Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; Faculty of Health Sciences, Universiti Teknologi MARA, Cawangan Pulau Pinang, Kampus Bertam, 13200 Kepala Batas, Penang, Malaysia.
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
Infect Genet Evol. 2020 Nov;85:104532. doi: 10.1016/j.meegid.2020.104532. Epub 2020 Sep 8.
Shigella is an intracellular bacterial pathogen that causes bacterial dysentery called shigellosis. The assessment of pro- and anti-inflammatory mediators produced by immune cells against this bacteria are vital in identifying the effectiveness of the immune reaction in protecting the host. In Malaysia, Shigella is ranked as the third most common bacteria causing diarrheal disease among children below 5 years old. In the present study, we aim to examine the differential cytokine gene expressions of macrophages in response to two types of clinical strains of Shigella flexneri 2a (S. flexneri 2a) isolated from patients admitted in Hospital Universiti Sains Malaysia, Kelantan, Malaysia. THP-1-derived macrophages, as the model of human macrophages, were infected separately with S. flexneri 2a mild (SH062) and virulence (SH057) strains for 6, 12, and 24 h, respectively. The gene expression level of inflammatory mediators was identified using real-time quantitative polymerase chain reaction (RT-qPCR). The production of nitric oxide (NO) by the macrophages was measured by using a commercialized NO assay kit. The ability of macrophages to kill the intracellular bacteria was assessed by intracellular killing assay. Induction of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1β, IL-6, IL-12, inducible NO synthase (iNOS), and NO, confirmed the pro-inflammatory reaction of the THP-1-derived macrophages in response to S. flexneri 2a, especially against the SH507 strain. The SH057 also induced a marked increase in the expression levels of the anti-inflammatory cytokine mRNAs at 12 h and 24 h post-infection. In the intracellular killing assay, both strains showed less viable, indicating the generation of pro-inflammatory cytokines in the presence of iNOS and NO was crucial in the stimulation of macrophages for the host defense against shigellosis. Transcription analysis of THP-1-derived macrophages in this study identifies differentially expressed cytokine genes that correlated with the virulence factor of S. flexneri 2a.
志贺氏菌是一种细胞内细菌病原体，可引起细菌性痢疾，称为志贺氏菌病。评估免疫细胞针对这种细菌产生的促炎和抗炎介质对于确定免疫反应在保护宿主方面的有效性至关重要。在马来西亚，志贺氏菌是导致 5 岁以下儿童腹泻病的第三大常见细菌。在本研究中，我们旨在检查对两种来自马来西亚吉兰丹大学医院住院患者的福氏志贺氏菌 2a 临床分离株（福氏志贺氏菌 2a）反应的巨噬细胞的差异细胞因子基因表达。THP-1 衍生的巨噬细胞作为人巨噬细胞的模型，分别感染福氏志贺氏菌 2a 弱毒株（SH062）和毒力株（SH057）6、12 和 24 小时。使用实时定量聚合酶链反应（RT-qPCR）鉴定炎症介质的基因表达水平。通过使用商业化的 NO 测定试剂盒测量巨噬细胞产生的一氧化氮（NO）。通过细胞内杀伤测定评估巨噬细胞杀死细胞内细菌的能力。肿瘤坏死因子-α（TNFα）、白细胞介素（IL）-1β、IL-6、IL-12、诱导型一氧化氮合酶（iNOS）和 NO 的诱导证实了 THP-1 衍生的巨噬细胞对福氏志贺氏菌 2a 的促炎反应，特别是对 SH507 株的反应。SH057 还在感染后 12 小时和 24 小时诱导抗炎细胞因子 mRNA 的表达水平显着增加。在细胞内杀伤测定中，两种菌株的活菌数均减少，表明在 iNOS 和 NO 的存在下产生促炎细胞因子对于刺激巨噬细胞抵抗志贺氏菌病的宿主防御至关重要。本研究中 THP-1 衍生巨噬细胞的转录分析确定了与福氏志贺氏菌 2a 的毒力因子相关的差异表达细胞因子基因。
