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. 2008 Sep;15(9):1450-5.
doi: 10.1128/CVI.00178-08. Epub 2008 Jun 25.

Expression and secretion of cathelicidin LL-37 in human epithelial cells after infection by Mycobacterium bovis Bacillus Calmette-Guérin

Patricia Méndez-Samperio  1 , Elena Miranda, Artemisa Trejo
Affiliations

Expression and secretion of cathelicidin LL-37 in human epithelial cells after infection by Mycobacterium bovis Bacillus Calmette-Guérin

Patricia Méndez-Samperio et al. Clin Vaccine Immunol. 2008 Sep.

Abstract

The antimicrobial cathelicidin LL-37 is considered to play an important role in the innate immune response to tuberculosis infection. However, little is known about the induction and secretion of this antimicrobial peptide in A549 epithelial cells after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), the world's most widely used tuberculosis vaccine. In this study, we investigated the effect of M. bovis BCG on LL-37 mRNA levels in A549 cells by real-time PCR and on protein levels by Western blotting. Treatment of cells with M. bovis BCG upregulates LL-37 mRNA expression in a dose- and time-dependent manner. The quantitative analysis of LL-37 gene expression correlated with our Western blotting results. Moreover, our results demonstrated that treatment of cells with the transcriptional inhibitor actinomycin D effectively inhibited in a concentration-dependent manner the ability of M. bovis BCG to induce LL-37 mRNA expression. Finally, inhibition of the MEK1/2 and p38 mitogen-activated protein kinase (MAPK) signaling pathways reduced M. bovis BCG-mediated LL-37 mRNA expression, a reduction that correlated with the observed high level of downregulation of LL-37 protein induction. Thus, these results indicate that the MEK1/2 and p38 MAPK signaling pathways play a critical role in the regulation of inducible LL-37 gene expression in A549 cells infected with M. bovis BCG.

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Figures

FIG. 1.
FIG. 1.
Induction of LL-37 gene expression by M. bovis BCG in A549 cells. A549 cells were left unstimulated (control) or were stimulated with M. bovis BCG at MOIs of 1:1, 5:1, and 10:1 for 18 h. The kinetics of LL-37 mRNA induction was determined as described above. Total RNA was extracted, and RT-PCR (A and B) or real-time PCR (C and D) was conducted to quantify LL-37 mRNA levels. As a negative control, RNA was omitted from reverse transcription and PCR amplification (no RNA). The β-actin gene was used as a housekeeping gene. In RT-PCR analysis, data shown are representative of four separate experiments. In real-time PCR analysis, results are represented as the means ± SD from at least three independent experiments. *, P < 0.05.
FIG. 2.
FIG. 2.
Effect of actinomycin D on M. bovis BCG-induced LL-37 mRNA expression. A549 cells were infected with M. bovis BCG at an MOI of 10:1 for 18 h after treatment with or without various concentrations of actinomycin D. Total RNA was extracted, and real-time PCR was conducted to quantify LL-37 mRNA levels, normalized to β-actin. Graphs show means ± SD from at least five independent experiments. *, P < 0.05, for comparison with M. bovis BCG cultures that did not receive actinomycin D.
FIG. 3.
FIG. 3.
Detection of LL-37 in cell extracts of A549 cells infected with M. bovis BCG by Western blot analysis. A549 cells were left unstimulated (control) or were stimulated with M. bovis BCG at MOIs of 1:1, 5:1, and 10:1 for 18 h. Total cell lysates were analyzed by Western blotting to observe LL-37 expression. The amount of protein from the lysates was determined by using the Bio-Rad assay kit, and equal amounts of protein were loaded in all wells. Parallel blotting was performed with an antibody to β-actin. Data from a representative experiment are presented, and similar results were obtained in four independent experiments.
FIG. 4.
FIG. 4.
Effect of specific MAPK inhibitors on M. bovis BCG-induced LL-37 mRNA expression. A549 cells were left unstimulated (control) or were stimulated with M. bovis BCG at an MOI of 10:1 for 18 h after preincubation with or without different concentrations of the MEK inhibitor U0126 (A) or PD98059 (B) or the p38 MAPK inhibitor SB203580 (C), and real-time PCR was conducted to quantify LL-37 mRNA levels, normalized to β-actin. Graphs show means ± SD from at least three independent experiments. *, P < 0.05, for comparison with M. bovis BCG cultures that did not receive an inhibitor.
FIG. 5.
FIG. 5.
Inhibitors of MAPK block M. bovis BCG-induced LL-37 protein expression. Cells were unstimulated (control) or stimulated with M. bovis BCG at an MOI of 10:1 after preincubation with or without the MEK inhibitor U0126 (20 μM) or PD98059 (10 μM) or the p38 MAPK inhibitor SB203580 (10 μM) or with vehicle (DMSO), and total cell lysates were analyzed by Western blotting to observe LL-37 protein expression. Parallel blotting was performed with an antibody to β-actin. Data from a representative experiment are presented, and similar results were obtained in three independent experiments.
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