Transcript Toll-like receptors: resent advances, open questions and

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.