Abstract
Macrophage apoptosis exerts an efficient mechanism in controlling intracellular infection during innate immune response against various pathogens including malaria parasites. This study was carried out to determine the apoptosis activity in mouse macrophage cell line J774A.1 infected with a Mycobacterium bovis bacille Calmette-Guerin (BCG) clone and a recombinant BCG clone expressing the C-terminus of merozoite surface protein-1 (BCG-MSP1C) of Plasmodium falciparum for 48 h. In this study, a parent BCG cells was used as a control. The nuclear staining with Hoechst 33342 showed that the BCG-MSP1C cells was capable of increasing the nuclear condensation and morphological stages of apoptosis in the infected cells compared to the BCG-infected cells and the lipopolysaccharide (LPS)-stimulated cells. The flow cytometric analysis using Annexin-V and Propidium iodide (PI) staining confirmed that the BCG-MSP1C cells significantly increased the percentage of early apoptotic activity in the infected macrophage higher than the one stimulated by the parent BCG cells and LPS. This apoptotic response corresponded with the reduction of the anti-apoptotic Bcl-2 protein expression and higher p53 expression. The colorimetric assay demonstrated that the BCG cells capable of stimulating higher production of caspase-1, –3, –8 and –9 while the BCG-MSP1C cells stimulated the expression of caspase-1 and -9 in the infected macrophages, suggesting the involvement of mitochondrial-mediated (intrinsic) pathway of apoptosis. In conclusion, both the BCG and BCG-MSP1C cells are capable of inducing macrophage apoptosis activity in the mouse macrophage cell line J774A.1. This mechanism is important for the elimination of pathogens such as malaria parasite during the phagocytosis activity of macrophage. However, the BCG-MSP1C cells showed higher apoptosis activity than those produced by the parent BCG cells.
Highlights
Malaria remains the public health concerns owing to the high rate of mortality and morbidity (Wiwanitkit 2011)
Mouse macrophage cell line J774A.1 (ATCC® TIB67TM) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, USA) supplemented with 1% of 100U/mL penicillin, 100 μg/ml streptomycin (Gibco,USA) and 10% fetal bovine serum (Gibco,USA). 5 × 105 cells/ml were grown in a 25 cm2 tissue culture flask and incubated at 37°C in the presence of 5% CO2 in a humidified incubator
The BCGMSP1C containing the MSP-1C of Plasmodium falciparum was constructed by Nurul (2007) using assembly Polymerase chain reaction (PCR) in the previous study.The stability of the MSP-1C gene of Plasmodium falciparum in bacille Calmette-Guerin (BCG)-MSP1C cells was determined using PCR and the protein expression was determined by immunocytochemistry (ICC) analysis using specific antibody against MSP-1C (Dhaniah et al 2014)
Summary
Malaria remains the public health concerns owing to the high rate of mortality and morbidity (Wiwanitkit 2011) It annually affects millions of people throughout the world, especially older people and pregnant ladies. Plasmodium falciparum causes the most serious pathologies of malaria disease in human due to its capability to multiply rapidly in the blood. Infections with this parasite can be lethal in the absence of quick detection of the disease (Sinden & Gilles 2002; Snow et al 2005; Ministry of Health Malaysia 2014; World Health Organization 2015)
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