Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Summary

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Abstract

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

Introduction

As a chronic demyelinating disease of the CNS, MS affects about 2.5 million people worldwide and lacks curative treatments¹. It is also considered an autoimmune disease, in which myelin antigen specific T lymphocytes initiate an inflammatory reaction and lead to demyelination and axonal injury in the CNS². Experimental autoimmune encephalomyelitis (EAE) has been widely used to investigate pathogenic mechanisms of MS as a classic autoimmune demyelination disease model in CNS³. There are two ways to induce EAE: one is to induce EAE actively by immunizing animals with myelin components, another is adoptive transfer by transferring encephalitogenic T cells into receptor^2,4,5. The susceptibilities to EAE are different in different animal strains⁶. In C57BL/6 mice, myelin oligodendrocyte glycoprotein (MOG) 35–55 challenge induces a monophasic disease with extensive demyelination and inflammation in the CNS, which is frequently used in experiments with gene-targeted mice⁷.

The generation of myelin-specific reactive T cells is required for the occurrence and development of disease in EAE and is an immunological sign of both EAE and MS. Activated autoreactive T lymphocytes cross the blood brain barrier (BBB) into the healthy CNS and initiate EAE disease. When MOG 35–55 Ag is encountered, these T lymphocytes induce inflammation and the recruitment of effector cells into the CNS, resulting in demyelination and axon destruction^8,9. In the EAE model, there is ample evidence that neuroantigen-specific CD4⁺ T cells can initiate and sustain neuroinflammation and pathology^3,10. Depending on the major cytokines produced, CD4⁺ T lymphocytes have been classified into different subsets: Th1 (characterized by the production of interferon-γ), Th2 (characterized by the production of interleukin 4), and Th17 (characterized by the production of interleukin 17). It is believed that activation of Th1 and Th17 cells contribute to the induction, maintenance, and regulation of inflammatory demyelination in EAE and MS by secreting effector cytokines IFN-γ and IL-17, which are capable of activating macrophages and recruiting neutrophils to the inflammatory sites to accelerate the lesions¹¹.

Because autoreactive T cells cross the BBB into the CNS and induce the development of disease in MS and EAE, it is very important to analyze T cells in the CNS. However, there are very few established protocols for the isolation of lymphocytes from the CNS¹². Therefore, a method optimized for isolating mononuclear cells from the brain and analyzing T lymphocytes with markers CD45, CD11b, CD3, CD4, INF-g, and IL-17 for flow cytometry was developed. The method uses MOG35–55 adjuvant Mycobacterium tuberculosis H37 Ra and Pertussis Toxin Working Solution (PTX) to induce an active immunization model of EAE in mice. Then, mechanical separation and density gradient centrifugation methods are used for the isolation of CNS mononuclear cells. Finally, an optimized flow cytometry gating strategy is used to identify T lymphocytes and subsets from the brain by staining multiple markers.

Protocol

All methods described here have been approved by the animal committee of the School of Basic Medical Sciences, Shanghai Jiao Tong University.

1. Preparation of the materials

1. Use the MEVGWYRSPFSRVVHLYRNGK sequence of MOG35–55 to obtain the lyophilized peptide from commercial sources. Ensure that the purity of the peptide is >95%. Prepare 10 mg/mL MOG stock solution in phosphate-buffered saline (PBS) and store at -20 °C.
2. Prepare a 4 mg/mL stock solution of M. tuberculosis H37 Ra by putting one 100 mg tube of M. tuberculosis H37 Ra into 25 mL of Complete Freund's Adjuvant (CFA) and mixing. Store the stock solution at -20 °C.
3. Prepare 1 ng/μL Pertussis Toxin Working Solution (PTX) by adding 50 μg of PTX into 50 mL of PBS. Store the working solution at -20 °C.
4. Store all antibodies (i.e., FITC anti-mouse CD3, PE/Cy7 anti-mouse CD4, PerCP/Cy5.5 anti-mouse CD11b, Alexa Fluor700 anti-mouse CD45.2, PE anti-mouse IL-17A, and APC anti-mouse IFN-γ) at 4 °C.
5. Make the Flow Cytometry Staining (FCS) Buffer by adding 2 mM ethylene diamine tetraacetic acid (EDTA) and 1% fetal bovine serum (FBS) into 500 mL of PBS.

2. Housing of C57BL/6 mice

1. Use female C57BL/6 mice at 8–12 weeks of age.
2. Acclimate C57BL/6J mice for at least 7 days prior to the injection.
3. House mice in an animal facility under pathogen-free conditions at constant temperature and humidity in a 12 h light/dark cycle and provide free access to water and standard pellet food.

3. Immunization of C57BL/6 mice

1. Leave all stock solutions for 15 min at room temperature (RT) to ensure complete rehydration.
2. Dilute 300 μL of MOG-peptide stock solution with 700 μL of PBS for preparing 3 mg/mL work solution.
3. Put 1 mL of M. tuberculosis H37 Ra stock solution and 1 mL of MOG35–55 peptides working solution into separate 10 mL syringes, then use a four-way stop cock to emulsify for at least 10 min. Ensure complete emulsification before injection.
4. Anesthetize mice at the peak of EAE with an intraperitonial injection of 1% sodium pentobarbital (50 mg/kg).
5. With a 1 mL syringe, subcutaneously inject mice with 100 μL of a MOG 35–55/CFA emulsion (300 µg/200 μL) at two sites, both at the back near the neck. Subcutaneously inject control mice with 200 μL of PBS.
6. On the same day (day 0) and on day 2 post immunization (PI), Intraperitoneally inject mice with 200 μL of 1 ng/μL PTX working solution. Intraperitoneally inject control mice with 200 μL of PBS.
7. Transfer the mice to their home cage with a warming pad.
8. Examine and grade all mice every day after the injection in a blinded manner for the neurological signs shown in Table 1^11,13. Euthanize the animals if the scores are worse than grade 4.
9. Record the weight changes during the disease course. This is a valuable additional measure for disease activity in the EAE model^11,13.
10. Add the first day of clinical signs for individual mice and divide by the number of mice in the group; the result is the onset. Add the first day of the maximum EAE score for individual mice and divide by the number of mice in the group; the result is the peak.

4. Single-cell suspension preparation from brain

1. Dilute density gradient medium in 9:1 ratio with PBS in a 15 mL conical tube to yield a final 100% solution.
2. Anesthetize mice at the peak of EAE with an intraperitonial injection of 1% sodium pentobarbital (50 mg/kg) and perfuse intracardially with 20 mL of sterile ice-cold PBS. Achieve this by slowly and steadily injecting PBS into the left ventricle of the heart using a 20 mL syringe and opening the right atrium.
3. Cut the cranium carefully from the nose to the neck, then remove the brain from the cranial box into 10 mL of RPMI in 50 mL conical tubes. Mix well to remove adherent red blood cells. Then remove the medium by aspiration and add 10 mL of RPMI.
4. Place the brains and medium in a 100 mm dish. Finely chop with a razor blade.
5. Transfer 6 mL from the dish to an ice-cold 7 mL sintered glass homogenizer with a clean pipette. Avoid leaving large quantities of tissue in the pipette. A small amount is unavoidable.
6. Grind the brain using the "loose" plunger of the pestle first, then use the "tight" plunger until the suspension is homogeneous, and pour into a prechilled 15 mL conical tube and keep on ice.
7. After all the samples are homogenized, estimate the volume. Adjust the volume with RPMI to 7 mL. Then place 3 mL of ice-cold 100% basement membrane matrix in a chilled 15 mL conical centrifuge tube and add 7 mL of the brain homogenate to yield a final 30% density gradient medium. Mix by inversion a couple of times. Do not vortex.
8. To ensure a sharp interface, carefully and slowly add 1 mL of 70% underlay density gradient medium in RPMI with a 3 mL pipette.
9. Centrifuge at 800 x g for only 20 min at 4 °C. Set the acceleration to 1 and deceleration to 0. After centrifugation, aspirate almost all of the top phase, being careful to completely remove the myelin at the top (Figure 1).
10. Remove the interface into a new 15 centrifuge tube. Adjust the volume to 10 mL with RPMI.
11. Centrifuge at 500 x g for 10 min. After centrifugation, aspirate the supernatant. Resuspend the pellet in ~200 μL of flow cytometry staining (FSC) buffer. The pellets are then ready to stain for FACS.

5. Flow cytometric analysis of single cells from brain

1. Use a hemocytometer and microscope to count the cells. Add 10 μL of the cells to 10 μL of trypan blue, mix well, and place 10 μL on a hemocytometer to count the cells. Then calculate the number of live cells per microliter under an inverted microscope (e.g., Olympus Inverted microscope).
2. Aliquot approximately 2 x 10⁶ of cells in RPMI into a single well of a 96 well plate.
3. Add 500x cell stimulation cocktail plus protein transport inhibitors to the wells.
4. Incubate the plate in the incubator at 37 °C for 4 h.
5. Centrifuge the cells at 400 x g for 5 min at RT. Discard the supernatant and resuspend the cells in 100 μL of FCS Buffer.
6. Preincubate the cells with anti-mouse CD16/CD32 Fc block (1:33) for 10 min at 4 °C before staining to block nonspecific Fc-mediated interactions.
7. Stain cell surface markers without washing. Add anti-mouse CD45.2 (1:200), anti-mouse CD11b (1:200), anti-mouse CD3 (1:200), and anti-mouse CD4 (1:200) antibodies.
   NOTE: To determine positive and negative gates, a fluorescence minus one (FMO) for each color and an isotype control antibody should be stained.
8. Incubate the plate for at least 30 min at 4 °C or on ice. Protect from light.
9. Wash the cells by adding FCS Buffer. Use 200 μL/well for microtiter plates. Centrifuge at 400 x g for 5 min at RT. Discard the supernatant.
10. Add 200 μL of intracellular (IC) fixation buffer to each well to fix the cells. Ensure the cells are fully resuspended in the solution.
11. Incubate 30–60 min at RT. Protect from light.
12. Centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.
13. Add 200 μL of 1x permeabilization buffer to each well and centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.
14. Resuspend the pellet in residual volume and adjust volume to about 100 μL with 1x permeabilization buffer.
15. Add anti-mouse IL-17A (1:200) and anti-mouse IFN-g (1:200) antibodies for detection of intracellular antigens to cells.
16. Incubate for at least 30 min at 4 °C. Protect from light.
17. Add 100 μL of 1x permeabilization buffer to each well and centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.
18. Resuspend the stained cells in 100 μL of flow cytometry staining buffer.
19. Analyze by flow cytometry.
    NOTE: The laser and compensation settings on the flow cytometer are adjusted, the samples are placed onto the cytometer, and all events are recorded as per the manufacturer’s recommendations.

6. Data analysis

1. Gate singlets using FSC-A vs. FSC-H and SSC-A vs. SSC-H.
2. Gate live cells using FSC-A and SSC-A based on size.
3. Next, identify the leukocytes, excluding the monocytes, by gating on CD45⁺ CD11b^- cells.
4. Then, identify the CD4 T lymphocytes by gating on CD3+CD4+ cells.
5. Lastly, identify the Th1 and Th17 subsets by gating on IFN-γ+ cells and IL-17+ cells separately, and determine the positive and negative populations using isotype controls and FMO.

Results

After immunization of C57BL/6 mice, all mice were weighed, examined, and graded daily for neurological signs. The representative clinical course of EAE should result in a disease curve as presented in Figure 2A and a change of body weight in the mouse as presented in Figure 2B. C57BL/6 mice immunized with MOG35-55 usually started to develop disease symptoms around day 10–12 and achieved the peak of disease around day 15–21 after active immunization (

Discussion

This study presents a protocol to induce and monitor EAE using MOG35-55 in C57BL/6 mice, which are considered a typical neuroimmunological experimental animal model of MS. EAE can be induced varying the mice strains or the type of protein used for induction according to the purpose of the study. For example, using PLP139–151 peptide in SJL mice can induce a relapsing-remitting EAE disease course that is especially well-suited for assessing therapeutic effects on relapses¹⁵. The experimental ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor700 anti-mouse CD45.2	eBioscience	56-0454-82
Anti-Mouse CD16/CD32 Fc block	BioLegend	101302
APC anti-mouse IFN-g	eBioscience	17-7311-82
BD LSRFortessa X-20	BD
Dounce homogenizer	Wheaton	353107542
eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) (500X)	eBioscience	00-4975-03
eBioscience Intracellular Fixation & Permeabilization Buffer Set	eBioscience	88-8824-00
FITC anti-mouse CD3	BioLegend	100203
FITC Rat IgG2b, κ Isotype Ctrl Antibody	BioLegend	400605
Freund's Adjuvant Complete (CFA)	Sigma-Aldrich	F5881
Mouse IgG2a kappa Isotype Control (eBM2a), Alexa Fluor 700, eBioscience	eBioscience	56-4724-80
Mycobacterium tuberculosis H37 Ra	Difco Laboratories	231141
PE anti-mouse IL-17A	eBioscience	12-7177-81
PE/Cy7 anti-mouse CD4	BioLegend	100422
PE/Cy7 Rat IgG2b, κ Isotype Ctrl Antibody	BioLegend	400617
Percoll	GE	17-0891-01
PerCP/Cy5.5 anti-mouse CD11b	BioLegend	101228
PerCP/Cy5.5 Rat IgG2b, κ Isotype Ctrl Antibody	BioLegend	400631
pertussis toxin (PTX)	Sigma-Aldrich	P-2980
Rat IgG1 kappa Isotype Control (eBRG1), APC, eBioscience	eBioscience	17-4301-82
Rat IgG2a kappa Isotype Control (eBR2a), PE, eBioscience	eBioscience	12-4321-80
Rat MOG35–55 peptides	Biosynth International		MEVGWYRSPFSRVVHLYRNGK