Guolong “Glenn” Zhang

Associate Professor

Department of Animal Science

International Symposium: Alternatives to antibiotics, Paris, France, Sept 25-28, 2012

Host Defense Peptide-Medicated Disease Control and Prevention through

Modulation of Innate Immunity

Effector Molecules

Amticrobial Peptides/proteins ROS and RNS Cytokines/Chemokines Costimulatory Molecules Adhesion Molecules Acute Phase Proteins

Regulation of Immunity

Direct Delivery of effector molecules

Indirect modulation of effector molecule production

Immunoregulatory Strategies

Direct Approach: Delivery of novel HDPs to boost innate immunity and disease resistance

Indirect Approach: Dietary modulation of HDP synthesis to boost innate immunity without triggering inflammatory response

Biological Functions of HDPs

Nature Biotech 24, 1551 - 1557 (2006)

3-D Structure of Fowlicidins

N

C

Fowlicidin-3 Fowlicidin-2

(FEBS J. 2006, 273: 2581)

(J. Innate Immun. 2009, 1: 168)

(FEBS J. 2007, 274: 418)

N

C

N

C

N

C

Fowlicidin-1

0 1 2 3 4 5 6 7 80

20

40

60

80

100 PBS (n=13)

F-1(6-26), 10 mg/kg (n=13)

Days Post Inoculation

Su

rviv

al (%

)

Protection of Mice From a Lethal MRSA Infection by Fowl-1(6-26)

**P < 0.001 (Log-rank Test)

(Peptides 2010, 31: 1225)

0 1 2 3 4 5 6 7 80

20

40

60

80

100

PBS

-7 day

-4 day

-2 day

-1 day

Days Post Inoculation

Su

rviv

al (%

)

Prevention of Mice From a Lethal MRSA Infection by Fowl-1(6-26)

**P < 0.01

(Log-rank Test)

**

**

0 2 5 100

20

40

60

80

100*

Fowl-1(6-26)-NH2 (mg/kg)

To

tal C

ell C

ou

nt

(x 1

04)

Neutrophils

Monocytes

Lymphocytes0

20

40

60

80HBSS

2 mg/kg

5 mg/kg

10 mg/kg

Cell C

ou

nt

(x 1

04)

**

**

**

In Vivo Recruitment of Leukocytes by Fowl-1(6-26)

IL-1

contr

ol

F1(6-

26)

F1(8-

26)

LL-37

IDR-1

0

1

2

3

4

Fo

ld In

du

cti

on

CCL2

contr

ol

F1(6-

26)

F1(8-

26)

LL-37

IDR-1

0

1

2

3

4

5

6

Fo

ld In

du

cti

on

0 0.4 2 PMA0

10000

20000

30000

40000 Oxidative Burst

F-1(6-26) (g/ml)

Re

lati

ve

Flu

ore

sce

nce

Activation of Macrophages by Fowl-1(6-26)

CCL3

contr

ol

F1(6-

26)

F1(8-

26)

LL-37

IDR-1

0

2

4

6

8

10

Fo

ld In

du

cti

on

CCL2

contr

ol

F1(6-

26)

F1(8-

26)

LL-37

IDR-1

0

1

2

3

4

5

6

Fo

ld In

du

cti

on

CCL2

contr

ol

F1(6-

26)

F1(8-

26)

LL-37

IDR-1

0

1

2

3

4

5

6

Fo

ld In

du

cti

on

CCL2

contr

ol

F1(6-

26)

F1(8-

26)

LL-37

IDR-1

0

1

2

3

4

5

6

Fo

ld In

du

cti

on

Summary

Direct delivery of fowlicidin-1(6-26) is capable of treating and preventing mice from lethal MRSA infections;

Prevention of MRSA infections by fowlicidin-1(6-26) is mediated in part by recruitment of neutrophils and activation of macrophages, but not by direct killing of bacteria;

Fowlicidin-1(6-26) enhances surface expression of co-stimulatory molecules on macrophages and potentiates unpolarized antibody response following immunization with a model antigen (ovalbumin).

Physiological Roles of SCFAs

Short chain fatty acids

Energy Source

pH

Wound Healing

Cell Proliferation

Sodium &

Water

Absorption

HDP

Induction

Mucous

Anti-

microbial

Bacterial

Invasion

Butyrate Induces Chicken β-Defensin 9 (AvBD9) Expression

0

1000

2000

3000

4000

5000

6000

7000

0 0.5 1 2 4 8

Butyrate (mM)

0

100

200

300

400

500

600

700

800

Butyrate (mM)

0 0.5 1 2 4 8

Fold

Incr

ease

0 0.5 1 2 40

100

200

300

400

500

600

700

800

Butyrate (mM)

3h 6h 12h

24h

48h0

1000

2000

3000

4000

Butyrate (4mM)

Fold

Incr

ease

HD11

Macrophages

HD11

Macrophages

Primary

Monocytes

Bone Marrow

Cells

0

50

100

150

Butyrate (mM)

AvBD9

0 1 2 4

Fold

Incr

ease

0

2

4

6

Butyrate (mM)

AvBD9

0 2 4

Fold

Incr

ease

Jejunum

Cecum

-5 0 5

0.5 1 2 4 0.5 1 2 4 8 Butyrate (mM)

HD11 Monocytes

Largely

Unaffected

Largely

Suppressed

Largely

Induced

Pattern of HDP Gene Induction by Butyrate

0 0.10.2 0 0.10.2 0 0.10.20

5

10

15

20

25

30

35

40 Crop

Cecal

TonsilCecum

Butyrate (%)

*

*Fold

Incr

ease

In vivo Induction of AvBD9 Expression by Butyrate

Butyrate Reduces Salmonella Colonization in the Cecum of Infected Chickens

0 0.10.2 0 0.10.2 0 0.10.20

5

10

15

20

25

30

35

40 Crop

Cecal

TonsilCecum

Butyrate (%)

*

*Fold

Incr

ease

Contr

ol

0.1%

Buty

rate

0.2%

Buty

rate

105

106

107

108

109

S.

en

teri

tidis

(CF

U/g

)

*P = 0.03

Correlation of Aliphatic Chain Length of Fatty Acids with HDP-Inducing Activity

0.1

1

10

100

1000

10000

AvBD

9 F

old

Incr

ease

SCFA

MCFA

LCFA

Aliphatic Chain

(CH2) (2-30C)

Carboxylic

Acid

1

10

100

1000

10000

IL-1

Fo

ld I

ncr

ea

se

Co

ntr

ol

Aceta

te (

C2)

Pro

pio

nate

(C

3)

Bu

tyra

te (

C4)

Hex

an

oa

te (

C6)

Oc

tan

oate

(C

8)

LP

S

Aceta

te (

C2)

Pro

pio

nate

(C

3)

Bu

tyra

te (

C4)

Hexan

oate

(C

6)

Octa

no

ate

(C

8)

LP

S

Minimum Impact of Fatty Acids on Proinflammatory Cytokine Expression

3 h

24 h

- + - - - - + + - + + +

- - + - - - + - + + + +

- - - + - + + +

Butyrate

Propionate

Acetate

0

20

40

60

80

AvB

D9

Fold

Cha

nge

Synergistic AvBD9 Induction by SCFAs

0

1000

2000

3000

4000

5000

AvB

D9

Fold

Cha

nge HD11 Cells

Monocytes

Contro

l

Butyra

te

Ace

tate

Prop

iona

te

Combi

natio

n

106

107

108

* ** * **

(CFU

/g)

CON SCFA1 SCFA2 SCFA3 MIX

*P < 0.05 **P < 0.01

Synergistic Reduction of Salmonella Colonization in Chickens by SCFAs

S. en

teri

tid

is T

iter

Butyrate - - - + + +

Extract (µM) - 25 50 - 25 50

0

500

1000

1500

2000

2500

AvB

D9 F

old

In

cre

ase

Synergistic Induction of HDP Expression Between Butyrate and a Plant Extract

Summary

SCFAs are potent inducers of chicken HDPs both in vitro and in vivo by acting as histone deacetylase inhibotors;

SCFAs synergistically enhance HDP synthesis and

reduce S. enteritidis colonization with a minimum impact on proinflammatory response;

A combination of natural SCFAs and herbal extracts may have potential to be explored as antibiotic alternatives;

See a poster entitled “Development of immune boosting dietary supplements as alternatives to antibiotics” in Session 2 (2.11).

Funding

Goal: Develop novel, highly efficient, cost-effective

immune boosting agents in disease control and

prevention for both farm animals and humans.