Selective regulation of T cell-mediated immune response by TCM drugs and

natural products

Qiang Xu, Ph.D.

State Key Laboratory of Pharmaceutical Biotechnology

School of Life Sciences

Nanjing University

T cell-mediated diseases Th1-associated

• Rheumatoid arthritis

• Multiple sclerosis

• Crohn’s disease

• Graft-versus host disease

• Insulin-dependent diabetes

• Crescentic glomerulonephritis

Th2-associated

• Ulcerative colitis

• Myasthenia gravis

• Systemic lupus erythematosus

• Rheumatic Fever

• Hemolytic Anemia

• Membranous glomerulonephritis

IL-12

IFN-IL-2

Th0

APCTh1

Th M

Th2B cell

Macrophage

activate

IL-10

(-)

IL-4

IL-4 IL-5

Humoral immunity

Cellular immunity

Inflammatorymolecules

Co-stimulatorymolecules

Antibodies production

T-bet

GATA-3

Immunosuppressive agents

Can we have a safe immunosuppressive therapy without harming innocents?

History of

immunosuppressive agents

Glucocorticoids

Cytotoxic anti-cancer agents

Cyclosporin A 、 FK-506

Novel immunosuppressants?

New way?

Why the progress in immunology does not accelerate the development of immune drugs?

APC

Reduced anti- infection potentialKidney injuryNeurotoxicityIncreased oncogenesis……

Low selectivityIL-12

IFN-IL-2

Th0

Th1

Th2Bcell

Macrophage

活化

IL-4

IL-4IL-5IL-13

TNF-

CsA

Why do we target T cells?

inflammation

Cellular immunity

Humoral immunity

Ab formation

Novel immunosuppressors？

Higher

selectivity !

Effects of several kinds of Chinese herbs and prednisolone on DTH and inflammation

Drugs Induction phase

Effector phase

inflammation others

Fructus Tribuli inhibition — —

Si-Ni-San inhibition — —

Radix Gentianae inhibition inhibition —

Caulis Spatholobi inhibition inhibition ND

Radix Sophorae Flavescentis

— inhibition ND

Fructus Kochiae — inhibition ND

Radix Paeoniae Rubra — inhibition ND

Rhizoma Smilacis — inhibition inhibition Enhancing humoral immunity

Cortex Dictamni — inhibition inhibition inhibiting humoral immunity but not immune organs

Lm-3 inhibition inhibition inhibition Increasing spleen and adrenal weights

Prednisolone/CsA inhibition inhibition inhibition inhibiting humoral immunity and immune organs

IL-12

IFN-IL-2

Th0

APC

Th1

Macrophage

activate

Cellular immunity

Inflammatorymolecules

Co-stimulatorymolecules

Ag

Differentiation, ProliferationAdhesion, migration…Molecule expressionCytokine production and signaling

1. Selective inhibition on 　 the activation of T lymphocytes

Function of activated T cellsActivation of T cells

Fusaruside

Isolated from Fusarium sp. IFB-121

Fusaruside selectively inhibits Th1- but not Th2-like cytokine production

Fusaruside inhibits IFN--mediated STAT1 phosphorylation but not STAT1

itself

fludarabine Ser/Thr phospharylation of both STAT1 and STAT3

Steroids

IL-2-activated STAT5 pathway

IL-12 -activated STAT4 pathway

IFN-γ -activated STAT1 pathway

Leptin-induced STAT3 phosphorylation

IL-12/STAT4 pathwayCurcuminPhpsphorylation of STAT3 and STAT5

STAT1 and STAT3 pathway15dPGJ2

PPAR- agonist

Int. Immunopharmacol. 2003; 3(6): 783-800

STAT signaling pathway in T cell activation

Fusaruside inhibits pSTAT1, increases pSTAT3, but does not influence pSTAT4 ， 5 and 6

Fusaruside enhances IFN--induced STAT3 phosphorylation in a manner independent of STAT1

129S1: 129S1/SvImJ

IFNR -/- : 129-Ifngrtm1Agt

STAT3-dependent

inhibition of STAT1 signaling by Fusaruside

STAT3-siRNA transfection and interference

a b

Inhibition of pSTAT1 by Fusaruside is dependent on the existence of STAT3

Fusaruside

Summary

STAT3 pSTAT3 pSTAT1, IFN-

2. Selective inhibitionon the activated T lymphocytes

IL-12

IFN-IL-2

Th0

APC

Th1

Macrophage

activate

Cellular immunity

Inflammatorymolecules

Co-stimulatorymolecules

Ag

Function of activated T cellsActivation of T cells

Adhesion,migrationCytotoxicityCytokine production

How to induce dysfunction?

T cells0

10

20

30

Control

astilbin

RSG ext% Apoptotic cells

HC

total Kuppfer

NPC

Activated T cells

Normal T cells

800 bp 800 bp

M C ast rsg M C ast rsg C ast rsg

IFN

-γ(O

D54

0)

0

0.04

0.08

0.12

0.16

norm cont ast CsA

Pharmacological Research 1997; 36:309-314. Planta Med. 1999; 65:56-59. Eur. J. Pharmacol. 1999; 377:93-100. Pharm. Pharmacol. Comm. 2000; 6: 41-47.Phytochemistry 2000; 53 ： 1051-1055. Pharmacol. Res. 2001; 44 ： 135-139. Chin. Chem. Lett. 2002; 537-538. J. Ethnopharmacol. 2003; 85(1):53-9.Inflamm Res. 2003;52(8):334-340. J. Pharm. Pharmacol. 2003; 55: 691-696.J. Chromatography B 2004; 805: 357-360.J. Pharm. Pharmacol. 2004; 56: 495-502.

o

O

OH

OH

O

HO

OH

O

H3C

OH

OH

OH

A C

B12

345

6

78

1'

2'3'

4'

5'6'

Astilbin

Astilbin, a flavanoid from Rhizoma Smilacis Glabrae

1 the T cell population but not other tissue cells;

2 the activated but not non-activated T cell populations;

3 Th1 but not Th2 cells.

selective targeting

Astilbin shows a strong inhibition on CIA

Negative cytokine regulation by Astilbin J Allergy Clin Immunol. 2005; 116(6): 1350-1356.

Astilbin selectively induces the apoptosisof activated T cells

40

30

20

10

0Jurkat Con A-activated Jurkat

Control

10-7 g/ml

10-6 g/ml

10-5 g/ml

B

1 2 3 4 5 6 7 8 9

500 bp

C

Jurkat Activated Jurkat

Control

10-7 g/ml

10-5 g/ml

10-6g/ml

A

a e

b f

c g

d h

D

Control

10-7 g/ml

10-5 g/ml

10-6g/ml

Jurkat Activated Jurkat

2.19%

2.99%

2.41%

3.13%

2.45%

12.86%

22.08%

35.72%

Astilbin selectively induces apoptosis of Con A-activated Jurkat T cells

How Astilbin targets activated T cells？

Streptolysin-O

Cytosol proteins

Incubation

Hoechst stain

DNA fragmentation

Cytosol protein Nuclei fro

m non-activated Jurkat

Incubation

DNAfragmentationelectrophoresis

PI stainFlow cytometry

1 2 3 4 5 6Astilbin

Astilbin does not target the cytosol or nuclear proteins

a b c1 2 3 4 5 6 1 2 3 4 5 61 2 3 4 5 6

Jurkat L929 ECV3040

10

20

30

40Astilbin Jurkat Jurkat + AstilbinJurkat + Con A Jurkat + Con A + Astilbin

Only the cytosol proteins from Con A-activated and astilbin-treated Jurkat cells induce apoptosis of non-activated Jurkat, L929, ECV cells

J u r k a t A c t iv a t e d J u r k at

C o nt r ol

1 0 - 7 g / m l

1 0 - 6 g / m l

1 0 - 5 g/ m l

A

0

0.1

0.2

0.3

0.4

0.5

Jurkat Activated Jurkat

Control

10-7 g/ml

10-5 g/ml

10-6 g/ml

B

C

87.3% 2.1%

1.8%

88.9% 0.9%

2.5%

90.5% 0.4%

3.5%

83.1% 7.9%

6.1%

80.7% 4.3%

5.4%

82.1% 7.9%

5.. 9%

51.7% 12.7%

28..4%

41.3% 10.4%

43..7%

Jurkat

Activated Jurkat

Control 10-7 g/ml 10-6 g/ml 10-5 g/ml

JC-1

Astilbin selectively disrupts mitochondrial transmembrane potential of Con A-activated Jurkat T cells

Astilbin selectively caused cytochrome c release from mitochondria to cytosol in Con A-activated Jurkat cells

D

Act ivated Jurkat Jurkat

Co ntro l

10 -7 g /ml10-6 g /ml10-5 g /ml

*

*

*

0

10

20

30

40

50

A

I sotype

C o nt rol

1 0- 7 g /m l

1 0- 5 g /m l

1 0- 6 g /m l

C o n A - +

Isotype

Control

10-7 g /m l

1 0-5 g /m l

1 0-6 g /m l

Con A - +B

C

M

C

C

Cyt

A IF

A st 0 10-7 10-6 10-5 0 10-7 10-6 10-5

Astilbin selectively translocated Bax and Bad but not Bid from cytosol to mitochondria in activated Jurkat cells

MC

MC

MC

Bad

Bax

Bid

0 10-7 10-6 10-5 0 10-7 10-6 10-5

Non-activated Activated

0 10-7 10-6 10-5 0 10-7 10-6 10-5

0 10-7 10-6 10-5 0 10-7 10-6 10-5

Release of cytochrome c from mitochondria

1. Mitochondria damage

2. Permeability transition pore (PTP) opening

3. Bcl-2 family homopolymers

There is an opener of VDAC in the mitochondria of activated T cells after astilbin treatment

DiOC6O(3)/FL-1

A

B

C E

F

Incubation Treatment DiOC6(3) Flow cytometryVDAC

From mouse liver

PTP

VDAC

ANT

liposome

0.25%SDS

5 uM Ca2+

10-6 g/ml Ast

10-5 g/ml Ast

Nor Mito Lysis

(Ast+Nor Mito) Lysis

Activ Mito Lysis

(Ast + Activ) Mito Lysis

Surface alterations; Nuclear changes; DNA fragmentation

astilbin

astilbin

Conclusion• For T cell activation

Si-Ni-San and its components

　 Jie-Xue-Hao, a Tibetan medicine

　 Fumigaclavine C, Fusaruside ……

• For the function of activated T lymphocytes

　 Fraxinellone and Obaculactone

　 Astilbin ……

• For both T cell activation and functions, as well as other cells

　 current immunosuppressants

1. Selective immunosuppressive therapy is possible.

2. A number of selective immunosuppressive agents will be benefitial for the treatment of immune diseases.

Natural Science Foundation of China 30230390 39925041 30070781 39970887 39470815 39270784

孫洋張先明費明健呉雪豊蔡宇 秦宇呉興新燕茹 趙蔚夏玉貴

Acknowledgements

陳　婷

譚仁祥

郭子建

Thanks!Thanks!

The ancient building in Nanjing University