Published online before print August 15, 2008

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(Journal of Leukocyte Biology. 2008;84:1434-1446.)
© 2008 by Society for Leukocyte Biology

TLR4-mediated activation of dendritic cells by the heat shock protein DnaK from Francisella tularensis

Amit R. Ashtekar^*, Ping Zhang, Jannet Katz, Champion C. S. Deivanayagam, Prasad Rallabhandi, Stefanie N. Vogel and Suzanne M. Michalek^*,1

^* Departments of Microbiology,
Pediatric Dentistry, and
Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, USA; and
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA

¹ Correspondence: Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, BBRB 258/5, Birmingham, AL 35294-2170, USA. E-mail: suemich{at}uab.edu

ABSTRACT

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-B in DC and the production of the proinflammatory cytokines IL-6, TNF-, 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-β (TRIF) that mediated differential responses. DnaK induced activation of MAPKs and NF-B 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.

Key Words: Toll-like receptors • MyD88 • TRIF • MAP kinases • NF-B

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