Toll-like receptors: recent advances, open questions and implications for aspergillosis

control.

Frank EbelMax-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+TLR2 bacterial lipopeptidesTLR2 lipopeptides, peptidoglycan, lipoteichoic acid,

zymosan, lipomannan, glycolipid, atypical LPS,Hsp60, porins

TLR2+TLR6 di-acyl lipopeptides, lipotechoic acid, zymosanTLR3 double stranded (viral) RNATLR4 LPS, glycan, lipopeptidophosphoglycan,

viral envelope proteins, Hsp60, TLR5 flagellinTLR7 ssRNA, imiquimodTLR8 ssRNA, imidazoquinolineTLR9 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 lipopeptidesTLR2 lipopeptides, peptidoglycan, lipoteichoic acid,

zymosan, lipomannan, glycolipid, atypical LPS,Hsp60, porins

TLR2+TLR6 di-acyl lipopeptides, lipotechoic acid, zymosanTLR3 double stranded (viral) RNATLR4 LPS, glycan, lipopeptidophosphoglycan,

viral envelope proteins, Hsp60, TLR5 flagellinTLR7 ssRNA, imiquimodTLR8 ssRNA, imidazoquinolineTLR9 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 genereporter

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-kBluciferase

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

0

100

200

300

400

500

600

unstim conidia hyphae spec agonist

RL

U

TLR1TLR2TLR3TLR4TLR5TLR6TLR7TLR8TLR9TLR10

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 differentA. fumigatus morphotypes

Resting conidia Swollen 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 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

.

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 witha blocking TLR2-specific monoclonal antibody showed reduced effciencyof 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 controlTLR4 can trigger a Th1-like response

inflammatory anti-inflammatory

Th1-like response Th2-like response

TNFα IL10

TLRs: implications for aspergillosis controlTLR4 can trigger a Th1-like response

inflammatory anti-inflammatory

Th1-like response Th2-like response

Tcell

TLR4 TLR2

Napolitani et al., Redecke et al.,Nature Immunol.2005 J. Immunol. 2004

DCLPS

TNFα IL10

TLRs: implications for aspergillosis control

inflammatory Th1-like response

Treg

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

Treg

Pam3Cys

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

Treg

Pam3Cys

TLR2

Sutmuller et al.,J. Clin. Invest. 2006

Treg

Treg

TLRs: implications for aspergillosis controlModulating granulocyte functions

inflammation fungizidal

tisssue damage activity

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)

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!

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.