A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Summary

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Abstract

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

Introduction

A breakdown of immunological tolerance can result in the generation of autoimmune disorders, such as multiple sclerosis (MS). It is estimated that 2.8 million people are living with MS worldwide¹. Although the exact cause of MS is still largely unknown, dysregulation of autoreactive T and B cells, as well as defects in Treg function, play important roles in the pathogenesis of the disease^2,3.

Animal models of autoimmune diseases are essential tools to investigate potential therapeutic modalities. The experimental autoimmune encephalomyelitis (EAE) model has been used for almost a century by researchers interested in MS⁴. In early experiments, the incidence of the disease was relatively low. The introduction of complete Freund's adjuvant (CFA), containing Mycobacterium and pertussis toxin, enabled the consistent induction of EAE in mice⁴. Most importantly, it is necessary to mix CFA with a central nervous system (CNS)-specific antigen to generate a homogenous water-in-oil emulsion for inducing EAE. The most common currently available EAE models are based on the active immunization of mice with encephalitogenic peptides. The genetic background of the mice plays an important role in disease susceptibility, with myelin oligodendrocyte glycoprotein (MOG_35-55) and myelin proteolipid protein (PLP_139-151) peptides used to induce EAE in C57BL/6J and SJL mice, respectively⁵. However, other mouse strains and CNS-derived peptides can also be used.

The quality of the CFA/peptide emulsion is a critical factor that determines disease penetrance in the active immunization EAE model⁶. A homogeneous water-in-oil emulsion must be prepared by mixing the encephalitogenic peptides dissolved in aqueous buffer with CFA, otherwise animals will not develop the disease. Numerous protocols have been published on the preparation of CFA/peptide emulsions. Examples include the use of a vortex⁷, sonication⁸, syringes and a three-way T connector⁹, or one syringe only⁵. However, all these methods are difficult to standardize and are often associated with lengthy and complicated protocols.

Compared to all the above methods, the simple method described here for emulsion preparation offers the advantages of having no person-to-person differences and being relatively fast. The emulsion is generated by a homogenizer shaking the reagents with a set speed, time, and temperature, ensuring fast and consistent results. In addition to inducing disease in the EAE model, this method can also be used to study other autoimmune disease models such as collagen-induced arthritis (CIA) and antigen induced arthritis (AIA)⁶. Therefore, it is anticipated that this method can be used to consistently induce disease in other animal models that depend on water-in-oil emulsions with autoantigens, such as experimental autoimmune neuritis (EAN)¹⁰, experimental autoimmune thyroiditis (EAT)¹¹, autoimmune uveitis (EAU)¹², and myasthenia gravis (MG)¹³. This method also induces general immune responses such as delayed-type hypersensitivity (DTH) consistently⁶, and could therefore be used for delivering cancer and malaria vaccines (see discussion).

Thus, a rapid (total preparation time ~30 min), simple (all reagents can be prepared in advance and stored), and standardized (the emulsion is accomplished using a shaking homogenizer) method has been developed and is presented here. The CFA/antigen emulsions prepared using this protocol consistently induce disease in autoimmune animal models.

Protocol

All animal procedures were performed according to the practices of the Swedish Board of Animal Research and were approved by the Animal Ethics Committee, Lund-Malmö, Sweden (Permit number: M126-16).

NOTE: A schematic flow of the method is described in Figure 1.

1. Material preparation

NOTE: Prepare all the reagents aseptically in a sterile hood, and aliquot and store at the indicated temperature. The reagents can be stored up to 2 years without losing their effect.

1. Prepare CFA containing 20 mg/mL Mycobacterium tuberculosis as described below.
   1. Add 100 mg of freeze-dried M. tuberculosis H37RA (see Table of Materials) and five 3.2 mm steel beads to a 7 mL tube (see Table of Materials). Shake the tube in a homogenizer (see Table of Materials) for 60 s at the highest speed setting (5,000 rpm).
   2. Add 5 mL of incomplete Freund's adjuvant (IFA) to the tube and shake again for 60 s at the highest speed. Transfer the slurry of homogenized M. tuberculosis in IFA (now named CFA) to a fresh tube with a pipette, leaving the beads behind, and store at 4 °C until use.
2. Add ultrapure water to the freeze-dried powder of MOG_35-55 peptide (see Table of Materials) to obtain a final peptide concentration of 10 mg/mL. Sterilize the solution by passing it through a 0.2 µm filter. Prepare 60 µL aliquots and store at -20 °C until use.
   NOTE: The MOG_35-55 peptide used for this experiment is of ImmunoGrade purity (~70%), is TFA-cleaved, dissolves easily in water, and contains a C-terminal amide (-NH₂) to increase in vivo stability¹⁴. Peptide aliquots were never thawed and re-frozen more than twice, and were stored at -20 °C until use.
3. Prepare 100 mL of pertussis toxin buffer: Dissolve 2.9 g of NaCl in 80 mL of 1x Ca^2+/Mg²⁺ free PBS and add 100 µL of 10% Triton X-100. Adjust the volume to 100 mL with PBS and sterilize the solution by passing it through a 0.2 µm filter. Prepare 10 mL aliquots and store at 4 °C until use.

2. Preparation of CFA/peptide emulsions

1. Calculate the amount of emulsion needed for immunization. In this standard protocol, each mouse receives 50 µg of MOG_35-55 peptide and 300 µg of M. tuberculosis dispersed in Freund's adjuvant (CFA). The amount of each reagent (MOG_35-55 peptide, PBS, CFA, and IFA) required for preparing the emulsions can be calculated using Supplemental File 1. An additional 20% of all reagents, to compensate for the small loss of emulsion, is included in the calculation.
   NOTE: The commercial emulsion kit (see Table of Materials) used in this study comprises a tube, screw cap, and a plunger in a sterilized pouch. Each tube of the emulsion kit holds a maximum of 9.6 mL, making it possible to prepare 8 x 1 mL syringes sufficient for immunizing 40 mice (or 80 mice if using 100 µL of emulsion per animal).
2. Place the screw capped tube (from the commercial emulsion kit) and reagents to be used on ice.
3. Add the emulsion components in the following order in the volumes calculated in step 2.1: PBS, then the peptide, then M. tuberculosis in CFA, and finally IFA.
4. Close the tube with the cap firmly by tightening and loosening it several times. Shake the tube vigorously for 5-10 s by hand to pre-mix the reagents.
5. Place the tube in the shaking homogenizer and secure it with the rod. Set the speed to the highest setting and the time to 60 s. Once the run is finished, place the tube on ice for 3 min. Repeat the run one or two more times with the same settings.
6. Centrifuge the tube at 300 x g for 1 min to remove trapped air and compact the emulsion.
7. Remove the cap from the tube, insert the plunger into the tube, and push it down slowly until it reaches the top of the emulsion. Remove the snap off closure at the bottom of the tube by twisting it.
8. Remove the plunger from an injection syringe (1 mL; see Table of Materials). Add a needle (preferably 25-27 G) to the injection syringe.
9. Attach the back end of the injection syringe to the dedicated lock at the bottom of the tube and lock it with a short twist.
10. Transfer the emulsion from the tube to the injection syringe by pushing the plunger gently. Stop when the emulsion reaches the 0.15 mL graduation of the injection syringe.
11. Separate the injection syringe from the tube and insert the plunger carefully, taking care that no air enters the syringe. Push the plunger until the emulsion comes out of the needle.
    NOTE: At this point, some of the emulsion can be used for quality control (see step 3).
12. Repeat steps 2.8-2.11 for the rest of the emulsion present in the tube.
    ​NOTE: To minimize the waste of expensive CFA/antigen emulsions, 1 mL syringes should be used. Other syringes that fit the dedicated lock at the bottom of the tube may be used; however, a larger volume of emulsion may need to be prepared. The CFA/MOG_35-55 peptide emulsion can be stored at 4 °C for up to 15 weeks without losing its EAE-inducing capacity.

3. Quality control of emulsion

1. Drop-test: Add a small drop of the emulsion into a sterile 50 mL conical tube filled with 20 mL of cold water. Close the tube and shake it by hand for a couple of seconds. The appearance of tiny distinct droplets confirms the formation of a water-in-oil emulsion.
2. Examine the emulsion using phase contrast microscopy.
   1. To analyze the size of the water-in-oil particles, add a tiny drop (2-3 µL) of the emulsion to a microscope slide, smear it out with a cover slip, and then push hard in a circular motion to flatten out the emulsion.
   2. Examine the smeared emulsion under a phase contrast microscope (see Table of Materials) with 400x magnification and focus on a field with a monolayer of the emulsion. Ensure that small uniform grey/white particles are visible (Figure 2A).
3. Analyze the emulsion particle size using laser diffraction.
   NOTE: Laser diffraction measures particle size distributions using a laser beam. This is the ultimate quality control test for emulsions. A representative result of particle size distributions using the method described here is shown in Figure 2B (for a detailed description, see Topping et al.⁶). Nevertheless, the drop test and microscope examination are sufficient to validate whether a satisfactory emulsion has been formed.
   1. Set the refractive indices for both red and blue lasers for the dispersant (see Table of Materials) to 1.456 and for the sample to 1.33 on the particle size analyzer (see Table of Materials). Set the absorbance of the refractive index to 0.02.
   2. Add one drop from the syringe containing the peptide emulsion to the dispersion unit containing light mineral oil. Start the data acquisition instantly after dispersion of the emulsion.
      ​NOTE: A general-purpose model was applied, and spherical particles were assumed, for the estimation of particle size distribution.

4. EAE induction using the MOG _35-55 peptide emulsion in C57BL6/J mice

NOTE: In all experiments, five 8-12-week-old female C57BL6/J mice were used.

1. Prior to immunization, anesthetize the mice using 2.5% vaporized isoflurane and confirm using a firm toe pinch.
2. Inject each mouse subcutaneously using 1 mL syringes into two different sites, on the right and left sides of the hind flank with 200 µL of emulsion containing 50 µg of MOG_35-55 peptide in a 1:1 (v/v) ratio of peptide/CFA.
3. At least 1.5 h after the immunization with the emulsion, and then again after 24 h, administer a total of 80 ng of Bordetella pertussis toxin in 200 µL of pertussis toxin buffer to each mouse, through intraperitoneal injections (100 µL on the left and 100 µL on the right site of the belly).
   NOTE: Precise localization of intraperitoneal injection with pertussis toxin has been shown to be essential for the activation of T cells in the draining lymph node¹⁵.
4. Optional: Inject MOG_35-55 peptide again on day 7 (as used in some protocols¹⁶) by repeating step 4.2.
   NOTE: Most importantly, make sure that the syringes and needles used for immunization containing MOG/CFA or pertussis toxin are adequately disposed of in biohazard bags/containers.
5. Evaluate the clinical score daily, using a scale from 0-6 as previously described⁶.

Results

The rapid, simple, and standardized protocol for the preparation of CFA/MOG emulsions is depicted in Figure 1. This method has recently been described elsewhere⁶. The CFA/MOG emulsions can also be prepared with other methods, such as the traditional syringe method or by vortexing. These methods were compared here by assessing the quality of the emulsions. All the methods produced water-in-oil emulsions; the homogeneity and quality of these emulsions were assessed by a...

Discussion

Water-in-oil emulsions, such as antigen/Freund's adjuvant, have been used for more than half a century to induce EAE¹⁷. There is currently no standardized method to prepare antigen emulsions that is independent of human influence. Manual mixing using syringes is standard for most laboratories, however this method is time consuming, often results in an excessive loss of material, and the quality differs depending on the scientist preparing it.

The method presented in...

Materials

Name	Company	Catalog Number	Comments
1 mL Injection syringe	B. Braun	9166017V
1 mL Injection syringe	Sigma-Aldrich	Z683531
7 ml empty tubes with caps	Bertin-Instruments	P000944LYSK0A.0	7 mL tube
50 mL sterile centrifuge tube	Fisher Scientific	10788561	50 mL tube
Bordetella pertussis toxin	Sigma-Aldrich	P2980	Store at -20 °C
Dispersant, light mineral oil	Sigma-Aldrich	M8410	Store at RT
Emulsion kit	Bertin-Instruments	D34200.10 ea	Containing a tube, cap, and plunger
Incomplete Freund's Adjuvant	Sigma-Aldrich	F5506	Store at +4 °C
Mycobacterium tuberculosis, H37RA	Fisher Scientific	DF3114-33-8	Store at +4 °C
Mastersizer 2000	Malvern Panalytical	N/A	Particle size analyzer
Minilys-Personal homogenizer	Bertin-Instruments	P000673-MLYS0-A	Shaking homogenizer
MOG 35-55 Peptide	Innovagen	N/A
Montanide ISA 51 VG	Seppic	36362Z	FDA-approved oil adjuvant
Pall Acrodisc Syringe Filters 0.2 μm	Fisher Scientific	17124381	Sterlie filter
PBS, Ca2+/Mg2+ free	Thermo Fisher Scientific	14190144	PBS
Phase-Constrast Microscope	Olympus	BX40-B
Steel Beads 3.2 mm	Fisher Scientific	NC0445832	Autoclave and store at RT
Triton X-100	Sigma-Aldrich	648463	Store at RT