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Evaluation of the effects of interleukin-27 transfected mesenchymal stem cells with RNA interference-IL-6 on certain neuroimmunological parameters in experimental autoimmune encephalomyelitis mice b y M. Shafi Mojadadi Supervisors: Dr. M. Ebtekar & Dr. M. Golkar Advisor: Dr. H. Khan Ahmad

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Experimental Autoimmune Encephalomyelitis (EAE) is a T-cell-mediated inflammatory demyelinating disease of the CNS and serves as the primary animal model for MS. Inflammation and demyelination are two main characteristics of MS and EAE. Suppression of inflammation and induction of remyelination are two strategies for the treatment of MS.

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Th1 and especially Th17 cells have central role in the pathogenesis of MS. IL-6, a proinflammatory cytokine, has a critical role in the development of Th17 cells. TH0 IL-6 + TGF-b Th17 Treg IL-6 + TGF-b

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RN interference (RNAi) is a powerful gene silencing mechanism. Suppression of IL-6 by using siRNA molecules can prevent Th17 development.

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Interleukin-27 (IL-27) has anti-inflammatory properties. IL-27 can suppress IL-23-mediated Th17 differentiation. Interestingly, IL-27 acts on activated differentiated helper T cells and converts them into IL -10-producing Tr1-like cells. Thus, IL-27 can suppress harmful TH17 inflammatory responses in MS.

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Mesenchymal stem cells (MSCs) having anti-inflammatory and neuroprotective properties can be a good candidate for treatment of inflammatory and neurodegenerative diseases.

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Evaluation of the therapeutic effects of IL-27-transfected MSCs in companion with siRNA-IL-6 in EAE model

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The therapeutic effects were investigated by measuring certain neuroimmunological parameters, including: 1.EAE progression and severity 2.Leukocyte infiltration into the CNS 3.Demyelination 4.Gliosis 5.Cytokine profile 6.Regulatory T cells (TregCD4+foxp3+ and Tr1CD4+IL-10+) population in the CNS and spleen

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IL-27-transfected BM-MSCs shRNA-IL6 (shRNA6) EAE mice Neuroimmunological measurements Work outline

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Part A: Generation of IL-27 transfected MSCs Part B: Design and efficacy evaluation of plasmid-based siRNA against IL-6 (shRNA6) Part C: Induction of EAE in C57BL/6 mice Part D: Grouping and treatment of the EAE mice Part E: Neuroimmunological measurements Part A: Generation of IL-27 transfected MSCs Part B: Design and efficacy evaluation of plasmid-based siRNA against IL-6 (shRNA6) Part C: Induction of EAE in C57BL/6 mice Part D: Grouping and treatment of the EAE mice Part E: Neuroimmunological measurements

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Part A: Generation of IL-27-transfected MSCs A-1. isolation of bone marrow-derived MSCs Part A: Generation of IL-27-transfected MSCs A-1. isolation of bone marrow-derived MSCs

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Part A: Generation of IL-27-transfected MSCs A-2. characterization of bone marrow-derived MSCs Part A: Generation of IL-27-transfected MSCs A-2. characterization of bone marrow-derived MSCs A-2a. differentiation assay

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Part A: Generation of IL-27-transfected MSCs A-2. characterization of bone marrow-derived MSCs Part A: Generation of IL-27-transfected MSCs A-2. characterization of bone marrow-derived MSCs A-2b. Immunophenotyping CD24 CD29 CD44 CD45 CD11b

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(1362 bp) A-3. Subcloning of mIL-27 gene into P240 plasmid Xma I BamH I Part A: Generation of IL-27-transfected MSCs

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mIL-27 (1362 bp) mIL-27 Part A: Generation of IL-27-transfected MSCs A-3. Subcloning of mIL-27 gene into P240 plasmid 1 3 2

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Colony PCR Enzyme digestion (Xma I & BamH I) mIL-27 (1362 bp) P240 (10 kb) Part A: Generation of IL-27-transfected MSCs A-3. Subcloning of mIL-27 gene into P240 plasmid Screening 1 2

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EndoFree P240-mIL27 plasmid Transfection of 293T cell Biological assay (IFN-g assay) Supernatant for ELISA Cell pellet for western blot Part A: Generation of IL-27-transfected MSCs A-3. Subcloning of mIL-27 gene into P240 plasmid

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Co-transfection of P240-mIL27, PsPAX2 and pMD2G.1 EndoFree plasmids Part A: Generation of IL-27-transfected MSCs A-4. Production of IL-27-coding lentiviral particles 1 2

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Virus concentration Virus Titration Part A: Generation of IL-27-transfected MSCs A-4. Production of IL-27-coding lentiviral particles

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BM-MSCs transduction (MOI: 50) Part A: Generation of IL-27-transfected MSCs A-5. Transduction of BM-MSCs

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BamHI/SalI G110046, u6shrna+ pBluescript II SK+ Cla I Part B: Design and efficacy evaluation of plasmid-based siRNA against IL-6 (shRNA6) B-1. Subcloning of shRNA6 construct into P240 plasmid ClaI-U6 promoter-shRNA6-loop-shRNA6-U6 terminatorClaI

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shRNA6 (325 bp) P240 (10 kb) Colony PCR Enzyme digestion (ClaI) Screening Part B: Design and efficacy evaluation of plasmid-based siRNA against IL-6 (shRNA6) B-1. Subcloning of shRNA6 construct into P240 plasmid 1 2

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B-2. Production of lentiviral vector-based shRNA6 B-3. Transduction of J774A.1 cells, a macrophage cell line (MOI: 10) B-4. Treatment of transduced cell line by LPS B-5. ELISA & Real-time PCR Part B: Design and efficacy evaluation of plasmid-based siRNA against IL-6 (shRNA6)

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50 Mice (female 8-12 week-old C57BL/6) 200 microgram MOG 35-55 in CFA (day 0) 600 ng pertussis toxin (day 0 and day 2) Part C: Induction of EAE in C57BL/6 mice

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1 4 2 3

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1 IL27-MSCs + shRNA6 2 MSCs 2 MSCs 3 P240-mIL27 3 P240-mIL27 4 P240-shRNA6 4 P240-shRNA6 5 PBS or 3T3 cells 5 PBS or 3T3 cells Part D: Grouping and treatment of the EAE mice

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Part E: Neuroimmunological measurements Score 1 Score 2 Score 3Score 4 E-1. The clinical symptoms of treated mice were evaluated daily and scored according to a standard 0-5 severity scale

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Part E: Neuroimmunological measurements E-2. At day 35, all mice were sacrificed and the following assays were performed: E-2a. Histopathological exams for determination of leukocyte infiltration into the CNS, gliosis and demyelination. E-2b. Immunological assays measurement of IL-4, IFN-, IL-17, IL-6 and IL-10, using ELISA measurement of CD4+Foxp3+ and CD4+IL10+ regulatory T cells population, using flowcytometry

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Part E: Neuroimmunological measurements E-2a. Histopathological exams 1 2 3 4

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Part E: Neuroimmunological measurements E-2a. Histopathological exams

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Part E: Neuroimmunological measurements E-2b. Immunological assays Cytokine assay Flow cytometry

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Part E: Neuroimmunological measurements E-2b. Immunological assays (IL-4, IFN-, IL-17, IL-6 and IL-10)

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Part E: Neuroimmunological measurements E-2b. Immunological assays

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1. independent t-test 2. One-Way ANOVA Tukey 3. Kruskal-Wallis H 4. Mann-Whitney U

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MSCs isolation and characterization 1. The cells could attach to the surface of cell culture flasks

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MSCs isolation and characterization 2. They could differentiate to adipocytes (a) and osteocytes (b) (a) (b)

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MSCs isolation and characterization 3. They expressed CD24, CD29 and CD44 surface markers but not CD11b and CD45

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Production of IL-27 coding lentiviral particles 1. mIL-27 gene was successfully subcloned into P240 plasmid. 2. P240-mIL27-transfected 293T cell (a) produced IL-27 (western blot assay) (b) (a) (b)

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3. mIL-27 was biologically active Production of IL-27 coding lentiviral particles

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4. mIL-27-coding lentiviral particles could efficiently transfect BM-MSCs (a) and (b) (a) (b) Production of IL-27 coding lentiviral particles

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5. mIL-27-coding lentiviral particles had no negative effect on the phenotype and differentiation potential of transfected MSCs. Surface markerBefore transductionAfter transduction CD24 78 275 4 CD29 93.1 397.66 7 CD44 484.6582.45 2 CD11b0 0.1 0.48 CD450 0.020.13 Production of IL-27 coding lentiviral particles

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Design and efficacy evaluation of shRNA6 1.shRNA6 was successfully subcloned into P240 plasmid.

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Design and efficacy evaluation of shRNA6 2. P240-shRNA6 could efficiently suppress the expression of IL-6 gene in J774A.1 cells, according to the results obtained from ELISA (a) and Real-time PCR (b). (a) (b)

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Evaluation of the EAE status in mice A. Disease severity and progression B. Mononuclear cells infiltration into the CNS C. Demyelination D. Gliosis E. Cytokine profile F. Regulatory T cells (TregCD4+foxp3+ and Tr1CD4+IL-10+) population in the CNS and spleen

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A. Disease severity A-1. shRNA6 (G4) and especially IL27 (G3) could efficiently suppress EAE severity, but MSCs couldn't. 1 2 3 4 5 2 5 > 1 4 > 3

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A. Disease severity Between-groups comparison of the mean scores of EAE severity 2 5 > 1 4 > 3

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A. Disease severity mean maximum scores of EAE severity A-2. The progression of disease was markedly attenuated in group 3.

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B. Mononuclear cells infiltration into the CNS B-1. A significant reduction in mononuclear cells infiltration was seen in group 3 and1.

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C. Demyelination C-1. A small degree of demyelination was seen in groups 1 and 3.

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D. Gliosis oligodendrocytosisastrocytosis oligodendrocytosis

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Histopathological findings

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E. Cytokine profile E-1. IL-4 significantly increased in groups 3 and 1. 5 < 2 4 1 < 3

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E. Cytokine profile E-2. IFN- significantly decreased in groups 3, 1 and 4. 2 5 < 4 < 1 < 3

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E. Cytokine profile E-3. IL-17significantly decreased in groups 3, 4 and 1. 5 2 < 1 < 4 < 3

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E. Cytokine profile E-4. IL-6 significantly decreased in groups 3, 4 and 1. 5 2 < 1 < 4 < 3

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E. Cytokine profile E-5. IL-10 significantly increased in groups 1, 3 and 2. 4 < 5 < 2 < 3 1

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F. Regulatory T cells population F-1. TregCD4+Foxp3+ population were significantly increased in the spleen of groups 3, 1 and 4. 5 2 < 1 4 < 3

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F. Regulatory T cells population F-2. Tr1CD4+IL10+ population were increased in the spleen of groups 3 and 1. 4 < 5 2 < 1 3

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F. Regulatory T cells population F-3. TregCD4+Foxp3+ population were significantly increased in the CNS of groups 3, 1 and 4. 2 5 < 4 < 1 3

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F. Regulatory T cells population F-4. No Tr1 cell was detected in the CNS of all groups

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1.MSCs were easily isolated from mouse bone marrow. 2. MSCs had dormant and immortal phases in their lifespan. 3. Lentiviral system but not nonviral methods could efficiently treansfect mouse MSCs. MSCs isolation, characterization and transduction

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4. Transduction had no negative effects on MSCs phenotype or differentiation potential. MSCs isolation, characterization and transduction

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A comparative view on the rate of MSCs transfection in different studies MSCs isolation, characterization and transduction MethodspeciesreagentRate of transfectionAuthor NonviralmouseLipofectamin ltx< 3%Our study Nonviralmouseturbofect< 3%Our study NonviralratEffecten< 9%Gheisari et al. NonviralratFuGENE HD< 6%Gheisari et al. Nonviralratpolyfect16%Gheisari et al. NonviralratLipofectamine 200020%Gheisari et al. NonviralHumanCationic liposomes2-35%Madeira et al. Viralmouselentivirus93%Our study Viralmouselentivirus61%Ricks et al. Viralhumanlentivirus70%Van Damme Viralmonkeylentivirus42%Lee Viralbaboonlentivirus72-99%Bartholomew

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1. BM-MSCs had no ameliorative effect in active EAE. 2. BM-MSCs were not found in the CNS of EAE mice. 3. BM-MSCs had no effect on cytokine profile of the EAE mice when compared with control group. MSCs and EAE

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In a similar study, injection of BM-MSCs at day 24 post immunization hasnt had any positive effect on EAE severity (Zappia et al 2005). On the contrary, it has been reported that intravenous injection of Adipose-derived MSCs at days 23 and 28 post immunisation has had ameliorative effects in EAE mice (Constantin et al. 2009) MSCs and EAE

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After I.V. infusion of human MSCs into injured mice, only a small number of the infused cells (0.01%) were detected 96 hours later in peripheral organs including the brain (Lee RH et al. 2009) MSCs and EAE

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The following factors may explain this discrepancy: source of MSCs time of injection number of injected cells (Liu et al. 2009) entrapment of intravenous injected MSCs MSCs and EAE

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1. IL-27: 1-1. reduced EAE severity 1-2. suppressed Th1 and Th17 responses 1-3. promoted Th2 response 1-4. enhanced CD4+Foxp3+ regulatory T cells population in the spleen and CNS IL-27 and EAE

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1-5. reduced mononuclear cells infiltration into the CNS 1-6. decreased demyelination IL-27 and EAE 1. IL-27:

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Sweeney et al. (2011): IL-27 mediates the response to IFN- b therapy in multiple sclerosis patients by inhibiting Th17 cells. Fitzgerald et al. (2007): IL-27 suppresses encephalitogenic Th17 responses and reduces their capacity to transfer EAE to naive animals. IL-27 and EAE

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Regulatory T cells play a key role in the control of self -antigen-reactive T cells and the induction of peripheral tolerance in vivo. Treg cells administered to mice can significantly reduce EAE severity (Grtner et al. 2006). Co-transfer of Treg cells with CD4+CD 25 T cells prevents the development of experimentally induced autoimmune diseases (Grtner et al). IL-27 and EAE

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1. We chose u6 promoter because of its efficacy. 2. Plasmid-based shRNA6 could effectively suppress the expression of IL-6 gene in vitro and in vivo. in a similar study, synthetic siRNA against IL-6 could suppress up to 75% of IL-6 expression (Khoury 2008). shRNA6: design, efficacy evaluation and therapeutic effects

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3. shRNA6 could ameliorate the clinical course of EAE mice. Combined siRNA against Il-1, IL-6 and IL-18 could suppress RA severity (Khoury et al. 2008). 5. By suppression of IL-6, shRNA6 could diminish TH17 development. shRNA6: design, efficacy evaluation and therapeutic effects

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6. shRNA6 enhanced Tregs population in the spleen and CNS of EAE mice. Although use of naked siRNA has been reported in many studies, it can be more effective if siRNA molecules delivered by using a suitable delivery systems. (e.g. cationic lipoplex, liposomes or cationic polymers). shRNA6: design, efficacy evaluation and therapeutic effects

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1. shRNA6 and especially IL-27 by shifting the immune responses from inflammatory Th1/Th17 towards anti-inflammatory Th2-type responses, as well as, by promoting Treg population may be considered a suitable candidate for the treatment of inflammatory diseases such as MS. 2. It seems that BM-MSCs have no therapeutic effect in EAE model that is in chronic phase, or when the disease has been stabilized. Final conclusion

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1.increasing the dose of MSCs 2.Changing the route of injection 3.Changing the time of injection 4.Use of other sources of MSCs 5.Further study about interactions between MSCs and inflammatory milieu

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6.Detailed studies about migratory patterns of MSCs in the body (the study of the migratory abilities of MSCs in vivo will elucidate the requirements for homing and engraftment ) 7. Targeting the migration of MSCs (e.g. lentiviral transduction of these cells with CXCR4 for homing of progenitor cells to ischemic tissues) 8. Increasing the dose and times of shRNA6 injection

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9. Use of a appropriate delivery system for shRNA6 (e.g. liposome, biodegradable polymers and ) 10. Targeting of siRNA molecules to a specific tissue or cell type by addition of a receptor ligand or antibody

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