Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund’s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.
Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund's adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne's disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP) strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.
Experimental autoimmune encephalomyelitis (EAE) is a common model for the study of human demyelinating disorders1. There are several models of EAE: active immunization using different myelin peptides in combination with potent adjuvants, passive immunization by in vitro transfer of myelin-specific CD4+ lymphocytes, and transgenic models of spontaneous EAE2. Each of these models has specific features that allow different aspects of EAE to be studied, such as the onset, effector phase, or chronic phase. The myelin oligodendrocyte glycoprotein (MOG) model of EAE is a good model to study the immune-mediated mechanisms of chronic neuroinflammation and demyelination, as it is characterized by mononuclear inflammatory infiltration, demyelination in peripheral white matter, and reduced recovery after the disease peak1.
MOG-EAE is induced by immunization of susceptible mice with the peptide MOG35-55 in complete Freund's adjuvant (CFA), followed by an intraperitoneal injection of pertussis toxin. This increases the permeability of the blood-brain barrier and allows myelin-specific T-cells activated in the periphery to reach the central nervous system (CNS), where they will be reactivated3. CFA plays a key role in the induction of EAE by enhancing the antigen uptake by antigen-presenting cells and the expression of cytokines related to humoral- and cell-mediated responses4. This mechanism is mainly due to the presence of killed Mycobacterium tuberculosis emulsified in oil, the components of which provide a strong stimulus for the immune system5. In fact, the induction of EAE is directly related to the amount of mycobacterium present during the antigenic challenge6.
The addition of other killed mycobacteria, such as Mycobacterium butyricum, to incomplete Freund's adjuvant7, as well as the effect of adjuvant combinations8, can modulate the clinical course of EAE and consequently influence the reproducibility of the results. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johne's disease in ruminants, has been associated with inflammatory disorders of the human CNS9, as its antigenic components are capable of eliciting a strong humoral- and cell-mediated response in patients with multiple sclerosis and neuromyelitis optica spectrum disorder9. Therefore, in this protocol, we show an alternative and reproducible method to induce MOG-EAE by replacing M. tuberculosis in CFA with M. paratuberculosis.
We demonstrated a robust alternative protocol to actively induce severe EAE in C57BL/6J mice using the peptide MOG35-55 emulsified in an adjuvant containing M. paratuberculosis10. The induction of EAE by this method resulted in a more severe disease than that induced by the common protocol with CFA. This difference could be due to the different lipidic components in the cell wall of the mycobacteria11. In fact, unlike other mycobacteria, M. paratuber…
The authors have nothing to disclose.
This work received support from a grant from the Japanese Society for the promotion of Science (grant no. JP 23K14675).
anti-mouse CD115 antibody | Biolegend, USA | 135505 | for cytofluorimetry 1:1,000 |
anti-mouse CD11b antibody | Biolegend, USA | 101215 | for cytofluorimetry 1:1,000 |
anti-mouse CD11c antibody | Biolegend, USA | 117313 | for cytofluorimetry 1:1,000 |
anti-mouse CD16/32 antibody | Biolegend, USA | 101302 | for cytofluorimetry 1:1,000 |
anti-mouse CD4 antibody | Biolegend, USA | 116004 | for cytofluorimetry 1:1,000 |
anti-mouse CD8a antibody | Biolegend, USA | 100753 | for cytofluorimetry 1:1,000 |
anti-mouse I-A/I-E antibody | Biolegend, USA | 107635 | for cytofluorimetry 1:1,000 |
anti-mouse Ly-6C antibody | Biolegend, USA | 128023 | for cytofluorimetry 1:1,000 |
BBL Middlebrook OADC Enrichment | Thermo Fisher Scientific, USA | BD 211886 | for isolation and cultivation of mycobacteria |
C57BL/6J mice | Charles River Laboratory, Japan | 3 weeks old, male and female | |
FBS 10279-106 | Gibco Life Techologies, USA | 42F9155K | for cell culture, warm at 37 °C before use |
Freeze Dryer machine | Eyela, Tokyo, Japan | FDU-1200 | for bacteria lyophilization |
incomplete e Freund’s adjuvant | Difco Laboratories, MD, USA | 263810 | for use in adjuvant |
Middlebrook 7H9 Broth | Difco Laboratories, MD, USA | 90003-876 | help in the growth of Mycobacteria |
Mycobacterium avium subsp. paratuberculosis K-10 | ATCC, USA | BAA-968 | bacteria from bovine origin |
Mycobacterium tuberculosis H37 Ra, Desiccated | BD Biosciences, USA | 743-26880-EA | for use in adjuvant |
Mycobactin J | Allied Laboratory, MO, USA | growth promoter | |
Myelin Oligodendrocyte Glycolipid (MOG) 35-55 | AnaSpec, USA | AS-60130-10 | encephalotigenic peptide |
Ovalbumin (257-264) | Sigma-Aldrich, USA | S7951-1MG | negative control antigen for proliferative assay |
pertussis toxin solution | Fujifilm Wako, Osaka Japan | 168-22471 | From gram-negative bacteria Bordetella pertussi, increases blood-brain barrier permeability |
Polytron homogenizer PT 3100 | Kinematica | for mixing the antigen with the adjuvant | |
RPMI 1640 with L-glutamine | Gibco Life Techologies, USA | 11875093 | For cell culture |
Thymidine, [Methyl-3H], in 2% ethanol, 1 mCi | PerkinElmer, Waltham, MA, USA | NET027W001MC | for proliferation assay, use (1 μCi/well) |
Zombie NIR Fixable Viability Kit | Biolegend, USA | 423105 | cytofluorimetry, for cell viability |