Invasive Aspergillosis has become a major cause of infection-related mortality in non-neutropenic patients after allogeneic stem cell transplantation (SCT). Activation of the innate immune system (granulocytes, monocytes) is a prerequisite for the successful control of invasive aspergillosis. In this study, we analysed the potential role of pattern-recognition receptors in the activation of dendritic cells. Peripheral blood derived monocytes were differentiated into immature dendritic cells (DC) by stimulation with IL-4 and GM-CSF, stimulated with different A. fumigatus antigen preparations and analysed for the expression of CD83, MHC and costimulatory molecules as well as the secretion of pro-inflammatory cytokines. 293-T cells were transiently transfected with cDNAs encoding for different toll-like receptors (tlr) and assessed for NFkB translocation after stimulation with A. fumigatus antigens. Moreover, we assessed the activation of DCs from wildtype, tlr4-deficient, tlr2 k.o., MyD88 k.o., as well as tlr2/tlr4 double-k.o. mice. Immature DCs were found to be activated and to differentiate into mature DCs in response to A. fumigatus antigens as characterized by CD83 expression, upregulation of MHC and costimulatory molecules, and the intracellular release of TNF-a. In the luciferase reporter assay, a significant translocation of NFkB was documented in tlr2 and tlr4 but not in tlr9 transfected 293-T cells. A complete blockade of IL-12 release in culture supernatants was documented in DCs from tlr2 and tlr4/tlr2-double k.o. mice only, whereas a mild reduction of the IL-12 release was observed in DCs from tlr4-deficient C3H/HeJ mice. The role of tlrs for the Aspergillus fumigatus-mediated activation of DCs was further supported by the blockade of cytokine release in DCs from MyD88 k.o. mice. The protein precipitate of A. fumigatus supernatant (PP-SAB) was found to provide the best stimulation of DCs when compared to a whole cellular extract (EC-SAB) and conidia of A. fumigatus, indicating that a factor secreted by the fungus provides the optimal stimulus for DC-activation. In conclusion, our results clearly indicate, that Aspergillus fumigatus provides a strong stimulus for the activation and maturation of immature DCs. According to our results, this activation is mediated via the tlr2. Future trials will have to assess, whether this pathway is inhibited in patients at high risk for invasive aspergillosis.
Full conference title:
44th Amercian Society of Hematology Annual Meeting
- ASH 44th (2002)