Role of CD4 T Cell in Relapsing-Remitting Experimental Autoimmune Encephalomyelitis

Summary

An experimental autoimmune encephalomyelitis (EAE) model in mice was employed to investigate the role of CD4 T cells in the initial and relapse of EAE from the perspective of the activation phase and immune effect function.

Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic animal model for studying MS. In this study, we aimed to investigate the role of CD4 T cells in the initiation and relapse of EAE, focusing on the activation phase and immune response. To create the EAE mice model, female mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 emulsified with complete Freund's adjuvant (CFA). Clinical scores were assessed daily, and results demonstrated that mice in the EAE group exhibited a classic relapsing-remitting pattern. Hematoxylin-eosin (H&E) and luxol fast blue (LFB) staining analysis revealed significant infiltration of inflammatory cells in the CNS and demyelination in EAE mice. Regarding the activation phase, both CD4⁺CD69⁺ effector T (Teff) cells and CD4⁺CD44⁺CD62L^- effector memory T (Tem) cells may contribute to the initiation of EAE, however, the relapse stage was probably dominated by CD4⁺CD44⁺CD62L^- Tem cells. Additionally, in terms of immune function, helper T (Th)1 cells are primarily involved in initiating the EAE. However, both Th1 and Th17 cells contribute to the relapse stage, and the immunosuppressive function of regulatory T (Treg) cells was inhibited during the EAE pathological process.

Introduction

Multiple sclerosis (MS) is an autoimmune disease that is characterized by the infiltration of the central nervous system (CNS) with immune cells and demyelination^1,2. A recent study has shown it to be the most common disabling disease affecting young people, with its incidence continuously increasing worldwide³. Experimental autoimmune encephalomyelitis (EAE) is a prototypic animal model for MS that simulates many aspects of the inflammatory phase of the human MS⁴. From the perspective of immune functions, the initial CD4 T cells that have not yet encountered antigens are referred to as helper T(Th) 0 cells. These cells undergo maturation and activation processes to exhibit various functions. CD4 T cells can be categorized into several subsets according to their specific functions, which include Th1, Th2, regulatory T (Treg), follicular helper T (Tfh), Th17, Th9, and Th22 cells⁵. It is a common consensus that Th1 and Th17 cell subtypes are crucial pathogenesis factors of EAE⁶. Th1 cells could secrete interferon γ (IFN-γ), and Th17 cells secrete interleukin (IL)-17 and other inflammatory factors, which could activate other immune cells like microglial cells and astrocytes. These cells produce inflammatory cytokines⁷, such as IL-18, IL-12, IL-23, and IL-1β, which could further induce Th1/Th17 cells' immune response, resulting in progressive demyelination and axonal damage. From the activation phase perspective, CD4 T cells possess a predetermined destiny to develop into distinct cell subsets and differentiated states, including naive, effector, and memory T cells⁸. The initial CD4 T cells proliferate and differentiate into a variety of effector subsets with different functions in different environments⁹. Most effector cells are short-lived, with a small population of T cells developing into memory T cells that exhibit rapid effector functions when re-encountering the same antigens and provide the host with highly potent and long-term protection^10,11.

Although current research suggests that CD4 T cells play a significant role in the development of EAE, it is still unclear how different CD4 T cell subsets, classified based on their activation phase and immune effect function, contribute to the initiation and relapse of EAE. In the present study, we established the EAE model in C57BL6/J mice by immunizing myelin oligodendrocyte glycoprotein (MOG)35-55 peptide and investigated the initiation and relapse of EAE, focusing on the activation phase and immune function of CD4 T cells.

Protocol

A total of 18 female C57BL/6J mice aged six to eight weeks were randomly divided into a control group (n = 6) and an EAE group (n = 12) for this study. Mice were purchased from the experiment animal center of Ningxia Medical University. Mice were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle, and free access to fresh water and food. Ethics approval was obtained from the Experimental Animal Ethics Committee of Ningxia Medical University before the study began.

1. Development of relapsing remitting EAE in C57BL/6 mice

1. Reagent dissolution
   1. Dissolve 5.4 mg MOG35-55 (Table of Materials) in 1.8 mL of phosphate buffered saline (PBS) solution. Weigh 12.6 mg Mycobacterium tuberculosis H37RA (Table of Materials) and add it into 1.8 mL of complete Freund's adjuvant (CFA) solution (Table of Materials). Dissolve 360 µL of pertussis toxin (PTX; Table of Materials) into 7.2 mL of PBS using a pipette (Table of Materials).
2. Preparation of the emulsion of MOG35-55 and CFA
   1. Pipette MOG35-55 solution and CFA solution using two 5 mL syringes prepared in step 1.1 and connect them with a tee valve. Mix continuously on ice for 1 h until a milky water-in-oil emulsion is obtained.
      NOTE: The emulsion is successfully prepared when it is floating on the water surface without dispersing after dropping it into the water.
3. Mouse anesthetization
   1. Unscrew the filling seal cap at the front end of the anesthesia machine evaporator. Slowly pour 20 mL of isoflurane along the sealing screw in the center and lock the filling seal cap. Switch the tee valve switch to ensure that the airflow from the anesthesia machine evaporator is communicated with the anesthesia induction box (Table of Materials).
   2. Connect the air pump power supply, turn on the air pump switch, and rotate and adjust the air source valve at the front end of the oxygen flow meter so that the output gas flow is 400 mL/min. Open the evaporator, adjust the induction concentration to 3%, and wait for the anesthetic to fill the induction box.
   3. About 1 min later, put the mice into the induction box, then close the induction box, and wait for the mice to be in deep sleep, muscle relaxation, and steady breathing; then, the mice could be determined as the ideal state of anesthesia (Table of Materials).
      NOTE: Switch the tee valve switch to ensure that the airflow from the anesthesia machine evaporator is communicated with the anesthesia mask until the mice are fully anesthetized.
4. Mouse immunization
   1. Subcutaneously inject 200 µL of emulsion prepared in step 1.2 using a 1 mL syringe with the needle at four sites on the mouse's back, about 0.5 cm on both sides of the spine in the axilla and groin plane¹² in the EAE group mice. Use 50 µL of emulsion at each site (Table of Materials).
5. PTX injection
   1. Take the mice out of the cage by catching the tail and place it on the cage wire. Hold the tail of mice with the 4th and 5th fingers and gently pull it up to maintain the trend of the body and limbs forward. Grasp the head of the mice and capture the skin on the back of the mice using the middle and ring fingers.
   2. At 0 h and 48 h after immunization in step 1.4, inject 200 µL of PTX intraperitoneally at the position of 0.5 cm along either side of the midabdominal line with the penetration depth of 1-2 cm using 1 mL syringe with needle (Table of Materials).
      NOTE: Before injecting PTX at 48 h after immunization of MOG35-55, it is necessary to anesthetize mice using the procedures described in step 1.3. The mice should be attended to until it has regained enough consciousness to maintain sternal recumbency.
   3. For the mice in control group, treat with saline using the same method and assess the clinical score daily.
      NOTE: Mice were assessed for signs of EAE according to the following scale¹³: 0 = no disease symptoms, 1 = complete paralysis of the tail, 2 = partial hind paralysis/weakness, 3 = complete hind limb paralysis, 4 = front and hind limb paralysis, and 5 = moribund state.

2. Histological staining analysis

1. Brain collection
   1. Anesthetize the mice following step 1.3. Euthanize the mice by cervical dislocation and open the chest to expose the heart fully. Cut the auricula dextra and perfuse slowly with a pre-cooled saline solution using a 20 mL syringe with needle at the left ventricle until the liver becomes gray and white.
   2. Perfuse slowly with 4% paraformaldehyde until the tail tip turns up, the body becomes stiff, and the liver becomes tough. Decapitate the head, cut the skull, collect the brain using tweezers, and fix it in 4% paraformaldehyde solution for 24 h (Table of Materials).
2. Preparation of paraffin sections
   1. Gradient dehydration: Immerse the brain tissues from step 2.1 successively in 70% ethanol for 1 h, 80% ethanol for 1 h, 95% ethanol for 1 h, anhydrous ethanol for 1 h, replace with fresh anhydrous ethanol, continue immersing for 1 h (Table of Materials).
   2. Transparency: Immerse the brain tissues from step 2.2.1 in xylene for 30 min, replace them with fresh xylene, and continue immersing until the tissues are transparent (Table of Materials).
   3. Embedding: Embed the brain tissues from step 2.2.2 in paraffin for 1 h, replace with fresh paraffin, continue embedding for 1 h, cut into 6 µm slices after mold release, place the slide in hot water, dry at 37 °C, store at room temperature (Table of Materials).
   4. Dewaxing: Immerse brain tissues from step 2.2.3 in xylene for 20 min, replace with fresh xylene, continue immersing for 20 min, place in anhydrous ethanol for 5 min, replace with fresh anhydrous ethanol, immerse for 5 min, 75% ethanol for 5 min, rinse with tap water (Table of Materials).
   5. Hematoxylin-eosin (H&E) staining: Stain tissues from step 2.2.4 with hematoxylin solution for 3-5 min and then rinse with tap water. Treat the tissues with a hematoxylin differentiation solution, briefly and rinse thoroughly with tap water. Treat the tissues with hematoxylin Scott tap bluing solution, and rinse with tap water. Immerse in 85% ethanol for 5 min and 95% ethanol for 5 min, and stain steps with eosin dye for 5 min (Table of Materials).
   6. Dehydrate: Immerse tissues from step 2.2.5 successively in anhydrous ethanol for 5 min, replace with fresh anhydrous ethanol, continue immersing for 5 min, replace with fresh anhydrous ethanol, immerse for 5 min, xylene for 5 min, replace with fresh xylene, immerse for 5 min, seal with neutral gum (Table of Materials).
   7. Observe using a microscope the brain tissues and perform image acquisition and analysis (Table of Materials).

3. LFB staining analysis

1. Myelin sheath collection: Cut the mice spine from the head terminal up 2 segments to the tail terminal down 2 segments, blow out the spinal cord to the tail terminal, and fix the spinal cord in 4% paraformaldehyde solution for 24 h.
   NOTE: The myelin sheath was harvested from the same mice in step 2.1, and the steps for heart perfusion are the same as in 2.1.
2. Paraffin sections deparaffinization and rehydration: Put the slides from step 3.1 successively into xylene for 20 min, replace with fresh xylene, continue immersing for 20 min, anhydrous ethanol for 5 min, replace with fresh anhydrous ethanol, immerse for 5 min, 75% alcohol for 5 min, wash with tap water.
3. Fast blue staining: Place luxol fast blue staining solution A in a 60 °C oven and preheat for 30 min. Put the slides from step 3.2 in solution A and cover them with a plastic membrane for 1 h to prevent slices from drying out, then take out the slides and quickly wash them with tap water.
4. Background differentiation: Put the slides from step 3.3 into luxol fast blue staining solution B for 2 s (while the dye is hot), and directly immerse in luxol fast blue staining solution C for differentiation for 15 s, wash with water until the myelin sheath is blue and the background is almost colorless.
5. Eosin counter staining: Put the slides from step 3.4 in the oven at 65 °C for drying. Once done, remove them from the oven and allow them to cool. Put the slides into 95% ethanol and eosin counterstaining for 1 min.
6. Dehydration and sealing: Put the slides from step 3.5 successively into anhydrous ethanol 5 min, replace with fresh anhydrous ethanol, continue immersing for 5 min, replace with fresh anhydrous ethanol again, continue immersing for 5 min, xylene 5 min, replace with fresh xylene, immerse for 5 min. Wash and dry the samples and seal them with neutral gum.
7. Microscope examination: Collect and analyze images using an upright optical microscope (Table of Materials).

4. Flow cytometry analysis

1. Open the abdominal cavity of mice. Extract the spleens using tweezers and mince into small pieces of 1.5-2.5 cm in PBS on ice using scissors. Grind the spleen in 10 mL of RPMI-1640 containing 2% fetal bovine serum (FBS) using a 2.5 mL syringe piston and push it through a 70 µm cell strainer to obtain single-cell suspension (Table of Materials).
   NOTE: The spleens were harvested from the same mice in step 2.1.
2. Transfer the suspension obtained in step 4.1 into 15 mL tubes and lyse using 5 mL of red blood cell lysis buffer on ice for 10 min. Wash with RPMI-1640 containing 2% FBS, centrifuge at 350 x g for 5 min, discard the supernatant, and resuspend cells with RPMI-1640 containing 2% FBS.
3. Count the cells with an automatic cell counter (Table of Materials) and adjust the concentration to 1 x 10⁶ cells/mL.
4. Pipette 100 µL of cell suspension into 1.5 mL tubes and incubate with 1 µL of anti-mouse CD16/32 antibody¹⁴ (0.5 mg/mL) for 30 min to block non-specific staining. Wash with RPMI-1640 and centrifuge at 350 x g for 5 min, discard the supernatant. Resuspend the cells in 100 µL of RPMI-1640 containing 2% FBS.
5. Stain with antibodies specific for CD45 (Alexa Fluor 700), CD3 (FITC), CD4 (APC), and CD69 (BV674) for Teff cells¹⁵ for 30 min, 2 µL for each antibody. Stain with antibodies specific for CD45 (Alexa Fluor 700), CD3 (FITC), CD4 (APC), CD44 (BV421), and CD62L (PE) for Tem cells¹⁶ for 30 min, 2 µL for each antibody. Wash with RPMI-1640 and centrifuge at 350 x g for 5 min, discard the supernatant and resuspend cells in 350 µL of PBS. Analyze cell proportion using a flow cytometer and the associated software (Table of Materials).

5. Analysis of cytokines by cytometric bead array

1. Use the cytometric bead array (Table of Materials) to measure IL-2, IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor (TNF)-α and IFN-γ in mice brains, methods as described below.
2. Collect mouse brain as described in step 2.1.
3. Extraction of total protein: Cut and weigh 50 mg of brain tissue (three brains in the control group, EAE group in the peak stage, and RR stage). Place brains in 1.5 mL tubes and add 500 µL of lysis buffer containing PMSF, protease inhibitor, and phosphatase inhibitors.
4. Cut brains into small pieces around 1 mm³, extract by ultrasonic homogenizer at 400 W, 5 s ON and 5 s OFF; repeat for 5x. Place the tube on ice for 10 min, centrifuge at 12000 x g for 5 min, and collect the supernatant (Table of Materials).
5. Prepare mouse Th1/Th2/Th17 cytokine standards as described below.
   1. Open a vial of lyophilized mouse Th1/Th2/Th17 standards. Transfer the standards into a 15 mL conical polypropylene tube. Label the tube top as standard.
   2. Reconstitute the standards in 2.0 mL of assay diluent. Allow the reconstituted standard to equilibrate for 15 min at room temperature. Gently mix the reconstituted protein by pipetting.
   3. Label 12 mm x 75 mm tubes and arrange them in the following order of dilution: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. Pipette 300 µL of assay diluent in each labeled tube.
   4. Perform serial dilutions: Transfer 300 µL of the top standard to the 1:2 dilution tube and mix thoroughly by pipetting. Continue serial dilutions by transferring 300 µL from the 1:2 tube to the 1:4 tube and so on till the 1:256 tube. Prepare a 12 mm x 75 mm tube containing only the assay diluent to serve as the 0 pg/mL negative control.
      NOTE: Mix thoroughly by pipetting. Do not vortex to prevent the antibody coupling on the microbeads from falling off.
   5. To mix the mouse Th1/Th2/Th17 cytokine capture beads, prepare 19 tubes, including standards with 3 samples in each group (control group, EAE group in peak stage, and RR stage) for the experiment. Vortex the capture bead suspension vigorously for 5 s before mixing. Add 190 µL of each capture bead into a single tube and label it as mixed capture beads. Vortex the bead mixture thoroughly.
      NOTE: The antibody-conjugated beads will settle out of suspension over time. It is necessary to vortex the vial before taking a bead-suspension aliquot.
   6. To perform the assay, vortex the mix capture beads. Add 50 µL of beads to all assay tubes. Add 50 µL of mouse Th1/Th2/Th17 cytokine standard dilutions to the control tubes as in Table 1.
   7. Add 50 µL of the mouse Th1/Th2/Th17 PE detection reagent to all assay tubes. Incubate the assay tubes for 2 h at room temperature and protect them from light. Add 1 mL of wash buffer to each assay tube and centrifuge at 200 x g for 5 min. Aspirate and discard the supernatant from each assay tube carefully.
   8. Add 300 µL of wash buffer to each assay tube to resuspend the bead pellet. Analyze mouse Th1/Th2/Th17 cytokine data using FCAP Array software (Table of Materials).

  

النتائج

Clinical scores assessment
As shown in Figure 1, the mice in the control group did not exhibit any clinical symptoms. The mice in the EAE group, which were immunized with MOG35-55, showed tail paralysis approximately 12 days after immunization. By day 16, the symptoms reached complete hind limb paralysis (defined as peak stage, Peak). After that, the symptoms gradually were remitted. Mice's clinical symptoms were aggravated at around day 25 and reached complete hin...

Discussion

MS is the most prevalent autoimmune demyelinating disease of the CNS, which affects millions of people worldwide¹⁷. EAE is the typical animal model for simulating the clinical pathological features of MS¹⁸. Studies have demonstrated that individuals with MS experience cognitive impairment and other disabilities due to the degeneration of axons and neurons in the CNS^19,20,21. At the...

Materials

Name	Company	Catalog Number	Comments
Anesthesia machine evaporator	Norvap	20-17368
Isoflurane	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	792632
70 μm cell strainer	XIYAN Co.,Ltd.	15-1070
Alexa Fluor 700 anti-mouse CD45 Antibody	Biolegend	103128
APC anti-mouse CD4 Antibody	Biolegend	100616
Automatic cell counter	Jiangsu JIMBIO technology Co., LTD	JIMBIO iCyta S2
BD* Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 CBA Kit	BD Biosciences	560485
Biotin anti-mouse CD16/32 Antibody	Biolegend	101303
Brilliant Violet 510 anti-mouse CD69 Antibody	Biolegend	104532
BV786 Rat Anti-Mouse CD62L(MEL-14)	BD Pharmingen	563109
Column Tissue&Cell Protein Extraction Kit	Shanghai Epizyme Biomedical Technology Co., Ltd	PC201Plus
Complete Freund's Adjuvant	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	SLCH4887
Dehydrator	DIAPATH	Donatello
Disposable sterilized syringe (1 mL)	Yikang Group	210820
Disposable sterilized syringe (2.5 mL)	Yikang Group	210820
Disposable sterilized syringe (5 mL)	Yikang Group	210820
Dyeing machine	DIAPATH	Giotto
Embedding machine	Wuhan Junjie Electronics Co., Ltd	JB-P5
Ethanol	SCRC	100092683
Fetal Bovine Serum	Procell Life Science&Technology Co.,Ltd.	FSP500
FITC Hamster Anti-Mouse CD3e(145-2C11)	BD Pharmingen	553062
Flow cytometer	Becton,Dickinson and Company	FACSCelesta
Flow cytometer	Becton,Dickinson and Company	Accuri C6
Flow Jo software	BD Biosciences	10.8.1
Frozen platform	Wuhan Junjie Electronics Co., Ltd	JB-L5
Glass slide	Servicebio	G6004
HE dye solution set	Servicebio	G1003
Hematoxylin-Eosin solution	Servicebio	G1002
High speed refrigerated centrifuge	Thermo Fisher Scientific	Mogafugo8R
Imaging system	Nikon	NIKON DS-U3
Luxol fast blue staining kit	Servicebio	G1030
Ms CD44 BV421 IM7	BD Pharmingen	564970
Mycobacterium tuberculosis H37RA	BD Pharmingen	231141
Myelin oligodendrocyte glycoprotein (MOG35-55)	AnaSpec	AS-60130-1
Neutral gum	SCRC	10004160
Organizer	KEDEE	KD-P
Oven	Labotery	GFL-230
Pathology slicer	Leica	RM2016
Pertussis Toxin from Bordetella pertussis	Sigma-Aldrich (Shanghai) Trading Co.Ltd.	P7208
Phosphate buffered saline	Servicebio	G4202
Pipette 0.5-10 μL	DLAB Scientific	7010101004
Pipette 100-1000 μL	DLAB Scientific	7010101014
Pipette 20-200 μL	DLAB Scientific	7010101009
RPMI-1640	Procell Life Science&Technology Co.,Ltd.	PM150110
Tee valve	Guangdong Kanghua Medical Co., LTD	A06
Tissue spreader	Zhejiang Kehua Instrument Co.,Ltd	KD-P
Upright optical microscope	Nikon	NIKON ECLIPSE E100
Xylene	SCRC	10023418