Tuberc Respir Dis.  2017 Jan;80(1):77-82. 10.4046/trd.2017.80.1.77.

Mycobacterium tuberculosis ESAT6 and CPF10 Induce Adenosine Deaminase 2 mRNA Expression in Monocyte-Derived Macrophages

Affiliations
  • 1Department of Physiology, Cell and Matrix Research Institute, BK21 Plus KNU Biomedical Convergence Program, Tumor Heterogeneity and Network (THEN) Research Center, Kyungpook National University School of Medicine, Daegu, Korea.
  • 2Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, Korea. kimch@knu.ac.kr jaelee@knu.ac.kr

Abstract

BACKGROUND
Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens.
METHODS
Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured.
RESULTS
CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-γ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor α and interleukin 10, respectively.
CONCLUSION
ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis.

Keyword

Adenosine; Cat Eye Syndrome Chromosome Region, Candidate 1 Protein, Human; Mycobacterium tuberculosis Antigen; Macrophage

MeSH Terms

Adenosine Deaminase*
Adenosine*
Animals
Cats
Granulocyte-Macrophage Colony-Stimulating Factor
Healthy Volunteers
Humans
Hypersensitivity, Delayed
Interleukin-10
Macrophage Colony-Stimulating Factor
Macrophages*
Monocytes
Mycobacterium tuberculosis*
Mycobacterium*
Pleural Effusion
RNA, Messenger*
Tumor Necrosis Factor-alpha
Adenosine
Adenosine Deaminase
Granulocyte-Macrophage Colony-Stimulating Factor
Interleukin-10
Macrophage Colony-Stimulating Factor
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

  • Figure 1 ESAT6 and CFP10 stimulation induces increased CECR1 mRNA expression in MDMs. (A) Monocytes isolated from human peripheral blood mononuclear cells were differentiated into MDMs in the absence of cytokines for 7 days, and then cultured with medium alone (control), ESAT6 (5 µg/mL) and CFP10 (5 µg/mL) for 12–18 hours (n=3). CECR1 mRNA expression levels were measured using real-time reverse transcription-polymerase chain reaction and normalized to the internal control human β-actin gene. (B) THP-1 cells were incubated with medium alone, ESAT6 (5 µg/mL) and CFP10 (5 µg/mL). Data was expressed for three independent experiments. (C, D) Monocytes were differentiated into MDMs in the presence of M-CSF (50 ng/mL) or GM-CSF (50 ng/mL) for 7 days, following which they were incubated with medium alone (control), ESAT6 (5 µg/mL), and CFP10 (5 µg/mL) for 12–18 hours (n=6). (E) M-CSF–treated MDMs from 13 different individuals were stimulated with either ESAT6 (5 µg/mL) or CFP10 (5 µg/mL) for 12–18 hours. (F) CECR1 mRNA expression levels in response to ESAT6 or CFP10 stimulation were compared in the presence and absence of IFN-γ (50 U/mL) (n=4). Each column and bar represent the mean and standard error values in Figure 1A–D. Differences were analyzed by Wilcoxon-paired signed rank test, *p<0.05. ESAT6: early secretory antigenic target protein 6; CFP10: culture filtrate protein 10; CECR1: cat eye syndrome chromosome region, candidate 1; MDM: monocyte-derived macrophage; M-CSF: macrophage colony-stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; IFN-γ: interferon γ.

  • Figure 2 Cytokine expression levels in MDMs in response to ESAT6 and CFP10 stimulation (A and D, n=3; B, C, E, and F, n=8). (A–C) IL-6, TNF-α, and IL-10 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10 than in controls. The human β-actin gene served as an internal control. (D–F) There were significantly positive correlations between CECR1 and TNF-α, as well as CECR1 and IL-10 mRNA expression levels. Each column and bar represent the mean and standard error values in Figure 2A–C. Differences were analyzed by Wilcoxon-paired signed rank test in Figure 2A–C and by Spearman correlation (ρ) analysis in Figure 2D–F, *p<0.05. NS: not significant; MDM: monocyte-derived macrophage; ESAT6: early secretory antigenic target protein 6; CFP10: culture filtrate protein 10; IL-6: interleukin 6; TNF-α: tumor necrosis factor α; IL-10: interleukin 10; CECR1: cat eye syndrome chromosome region, candidate 1.


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