J Bacteriol Virol.  2012 Jun;42(2):121-132. 10.4167/jbv.2012.42.2.121.

Intracellular Signaling Pathways that Regulate Macrophage Chemokine Expression in Response to Mycobacterium abscessus

• Kim TS
• Lee HM
• Yoo H
• Park YK
• Jo EK

• KMID: 1717683
• DOI: http://doi.org/10.4167/jbv.2012.42.2.121

Abstract

Mycobacterium abscessus (Mabc) is an emerging human pathogen. Less is known about the host immune response to Mabc than to M. tuberculosis. Here, we examined the intracellular signaling pathways that govern the expression of chemokines including (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 2 (CXCL2) in macrophages after infection with Mabc. Specifically, Mabc triggered the generation of reactive oxygen species (ROS) and the production of CCL2 and CXCL2 in murine bone marrow-derived macrophages (BMDMs). Mabc-induced CCL2, but not CXCL2, was dependent on the generation of ROS. Toll-like receptor (TLR) 2, MyD88, but not TRIF, was required for Mabc-induced CCL2 and CXCL2 expression. Additionally, Mabc infection significantly induced nuclear factor (NF)-kappaB nuclear translocation and luciferase activity. The activation of NF-kappaB was required for Mabc-induced CCL2, but not CXCL2 expression. Moreover, Mabc-induced ROS generation was required for NF-kappaB activation. Treatment of BMDMs with Mabc rapidly induced the activation of mitogen-activated protein kinase (MAPKs) pathways. Interestingly, CCL2 expression was dependent on the activation of JNK and ERK1/2 pathways, whereas it was negatively regulated by the p38 MAPK pathway. In contrast, Mabc-dependent CXCL2 expression was not regulated by MAPK pathways. These data suggest that intracellular ROS generation is required for innate and inflammatory responses during Mabc infection of macrophages.

Keyword

Mycobacterium abscessus; CCL2; CXCL2; TLR; MyD88

MeSH Terms

Chemokines
Humans
Luciferases
Macrophages
Mycobacterium
NF-kappa B
p38 Mitogen-Activated Protein Kinases
Protein Kinases
Reactive Oxygen Species
Toll-Like Receptors
Tuberculosis
Chemokines
Luciferases
NF-kappa B
Protein Kinases
Reactive Oxygen Species
Toll-Like Receptors
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 Mabc induce intracellular ROS generation and production of CCL2 and CXCL2 in BMDMs. (A) BMDMs were infected with Mabc (moi = 3) for 30 min in the absence or presence of DPI (10 µM, 45 min). Cells were incubated with DHE (10 µM) for 30 min and subjected to FACS analysis for superoxide production. (B and C) BMDMs were infected with Mabc (moi = 3) for the indicated times. (B) Cells were harvested (0~18 h) and subjected to semi-quantitative RT-PCR analysis of Ccl2 and Cxcl2 mRNA expression. Top, representative gel images. Bottom, densitometry values for Ccl2 and Cxcl2 were normalized to β-actin mRNA levels. Data are from a representative of three independent experiments. (C) Supernatants were harvested (0~48 h) and subjected to ELISA analysis for CCL2 and CXCL2 production. The results are the means ± SD of three independent experiments. U, uninfected; Mabc, M. abscessus.

• 

  Figure 2 Intracellular ROS formation is involved in Mabc-induced chemokine expression in BMDMs. BMDMs were infected with Mabc (moi = 3) in the presence or absence of antioxidant (NAC; 10, 20 or 30 mM), NADPH oxidase inhibitor (DPI; 1, 5 or 10 µM), or Tiron (10, 20 or 30 mM) for 45 min. (A) After 6 h, the cells were harvested and subjected to semi-quantitative RT-PCR analysis of Ccl2 and Cxcl2 mRNA expression. Top, representative gel images. Bottom, densitometry values for Ccl2 and Cxcl2 were normalized to β-actin mRNA levels. Data are from a representative of three independent experiments. (B) After 18 h, the supernatants were harvested and subjected to ELISA analysis of CCL2 and CXCL2 production. The results are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, **p < 0.001 compared with control cultures. U, uninfected; Mabc, M. abscessus; D, solvent control (0.05% DMSO).

• 

  Figure 3 TLR2 and MyD88, but not TRIF, are essential for Mabc-induced chemokine production in BMDMs. BMDMs from (A) TLR2 WT and TLR2 KO or (B) WT, MyD88 KO, and TRIF KO mice were infected with Mabc (moi = 3) for 18 h. The supernatants were harvested and subjected to ELISA analysis of CCL2 and CXCL2 production. The results are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with each stimulator (Mabc, LPS, Pam3CSK4). U, uninfected; L, LPS; P, Pam3CSK4.

• 

  Figure 4 Mabc rapidly induces NF-κB activation in BMDMs. (A) BMDMs were transduced with a NF-κB-Luc reporter plasmid for 36 h, and infected with Mabc (moi = 1, 3 or 9) for 6 h. The cells were harvested and luciferase reporter activity was determined. Luciferase activity results are representative of three independent experiments. (B) BMDMs were infected with Mabc (moi = 3) for 30 min and immunostained with anti-NF-κB p65 and anti-rabbit-Alexa Fluor 488. Top, representative immunofluorescence images. Scale bars = 20 µm. Bottom, relative fluorescence intensities are presented as the means ± SD of three independent samples, with each experiment including at least 200 cells scored in six random fields. (C) BMDMs were infected with Mabc (moi = 3) for the indicated times (0~120 min). The cells were harvested and subjected to Western blot analysis for phosphorylated IKK-α/β and IκBα. β-actin was probed as a loading control. The results are the means ± SD of three independent experiments. U, uninfected; Mabc, M. abscessus

• 

  Figure 5 NF-κB and Mabc-induced chemokine expression. (A and B) BMDMs were infected with Mabc (moi = 3) in presence or absence of BAY 11-7082 (BAY; 0.3, 1 or 3 µM, 45 min) or CAPE (1, 5 or 10 µM, 2 h). (A) After 6 h, cells were harvested and subjected to semi-quantitative RT-PCR analysis for detection of Ccl2 and Cxcl2 mRNA expression. (B) After 18 h, the supernatants were harvested and subjected to ELISA analysis of CCL2 and CXCL2 production. (C) BMDMs were transduced with NF-κB-Luc reporter plasmid for 36 h. Cells were infected with Mabc (moi = 3) for 6 h in presence or absence of NAC (10, 20 or 30 mM), DPI (1, 5 or 10 µM), or Tiron (10, 20 or 30 mM) for 45 min. Cells were harvested and luciferase reporter activity was determined. Luciferase activity results are representative of three independent experiments. The results are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control cultures. U, uninfected; D, solvent control (0.05% DMSO).

• 

  Figure 6 MAPK phosphorylation and Mabc-induced chemokine expression. (A) BMDMs were infected with Mabc (moi = 3) for the indicated times (0~480 min). Cells were harvested and subjected to Western blot analysis of phosphorylated MAPK (JNK, ERK1/2, and p38). β-actin was probed as a loading control. (B) BMDMs were infected with Mabc (moi = 3) for 18 h in presence or absence of JNK inhibitor (SP600125; 5, 20 or 30 µM), MEK-1 inhibitor (U0126; 5, 10 or 20 µM), or p38 inhibitor (SB203580; 1, 5 or 10 µM) for 45 min. The supernatants were harvested and subjected to ELISA analysis of CCL2 and CXCL2 production. The results are the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control cultures. U, uninfected; SP, SP600125; SB, SB203580; D, solvent control (0.05% DMSO).

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