Activation Analysis Of Human Dendritic Cells Stimulated With Antigens Of Coxiella Burnetii

Coxiella burnetii, the etiological agent of human Q fever, is an obligateintracellular Gram-negative acidophile. C. burnetii is one of the major bio-warfareand potential bio-terrorism agents, which is high pathogenic and has an ability toresist the adverse environments and physical or chemical factors. Inoculation ofvaccines against Q fever is an effective means for the prevention of C. burnetiiinfection. Although the Q fever vaccine derived from inactivated C. burnetiiwhole-cells has the capability to induced protective immunity against Q fever inhumans, and however the strong side effects elicited by its immunization limit itswidespread application. Q fever vaccine study is currently covered by attenuatedvaccines, subunit vaccines, and molecular vaccines. The development of a definedmolecular vaccine against Q fever involves the determination of candidate moleculesthat have capabilityto elicit protective immunityagainst C. burnetii.Dendritic cells (DCs) are potent antigen-presenting cells (APCs) which bridgethe innate and adaptive immune responses through direct pathogen neutralization,cytokine production, and T cell activation. Immature DCs (iDCs) reside in theperipheral epithelial tissues where they serve as sentinels against invadingmicroorganisms. Contacting with a pathogen typically results in stimulation of DCsvia pattern recognition receptors such as the toll-like receptor (TLR) and subsequentconversion of iDCs into mature DCs (mDCs). mDCs process antigens, expresslymphocyte co-stimulatory molecules, migrate to lymphoid organs and secretecytokines to initiate immune responses. mDCs are ideal biological immune adjuvantsand antigen carriers.In this study, monocytes, which were isolated from human peripheral bloodmononuclear cells, were cultured in medium containing IL-4 and GM-CSF. After 5～6 days of culture, more than 95% of cells converted into iDCs with phenotype ofCD11c+, CD54low, CD83low, CD40low, CD58low, CD86low, and CD80low, which wasdetermined by flow cytometry. Human iDCs were stimulated with phase I and phaseII strains of C. burnetii that differ in LPS length. After 24 h of antigen stimulation, theexpression of co-stimulatory molecules (CD40, CD58, CD80, CD83, and CD86) ofDCs was analyzed by flow cytometry. Stimulation with the Xinqiao strain (phase I), a high virulent C. burnetii organism with full-length LPS, did not result in completematuration of DCs. In contrast, stimulation with Grita strain (phase II), a low virulentC. burnetii organism with truncated LPS, resulted in complete maturation of DCs withhigher expression of co-stimulatory molecules. Furthermore, LPS-removed Xinqiaostrain dramatically increased the ability to induce DC maturation. In addition, theiDCs were stimulated with LPS extracted from Xinqiao strain and LPS of E. coli,respectively. Like Xinqiao strain-pulsed DCs, DCs pulsed with LPS from Xinqiaostrain exhibited dramatically lower levels of co-stimulatory molecules compared withE. coli LPS-pulsed DCs. The results suggest that LPS blocks the surface proteinmolecules of C. burnetii which are necessary for activation ofDCs and the LPS maskis an immune evasion strategy for the high virulent C. burnetii strains in animmunocompetent host.The C. burnetii genes, com1, icmO, enhA, secB, lo1A, and hspB, encode outermembrane protein (Com1), type IV secretion system protein (IcmO), enhanced entryprotein A (EnhA), protein-export protein B (SecB), outer membrane lipoproteincarrier protein A(Lo1A), and heat shock protein B (HspB), respectively. In this study,in order to investigate capabilites of these proteins to activate DCs, recombinantprotein Com1, IcmO, EnhA, SecB, Lo1A, and HspB were used to stimulate iDCs,respevtively. Com1-activated DCs and SecB-pulsed DCs exhibited significantlyhigher levels of co-stimulatory molecule expression compared with DCs pulsed withother proteins. In contrast, DCs pulsed with HspB exhibited the lowest levels ofco-stimulatory molecule expression. These results indicate that Com1 and SecB canefficiently activate DCs, but HspB can not. In addition, IL-12p70 and IL-10 weredetermined on Com1-, SecB-, or HspB-pulsed DCs by flow cytometric analysis andthe results shown the level of IL-12p70 was greatly elevated after Com1 or SecBstimulation, and however IL-10 was greatly increased after HspB stimulation. Theseresults suggest that Com1- or SecB-activated DCs can drive T cells toward Th1 celldevelopment due to a high level of IL-12 production.To demonstrate T cell activation by Com1-, SecB-, or HspB-plused DCs, the Tcell activation marker CD69 and cytokines (IFN-γand TNF-α) expressed by CD4+and CD8+ T cells were determined in T cells co-cultured with the antigen-plused DCsor their supernatant, respectively. After stimulation of Com1- or SecB-activated DCsor their culture supernatants, both CD4+ and CD8+ T cells exhibited significantlyhigher expression of CD69, IFN-γand TNF-αcompared with HspB-plused DCs. These results suggest that Com1 and SecB can efficiently activate DCs and theactivated DCs have a capability to induce a specific cellular immune response.Moreover, T lymphocyte proliferation assay showed that the proliferation levels ofCD4+ and CD8+ T cells in T cells interacted with Com1-activated DCs orSecB-activated DCs were significantly higher than that interacted with mock-pulsedDCs or HspB-plused DCs, strongly suggesting that the expanded populations of CD4+and CD8+ T cells were well developed into CD4+ Th1 and CD8+ Tcl cells by directionof Com1-activated DCs or SecB-activated DCs, and the activated CD4+ and CD8+ Tcells produced a large amount of IFN-γand TNF-αwhich would efficiently activate and regulate host cells (macrophages) to kill intracellular C. burnetii organisms. Theseresults indicate that Com1 and SecB are potential protective antigens of C. burnetii,which have capabilityto elicit specific cellular immune response.