Disclosure Information 3 H 1, 2 Dithiole 3 Thione (D3T) as a - - PowerPoint PPT Presentation

SLIDE 1

3H‐1, 2‐Dithiole‐3‐Thione (D3T) as a Novel Therapeutic Agent for the Treatment of Experimental Autoimmune Encephalomyelitis

Jui‐Hung (Jimmy) Yen, Ph.D.

Department of Microbiology and Immunology Indiana University School of Medicine

Disclosure Information

• All authors declare that there are no

conflicts of interest.

Learning Objectives

• Examine the potential beneficial effect of D3T in

the treatment of EAE, an animal model of MS.

• Investigate the molecular mechanisms

underlying the protective effect of D3T in EAE.

Multiple Sclerosis (MS)

• A chronic autoimmune, inflammatory neurological

disease of central nervous system (CNS).

▫ More then 2.3 million people affected by MS worldwide.

‐ National Multiple Sclerosis Society website.

• Experimental autoimmune encephalomyelitis (EAE)

is a widely used animal model for MS.

SLIDE 2

3H‐1,2‐Dithiole‐3‐Thione (D3T)

• D3T, a natural compound, can be found in cruciferous vegetables.
• D3T is the parent compound of dithiolethiones, which is a well‐known

class of cancer chemopreventive agents.

‐ Mol Cancer Ther (2008)

• D3T provides antioxidant activity through Nrf2 pathway.

‐ Oncol Res (1997); Cell Mol Neurobiol (2008); Exp Bio Med (2008)

• Nrf2 pathway has been reported to provide anti‐inflammatory effects.

( Nrf2 / HO‐1 / NF‐B pathway )

‐ J Pharmacol Exp (2006); J Exp Med (2011); J Immunol (2014)

S S S

D3T

O

Anethole dithiolethione (ADT) Oltipraz

To investigate whether D3T would provide a beneficial effect in EAE through its anti‐inflammatory property

Hypothesis

D3T  Nrf2  anti‐inflammation The effect of D3T in the autoimmune inflammatory disease MS/EAE is unknown

5 10 15 20 25 30 35 40 45 50 55 60 1 2 3 4 5 *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ** * * * * * * * * * * ** * *

Days post immunization Clinical Scores Vehicle D3T D3T 10mg/kg Every Day D3T Withdrawal

D3T Reduces EAE Severity

s.c. MOG immunization i.p. D3T 10mg/kg Every day starting from day 1 30d Stop Treatment C57BL/6 female 30d Clinical scores

*p<0.05; **p<0.01; ***p<0.001

Score Clinical observations

1 2 3 4 5 No obvious changes. Limp tail. Limp tail and weakness of hind legs. Limp tail and partial paralysis of hind legs. Limp tail, complete hind leg and partial front leg paralysis. Mouse is found dead due to paralysis. Vehicle D3T D3T 30mg/kg Every Other Day Clinical Score Days post immunization ** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** * 1 2 3 4 5 5 10 15 20 25 30

D3T Reduces EAE Severity

i.p. D3T 30mg/kg Every other day starting from day 3 30d C57BL/6 female Clinical scores

*p<0.05; **p<0.01; ***p<0.001

SLIDE 3

• 12 -10 -8
• 6
• 4
• 2

2 4 6 8 10 12 14 16 18 20 1 2 3 4 5 ** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ** *** *** ***

Days post treatment Clinical Scores

D3T 30mg/kg Every Other Day D3T Vehicle

D3T Inhibits EAE Progression

i.p. D3T 30mg/kg Every other day 20d EAE mice

Disease score 1.5‐2

Clinical scores

**p<0.01; ***p<0.001

Th1 Th17

CNS BBB

MG

Peripheral lymphoid

• rgan

Neuron

Myelin sheath Cytokines Cytokines IL‐12 IL‐23 IFN‐ Cytokines Chemokines IL‐17

Th1 Th17

Pathogenesis of MS/EAE

Periphery

DCs T Cells

DCs : dendritic cells MG : microglia BBB : blood‐brain barrier

D3T Inhibits DC Activation

0.0 0.5 1.0 1.5 2.0 *** ***

Il-12p40 / β-actin

LPS 3h LPS 6h MED 0.0 0.5 1.0 1.5 2.0 * ***

Il-12p35 / β-actin

1 2 3 4 5 ***

Il-23p19 / β-actin

D3T Vehicle 0.4% 1% 30% 14% 12% 12%

CD40

0% 61% 72% 52% 51% 51%

CD80

0% 18% 39% 30% 27% 26%

CD86 ISO MED LPS 100 75 50 Counts LPS + D3T (µM) Fluorescence Intensity

IL‐23p19 IL‐12p35 IL‐12p40

LPS 3h LPS 6h MED LPS 3h LPS 6h MED 1 2 3 *** *** ***

IL-23 ng/ml

MED 100 75 50 D3T (µM) LPS

IL‐23

1 2 3 4 5 *** IL-12 ng/ml *** *** D3T Vehicle

IL‐12

MED 100 75 50 D3T (µM) LPS

*p<0.05; ***p<0.001

BM‐DCs / FACS BM‐DCs / mRNA BM‐DCs / Protein

Nrf2 β-actin

Nrf2+/-

LPS

• +

+ D3T

• +

D3T Inhibits DC Activation

MED LPS MED LPS 0.0 0.5 1.0 1.5 2.0 2.5 *** IL-23 ng/ml D3T Vehicle

Nrf2+/- Nrf2-/-

1 2 3 *** *** IL-12 ng/ml

IL‐23 IL‐12

Nrf2 pathway  anti‐inflammation

*** P<0.001

BM‐DCs / WB BM‐DCs / ELISA

Nrf2-/-

• +

+

• +

SLIDE 4

Pathogenesis of MS/EAE

DCs

Periphery CNS BBB

MG

Peripheral lymphoid

• rgan

Neuron

Cytokines Chemokines Myelin sheath Cytokines Cytokines IL‐12 IL‐23 IFN‐ IL‐17

D3T

Th1 Th17 Th1 Th17 T Cells

DCs : dendritic cells MG : microglia BBB : blood‐brain barrier

CD4 IFNγ

16.8% 1.4% 1.7% 6.5% 0.01%

Th1

6.4% 0.7% 2.2% 5.8% 0%

Th17 IL-17 ISO

100 75 50 D3T (µM) Vehicle

D3T Inhibits Th1 and Th17 Differentiation

3 6 9 12 *** *** * IFNγ ng/ml

Th1

Vehicle 100 D3T (µM) 75 50

CD4+ IFN+

0.0 0.1 0.2 0.3 0.4 0.5 *** *** ** IL-17 ng/ml Vehicle 100 D3T (µM) 75 50

Th17

CD4+ IL‐17+ CD4+ T cells

*p<0.05; **p<0.01; ***p<0.001

D3T Reduces Peripheral Th1/Th17 Differentiation in EAE

Vehicle D3T CD4 IFNʏ IL-17

Spleen

Th1 Th17 8d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice Splenocytes Spleen

Vehicle D3T 3 6 9 12 15 ***

CD4+IFNʏ+ cells (%)

Vehicle D3T 1 2 3 4 5 6 ***

CD4+IL-17+ cells (%)

Spleen

CD4+ IFN+ CD4+ IL‐17+

Th1 Th17 ***p<0.001

D3T Decreases CNS Th1/Th17 Infiltration in EAE

CD4+ IFN+ CD4+ IL‐17+

# of CD4+IFNγ+ Cells (x 100)

6 12 18 24 30 ** Brain Spinal Cord 10 20 30 40 50 **

# of CD4+IL-17+ Cells (x 100)

3 6 9 12 15 **

D3T Vehicle D3T Vehicle

5 10 15 20 25 **

Th1 Th17

12d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice Mononuclear cells ( 30/70 Percoll )

CD4+

SSC Brain Spinal Cord Vehicle D3T CD4

# of CD4+ Cells (x 1000)

Brain Spinal Cord

3 6 9 12 15 **

D3T Vehicle

2 4 6 8 10 **

D3T Vehicle

**p<0.01

SLIDE 5

Pathogenesis of MS/EAE

DCs

Periphery CNS BBB

Peripheral lymphoid

• rgan

Neuron

Cytokines Chemokines Myelin sheath Cytokines Cytokines IL‐12 IL‐23 IFN‐ IL‐17

D3T

Th1 Th17 Th1 Th17

D3T D3T

MG T Cells

DCs : dendritic cells MG : microglia BBB : blood‐brain barrier

MED LPS LPS+D3T Iba1/DAPI CD80

2% 12% 43% 24%

CD86

1% 22% 60% 49% Fluorescence Intensity Counts ISO MED LPS LPS+D3T

D3T Inhibits MG Activation

Ramified form (resting) Amoeboid form (activated)

pMG: Primary microglia generated from neonatal mouse brain

1 2 3 4 5 *** Il-23p19 / β-actin *** 1 2 3 4 *** *** Il-12p35 / β-actin LPS 1.5h LPS 3h MED 0.0 0.5 1.0 1.5 2.0 2.5 *** *** Il-12p40 / β-actin D3T Vehicle LPS 1.5h LPS 3h MED LPS 1.5h LPS 3h MED

IL‐23p19 IL‐12p35 IL‐12p40

1 2 3 *** iNos / β-actin D3T Vehicle 0.0 0.5 1.0 1.5 2.0 2.5 ** Gm-csf / β-actin 0.0 0.5 1.0 1.5 2.0 *** Il-1β / β-actin LPS 1.5h LPS 3h MED 0.0 0.5 1.0 1.5 *** *** Il-6 / β-actin LPS 1.5h LPS 3h MED LPS 1.5h LPS 3h MED LPS 1.5h LPS 3h MED

iNOS GM‐CSF IL‐1 IL‐6

D3T Inhibits MG Activation

**p<0.01; ***p<0.001

D3T Suppresses MG Activation in the CNS of EAE Mice

CD80+ MG CD86+ MG

Brain Spinal Cord

CD80+CD45intCD11b+ Cells (%) 20 40 60 80 100 ** 10 20 30 40 50 60 ** Naïve Vehicle D3T Vehicle D3T Naïve 10 20 30 40 50 5 10 15 20 25 * CD86+CD45intCD11b+ Cells (%) Naïve Vehicle D3T Vehicle D3T Naïve

Brain Spinal Cord

MG

CD45int CD11b+

CD45 CD11b 12d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice Mononuclear cells ( 30/70 Percoll ) * P<0.05, ** P<0.01

Mononuclear cells / FACS

CD86+ CD45int CD11b+

CD80+ CD45int CD11b+

CD80 CD11b CD86 Brain Spinal Cord Naïve Vehicle D3T Brain Spinal Cord

SLIDE 6

D3T Reduces Inflammatory Cytokine Expression in the CNS of EAE

Il-23p19 / β-actin

0.0 0.5 1.0 1.5

* Vehicle D3T

0.0 0.5 1.0 1.5

*

Il-12p35 / β-actin

0.0 0.5 1.0 1.5 2.0

***

Il-12p40 / β-actin

0.0 0.5 1.0 1.5 2.0

***

0.0 0.2 0.4 0.6 0.8 1.0

***

0.0 0.5 1.0 1.5

** Vehicle D3T Brain

Spinal Cord

IL‐23p19 IL‐12p35 IL‐12p40

1 2 3 4 5

***

Gm-csf / β-actin

0.0 0.5 1.0 1.5

***

Il-1β / β-actin

0.0 0.5 1.0 1.5 2.0

**

Il-6 / β-actin

Brain Spinal Cord

0.0 0.5 1.0 1.5 2.0

***

0.0 0.5 1.0 1.5

***

0.0 0.5 1.0 1.5

*** Vehicle D3T Vehicle D3T

GM‐CSF IL‐1 IL‐6

12d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice

Q‐PCR *p<0.05; **p<0.01; ***p<0.001

DCs : dendritic cells MG : microglia BBB : blood‐brain barrier

DCs

Periphery CNS BBB

Peripheral lymphoid

• rgan

Neuron

Cytokines Chemokines Myelin sheath Cytokines Cytokines IL‐12 IL‐23 IFN‐ IL‐17

D3T

Th1 Th17 Th1 Th17

D3T D3T

MG

Conclusion

D3T

T Cells

Vehicle D3T D3T 30mg/kg Every Other Day

Clinical Score Days post immunization

** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** * 1 2 3 4 5 5 10 15 20 25 30

Acknowledgments

 Department of Microbiology and Immunology Ping-Chang Kuo, PhD Barbara Scofield, BS  Department of Anatomy and Cell Biology I-Chen Yu, PhD

 Department of Pharmaceutical Sciences

Dennis Brown, PhD  Department of Neurology Fen-Lei Chang, MD, PhD

SLIDE 7

ALT: alanine transaminase

Blank Positive Naïve Vehicle D3T 25 50 75 100 125 150

ALT activity (U/L)

EAE

Serum

i.p. D3T 30mg/kg Every other day starting from day 3 30d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice Serum

Th1 IFN

Vehicle D3T DMF

CD4+ T cells

IL‐17 CD4 Th17

8.4% 1.7% 0.2% 1.1% 0.2% 0.0%

D3T : 100M DMF : 100M Dimethyl fumarate (DMF) C6H8O4 MW : 144.127 g/mol 3H‐1, 2‐dithiole‐3‐thione (D3T) C3H2S3 MW : 134.243 g/mol

CD4+ T cells / FACS

IL‐12p40 IL‐12p35 IL‐23p19

1 2 3 4 5

Il-23p19 / β-actin

LPS 3h MED D3T Vehicle DMF 0.0 0.5 1.0 1.5 2.0

Il-12p35 / β-actin

LPS 3h MED D3T Vehicle DMF 0.0 0.5 1.0 1.5 2.0

Il-12p40 / β-actin

LPS 3h MED D3T Vehicle DMF

D3T : 100M DMF : 100M LPS : 100ng/ml

BM‐DCs / Q‐PCR

i.p. D3T 30mg/kg Every other day starting from day 3 30d

Nrf2+/‐, Nrf2‐/‐ female

Clinical scores

Nrf2+/- Nrf2-/-

Vehicle D3T (30mg/kg) Vehicle D3T (30mg/kg) Incidence 14/14 9/9 10/10 13/13 Mortality 1/14 0/9 0/10 0/13 Maximum score 3.9±0.2 1.5±0.3 3.7±0.4 2.1±0.3 Cumulative score 50.1±2.3 9.9±2.5 55.6±5.5 19.7±3.4* Vehicle-Nrf2+/- Vehicle-Nrf2-/- D3T-Nrf2+/- D3T-Nrf2-/- 5 10 15 20 25 30 1 2 3 4 5

Days post immunization Clinical Scores

D3T 30mg/kg Every Other Day D3T-Nrf2-/- D3T-Nrf2+/- Vehicle-Nrf2-/- Vehicle-Nrf2+/-

* * *

* P<0.05

SLIDE 8

Nrf2 β-actin

Nrf2+/-

LPS

• +

+ D3T

• +

D3T Inhibits DC Activation

MED LPS MED LPS 0.0 0.5 1.0 1.5 2.0 2.5 *** IL-23 ng/ml D3T Vehicle

Nrf2+/- Nrf2-/-

1 2 3 *** *** IL-12 ng/ml

IL‐23 IL‐12

Nrf2 pathway  anti‐inflammation

*** P<0.001

BMDCs / WB BMDCs / ELISA

Nrf2-/-

• +

+

• +

CD4+ T cells / FACS

Th1

CD4+IFNʏ+ cells (%)

D3T Vehicle

Nrf2+/-

CD4+ IFN+

50 100 150

Vehicle D3T Vehicle D3T Nrf2-/-

Th17

CD4+IL-17+ cells (%)

CD4+ IL‐17+

50 100 150

Vehicle D3T Vehicle D3T

D3T Vehicle

Nrf2+/- Nrf2-/-

MED LPS LPS+D3T ISO

Nrf2+/- Nrf2-/-

0.1%

CD80

59% 91% 90% 70% 59% 39%

CD40

36% 52% 1% 0.3% 48% 53% 3%

Nrf2+/- Nrf2-/- Nrf2-/- CD86

43% 7% 0.6% 34%

Nrf2+/-

60% 56% 23%

Nrf2+/‐ & Nrf2‐/‐ BM‐DCs / FACS

1h 3h 5h 7h LPS

• +

+ + + + + + + D3T

• +
• +
• +
• +

Nrf2+/- Nrf2 HO-1 β-actin Nrf2-/- Nrf2 HO-1 β-actin

WB

Nrf2 HO-1 β-actin C57BL/6wt

BM‐DCs WB

SLIDE 9

Nrf2 Pathway

• Nuclear factor E2‐related factor 2 (Nrf2) is a basic leucine

zipper transcription factor.

Keap1 : Kelch‐like ECH‐associated protein 1 ARE : Antioxidant Response Element HO‐1 : Heme oxygenase 1 GCLC : Glutamate‐cysteine ligase, catalytic subunit NQO1 : NAD(P)H dehydrogenase, quinone 1

Nrf2

Cytoplasm

Nucleus

Unstressed condition Nrf2

26S proteasome

Nrf2 degradation Oxidative stress

ARE

Maf

Nrf2

P

Phase II antioxidant proteins ( HO‐1, GCLC, NQO1… )

Anti‐oxidant and anti‐inflammatory activities Keap1 Cul3 Keap1 Nrf2

P

Cul3

D3T

SH

D3T

SH LPS 3h LPS 6h 1 2 3 4 5 *** Gm-csf / β-actin MED

BM‐DCs / Q‐PCR

0.0 0.5 1.0 1.5 2.0 *** *** Il-1β / β-actin D3T Vehicle LPS 3h LPS 6h MED 0.0 0.5 1.0 1.5 2.0 * Il-6 / β-actin LPS 3h LPS 6h MED

GM‐CSF IL‐1 IL‐6

* P<0.05, *** P<0.001 3 6 9 12 15 Vehicle D3T

CD4+Foxp3+ cells (%)

CD4+ Foxp3+

Tregs

CD4 Foxp3 Vehicle D3T Tregs

8d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice Splenocytes

Splenocytes / FACS Splenocytes / FACS

(n=7) (n=7)

IL‐27  IL‐10+ Tr1 TGF‐  Foxp3+ Tregs

BM‐DCs / Q‐PCR

*** P<0.001

SLIDE 10

0.3% 0.3% 1.8% 0.9% 1.6% 4.9% MED LPS Annexin V 0.3% 0.5% 1.9% 0.4% 0.6% 1.7% 0.2% 0.4% 1.3% LPS+D3T (μM) 100 75 50 PI

BM‐DCs / FACS

D3T Promotes Phase II Anti‐oxidant Enzyme Induction in MG

1 2 3 4 5 *** Gclc / β-actin *** ** 0.0 0.5 1.0 1.5 2.0 2.5 Ho-1 / β-actin *** *** *** LPS 1.5h LPS 3h LPS 5h MED 2 4 6 * *** Nqo1 / β-actin * D3T Vehicle LPS 1.5h LPS 3h LPS 5h MED LPS 1.5h LPS 3h LPS 5h MED

NQO1 GCLC HO‐1

* P<0.05, ** P<0.01, *** P<0.001

pMG / Q‐PCR

8d Vehicle‐ or D3T‐ treated C57BL/6 EAE mice Splenocytes

Vehicle D3T

Spleens

LPS D3T β-actin Nrf2 2h

mRNA Level (arbitrary Units)

Nrf2

LPS D3T 5 4 3 2 1 1.5h

BV2 Cells

SLIDE 11

D3T Promotes Phase II Anti‐oxidant Enzyme Induction in MG

1 2 3 4 5 *** Gclc / β-actin *** ** 0.0 0.5 1.0 1.5 2.0 2.5 Ho-1 / β-actin *** *** *** LPS 1.5h LPS 3h LPS 5h MED 2 4 6 * *** Nqo1 / β-actin * D3T Vehicle LPS 1.5h LPS 3h LPS 5h MED LPS 1.5h LPS 3h LPS 5h MED

NQO1 GCLC HO‐1

*p<0.05; **p<0.01; ***p<0.001