Cholesterol oxidase (ChoD) is an enzyme that is involved but is dispensable in the process of cholesterol degradation by Mycobacterium tuberculosis (Mtb). Interestingly, ChoD is a virulence factor of Mtb, and it strongly modulates the function of human macrophages in vitro, allowing the intracellular survival of bacteria. Here, we determined the immunogenic activity of recombinant ChoD from Mtb in a mouse model. We found that peritoneal exudate cells obtained from mice injected i.p. with ChoD but not those from mice injected with PBS responded in vitro with highly spontaneous, as well as phorbol 12-myristate 13-acetate (PMA)-stimulated, production of reactive oxygen species (ROS). However, ChoD significantly reduced the ROS response to PMA in re-stimulated cells in vitro. The cytokine secretion pattern in mice immunized s.c. with ChoD emulsified with incomplete Freund’s adjuvant (IFA) showed evidence of Th2-induced or proinflammatory immune responses. The main cytokines detected in sera were interleukin (IL) 6 and 5, tumour necrosis factor α (TNF-α) and monocyte chemoattractant protein 1, while IL-2 and IL-12 as well as interferon γ were undetectable. Similarly, ChoD protein alone activated THP-1-derived macrophages to release proinflammatory IL-6, IL-8 and TNF-α, in vitro. Moreover, a statistically significant predominance of the IgG1 isotype over that of IgG2a in the sera of mice immunized with ChoD/IFA was observed. In conclusion, we demonstrated here that ChoD of Mtb is an active protein, which is able to induce the immune response both in vivo and in vitro.

胆固醇氧化酶 (ChoD) 是一种酶，在结核分枝杆菌(Mtb)的胆固醇降解过程中参与但不是必需的。有趣的是，ChoD 是 Mtb 的毒力因子，它在体外强烈调节人类巨噬细胞的功能，使细菌在细胞内存活。在这里，我们在小鼠模型中确定了来自 Mtb 的重组 ChoD 的免疫原活性。我们发现从腹腔注射 ChoD 的小鼠获得的腹膜渗出液细胞，而不是注射了 PBS 的小鼠的腹膜渗出液细胞在体外有反应具有高度自发性以及佛波醇 12-肉豆蔻酸酯 13-乙酸酯 (PMA) 刺激的活性氧 (ROS) 产生。然而，在体外再刺激的细胞中，ChoD 显着降低了对 PMA 的 ROS 反应。用不完全弗氏佐剂 (IFA) 乳化的 ChoD 皮下免疫小鼠的细胞因子分泌模式显示出 Th2 诱导或促炎免疫反应的证据。血清中检测到的主要细胞因子是白细胞介素（IL）6和5、肿瘤坏死因子α（TNF-α）和单核细胞趋化蛋白1，而IL-2和IL-12以及干扰素γ未检测到。类似地，单独的 ChoD 蛋白在体外激活 THP-1 衍生的巨噬细胞以释放促炎性 IL-6、IL-8 和 TNF-α 。此外，观察到在用 ChoD/IFA 免疫的小鼠的血清中 IgG1 同种型比 IgG2a 具有统计学显着优势。总之，我们在这里证明 Mtb 的 ChoD 是一种活性蛋白，它能够在体内和体外诱导免疫反应。