Avian cathelicidins: paradigms for the development of anti-infectives

Henk P. Haagsman Division of Molecular Host Defence Department of Infectious Diseases and Immunology

Organization of the lecture

•  Introduction

• Host defense peptides (HDPs)

• Chicken cathelicidin 2 (CATH-2)

• CATH-2 based anti-infectives

• Discussion

Ports of entry of microorganisms

150 m2 400 m2 2 m2

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

Molecular host defence at the interface

Herias et al., 2007

Lung, trachea Lung, bronchioli Small intestine

Effector molecules

•  Host Defence Peptides (HDPs) •  Defensins •  Cathelicidins

•  Large soluble pattern recognition molecules •  Collectins

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

Defensins

K R

H

hydrophobic

(+)

Chicken β-defensin AvBD9

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

S. typhimurium adheres to (and invades) porcine intestinal IPI-2 cells

Veldhuizen et al., 2009

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

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

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

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

CATH-2 is produced by chicken heterophils

Heterophils

Mononuclear cells

Giemsa Anti-CATH-2

Van Dijk et al., 2009a

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

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

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

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

Conclusions 3

•  CATH-2 is microbicidal against Gram (-), Gram (+) bacteria, yeasts and fungi

•  Truncated CATH-2 analogs are antimicrobial

 

CATH-­‐2  HD11  cell  line  

Experimental set-up

4h 24h

chemokine expression

(qPCR)

Direct effects of CATH-2 on chemokine expression in HD11 cells

Conclusions 4

•  CATH-2 and derived peptides induce cytokine production in a chicken macrophage cell line

•  CATH-2 dampens the LPS-induced inflammatory response

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)

Challenges (discussion)

•  Improve specific activities of compounds

•  Improve stability of lead structures

•  Development carrier/delivery systems

•  Pharmacokinetics

•  Cost-effective large scale production

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