Passive induction of experimental autoimmune encephalomyelitis by activated or tolerogenic dendritic cells

doi:10.1038/protex.2017.136

Method Article

Passive induction of experimental autoimmune encephalomyelitis by activated or tolerogenic dendritic cells

https://doi.org/10.1038/protex.2017.136

This work is licensed under a CC BY 4.0 License

This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.

Version 1

posted

You are reading this latest protocol version

Experimental autoimmune encephalomyelitis (EAE) is a widely accepted murine model for studying pathological features of multiple sclerosis (MS). Induction of EAE results infiltration of T cells and demyelination of axon in central nervous system, further leading the clinical symptoms of MS. Beside T cells, dendritic cells (DCs) is a key importance for initiation of EAE through their antigen presentation ability. Because of the plasticity of DCs, agents (CDDO-DFPA in our lab) that repress activated DCs confer a tolerogenic DC (TolDC) phenotype. TolDCs have emerged as clinical targets for the treatment of autoimmunity. This protocol describes a method for passive induction of EAE by adoptive transfer of DCs primed with myelin antigen into naïve mice. We have shown the ability of inducing EAE between activated DCs and TolDCs in associated publication “A unique tolerizing dendritic cell phenotype induced by the synthetic triterpenoid CDDO-DFPA (RTA-408) is protective against EAE” in Scientific report.

Cell biology

Developmental biology

Biological techniques

CDDO-DFPA

Dendritic cell tolerance

Experimental autoimmune encephalomyelitis

Multiple sclerosis

Both MS and EAE are characterized by the infiltration of inflammatory cells, including T cells, B cells, macrophages and DCs and demyelination of axonal tracks in the central nervous system.¹ EAE can be initiated by 1. active induction: immunization with myelin antigens in adjuvant² or 2. passive induction: adoptive transfer of myelin-specific T cells.³ Both methods depend on the same fundamental principles. Beside T cells, evidences from clinical researches and basic experimental models indicate that DCs are involved in the pathogenesis of most autoimmune diseases.⁴ In addition, among the antigen-presenting cells (APCs), DCs alone are sufficient to prime myelin-reactive T cells to induce EAE.⁵ Therefore, due to plasticity of DCs, agents that alter the DC differentiation from activation to tolerance, have been emerged and applied on the clinical trials.^6-8 This results TolDCs to become a potential therapy in the clinical treatment of autoimmune disorders.^9-11

In order to test the function of agents in inducing TolDC phenotype, the protocol has been established to show the passive induction of EAE by adoptive transfer of DCs primed with myelin antigen into naïve mice. Hematopoietic stem cells (HSCs) were isolated from mice bone marrow and cultured with differentiation factors to generate bone marrow-derived DCs (BMDCs). BMDCs were treated with LPS for activation before received agents (CDDO-DFPA in our publication) for tolerogenic phenotypes or PBS. BMDCs were subcutaneously injected into C57BL/6 mice once each week for four consecutive weeks. Mice were monitored daily symptoms of EAE, using standard criteria (Figure 1).¹²

GM-CSF (Peprotech Inc.)

IL-4 (Peprotech Inc.)

lipopolysaccharides (LPS) (Sigma Aldrich Inc.)

β-mercaptoethanol (Sigma Aldrich Inc.)

pertussis toxin (PTX) (BD bioscience)

MOG (35-55) peptide (21stCentury Biochemicals)

ACK buffer (Gibco)

RPMI-1640 plus L-glutamine (Gibco)

FBS (premium select, low endotoxin) (Atlanta biologicals)

Penicillin/streptomycin (Gibco)

CD11c antibody for Flow cytometry (Becton Dickinson & Co.)

Basic cell culture equipment

Dissecting tools

3 ml syringe with 21 gauge (Becton Dickinson & Co.)

Centrifuge with temperature control

Cell strainer (40 μm)

Cell incubator

Flow cytometry

1 ml syringe with 26 gauge (Becton Dickinson & Co.)

Preparation and characterization of bone marrow-derived dendritic cells

Bone marrow-derived dendritic cells (BMDCs) were expanded from hematopoietic progenitors isolated from C57BL/6 mice.¹³ After red cell lysis with ACK buffer, progenitors were cultured in RPMI-1640 plus L-glutamine, 10% FBS, 50 nM β-mercaptoethanol, and 5% penicillin/streptomycin. Fresh medium with 15 ng/ml of GM-CSF and 10 ng/ml of IL-4 were added to cultures on day 0, 3, and 5. BMDCs were harvested on day 7 and analyzed by flow cytometry for CD11c expression (70-80% of the expanded cell population, Figure 2).

Preparation of mature or tolerogenic DCs

BMDCs were harvested on day 7 and pre-treated with the absence or presence of CDDO-DFPA (400 nM) for 4 hours before LPS (100 ng/ml) treatment for 24 hours. BMDCs were then treated with MOG (35-55) (100 μg/ml) for 4 hours prior to the administration.

Induction and evaluation of EAE

EAE was induced in 8-10 week old female C57BL/6 mice. Methods for passive induction of EAE by ex vivo expanded BMDCs were adopted from previously described protocol.¹⁴ In brief, mice received 2×10⁶ LPS-stimulated, MOG-pulsed CD11c+ BMDCs at a total volume of 200 μL injected subcutaneously (100μ L into each hind leg). On the day of BMDC transfer and 48 hours later, each mouse also received 200 ng of pertussis toxin (PTX), and DCs were injected into C57BL/6 mice once each week for four consecutive weeks. Mice were monitored daily for survival and symptoms of EAE, using standard criteria (Video 1).¹²

Preparation and characterization of bone marrow-derived dendritic cells- 7 days

Preparation of matured or tolerogenic DCs- 1 days

Induction of EAE- 4 weeks

Evaluation of EAE- 35 days

1 Constantinescu, C. S., Farooqi, N., O'Brien, K. & Gran, B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol 164, 1079-1106, doi:10.1111/j.1476-5381.2011.01302.x (2011).

2 Stromnes, I. M. & Goverman, J. M. Active induction of experimental allergic encephalomyelitis. Nat Protoc 1, 1810-1819, doi:10.1038/nprot.2006.285 (2006).

3 Stromnes, I. M. & Goverman, J. M. Passive induction of experimental allergic encephalomyelitis. Nat. Protocols 1, 1952-1960 (2006).

4 Ganguly, D., Haak, S., Sisirak, V. & Reizis, B. The role of dendritic cells in autoimmunity. Nat Rev Immunol 13, 566-577, doi:10.1038/nri3477 (2013).

5 Greter, M. et al. Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis. Nat Med 11, 328-334, doi:10.1038/nm1197 (2005).

6 Dhodapkar, M. V., Steinman, R. M., Krasovsky, J., Munz, C. & Bhardwaj, N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 193, 233-238 (2001).

7 Giannoukakis, N., Phillips, B., Finegold, D., Harnaha, J. & Trucco, M. Phase I (safety) study of autologous tolerogenic dendritic cells in type 1 diabetic patients. Diabetes Care 34, 2026-2032, doi:10.2337/dc11-0472 (2011).

8 Hilkens, C. M. & Isaacs, J. D. Tolerogenic dendritic cell therapy for rheumatoid arthritis: where are we now? Clin Exp Immunol 172, 148-157, doi:10.1111/cei.12038 (2013).

9 Raiotach-Regue, D. et al. Stable antigen-specific T-cell hyporesponsiveness induced by tolerogenic dendritic cells from multiple sclerosis patients. European journal of immunology 42, 771-782, doi:10.1002/eji.201141835 (2012).

10 Harry, R. A., Anderson, A. E., Isaacs, J. D. & Hilkens, C. M. Generation and characterisation of therapeutic tolerogenic dendritic cells for rheumatoid arthritis. Ann Rheum Dis 69, 2042-2050, doi:10.1136/ard.2009.126383 (2010).

11 Li, X. et al. Induction of type 2 T helper cell allergen tolerance by IL-10-differentiated regulatory dendritic cells. American journal of respiratory cell and molecular biology 42, 190-199, doi:10.1165/rcmb.2009-0023OC (2010).

12 Pareek, T. K. et al. Triterpenoid modulation of IL-17 and Nrf-2 expression ameliorates neuroinflammation and promotes remyelination in autoimmune encephalomyelitis. Scientific reports 1, 201, doi:10.1038/srep00201 (2011).

13 Madaan, A., Verma, R., Singh, A. T., Jain, S. K. & Jaggi, M. A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells. JBM 1, e1, doi:10.14440/jbm.2014.12 (2014).

14 Aghdami, N., Gharibdoost, F. & Moazzeni, S. M. Experimental autoimmune encephalomyelitis (EAE) induced by antigen pulsed dendritic cells in the C57BL/6 mouse: influence of injection route. Exp Anim 57, 45-55 (2008).

We thank Reata Pharmaceuticals for providing RTA-408 (CDDO-DFPA). We also acknowledge the support of the Jane and Lee Seidman Chair in Pediatric Cancer Innovation (John Letterio). This work was supported by Department of Defense [W81XWH-12-1-0452]; the Angie Fowler Adolescent and Young Adult Cancer Research Initiative at the Case Comprehensive Cancer Center; and the Callahan Graduate Scholar Award for Hsi-Ju Wei from F.J. Callahan Foundation.

The authors have no conflicting financial interests.

supplement0.m4v
Video 1 Induction and evaluation of EAE

Download PDF

Version 1

posted

You are reading this latest protocol version

Passive induction of experimental autoimmune encephalomyelitis by activated or tolerogenic dendritic cells

Status:

Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Timing

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Passive induction of experimental autoimmune encephalomyelitis by activated or tolerogenic dendritic cells

Status:

Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Timing

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Manage Cookie Preferences