Nkwouano Vanesa, Witkowski Sven, Rehberg Nidja, Kalscheuer Rainer, Nausch Norman, Mayatepek Ertan, Jacobsen Marc
Department of General Pediatrics, Neonatology, and Pediatric Cardiology, University Children's Hospital, Heinrich Heine University, Duesseldorf, Germany.
Institute for Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University, Duesseldorf, Germany.
PLoS One. 2017 Feb 15;12(2):e0171817. doi: 10.1371/journal.pone.0171817. eCollection 2017.
Macrophages are natural host cells for pathogenic mycobacteria, like Mycobacterium tuberculosis (M.tb). Immune surveillance by T cells and interaction with M.tb infected macrophages is crucial for protection against M.tb reactivation and development of active tuberculosis. Several factors play a role in the control of M.tb infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional in vitro assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional in vitro assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a Mycobacterium bovis BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated in vitro and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. M.tb protein specific CD4+ and CD8+ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4+ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional in vitro assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.
巨噬细胞是致病性分枝杆菌（如结核分枝杆菌）的天然宿主细胞。T细胞的免疫监视以及与结核分枝杆菌感染的巨噬细胞的相互作用对于预防结核分枝杆菌再激活和活动性结核病的发展至关重要。有几个因素在控制结核分枝杆菌感染中发挥作用，但可靠的生物标志物仍然难以捉摸。一个主要障碍是缺乏功能性体外检测方法，这些方法能够同时测定：i）分枝杆菌的根除；ii）对宿主巨噬细胞的细胞毒性作用；以及iii）效应T细胞功能。我们基于对感染了含有四环素诱导型活/死报告质粒（LD-BCG）的牛分枝杆菌卡介苗菌株的单核细胞衍生巨噬细胞（MDM）进行流式细胞术分析，建立了一种新型功能性体外检测方法。在体外生成健康人类供体的MDM，并感染确定数量的LD-BCG。在MDM/LD-BCG与自体效应T细胞短期共孵育后或在抗生素存在的情况下，通过流式细胞术确定含有活的或死的LD-BCG的MDM比例。同时测量添加的特定数量的珠子，以便比较样品之间MDM的绝对数量。确定了T细胞亚群对抗分枝杆菌细胞毒性和MDM凋亡的不同影响。用利福平处理的MDM/LD-BCG的流式细胞术测量结果与分枝杆菌菌落形成单位和荧光显微镜结果高度相关。与预激活的效应T细胞共培养以浓度依赖的方式降低了LD-BCG和MDM的活力。结核分枝杆菌蛋白特异性CD4+和CD8+ T细胞对抗分枝杆菌细胞毒性的贡献相似，但CD4+ T细胞在感染的MDM中诱导更高水平的凋亡。这种新型检测方法能够快速定量抗分枝杆菌细胞毒性并表征效应功能。我们的功能性体外检测方法有可能有助于鉴定针对结核病的保护性T细胞反应的生物标志物。
