6/18/215 Therapeutic developments for autoimmune demyelinating diseases: Musings from a MD (Mouse Doctor) Michael K. Racke, M.D. May 28, 215 Relevant Disclosures Editorial Boards for Journal of Neuroimmunology, JAMA Neurology, and Annals of Neurology Grant support from National Multiple Sclerosis Society and National Institutes of Health Consultant for Accorda, Biogen Idec, Diogenix, Genentech, Questcor, Revalesio, Novartis, Roche and Teva Neuroscience 1
6/18/215 Multiple Sclerosis Worked on the mechanism of glatiramer acetate (copolymer 1) as a resident Copolymer 1 first treatment derived from EAE model Bob Fritz s relationship with Dale results in my heading to Bethesda McFarland and McFarlin 2
6/18/215 To understand MS, you need to work on patients! 3
6/18/215 1=limp tail 2=moderate hind limb weakness 3=severe hind limb weakness 4=hind limb paralysis 5=hind and fore limb paralysis 6=death due to EAE Clinical Score Evaluation in EAE 4
6/18/215 Role of adjuvants Component of pathogen that engages Toll-like receptors IRAK-1 is important molecule to deliver signal from TLR to nucleus Immune genes are activated as part of the innate immune response 3 WT IRAK-1-/- EAE score 2 1 13/13 3/13 1 2 3 4 Time (days) Immunize with MOG 35-55/CFA 5
6/18/215 Differentiation of Naïve T Cells is Primarily Determined by Cytokine Environment IFNγ and IL-12 Th1 Cell Secrete proinflammatory cytokines: IFNγ, LT, and IL-2 Mediate MS. Naïve CD4+ T Cell IL-4 Th2 Cell Secrete Anti- Inflammatory cytokines: IL-4, IL- 5, IL-1, and IL-13 Protect from MS. Transcriptional Regulation of T Cell Differentiation IL-23 IL-12R IL-12 MHC TCR CD3 B7 CD28 IL-4 IL-4R IL-23R IFN-γ IFN-γR STAT1 T-bet STAT6 STAT3 STAT4 GATA3 RORγt RORγt ligand unknown IL-17 IFN-γ IL-4 6
6/18/215 Development of an sirna specific for T-bet Remove spleen Transfect cells with sirna Culture splenocytes with MBP Ac1-11 Isolate protein from nucleus Transgenic mice with T cell receptor specific for MBP Ac1-11 Western Blot T-bet Actin ------ 24 hr ----- ------ 48 hr ----- MBP Ac1-11 + + - - + + - - sirna-tbet + - + - + - + - Mock-transfected - + - + - + - + Relative Protein Expression (T-bet/ Actin) -------- 24 hr ------- ------- 48 hr -------- 1..75.5.25. sirna-tbet, +Ag Mock, +Ag sirna-tbet, -Ag Mock, -Ag sirna-tbet, +Ag Mock, +Ag sirna-tbet, -Ag Mock, -Ag Lovett-Racke et al (24) Immunity 21:719-731. Silencing T-bet Inhibits IFNγ Production and Th1 Differentiation Transgenic mice with T cell receptor specific for MBP Ac1-11 Remove spleen Transfect cells with sirna Culture splenocytes with MBP Ac1-11 or no antigen Collect supernatants at 48, 72, and 96 hr. ELISA Interferon- [pg/ml] 15 1 5 Mock -Ag Mock +Ag sirna-tbet -Ag sirna-tbet +Ag 48 hr 72 hr 96 hr Lovett-Racke et al (24) Immunity 21:719-731. 7
6/18/215 Myelin-specific T cells in which T-bet has been Silenced Fail to Transfer Experimental Autoimmune Encephalomyelitis Transgenic mice with T cell receptor specific for MBP Ac1-11 Remove spleen Transfect with sirna Culture splenocytes with MBP Ac1-11 Inject cells into wild-type mice Mean Clinical Score 3 Non-transfected (3/5) sirna-ns (3/3) sirna-tbet (1/6) AS-Tbet (1/6) AS-GATA3 (2/2) 2 1 5 1 15 2 25 3 Days Post Transfer Lovett-Racke et al (24) Immunity 21:719-731. Silencing T-bet Ameliorates EAE Transgenic mice with T cell receptor specific for MBP Ac1-11 Remove spleen Culture splenocytes with MBP Ac1-11 plus IL-12 Inject cells into wild-type mice Inject sirna via tail vein. 4 sirna-ns (9/9) sirna-tbet (8/1) Mean Clinical Score 3 2 1 5 1 15 2 25 3 35 4 45 5 55 6 Days Post Transfer 8
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6/18/215 3 HC1 Naïve T cells require costimulation 25 2 15 1 5 No Ag MBP TT No Ag MBP TT ------------------ Control Ab ----------------------------------- Anti-CD28 ----------------- Lovett-Racke et al. J Clin Invest 1998 4 3 Memory/activated T cells do not require costimulation MS9 2 1 5 4 3 2 1 No Ag MBP TT No Ag MBP TT ----------------- Control Ab ----------------- ------------------ Anti-CD28 ----------------- Lovett-Racke et al. J Clin Invest 1998 1
6/18/215 T Cells in Multiple Sclerosis MHC peptide TCR IFNγ IL-12 T H 1 T-bet T H 2 EAE/MS Perivascular Inflammation in MS brain APC B7- CD28 Naïve CD4 T Cell IL-4 IL-6 GATA3 T H 17 No EAE Demyelination in MS brain RORγt T-bet EAE/MS Activation Differentiation Effector Function Intrinsic defects in MS patients CD4 T cells make them more susceptible to develop into actively pathogenic cells Can these defects translate into clinically useful biomarkers? Isolation of Naïve CD4 T Cells Peripheral Blood Mononuclear Cells Healthy (n=16) RRMS (n=12) SPMS (n=5) PPMS (n=5) Magnetic Bead Sorting (Negative Selection) CD45RA <APC-A>: CD45RA 1 5 1 4 1 3 PRE-COLUMN POST-COLUMN 45 37 37 1 5 <APC-A>: CD45RA 1 4 1 3 1 2 1 2 2.8 15 1.9 97 2.4 97 1.7 Naïve CD4 T cells (CD4+CD45RA+) 1 2 1 3 1 4 1 5 <Pacific Blue-A>: CD4 CD4 1 2 1 3 1 4 1 5 <Pacific Blue-A>: CD4 CD4 11
6/18/215 Differential Expression of mirna in MS 55 significantly different mirnas (P<.5, StatMiner, parametric Limma test) in one or more type of MS 23 up-regulated 32 down-regulated Disability RRMS Time 9 11 4 3 2 1 12 Disability SPMS Time (follows RRMS) Disability PPMS Time mirs Target Bmi-1 and GATA3 Transfect with mirna Flow Cytometry of Bmi-1 and GATA3 Naïve human CD4 T cells Healthy Donor Anti-CD3/CD28 1 5 Control mir-27a mir-27b mir-128 mir-34 All mirs 38 26 1 5 37 2 1 5 4 2 1 5 42 17 1 5 38 12 1 5 37 8.6 <APC-A>: BMI1 1 4 1 3 <APC-A>: BMI1 1 4 1 3 <APC-A>: BMI1 1 4 1 3 <APC-A>: BMI1 1 4 1 3 <APC-A>: BMI1 1 4 1 3 <APC-A>: BMI1 1 4 1 3 Bmi1 1 2 36.2 1 2 42.2 1 2 1 2 1 2 1 2 4.3 41.3 5.4 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 <PE-A>: GATA-3 <PE-A>: GATA-3 <PE-A>: GATA-3 <PE-A>: GATA-3 <PE-A>: GATA-3 <PE-A>: GATA-3 GATA3 54.4 Guerau-de-Arellano et al. Brain 211 12
6/18/215 mirs Enhance Encephalitogenicity TCR transgenic mouse Isolate spleen T Cells Transfect cells with mirs Activate with MBP Ac1-11 EAE Mean Clinical Score 4 3 2 1 Control mir (n=7) mir-27ab/128/34 (n=7) p=.12 2 4 6 8 1 12 Days post-transfer T Cells in Multiple Sclerosis MHC peptide TCR IFNγ IL-12 T H 1 T-bet T H 2 EAE/MS Perivascular Inflammation in MS brain APC B7- CD28 Naïve CD4 T Cell mir-27ab mir-128 mir-34 IL-4 IL-6? GATA3 T H 17 RORγt T-bet No EAE EAE/MS Sclerotic plaques in MS brain Activation Differentiation Effector Function mir-27ab mir-128 mir-34 Bmi-1 GATA3 Th1 Pathway Susceptibility to EAE/MS 13
6/18/215 Fumarate Effect on Psoriasis Psoriasis Area-and Severity Index 25 2 15 1-5 <6 <24 >24 Week IFN- to IL-4 ratio in CD4+ T cells 6 4 2 <6 <24 >24 Week Ghoreschi et al. J Exp Med 211 14
6/18/215 EAE score 4 3 2 1 Ac1-11+MMF Ac1-11 1 2 3 4 Days after adoptive transfer EAE incidence 15 1 5 1 2 3 4 days after adoptive transfer Ac1-11 Ac1-11+MMF MMF treated TCR Tg lymphocytes adoptively transfer a milder course of EAE. Knocking down HO-1 successfully restored p19 activity Luciferase activity (Fold change) 3 Control HO-1 sirna 2 1 LPS DMF + + + + + + 15
6/18/215 HO-1 interacts with p19 promoter Journal of Experimental Medicine, Racke and Rocken lab. 211 Effect of DMF on APCs Peng et al. JBC 212 16
6/18/215 My first paper in 1991 Transforming growth factor-beta Worked on transforming growth factor (TGF)-beta as treatment for EAE and MS patients as a fellow This is how it is supposed to work, treatment works in EAE and then you try it in people with MS 17
6/18/215 TGF-beta for SPMS failed mirnas in MS target TGF- signaling mir-let-7 mir-27 mir-128 mir-545 mir-573 mir-642 TGFβ mir-34 mir-573 mir-let-7 mir-18 mir-27 mir-128 mir-34 mir-21 mir-33 mir-181 mir-519 TGFβR1 Smad2 Smad7 Cytoplasm TGFβR2 Smad2 P Smad2 P Smad4 mir-27 mir-128 mir-34 mir-545 mir-573 Nucleus Smad2 P Smad4 Smad binding domains 18
6/18/215 Conclusions Differences in immune response in MS patients and healthy controls will define therapeutic targets Micro RNA may help define susceptibility and provide new therapeutic targets for a number of diseases, including MS EAE can provide insights into pathogenesis and help test new therapies What a crew! 19