Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Summary

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.

Abstract

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., MOG_35-55) 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.

Introduction

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 MOG_35-55 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.

Protocol

All mouse experiments were approved by the Institutional Animal Care and Use Committee of the Juntendo University School of Medicine (Approval Number 290238) and were conducted in accordance with the National Institutes of Health Guidelines for Animal Experimentation.

1. General comments on the experiment

1. House the mice in individual cages in the animal facility under controlled, pathogen-free conditions at 23 °C ± 2 °C with 50% ± 10% humidity, and a 12 h light/dark cycle with ad libitum access to food and water.
2. Inject the mice with phosphate-buffered saline (PBS) without antigen and CFA, and use them as negative and positive controls, respectively.

2. Preparation of mycobacterial antigens

1. Grow the M. paratuberculosis K-10 strain in Middlebrook liquid medium 7H9 enriched with 10% OADC (oleic acid, albumin, dextrose, catalase), 0.005% Tween 80, and 2 mg/L of mycobactin J for 2 weeks in T25 tissue culture flasks at 37 °C. Assess the colony growth regularly by visual inspection.
   CAUTION: Handle M. paratuberculosis in a Biosafety Level 2 (BSL-2) facility.
2. Grow the enrichment cultureina 500 mL Erlenmeyer flask with a suspension of 1.7 × 10⁵ colony-forming units (CFU)/mL in a final volume of 200 mL (same medium as step 2.1) in a shaking incubator for 1 week.
   NOTE: As contaminating organisms may affect the results, the bacteria must be inoculated onto Middlebrook 7H10 solid media to determine colony morphology and to verify purity by conventional polymerase chain reaction (PCR) detection kits for M. paratuberculosis.
3. Inactivate the bacterial suspensions for 5-10 min at 70 °C and centrifuge at 3,000 × g for 10 min. Washed the weighed pellet in PBS twice, disrupt by sonication, and store at -20 °C.
4. Transfer the pellet to a sterile container and freeze it with dry ice or liquid nitrogen. Transfer immediately to a freeze-drying chamber.
5. Freeze-dry (4 h at -50 °C under vacuum) M. paratuberculosis according to the machine manual.
6. At the end of lyophilization, remove the stainless-steel container from the freeze-dryer and transfer it to a bio-cleaning bench. Use a stainless-steel spatula to dislodge the dried MAP lumps adhering to the inner wall of the stainless-steel container and pulverize them as finely as possible.
7. Weigh the pulverized cells using an electronic balance and manually place them in an aseptic 10 mL vial at a concentration of 10 mg/mL.
   NOTE: The cell density was quantified as grams of dry weight per liter of sample. After 3 weeks, 1 mg (wet weight) of bacterial cell pellet contains approximately 2.5 × 10⁸ CFU.

3. Preparation of MOG _35-55 emulsion

1. Grind the contents of one vial of dried M. paratuberculosis (10 mg) to a fine powder with a mortar and pestle and add 10 mL to incomplete Freund's adjuvant to obtain a 10 mg/mL stock solution. Store at -4 °C.
2. Prior to immunization, dilute the stock solution to a final concentration of 4 mg/mL.
3. Dilute the peptide MOG_35-55 in ddH₂O to a final concentration of 2 mg/mL and store at -20 °C.
4. Using a homogenizer, mix the MOG_35-55 solution (2 mg/mL) with an equal volume of adjuvant (4 mg/mL) from step 3.2 in a 5 mL tube until a thick emulsion is formed. After every 10 s of mixing, place the solution on ice for 20 s, and spin the tube to recover all the solution.
5. Transfer the emulsion into a 1 mL syringe, remove all air, and add a 27 G needle. The emulsion is now ready for injection.
   NOTE. Since some loss of the viscous emulsion of MOG_35-55 occurs during preparation, it is best to prepare 1.5 times the amount needed.

4. Animal immunization

1. Anesthetize the animals with inhalation of isoflurane to minimize stress on the animals.
2. Inject the emulsion (200 µL containing 200 µg/mouse of MOG_35-55) subcutaneously into the lower back.
3. Administer a dose of pertussis toxin (100 µL containing 200 ng/mouse) intraperitoneally on days 0 and 2 after immunization.
   ​CAUTION: Pertussis toxin has a multiple suppressive effect on the immune system; avoid ingestion and contact with eyes and skin.

5. Clinical assessment

1. Monitor the mice daily for weight and clinical signs. Use the following scoring system: 0 = no clinical signs; 1 = flaccid tail; 2 = impairment of the righting reflex and weakness of the hind limbs; 3 = complete paralysis of the hind limbs; 4 = complete paralysis of the hind limbs with partial paralysis of the forelimbs; 5 = moribund.
   NOTE: Upon the initial observation of clinical signs, animals are provided with increased access to water and food. Rodent chow is softened and moistened, then placed on the cage floor from the food hopper. In the case of dehydrated animals, subcutaneous fluid supplementation is administered, or a rodent chow slurry is given via oral gavage. If a mouse necessitates this type of assistance for over 3 days, euthanasia will be conducted.
2. To avoid or minimize animal pain and distress, use the following human endpoints: body weight loss greater than 20%, clinical score ≥4.0, absence of the righting reflex at score 3, and when the animal is unable to access feed or water for 24 h.
   NOTE: The mice were euthanized on day 30 after EAE induction by intraperitoneal injections of pentobarbital (≥150 mg/kg).

Results

Groups of C57BL/6 mice (total n = 15/group) were immunized with MOG_35-55 in an emulsion containing M. paratuberculosis or by the common method with CFA. All groups of mice manifested an acute monophasic disease characterized by a single peak of disability observed at 14-17 days, followed by a partial recovery of symptoms over the next 10 days (Figure 1A). Mice immunized with the adjuvant containing M. paratuberculosis, irrespectiv...

Discussion

We demonstrated a robust alternative protocol to actively induce severe EAE in C57BL/6J mice using the peptide MOG_35-55 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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