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

Disclosure Information 3 H ‐ 1, 2 ‐ Dithiole ‐ 3 ‐ Thione (D3T) as a Novel Therapeutic Agent for the Treatment of Experimental • All authors declare that there are no conflicts of interest. Autoimmune Encephalomyelitis Jui ‐ Hung (Jimmy) Yen, Ph.D. Department of Microbiology and Immunology Indiana University School of Medicine Multiple Sclerosis (MS) Learning Objectives • A chronic autoimmune, inflammatory neurological • Examine the potential beneficial effect of D3T in disease of central nervous system (CNS). the treatment of EAE, an animal model of MS. ▫ More then 2.3 million people affected by MS worldwide. ‐ National Multiple Sclerosis Society website. • Investigate the molecular mechanisms • Experimental autoimmune encephalomyelitis (EAE) underlying the protective effect of D3T in EAE . is a widely used animal model for MS.

3 H ‐ 1,2 ‐ Dithiole ‐ 3 ‐ Thione (D3T) Hypothesis D3T, a natural compound, can be found in cruciferous vegetables. • The effect of D3T in the autoimmune D3T is the parent compound of dithiolethiones, which is a well ‐ known • inflammatory disease MS/EAE is unknown class of cancer chemopreventive agents. ‐ Mol Cancer Ther (2008) S D3T  Nrf2  anti ‐ inflammation S Anethole O S dithiolethione D3T (ADT) Oltipraz To investigate whether D3T would D3T provides antioxidant activity through Nrf2 pathway. • provide a beneficial effect in EAE through ‐ Oncol Res (1997); Cell Mol Neurobiol (2008); Exp Bio Med (2008) its anti ‐ inflammatory property 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) D3T Reduces EAE Severity D3T Reduces EAE Severity *p<0.05; **p<0.01; ***p<0.001 C57BL/6 female 5 C57BL/6 Vehicle *** *** *** 5 *** *** *** *** female *** D3T *** *** *** *** *** Vehicle *** *** *** *** *** *** *** *** 4 *** *** *** *** *** * * * *** *** * ** * D3T *** * *** *** *** * * 4 * Clinical Scores * * *** * *** s.c. MOG immunization 3 *** i.p. D3T 10mg/kg 3 *** Clinical Score Every day starting from i.p. D3T 30mg/kg *** day 1 2 *** D3T Every other day 10mg/kg 2 D3T *** Every Day starting from day 3 30mg/kg 30d D3T Withdrawal *** ** Every Other 1 Day 30d 1 Stop Treatment ** * 0 30d Clinical scores 0 5 10 15 20 25 30 35 40 45 50 55 60 0 Days post immunization 0 5 10 15 20 25 30 Clinical scores Days post immunization Score Clinical observations 0 No obvious changes. 1 Limp tail. 2 Limp tail and weakness of hind legs. 3 Limp tail and partial paralysis of hind legs. 4 Limp tail, complete hind leg and partial front leg paralysis. 5 Mouse is found dead due to paralysis. *p<0.05; **p<0.01; ***p<0.001

Pathogenesis of MS/EAE D3T Inhibits EAE Progression Periphery CNS BBB Neuron EAE mice 5 *** Disease score 1.5 ‐ 2 *** *** *** Vehicle *** *** *** *** *** *** *** *** D3T *** *** *** *** *** ** 4 Chemokines Th1 Th17 Clinical Scores ** Peripheral Th1 IL ‐ 12 3 lymphoid Myelin Cytokines DCs organ IL ‐ 17 sheath D3T i.p. D3T 30mg/kg 30mg/kg 2 Every IFN ‐  Every other day Other Day Cytokines 1 20d Cytokines Th17 T Cells IL ‐ 23 MG Clinical scores 0 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 Days post treatment DCs : dendritic cells **p<0.01; ***p<0.001 MG : microglia BBB : blood ‐ brain barrier D3T Inhibits DC Activation D3T Inhibits DC Activation BM ‐ DCs / mRNA Nrf2 pathway  anti ‐ inflammation BM ‐ DCs / FACS BM ‐ DCs / Protein IL ‐ 23p19 5 Il-23p19 / β -actin Vehicle *** 4 D3T IL ‐ 23 CD40 CD80 CD86 BM ‐ DCs / ELISA 3 BM ‐ DCs / WB 3 Vehicle 2 0.4% 0% 0% D3T ISO 1 IL-23 ng/ml 2 Nrf2 +/- Nrf2 -/- *** 0 *** MED *** LPS 3h LPS 6h 1 Nrf2 +/- Nrf2 -/- 1% 61% 18% MED 2.5 Vehicle 0 LPS - + + - + + 2.0 IL ‐ 12p35 D3T IL-23 ng/ml MED 100 75 50 2.0 D3T - - + - - + 1.5 72% D3T (µM) 39% LPS Il-12p35 / β -actin *** *** IL ‐ 23 30% 1.5 LPS 1.0 1.0 * Nrf2 0.5 52% 0.5 30% 100 14% LPS + D3T (µM) IL ‐ 12 0.0 0.0 MED LPS 3h LPS 6h 5 3 *** *** β -actin 51% 4 IL-12 ng/ml 27% 75 IL-12 ng/ml 12% 3 *** IL ‐ 12p40 2 *** *** 2 Counts 2.0 IL ‐ 12 *** 1 51% Il-12p40 / β -actin 26% 50 1.5 *** 1 12% 0 MED 100 75 50 1.0 Fluorescence Intensity D3T (µM) 0.5 0 LPS MED MED 0.0 LPS LPS MED LPS 3h LPS 6h *p<0.05; ***p<0.001 *** P<0.001

D3T Inhibits Th1 and Th17 Pathogenesis of MS/EAE Differentiation CD4 + IFN  + CNS Periphery BBB Neuron 12 CD4 + T cells IFN γ ng/ml 9 Th1 D3T (µM) D3T 6 * ISO Vehicle 75 50 100 Chemokines 0.01% 16.8% 1.4% 1.7% 6.5% 3 *** Th1 Th17 *** IFN γ Th1 Peripheral IL ‐ 12 Th1 0 lymphoid Myelin Vehicle 100 75 50 Cytokines DCs IL ‐ 17 sheath organ D3T (µM) 6.4% 0.7% 2.2% 0% 5.8% IFN ‐  IL-17 Th17 CD4 + IL ‐ 17 + Cytokines 0.5 Cytokines CD4 Th17 T Cells Th17 IL ‐ 23 MG 0.4 IL-17 ng/ml ** 0.3 *** 0.2 *** 0.1 DCs : dendritic cells 0.0 MG : microglia Vehicle 100 75 50 D3T (µM) BBB : blood ‐ brain barrier *p<0.05; **p<0.01; ***p<0.001 D3T Decreases CNS Th1/Th17 D3T Reduces Peripheral Th1/Th17 Infiltration in EAE Differentiation in EAE CD4 + Vehicle ‐ or D3T ‐ treated C57BL/6 Spinal Brain Th17 EAE mice Cord Th1 Vehicle ‐ or D3T ‐ Spinal Brain Cord treated C57BL/6 Spleen CD4 + IFN  + CD4 + IL ‐ 17 + EAE mice Vehicle ** 30 50 ** # of CD4 + IFN γ + Cells (x 100) Vehicle D3T 24 Th1 40 Spleen Spleen 18 30 CD4 + IFN  + 12d IFN ʏ D3T Th1 15 6 12 20 *** *** CD4 + IL-17 + cells (%) SSC CD4 + IFN ʏ + cells (%) 5 6 10 12 8d 0 0 4 CD4 9 3 IL-17 Spinal Th17 Brain 6 Cord 2 15 ** 25 ** # of CD4 + IL-17 + Cells (x 100) 3 1 ** ** # of CD4 + Cells (x 1000) 15 10 12 20 Th17 CD4 0 0 12 8 9 15 CD4 + IL ‐ 17 + Vehicle D3T Vehicle D3T Splenocytes 9 6 6 10 Mononuclear cells 6 4 3 5 ( 30/70 Percoll ) 3 2 0 0 ***p<0.001 0 0 Vehicle D3T Vehicle D3T Vehicle D3T Vehicle D3T **p<0.01

Pathogenesis of MS/EAE D3T Inhibits MG Activation MED LPS LPS+D3T CNS Periphery BBB Neuron Iba1/DAPI D3T pMG: Primary microglia generated from neonatal D3T Chemokines mouse brain Th1 Th17 Peripheral Th1 IL ‐ 12 lymphoid Myelin Cytokines DCs organ IL ‐ 17 sheath Ramified form Amoeboid form MED IFN ‐  ISO LPS LPS+D3T (resting) (activated) Cytokines Cytokines 2% 12% 43% 24% CD80 Th17 T Cells IL ‐ 23 MG Counts 22% 60% 49% CD86 D3T 1% DCs : dendritic cells MG : microglia Fluorescence Intensity BBB : blood ‐ brain barrier D3T Suppresses MG Activation in D3T Inhibits MG Activation the CNS of EAE Mice Vehicle ‐ or D3T ‐ treated C57BL/6 EAE mice Mononuclear cells / FACS IL ‐ 23p19 IL ‐ 12p35 IL ‐ 12p40 CD80 + MG CD80 + CD45 int CD11b + 4 *** 2.5 Il-12p35 / β -actin *** Brain Il-23p19 / β -actin 5 *** Vehicle Spinal Cord Il-12p40 / β -actin D3T 12d 3 2.0 4 *** Naïve Vehicle D3T CD80 + CD45 int CD11b + ** 100 ** 60 1.5 3 *** 2 50 *** 80 Cells (%) 1.0 Brain 2 40 1 60 0.5 1 30 40 0 0 0.0 20 MED MED MED LPS LPS LPS LPS LPS LPS 20 10 Spinal 1.5h 3h 1.5h 3h 1.5h 3h CD80 Cord 0 0 Mononuclear cells D3T Naïve Vehicle D3T Naïve Vehicle IL ‐ 1  iNOS GM ‐ CSF IL ‐ 6 ( 30/70 Percoll ) CD86 + MG CD86 + CD45 int CD11b + *** 3 Vehicle 2.5 2.0 1.5 D3T Gm-csf / β -actin ** Il-1 β / β -actin *** Brain Spinal Cord iNos / β -actin Il-6 / β -actin 2.0 MG 1.5 *** CD86 + CD45 int CD11b + Cells 2 *** 1.0 Brain CD45 int CD11b + 50 1.5 25 1.0 * 1.0 40 1 20 0.5 0.5 0.5 (%) 30 15 CD45 0 0.0 0.0 0.0 Spinal 20 10 Cord MED MED MED MED CD86 LPS LPS LPS LPS LPS LPS LPS LPS 5 10 1.5h 3h 1.5h 3h 1.5h 3h 1.5h 3h 0 0 CD11b Naïve Vehicle D3T Naïve Vehicle D3T CD11b **p<0.01; ***p<0.001 * P<0.05, ** P<0.01

D3T Reduces Inflammatory Cytokine Conclusion Expression in the CNS of EAE Vehicle ‐ or D3T ‐ CNS Periphery BBB Neuron treated C57BL/6 Spinal Cord Brain Spinal Cord Brain D3T EAE mice 5 *** *** 1.5 2.0 2.0 *** *** *** Vehicle *** *** *** *** Il-23p19 / β -actin 5 *** *** * *** *** Gm-csf / β -actin *** *** *** 1.5 4 1.5 D3T 1.0 D3T *** IL ‐ 23p19 GM ‐ CSF 3 4 1.0 1.0 *** Chemokines 0.5 2 0.5 0.5 Th1 Th17 1 *** Clinical Score 0.0 0.0 0 0.0 Th1 IL ‐ 12 Peripheral 3 12d *** *** *** Myelin 1.5 1.5 lymphoid DCs Cytokines 1.5 * 1.0 *** Il-12p35 / β -actin Il-1 β / β -actin IL ‐ 17 sheath 0.8 organ *** IL ‐ 1  IL ‐ 12p35 1.0 1.0 1.0 0.6 IFN ‐  2 D3T *** 0.4 0.5 0.5 0.5 30mg/kg 0.2 *** Cytokines 0.0 0.0 Every Other 0.0 0.0 Day *** 1.5 1 Cytokines 2.0 2.0 *** 1.5 ** ** ** Th17 Il-12p40 / β -actin T Cells IL ‐ 23 Il-6 / β -actin 1.5 MG 1.5 1.0 * 1.0 IL ‐ 6 IL ‐ 12p40 1.0 1.0 0.5 D3T 0.5 0 0.5 0.5 0 5 10 15 20 25 30 Q ‐ PCR 0.0 0.0 0.0 0.0 Vehicle D3T Vehicle D3T Vehicle D3T Vehicle D3T DCs : dendritic cells Days post immunization D3T MG : microglia BBB : blood ‐ brain barrier *p<0.05; **p<0.01; ***p<0.001 Acknowledgments  Department of Microbiology and Immunology Ping-Chang Kuo, PhD Barbara Scofield, BS  Department of Anatomy and Cell Biology  Department of Neurology I-Chen Yu, PhD Fen-Lei Chang, MD, PhD  Department of Pharmaceutical Sciences Dennis Brown, PhD