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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 TLR TNF IL-6 The signalling pathways of Toll and TLR4 Drosophila (Toll) Human (TLR4) Silverman & Maniatis, 2001 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 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 ? -------------------------------------------------------------------------- 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 TLR gene reporter Transfection of HEK293 cells with different human Toll-like receptor genes PAMP TLR luciferase NF-kB NF-kB-dependent luciferase gene Controls: 1. expression verified by immunoblot 2. function tested using available positive controls Transfection of HEK293 cells with different human TLR genes 600 TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 500 RLU 400 300 200 100 0 unstim conidia hyphae spec agonist 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 Involvement of TLR2 and/or TLR4 in the immunological response of murine macrophages to A. fumigatus Meier et al., Cell. Microbiol. 2003 PRRs must recognize different A. fumigatus morphotypes Resting conidia Germlings Swollen conidia Hyphae Macrophage A. fumigatus TLR2 TLR4 morphotype -------------------------------------------------------------------------------------------------Wang et al., 2001 HMDMΦ Hy ++ Reference Mambula et al.,2002 pMΦ pMΦ pMΦ RC SC Hy ++ ++ ++ - Netea et al., 2003 pMΦ pMΦ RC Hy + + ++ - Meier et al., 2003 pMΦ pMΦ RC Hy + + ++ ++ Bellocchio et al., 2004 hPMN hPMN RC Hy + + ++ ++ Hohl et al., 2005 RC + n.d. BMDMΦ Steele et al., 2005 AMΦ Hy ++ n.d. --------------------------------------------------------------------------------------------------RC = resting conidia, SC = swollen conidia, Hy = hyphae, n.d. = not determined 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 . Dectin-1: an important PRR in antifungal 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). Dectin-1 and TLR2 are both required for efficient phagocytosis of A. fumigatus conidia by 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. 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 and in addition for an efficient phagocytosis of A. fumigatus conidia. TLRs: implications for aspergillosis control TLR4 can trigger a Th1-like response inflammatory Th1-like response TNFα anti-inflammatory Th2-like response IL10 TLRs: implications for aspergillosis control TLR4 can trigger a Th1-like response inflammatory Th1-like response anti-inflammatory Th2-like response TNFα IL10 Tcell Napolitani et al., Nature Immunol.2005 LPS Redecke et al., J. Immunol. 2004 TLR4 DC TLR2 TLRs: implications for aspergillosis control Treg TLR2 inflammatory Th1-like response Sutmuller et al., J. Clin. Invest. 2006 TLRs: implications for aspergillosis control TLR2 can transiently induce a Th1-like response Treg TLR2 inflammatory Th1-like response Sutmuller et al., J. Clin. Invest. 2006 Pam3Cys TLRs: implications for aspergillosis control TLR2 can transiently induce a Th1-like response Treg Treg TLR2 inflammatory Th1-like response Sutmuller et al., J. Clin. Invest. 2006 Treg Pam3Cys TLRs: implications for aspergillosis control Modulating granulocyte functions inflammation tisssue damage fungizidal activity TLRs: implications for aspergillosis control Modulating granulocyte functions inflammation tissue damage TLR2 amphotericin B Bellocchio et al., JAC (2005) fungizidal activity TLR4 liposomal amphotericin B liposomes TLRs: implications for aspergillosis control Effecting granulocyte functions inflammation tissue damage TLR2 amphotericin B Bellocchio et al., JAC (2005) fungizidal activity TLR4 protective! liposomal amphotericin B liposomes Outlook Targeted activation of TLR2- and/or TLR4signalling 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.
