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

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.

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).

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, irrespective of sex, showed an earlier onset from 8 to 9 days after immunization and greater severity in the acute phase than mice immunized with CFA (Figure 1B).There was no significant difference in body weight among the groups (Figure 1C). Cytofluorimetric analysis of spleen cells from EAE mice and proliferative activity of T-lymphocytes assessed by ³H-thymidine incorporation were performed, as previously published¹¹. All spleen cells of EAE mice, regardless of the adjuvant used, showed a strong proliferative response to the peptide MOG_35-55, whereas they did not proliferate in response to the control peptide ovalbumin (Figure 1D). M. paratuberculosis-immunized EAE mice showed an increased proportion of T-lymphocytes (CD4⁺ CD27⁺), dendritic cells (CD11c⁺I-A/I-E⁺), and monocytes (CD11b⁺CD115⁺) in the spleen compared to CFA-immunized mice during the acute phase of EAE (Figure 1E). Histological analysis by hematoxylin-eosin revealed a typical perivascular and meningeal mononuclear inflammatory infiltration in the brain and spinal cord (Figure 1F).

Figure 1: Representative EAE results. (A) Clinical scores of mice evaluated daily; (B) differences in disease course; (C) body weight; (D) T-cell proliferative response to the MOG_35-55 peptide; (E) immune cell populations in splenocytes during the acute phase; (F) histological images of spinal cord sections (4x magnification) stained with hematoxylin-eosin(scale bar = 200 µm) during the acute phase. Arrows indicate inflammatory infiltrates. The data show combined results of three independent experiments (n = 5 mice/group/experiment). Data are expressed as mean ± standard deviation calculated using a one-way ANOVA followed by Bonferroni's post hoc test or Mann-Whitney U test. Abbreviations: EAE = experimental autoimmune encephalomyelitis; MOG = myelin oligodendrocyte glycoprotein; PBS = phosphate-buffered saline; CFA = complete Freund's adjuvant. Please click here to view a larger version of this figure.

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. paratuberculosis produces a lipopentapeptide antigen on the cell wall surface instead of glycopeptidolipids¹¹. This lipopentapeptide did not cross-react with other closely related species and has been shown to be a target for a strong specific humoral response in patients with autoimmune disorders¹². Mycobacteria possess pathogen-associated molecular patterns that play different roles in immune responses⁹. For instance, we have recently shown that oral immunization with M. paratuberculosis increases the severity of MOG-EAE¹³, whereas vaccination with bacillus Calmette-Guerin (BCG)-Tokyo-172 appears to have a protective role in the progression of active and spontaneous EAE models¹⁴.

Potential limitations of the protocol include the self-limited monophasic course of EAE, which may became chronic if the concentrations of mycobacterium in the adjuvant and pertussis toxin are doubled. This aspect is very important to consider, especially when studying the neurodegenerative aspect of EAE using knockout mice, as the absence of recovery phase may be caused by the high doses of the reagents used and not by the lack of function of certain genes. Furthermore, although the protocol described above can be applied to other EAE protocols by varying the strains and age of mice or the type and amount of protein, to ensure that EAE experiments are performed in a methodologically correct manner, experimental groups must be matched for age and sex.

Since the incidence of the disease may vary, recommendations for troubleshooting are: the optimal M. paratuberculosis concentration can range from 2 to 4 mg/mL, and a three-way connector can be used for mixing small volumes of the antigen in the aqueous phase. We have described a method for preparing the emulsion by using a homogenizer; however, an alternative and simple method for mixing small volumes of antigen in the aqueous phase, in the oil phase, involves the use of a three-way connector fitting, to which two glass syringes are connected. In conclusion, we identified M. paratuberculosis as a potent adjuvant candidate in the induction of active EAE. Further studies on the mode of action and the type of immunity induced in different animals species will increase confidence in the possibility of adopting this alternative method to understand the mechanism of neuroinflammation.