Apoptosis Activity of the Mouse Macrophage Cell Line J774A.1 Infected with a Recombinant BCG consisting the C-Terminus of Merozoite Surface Protein-1 of Plasmodium falciparum
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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.
Apoptosis makrofaj merupakan mekanisme yang berkesan dalam mengawal jangkitan intrasel semasa gerak balas imun semulajadi terhadap pelbagai patogen termasuk parasit malaria. Kajian ini dijalankan untuk menentukan aktiviti apoptosis dalam sel makrofaj mencit J774A.1 yang dijangkiti klon Mycobacterium bovis BCG dan BCG rekombinan yang mengekspreskan terminus C protein permukaan merozoit-1 (BCG-MSP1C) daripada Plasmodium falciparum selama 48 jam. Kajian ini menggunakan sel BCG sebagai kawalan. Pewarnaan nukleus menggunakan Hoest 33342 menunjukkan bahawa sel BCG-MSP1C berupaya meningkatkan kondensasi nuklear dan peringkat morfologi apoptosis dalam sel makrofaj yang dijangkiti secara signifikan berbanding sel yang dijangkiti oleh sel BCG dan sel yang dirangsang dengan LPS. Analisis flow sitometri menggunakan pewarnaan Annexin-V dan PI membuktikan bahawa sel BCG-MSP1C meningkatkan peratusan aktiviti apoptotik awal didalam sel makrofaj mencit yang dijangkiti berbanding sel yang dijangkiti oleh BCG dan dirangsang dengan LPS. Gerak balas apoptosis yang ditunjukkan ini seiring dengan pengurangan pengekpresan protein anti-apoptotik Bcl-2 dan peningkatan pengekspresan protein p53. Ujian permeteran warna menunjukkan sel BCG berupaya meningkatkan mengekspreskan aktiviti kaspase-1, -3, -8 dan -9 manakala sel BCG-MSP1C hanya mengaktifkan pengekspresan kaspase-1 and -9 di dalam sel makrofaj yang dijangkiti, mencadangkan penglibatan laluan apoptosis mitokondria (intrinsik). Sebagai kesimpulan, kedua-dua sel BCG dan BCG-MSP1C berupaya meningkatkan aktiviti apoptosis di dalam sel makrofaj mencit, J774A.1. Mekanisme ini adalah penting untuk menyingkirkan patogen seperti parasit malaria semasa aktiviti fagositosis makrofaj. Walaubagaimanapun, sel BCGMSP1C menunjukkan aktiviti apoptosis yang lebih tinggi berbanding sel BCG.
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