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. 2011;6(12):e29148.
doi: 10.1371/journal.pone.0029148. Epub 2011 Dec 21.

Mycobacterium Abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HβD2 Gene Expression and IL-8 Release

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Mycobacterium Abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HβD2 Gene Expression and IL-8 Release

Lisa B Davidson et al. PLoS One. .
Free PMC article

Abstract

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A549 cells increase HβD2 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 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. * 390S versus 390R and 390V; P<0.05, t-test.
Figure 2
Figure 2. A M. abscessus 390SΔmmpL4b deletion mutant lacking GPL has acquired the ability to stimulate HBD2 gene expression in A549 cells.
(A) A549 cell monolayers were uninfected or challenged with M. abscessus variants 390V, 390S or 390SΔmmpL4b, a deletion mutant lacking the mmpL4b 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. * 390SΔmmpL4b mutant vs 390S wild type; P<0.05, t-test. (B) A549 cell monolayers were uninfected or challenged with M. abscessus 390SΔmmpL4b, or the complemented 390SΔmmpL4b 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. * 390SΔmmpL4b complemented versus 390SΔmmpL4b mutant; P<0.05, t-test.
Figure 3
Figure 3. TLR2 siRNA treatment decreases TLR2 gene expression in uninfected and M. abscessus -infected A549 cells.
As a first step in assessing the role of TLR2 in respiratory epithelial responses to M. abscessus, the effect of transfection of A549 cells with TLR2 siRNA on TLR2 gene expression was assessed. (A) Uninfected A549 cells were transfected with scrambled RNA or TLR2 siRNA. After 48 h TLR2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative difference in TLR2 gene expression using the A549 monolayers receiving scrambled RNA as the reference, with data presented as mean +/− SD of measurements from the same experiment performed in triplicate. *TLR2 transfected cells versus cells receiving scrambled RNA P<0.05, t-test. (B) Western blotting of A549 cell extracts from (A) demonstrates decreased TLR2 in cells treated with TLR2 siRNA. (C) A549 cell monolayers were either untreated or transfected with RNA, and either uninfected or challenged with M. abscessus 390SΔmmpL4b. TLR2 gene expression was quantified by real-time PCR. The results of real-time PCR are expressed as the relative difference in TLR2 gene expression using uninfected A549 monolayers receiving scrambled RNA as the reference, with data presented as mean +/− SD of measurements from the same experiment performed in triplicate. * TLR2 siRNA + M. abscessus 390SΔmmpL4b versus scrambled RNA + M. abscessus 390SΔmmpL4b; P<0.05, t-test.
Figure 4
Figure 4. TLR2 siRNA treatment decreases HβD2 gene expression 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 390SΔmmpL4b. 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. * TLR2 siRNA + M. abscessus 390SΔmmpL4b versus scrambled RNA + M. abscessus 390SΔmmpL4b; P<0.05, t-test.
Figure 5
Figure 5. Antibody to TLR2 decreases IL-8 release from A549 cells in response to the M. abscessus 390SΔmmpL4b 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 390SΔmmpL4b 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. * 390SΔmmpL4b + anti-TLR2 antibody versus 390SΔmmpL4b alone and 390SΔmmpL4b + isotype antibody; p<0.01, t-test.
Figure 6
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 ± 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 390SΔmmpL4b deletion mutant or the 390SΔmmpL4b 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. * 390SΔmmpL4b deletion mutant + anti-TLR2 antibody versus 390SΔmmpL4b deletion mutant alone and versus 390SΔmmpL4b deletion mutant + isotype antibody; p<0.01. * 390SΔmmpL4b complemented mutant versus 390SΔmmpL4b deletion mutant; p<0.01.

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