Supplementary Material for: Attenuated Activation of Macrophage TLR9 by DNA from Virulent Mycobacteria

Alveolar macrophages are the first line of host defence against mycobacteria, but an insufficient host response allows survival of bacteria within macrophages. We aimed to investigate the role of Toll-like receptor 9 (TLR9) activation in macrophage defence against mycobacteria. Human in vitrodifferentiated macrophages as well as human and mouse alveolar macrophages showed TLR9 mRNA and protein expression. The cells were markedly activated by DNA isolated from attenuated mycobacterial strains (H37Ra and <i>Mycobacterium bovis</i> BCG) as assessed by measuring cytokine expression by real-time PCR, whereas synthetic phosphorothioate-modified oligonucleotides had a much lower potency to activate human macrophages. Intracellular replication of H37Ra was higher in macrophages isolated from TLR9-deficient mice than in macrophages from wild-type mice, whereas H37Rv showed equal survival in cells from wild-type or mutant mice. Increased bacterial survival in mouse macrophages was accompanied by altered cytokine production as determined by Luminex bead assays. In vivo infection experiments also showed differential cytokine production in TLR9-deficient mice compared to wild-type animals. Both human monocyte-derived macrophages as well as human alveolar macrophages showed reduced activation upon treatment with DNA isolated from bacteria from virulent (<i>M. bovis </i>and H37Rv) compared to attenuated mycobacteria. We suggest attenuated TLR9 activation contributes to the insufficient host response against virulent mycobacteria.

肺泡巨噬细胞是宿主对抗分枝杆菌的第一道防线，但宿主应答不足会使细菌得以在巨噬细胞内存活。本研究旨在探讨Toll样受体9（Toll-like receptor 9，TLR9）激活在巨噬细胞抗分枝杆菌防御过程中的作用。实验中，人体外分化巨噬细胞、人及小鼠肺泡巨噬细胞均检测到TLR9的mRNA与蛋白表达。通过实时聚合酶链反应（real-time PCR）检测细胞因子表达水平发现，减毒分枝杆菌菌株（H37Ra与牛分枝杆菌卡介苗（Mycobacterium bovis BCG））提取的DNA可显著激活上述细胞；而合成硫代磷酸酯修饰寡核苷酸激活人巨噬细胞的能力则显著较弱。从TLR9缺陷型小鼠体内分离的巨噬细胞中，H37Ra的胞内复制水平显著高于野生型小鼠来源的巨噬细胞；而H37Rv在野生型与突变型小鼠巨噬细胞内的存活水平无明显差异。小鼠巨噬细胞内细菌存活增加的同时，伴随细胞因子生成的改变，该结果通过Luminex微球检测技术（Luminex bead assays）得以验证。体内感染实验同样显示，与野生型小鼠相比，TLR9缺陷型小鼠体内的细胞因子生成存在差异。相较于减毒分枝杆菌，从强毒菌株（牛分枝杆菌（Mycobacterium bovis）与H37Rv）提取的DNA处理后，人单核细胞来源巨噬细胞及人肺泡巨噬细胞的激活水平均有所降低。本研究提示，TLR9激活不足是宿主对抗强毒分枝杆菌应答不足的潜在机制之一。