Hye-Mi Lee; Dong-Min Shin; Eun-Kyeong Jo

doi:10.4167/jbv.2009.39.1.1

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Lee, Shin, and Jo: Mycobacterium tuberculosis Induces the Production of Tumor Necrosis Factor-α, Interleukin-6, and CXCL8 in Pulmonary Epithelial Cells Through Reactive Oxygen Species-dependent Mitogen-activated Protein Kinase Activation

Original Article

J Bacteriol Virol 2009;39(1):1-10.

Published online: 18 January 2009

DOI: https://doi.org/10.4167/jbv.2009.39.1.1

Mycobacterium tuberculosis Induces the Production of Tumor Necrosis Factor-α, Interleukin-6, and CXCL8 in Pulmonary Epithelial Cells Through Reactive Oxygen Species-dependent Mitogen-activated Protein Kinase Activation

Hye-Mi Lee, Dong-Min Shin, Eun-Kyeong Jo^∗

Department of Microbiology and Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon, Korea

^∗Corresponding author: Dr. Eun-Kyeong Jo. Department of Microbiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-ku, Daejeon 301-747, Korea. Phone: +82-42-580-8243, Fax: +82-42-585-3686 e-mail: hayoungj@cnu.ac.kr

^∗∗ This work was supported by a grant of the Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National University. The authors declare that they have no competing financial interests.

Revised 9 January 200920 February 2009 Accepted 24 February 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of inflammatory cytokines/chemokines. In this study, we found that Mycobacterium tuberculosis (Mtb)-induced reactive oxygen species (ROS) production is essential for the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and CXC-chemokine ligand (CXCL) 8 through the activation of mitogen-activated protein kinases [MAPKs; extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK] in A549 cells representing alveolar epithelial cells. We observed that Mtb rapidly enhanced ROS production after stimulation in a toll-like receptor (TLR) 2-dependent manner. In addition, Mtb triggered ERK1/2 and p38 MAPK signaling pathways which were dependent on ROS generation in A549 cells. Moreover, Mtb stimulation significantly increased the secretion of TNF-α, IL-6, and CXCL8 over that in untreated controls. Pretreatment of A549 cells with the antioxidant, N-acetylcysteine and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenylene iodonium, substantially inhibited Mtb-induced production of TNF-α, IL-6, and CXCL8. Studies using inhibitors selective for ERK1/2 and p38 MAPK pathways showed that both pathways play an essential role in the induction of TNF-α, IL-6, and CXCL8 at transcriptional levels in A549 cells. Collectively, our findings indicate the critical role of TLR2-dependent ROS in the Mtb-induced inflammatory cytokine/chemokine production in alveolar epithelial cells through MAPK-dependent signaling pathways.

Keywords: Mycobacterium tuberculosis, Tumor necrosis factor-α, Interleukin-6, CXCL8, Alveolar epithelial cells, Reactive oxygen species

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Figure 1.

Mtb-induced ROS generation is dependent on the TLR2, but not TLR4, in A549 cells. (A) The A549 cells were stimulated with Mtb (MOI = 5) for 15 to 30 min and then DHE assays were performed. Representative immunofluorescence images are shown. The data shown are representative of three experiments. (B) The experimental conditions follow the same pattern as outlined in Panel A. The A549 cells were pre-incubated with an anti-TLR2 (αTLR2), anti-TLR4 (αTLR4; both 5, 10 μg/ml) or an isotype control mAb (10 μg/ml), followed by the stimulation with Mtb (MOI = 5) for 30 min. The quantitative data for DHE (for superoxide) fluorescence shown are the mean ± SD of three experiments. Significant differences (∗∗∗, p < 0.001). UN, unstimulated; Iso, isotype control.

Figure 2.

Mtb rapidly induces the phosphorylation of MAPKs by A549 cells. The A549 cells were stimulated with Mtb (MOI = 5) for the indicated times (0~480 min). The cells were harvested and subjected to Western blot analysis for phosphorylated ERK1/2 and p38 MAPK. The same blots were washed and blotted for α-actin as the loading controls. Data are representative of five independent experiments with similar results.

Figure 3.

Intracellular ROS production is essential for Mtb-induced MAPK activation by A549 cells. The A549 cells were pretreated with or without NAC (10, 50, 100 mM), DPI (5, 10, 20 μM) or rotenone (5, 10, 20 μM) for 45 min before stimulation with Mtb (MOI = 5). The cells were harvested after 15 min and subjected to Western blot analysis for phosphorylated ERK1/2 and p38 MAPK. The same blots were washed and blotted for α-actin as the loading controls. Data are representative of three independent experiments with similar results. un, unstimulated; D, solvent control (0.1% DMSO).

Figure 4.

Mtb induces pro-inflammatory cytokines and chemokine production by A549 cells. The A549 cells were stimulated with Mtb (MOI = 5) for the indicated times and then performed ELISA analysis (for TNF-α, IL-6 and CXCL8). Data are the mean ± SD of three experiments.

Figure 5.

The roles of ROS in pro-inflammatory cytokine and chemokine production in response to Mtb stimulation in A549 cells. The A549 cells were pretreated with NAC (10, 50, 100 mM), DPI (5, 10, 20 μM) or rotenone (5, 10, 20 μM) for 45 min, followed by stimulation with Mtb (MOI = 5) for 18 h. The supernatants were harvested at 18 h, and the production of pro-inflammatory cytokines (for TNF-α and IL-6) and chemokine (for CXCL8) was measured by ELISA. Data are the mean ± SD of three experiments. un, unstimulated; D, solvent control (0.1% DMSO). Significant differences (∗∗, p < 0.01;∗∗∗, p < 0.001)

Figure 6.

The roles of MEK1/2 or p38 MAPK on the mRNA expression of TNF-α, IL-6, and CXCL8 in A549 cells. The A549 cells were pretreated in the presence or absence of U0126 (5, 10, 20 μM) or SB203580 (1, 5, 10 μM) for 45 min, and then stimulated with Mtb (MOI = 5) for 6 h. The mRNA expression of TNF-α, IL-6, and CXCL8 was detected by semi-quantitative RT-PCR analysis by using the specific primers. The data shown are representative of three experiments. un, unstimulated; D, solvent control (0.1% DMSO).

Table 1.

Primer sequences

Gene	Primer Sequences
hTNF-α	forward: 5′-CAGAGGGAAGAGTTCCCCAG-3′ reverse: 5′-CCTTGGTCTGGTAGGAGACG-3′
hIL-6	forward: 5′- TGACCCAACCACAAATGC-3′ reverse: 5′-CGAGCTCTGAAACAAAGGAT-3′
hCXCL8	forward: 5′-CATGACTTCCAAGCTGGCCG-3′ reverse: 5′-TTTATGAATTCTCAGCCCTC-3′
β-actin	forward: 5′- ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ reverse: 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGCG-3′

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