J Bacteriol Virol.  2007 Jun;37(2):91-103. 10.4167/jbv.2007.37.2.91.

Identification and Functional analysis of Gene Expression in Mycobacterium tuberculosis-infected Human Monocytic Cells Under Hypoxic Conditions

• Lee JS
• Oh JH
• Son JW
• Song CH
• Kim HJ
• Park JK
• Paik TH
• Jo EK

• KMID: 2055043
• DOI: http://doi.org/10.4167/jbv.2007.37.2.91

Abstract

Mycobacterium tuberculosis-induced granulomatous lesions, particularly those undergoing central caseation, are known as hypoxic. To analyze the host genes associated with hypoxic conditions from cells infected with M. tuberculosis, we performed GeneChip analyses on mRNA from M. tuberculosis H37Rv-treated human monocytic THP-1 cells cultured in oxygen-depleted status for 18 h. The expression of 99 genes was altered, including those involved in intracellular signaling, energy production, and protein metabolism, as revealed by stringent microarray data analysis. Most notably, mRNA expression of chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) was significantly up-regulated in M. tuberculosis-infected cells under hypoxic conditions. We further analyzed the CCL20 expression in peripheral blood mononuclear cells (PBMCs) and monocyte derived macrophages (MDMs) from healthy controls and TB patients. A comparative analysis has revealed that the mRNA and protein expression of CCL20 were prominently up-regulated in PBMCs, and MDMs from TB patients, compared with healthy controls. Collectively, these data show that the gene expression of CCL20 was up-regulated in M. tuberculosis H37Rv-infected human monocytic THP-1 cells cultured in hypoxic conditions. In addition, the production of CCL20 is substantially increased in cells from TB patients than in healthy controls, suggesting an important role of CCL20 in the immunopathogenesis during TB infection.

Keyword

Mycobacterium tuberculosis; Hypoxic; CCL20; 30-kDa antigen

MeSH Terms

Gene Expression*
Humans*
Macrophages
Metabolism
Mycobacterium tuberculosis
Mycobacterium*
RNA, Messenger
Statistics as Topic
Tuberculosis
RNA, Messenger

Figure

• 

  Figure 1. Comparative analysis of human cathepsin D and CCL20 mRNA expression in M. tuberculosis-infected THP-1 cells under normoxic and hypoxic conditions. (A) RT-PCR analysis was used to determine the differential expression of cathepsin D and CCL20 genes in THP-1 cells cultured under normoxic and hypoxic conditions. normoxic, aerobically incubated (20% O2); Hypoxic, incubated in hypoxic conditions (2% O2); Un, unstimluated. Rv, M. tuberculosis H37Rv-infected; Ra, M. tuberculosis H37Ra-infected. (B) Values are ratios of band density to band density of GAPDH mRNA at each condition and are mean ± SEM. ∗∗, p<0.01; ∗∗∗, p<0.001

• 

  Figure 2. Kinetics of CCL20 induction in response to 30-kDa Ag. PBMCs and MDMs were incubated with different doses of 30-kDa Ag (A) and different time point (B). Supernatants were harvested after the times indicated and CCL20 levels were measured by ELISA.

• 

  Figure 3. CCL20 expressions in PBMCs and MDMs from healthy controls (HC) and TB patients (E-TB and Cr-TB) in response to 30-kDa Ag. (A) The 30-kDa Ag was added to PBMCs at 1.0 μg/ml for 6 hr. RT-PCR analysis was done to determine the comparative expression of CCL20 mRNA from PBMCs from HCs and TB patients. PBMCs (B) and MDMs (C) from HCs and TB patients were stimulated by 30-kDa Ag. After 18-hr stimulation, the supernatants were harvested and CCL20 protein levels were measured by ELISA. Closed circle, unstimulated; open circle, stimulated with the 30-kDa Ag. E-TB, early diagnosed pulmonary tuberculosis patients; Cr-TB, chronic tuberculosis patients. ∗, p<0.05; ∗∗, p<0.01; ∗∗∗, p<0.001

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