Ashtekar Amit R, Zhang Ping, Katz Jannet, Deivanayagam Champion C S, Rallabhandi Prasad, Vogel Stefanie N, Michalek Suzanne M
Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, BBRB 258/5, Birmingham, AL 35294-2170, USA.
J Leukoc Biol. 2008 Dec;84(6):1434-46. doi: 10.1189/jlb.0308215. Epub 2008 Aug 15.
Francisella tularensis is the causative agent of tularemia, a severe, debilitating disease of humans and other mammals. As this microorganism is also classified as a "category-A pathogen" and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis, including the heat shock protein DnaK, have been proposed for use in a potential subunit vaccine. In this study, we characterized the innate immune response of murine bone marrow-derived dendritic cells (DC) to F. tularensis DnaK. Recombinant DnaK was produced using a bacterial expression system and purified using affinity, ion-exchange, and size-exclusion chromatography. DnaK induced the activation of MAPKs and NF-kappaB in DC and the production of the proinflammatory cytokines IL-6, TNF-alpha, and IL-12 p40, as well as low levels of IL-10. DnaK induced phenotypic maturation of DC, as demonstrated by an up-regulation of costimulatory molecules CD40, CD80, and CD86. DnaK stimulated DC through TLR4 and the adapters MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) that mediated differential responses. DnaK induced activation of MAPKs and NF-kappaB in a MyD88- or TRIF-dependent manner. However, the presence of MyD88- and TRIF-dependent signaling pathways was essential for an optimal, DnaK-induced cytokine response in DC. In contrast, DnaK induced DC maturation in a TRIF-dependent, MyD88-independent manner. These results provide insight about the molecular interactions between an immunodominant antigen of F. tularensis and host immune cells, which is crucial for the rational design and development of a safe and efficacious vaccine against tularemia.
土拉弗朗西斯菌是兔热病的病原体，兔热病是一种严重的、使人衰弱的人类和其他哺乳动物疾病。由于这种微生物也被归类为“A类病原体”和潜在的生物战剂，因此需要一种有效的疫苗。土拉弗朗西斯菌的几种抗原，包括热休克蛋白DnaK，已被提议用于潜在的亚单位疫苗。在本研究中，我们表征了小鼠骨髓来源的树突状细胞（DC）对土拉弗朗西斯菌DnaK的天然免疫反应。使用细菌表达系统产生重组DnaK，并通过亲和、离子交换和尺寸排阻色谱法进行纯化。DnaK诱导DC中MAPK和NF-κB的激活以及促炎细胞因子IL-6、TNF-α和IL-12 p40的产生，以及低水平的IL-10。DnaK诱导DC的表型成熟，共刺激分子CD40、CD80和CD86的上调证明了这一点。DnaK通过TLR4以及介导不同反应的衔接蛋白MyD88和含Toll/IL-1R结构域的衔接蛋白诱导IFN-β（TRIF）刺激DC。DnaK以MyD88或TRIF依赖的方式诱导MAPK和NF-κB的激活。然而，MyD88和TRIF依赖的信号通路的存在对于DC中DnaK诱导的最佳细胞因子反应至关重要。相反，DnaK以TRIF依赖、MyD88非依赖的方式诱导DC成熟。这些结果提供了关于土拉弗朗西斯菌的一种免疫显性抗原与宿主免疫细胞之间分子相互作用的见解，这对于合理设计和开发一种安全有效的兔热病疫苗至关重要。
