M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression

Abstract

Background: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG.

Results: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA.

Conclusion: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.

Figure 1

Mycobacterium-induced cell death in THP-1 cells. THP-1 cells were plated on coverslips at 2 × 10⁵ per well and infected with either M. leprae or BCG at a multiplicity of infection (MOI) of 10 and 20 per cell for 18 h. Cells were stained for viability using acridine orange – ethidium bromide under which, viable cells appear green while apoptotic cells appear red/orange. A. unstimulated cells, B – C. M. leprae MOI-10 and MOI-20, D – E. BCG MOI-10 and MOI-20.

Figure 2

M. leprae induces a lower rate of cell death as compared with BCG. THP-1 cells were infected with M. leprae and BCG as described in Fig. 1. A. Cellular viability was assessed after fluorescent staining at 18 and 48 h post-infection. Graphs show the results from 5 independent experiments with SD shown as 'y' error bar. '*' denotes significant differences (P < 0.05). (i) M. leprae, (ii) BCG. B. Agarose gel electrophoresis of internucleosomal DNA fragmentation in THP-1 cells shows spontaneous (lane 1), or BCG-induced apoptosis at MOI-10 (lane 2) and MOI-20 (lane 3) after 18 h of culture. The molecular markers are indicated to the left (lane M).

Figure 3

Mycobacterium-induced TNFα, Bcl-2, Bad and Bak gene expression. THP-1 cells were infected with M. leprae and BCG as described previously and total cellular RNA was extracted, reverse transcribed and PCR was carried out for β-actin, TNFα, Bcl-2, Bad and Bak genes. Gene expression was quantified using scanning densitometry. Panels illustrate gene expression in unstimulated cells 'sp', M. leprae 'ML10', 'ML20', and BCG 'BCG10' and 'BCG20' infected cells at 18 h post-stimulation in a representative experiment. Graphs illustrate expression of TNFα, Bcl-2, Bad and Bak mRNA relative to β-actin in each case.

Figure 4

M. leprae-, and BCG- induced time course expression of Bad and genes. THP-1 cells were infected with M. leprae, live and heat-killed BCG for 6, 18 and 48 h at MOI of 10 each. RNA was extracted, converted to cDNA and PCR carried out for Bad, Bak and β-actin genes. Graphs illustrate results obtained from the mean of 3 independent experiments. A, D. M. leprae-, B, E. live BCG- and C, F. heat killed BCG-induced Bad/βactin and Bak/βactin mRNA expression ratios; Spon (unstimulated cells).

Figure 5

Differential downregulation of Bad/Bcl-2 and Bak/Bcl-2 mRNA by M. leprae as compared with BCG. Gene expression was determined in cells infected with either M. leprae or BCG at 18 h post-infection. mRNA expression was quantified using densitometry and graphs illustrate the mean pro-/anti-apoptotic gene ratios of 3 independent experiments. A. Bad/Bcl-2 and B. Bak/Bcl-2 mRNA expression.

Figure 6

M. leprae upreglates Mcl-1 mRNA in THP-1 cells. THP-1 monocytes were infected with M. leprae or BCG at MOI-10 and RNA was harvested after 18 and 48 h of culture. mRNA was reverse transcribed and used for real-time PCR quantification of Mcl-1 and HuPO genes. Relative quantification data of 3 independent experiments calculated using the C_T method is illustrated.