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 TNF IL-6 TLR

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

TLRs and the corresponding PAMPs TLR1+TLR2bacterial lipopeptides TLR2lipopeptides, peptidoglycan, lipoteichoic acid, zymosan, lipomannan, glycolipid, atypical LPS, Hsp60, porins TLR2+TLR6di-acyl lipopeptides, lipotechoic acid, zymosan TLR3double stranded (viral) RNA TLR4LPS, glycan, lipopeptidophosphoglycan, viral envelope proteins, Hsp60, TLR5flagellin TLR7ssRNA, imiquimod TLR8ssRNA, imidazoquinoline TLR9non-methylated CpG-DNA TLR10? adapted from Ishii et al., 2005

TLR2 and 4 are engaged in recognition of PAMPs localized on microbial surfaces TLR1+TLR2bacterial lipopeptides TLR2lipopeptides, peptidoglycan, lipoteichoic acid, zymosan, lipomannan, glycolipid, atypical LPS, Hsp60, porins TLR2+TLR6di-acyl lipopeptides, lipotechoic acid, zymosan TLR3double stranded (viral) RNA TLR4LPS, glycan, lipopeptidophosphoglycan, viral envelope proteins, Hsp60, TLR5flagellin TLR7ssRNA, imiquimod TLR8ssRNA, imidazoquinoline TLR9non-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 Controls: 1.expression verified by immunoblot 2.function tested using available positive controls NF-kB-dependent luciferase gene TLR NF-kB luciferase PAMP

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

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 conidiaSwollen conidia Germlings Hyphae

Reference Macrophage A. fumigatus TLR2 TLR4 morphotype Wang et al., 2001 HMDMΦ Hy - ++ Mambula et al.,2002 pMΦ RC ++- pMΦ SC -++ pMΦ Hy ++- Netea et al., 2003 pMΦ RC + ++ pMΦ Hy +- Meier et al., 2003 pMΦ RC + ++ pMΦ Hy + ++ Bellocchio et al., 2004 hPMN RC + ++ hPMN Hy + ++ Hohl et al., 2005 BMDMΦ RC + n.d. Steele et al., 2005 AMΦ Hy ++n.d RC = resting conidia, SC = swollen conidia, Hy = hyphae, n.d. = not determined

Reference Immunosuppression TLR2 TLR4 TLR 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. Analysis of TLR2 and TLR4 in murine models of invasive aspergillosis

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).

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.

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. Summary

TLRs: implications for aspergillosis control TLR4 can trigger a Th1-like response inflammatory anti-inflammatory Th1-like response Th2-like response TNFα IL10

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

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

TLRs: implications for aspergillosis control TLR2 can transiently induce a Th1-like response inflammatory Th1-like response T reg Pam 3 Cys TLR2 Sutmuller et al., J. Clin. Invest. 2006

TLRs: implications for aspergillosis control TLR2 can transiently induce a Th1-like response inflammatory Th1-like response T reg Pam 3 Cys TLR2 Sutmuller et al., J. Clin. Invest T reg

TLRs: implications for aspergillosis control Modulating granulocyte functions inflammation fungizidal tisssue damage activity

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

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

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.