selective regulation of t cell-mediated immune response by tcm drugs and natural products qiang xu,...
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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
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