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Toll-like receptors: recent advances, open questions and implications for aspergillosis control. Frank Ebel Max-von-Pett

Toll-like receptors: recent advances, open questions and implications for aspergillosis control. Frank Ebel Max-von-Pettenkofer-Institut, Munich, Germany. Pattern recognition receptors (PRR) recognize pathogen-associated molecular patterns (PAMP) (Janeway & Medzhitov, 1998). activation.

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Toll-like receptors: recent advances, open questions and implications for aspergillosis control. Frank Ebel Max-von-Pett

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  1. Toll-like receptors: recent advances, open questions and implications for aspergillosis control.Frank EbelMax-von-Pettenkofer-Institut, Munich, Germany

  2. Pattern recognition receptors (PRR) recognize pathogen-associated molecular patterns (PAMP) (Janeway & Medzhitov, 1998) activation TLR TNF IL-6

  3. The signalling pathways of Toll and TLR4 Drosophila (Toll) Human (TLR4) Silverman & Maniatis, 2001

  4. TLRs and the corresponding PAMPs ------------------------------------------------------------------------- TLR1+TLR2 bacterial lipopeptides TLR2 lipopeptides, peptidoglycan, lipoteichoic acid, zymosan, lipomannan, glycolipid, atypical LPS, Hsp60, porins TLR2+TLR6 di-acyl lipopeptides, lipotechoic acid, zymosan TLR3 double stranded (viral) RNA TLR4 LPS, glycan, lipopeptidophosphoglycan, viral envelope proteins, Hsp60, TLR5 flagellin TLR7 ssRNA, imiquimod TLR8 ssRNA, imidazoquinoline TLR9 non-methylated CpG-DNA TLR10 ? -------------------------------------------------------------------------- adapted from Ishii et al., 2005

  5. TLR2 and 4 are engaged in recognition of PAMPs localized on microbial surfaces ------------------------------------------------------------------------- TLR1+TLR2 bacterial lipopeptides TLR2 lipopeptides, peptidoglycan, lipoteichoic acid, zymosan, lipomannan, glycolipid, atypical LPS, Hsp60, porins TLR2+TLR6 di-acyl lipopeptides, lipotechoic acid, zymosan TLR3 double stranded (viral) RNA TLR4 LPS, glycan, lipopeptidophosphoglycan, viral envelope proteins, Hsp60, TLR5 flagellin TLR7 ssRNA, imiquimod TLR8 ssRNA, imidazoquinoline TLR9 non-methylated CpG-DNA TLR10 ? --------------------------------------------------------------------------

  6. Identification of human TLRs that recognize A. fumigatus in transfected cells - human HEK293 cells lack expression of all TLRs - co-transfection of a NF-kB-dependent luciferase construct reporter TLR gene

  7. Transfection of HEK293 cells with different human Toll-like receptor genes • Controls: • expression verified by immunoblot • function tested using available positive controls PAMP TLR luciferase NF-kB NF-kB-dependent luciferase gene

  8. Transfection of HEK293 cells with different human TLR genes Activation through TLR4 and TLR2(TLR1?) Further experiments revealed no evidence for an involvement of TLR1/TLR2- or TLR2/TLR6-heterodimers Meier et al., Cell. Microbiol. 2003

  9. Involvement of TLR2 and/or TLR4 in the immunological response of murine macrophages to A. fumigatus Meier et al., Cell. Microbiol. 2003

  10. PRRs must recognize differentA. fumigatus morphotypes Resting conidia Swollen conidia Germlings Hyphae

  11. Reference Macrophage A. fumigatus TLR2 TLR4 morphotype -------------------------------------------------------------------------------------------------- Wang et al., 2001HMDMΦHy - ++ Mambula et al.,2002 pMΦRC ++ - pMΦSC - ++ pMΦHy ++ - Netea et al., 2003 pMΦRC + ++ pMΦHy + - Meier et al., 2003 pMΦRC + ++ pMΦHy + ++ Bellocchio et al., 2004 hPMN RC + ++ hPMN Hy + ++ Hohl et al., 2005 BMDMΦRC + n.d. Steele et al., 2005 AMΦHy ++ n.d. --------------------------------------------------------------------------------------------------- RC = resting conidia, SC = swollen conidia, Hy = hyphae, n.d. = not determined

  12. Analysis of TLR2 and TLR4 in murine models of invasive aspergillosis Reference Immunosuppression TLR2 TLR4 TLR9 ----------------------------------------------------------------------------------------------------- Bellocchio et al., 2004 cyclophosphamide+ ++ - Balloy et al., 2005 vinblastine ++ n.d. n.d. ------------------------------------------------------------------------------------------------------ TLR4 and TLR2 are both required for an efficient immune response to A. fumigatus .

  13. Dectin-1: an important PRR in anti-fungal immunity Dectin-1 is a surface PRR on dendritic cells and macrophages that specifically recognizes ß1-3 glucan (Brown et al., 2003). Dectin-1 and TLR2 co-operate in the inflammatory response to A. fumigatus (Hohl et al., 2005; Steele et al., 2005). Dectin-1 is engaged in phagocytosis of Candida albicans (Herre et al., 2004; Gantner et al., 2005).

  14. Dectin-1 and TLR2 are both required for efficient phagocytosis of A. fumigatus conidiaby murine macrophages Our unpublished data suggest that dectin-1 is required for efficient phagocytosis of A. fumigatus conidia. Macrophages lacking TLR2 or wild type macrophages preincubated with a blocking TLR2-specific monoclonal antibody showed reduced effciency of phagocytosis of A. fumigatus conidia.

  15. Summary Several lines of evidence suggest that TLR2 and TLR4 are both engaged in recognition of A. fumigatus, but conflicting data exist with respect to the their relative importance for recognition of certain A. fumigatus morphotypes Recent data suggest a cross-talk between TLR2 and dectin-1 that seems to be important for the inflammatory response to A. fumigatus andin addition for an efficient phagocytosis of A. fumigatus conidia.

  16. TLRs: implications for aspergillosis controlTLR4 can trigger a Th1-like response inflammatory anti-inflammatory Th1-like response Th2-like response TNFα IL10

  17. Tcell TLRs: implications for aspergillosis controlTLR4 can trigger a Th1-like response inflammatory anti-inflammatory Th1-like response Th2-like response TNFα IL10 Napolitani et al., Redecke et al., Nature Immunol.2005 J. Immunol. 2004 DC LPS TLR4 TLR2

  18. Treg TLRs: implications for aspergillosis control inflammatory Th1-like response TLR2 Sutmuller et al., J. Clin. Invest. 2006

  19. Treg TLRs: implications for aspergillosis controlTLR2 can transiently induce a Th1-like response inflammatory Th1-like response TLR2 Pam3Cys Sutmuller et al., J. Clin. Invest. 2006

  20. Treg Treg Treg TLRs: implications for aspergillosis control TLR2 can transiently induce a Th1-like response inflammatory Th1-like response TLR2 Pam3Cys Sutmuller et al., J. Clin. Invest. 2006

  21. TLRs: implications for aspergillosis controlModulating granulocyte functions inflammation fungizidal tisssue damage activity

  22. TLRs: implications for aspergillosis controlModulating granulocyte functions inflammation fungizidal tissue damage activity TLR2 TLR4 amphotericin B liposomal amphotericin B liposomes Bellocchio et al., JAC (2005)

  23. TLRs: implications for aspergillosis controlEffecting granulocyte functions inflammation fungizidal tissue damage activity TLR2 TLR4 amphotericin B liposomal amphotericin B liposomes Bellocchio et al., JAC (2005) protective!

  24. Outlook Targeted activation of TLR2- and/or TLR4-signalling in the lung of patients at risk to develop invasive aspergillosis seems to be a promising strategy to protect such patients from A. fumigatus infections.

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