paradigms for the development of anti-infectives · length (m) relative gene expression (a.u.) 0 0...
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Avian cathelicidins: paradigms for the development of anti-infectives
Henk P. Haagsman Division of Molecular Host Defence Department of Infectious Diseases and Immunology
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Organization of the lecture
• Introduction
• Host defense peptides (HDPs)
• Chicken cathelicidin 2 (CATH-2)
• CATH-2 based anti-infectives
• Discussion
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Ports of entry of microorganisms
150 m2 400 m2 2 m2
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Innate Host Defence
• Present in all organisms
• Limited repertoire of molecules
• Rapid
• Broad specificity • Ancient
The first line of defence against infection
Cellular defences
Effector molecules
Neutrophils (heterophils) Macrophages
Enzymes, HDPs Collectins
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Molecular host defence at the interface
Herias et al., 2007
Lung, trachea Lung, bronchioli Small intestine
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Effector molecules
• Host Defence Peptides (HDPs) • Defensins • Cathelicidins
• Large soluble pattern recognition molecules • Collectins
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Host defence peptides
• Between 10 and 50 amino acids • Positive charge • Amphipathic • Rapid acting; potent; broad spectrum of
antimicrobial activity • Immunomodulatory effects • Constitutive and stimulated secretion
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Defensins
K R
H
hydrophobic
(+)
Chicken β-defensin AvBD9
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Differential expression of 2 β-defensin genes in porcine intestine
0 2 4 6 8 100
10
20
30Ile.Jej.Dd.
a
b b
a
a
Length (m)
Rel
ativ
e ge
ne e
xpre
ssio
n (a
.u.)
0 2 4 6 8 100
5
10
15 Dd. Jej. Ile.a
b
b
bb
b
bb
b
Length (m)
Rel
ativ
e ge
ne e
xpre
ssio
n (a
.u.)
Veldhuizen et al., 2007
pBD-1 pBD-2
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S. typhimurium adheres to (and invades) porcine intestinal IPI-2 cells
Veldhuizen et al., 2009
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S. typhimurium induces pBD-2 in porcine intestinal epithelial cells
6 12 24 6 12 24 6 12 24 6 12 240
10
20
30
ControlColistinHeatS. typh.
*
*R
elat
ive
pBD
-2 g
ene
expr
essi
on
Veldhuizen et al., 2009
h
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AvBD-9 induces morphological changes in C. perfringens
Control 1.5 µg/ml 6.2 µg/ml
12.5 µg/ml 12.5 µg/ml 25 µg/ml
1 µm Van Dijk et al., 2007
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Conclusions 1
• Defensins are differentially expressed in porcine digestive tract
• Upregulation of porcine β-defensin 2 in epithelial cells requires uptake of live bacteria
• HDPs may exert intracellular antimicrobial effects at low concentrations
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Chicken cathelicidin 2 (Cath-2)
• Cationic (+9) • 26 amino acids • Amphipathic RFGRFLRKIRRFRPKVTITIQGSARF-NH2
α α Hinge region
N
C
Van Dijk et al., 2005 Xiao et al., 2006
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CATH-2 is produced by chicken heterophils
Heterophils
Mononuclear cells
Giemsa Anti-CATH-2
Van Dijk et al., 2009a
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Salmonella enteritidis challenge results in recruitment of CATH-2 containing heterophils
Control 8h p.i.
Control 48h p.i.
S. enteritidis 8h p.i.
S. enteritidis 48h p.i.
S. enteritidis 48h p.i. (Giemsa)
S. enteritidis 48h p.i. (preimmune)
Van Dijk et al., 2009a
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Conclusions 2
• CATH-2 is produced by heterophils but not by monocytes, lymphocytes, thrombocytes or red blood cells
• Salmonella enteritidis infections of chickens result in accumulation of CATH-2 positive heterophils at the site of infection
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Antibacterial activities of truncated CATH-2 analogs
E. coli
0 10 20
<22345678
C1-27C1-26*C1-21
40
C1-15C12-26*
Peptide (µM)
Log
CFU
/ml
S. enteritidis
0 5 10 15 20
<22345678
C1-27C1-26*C1-21
40
C1-15C12-26*
Peptide (µM)
Log
CFU
/ml
S. aureus
0 5 10 15 20
<22345678
C1-27C1-26*C1-21
40
C1-15C12-26*
Peptide (µM)
Log
CFU
/ml
B. globigii
0 5 10 15 20
<22345678
C1-27C1-26*C1-21
40
C1-15C12-26*
Peptide (µM)
Log
CFU
/ml
Van Dijk et al., 2009b
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Killing curves of truncated CATH-2 analogs
S. enteriditis 13367
0 10 20 30 40 50 60
<22345678
C1-27C1-26*C1-21C1-15C12-26*medium
time (min)
Log
CFU
/ml
Van Dijk et al., 2009b
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Conclusions 3
• CATH-2 is microbicidal against Gram (-), Gram (+) bacteria, yeasts and fungi
• Truncated CATH-2 analogs are antimicrobial
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CATH-‐2 HD11 cell line
Experimental set-up
4h 24h
chemokine expression
(qPCR)
Direct effects of CATH-2 on chemokine expression in HD11 cells
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Conclusions 4
• CATH-2 and derived peptides induce cytokine production in a chicken macrophage cell line
• CATH-2 dampens the LPS-induced inflammatory response
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General conclusions
• Based on host defense peptides of chicken (and pig), small peptides were produced that exhibit strong, broad-range, antibacterial activity
• Peptides were produced that are very active towards bacteria but are not toxic to host cells
• Peptides were produced that show immunomodulatory activities
• Prophylactic or therapeutic treatment of chickens by CATH-2 significantly reduced Salmonella survival in the crop
See also posters by Van Dijk (2.15) and Veldhuizen (1.33)
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Challenges (discussion)
• Improve specific activities of compounds
• Improve stability of lead structures
• Development carrier/delivery systems
• Pharmacokinetics
• Cost-effective large scale production
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Division Molecular Host Defence
Department of Infectious Diseases & Immunology
Albert van Dijk Margo Molhoek Floris Bikker Hanne Tjeerdsma Edwin Veldhuizen
Maarten Coorens Tryntsje Cuperus Soledad Ordonez Andreas Peschel Viktoria Schneider
ALTANT