stimulate HBD2 gene expression in A549 cells.

<p>(A) A549 cell monolayers were uninfected or challenged with <i>M. abscessus</i> variants 390V, 390S or 390SΔ<i>mmpL4b</i>, a deletion mutant lacking the <i>mmpL4b</i> 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 HβD2 gene expression over that of the untreated group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> 390SΔ<i>mmpL4b</i> mutant vs 390S wild type; <i>P</i><0.05, <i>t</i>-test. (B) A549 cell monolayers were uninfected or challenged with <i>M. abscessus</i> 390SΔ<i>mmpL4b</i>, or the complemented 390SΔ<i>mmpL4b</i> mutant. After 8 hours, HβD2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative fold increase in HβD2 gene expression over that of the untreated group and presented as mean +/− SD of measurements from the same experiment performed in triplicate. <b>*</b> 390SΔ<i>mmpL4b</i> complemented versus 390SΔ<i>mmpL4b</i> mutant; <i>P</i><0.05, <i>t</i>-test.</p