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

Published: January 19, 2024

doi:

Jin Hu*^1,2, Xiangyu Zhou*³, Yikun Cao*³, Huili Tian*⁴, Na Li, Xiuli Cheng, Xiaoying Yang, Hongwan Dang⁶

¹School of Basic Medicine,Ningxia Medical University, ²Preparation Center,General Hospital of Ningxia Medical University, ³Institute of Neurological Diseases,First People’s Hospital of Shizuishan, ⁴Department of Neurology,Xianyang Hospital of Yan’an University, ⁵Department of Pharmacy,People’s Hospital of Ningxia Hui Autonomous Region, ⁶Department of Pharmacy,General Hospital of Ningxia Medical University

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 demyelination¹^,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 protection¹⁰^,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. <p class="jove_title"…

Representative Results

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 CNS¹⁹^,²⁰^,²¹. At the…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Natural Science Foundation of Ningxia (2022AAC03601 and 2023AAC02087) and Research Foundation of Ningxia Medical University (XM2019052). Thanks for the support of the Medical Science and Technology Research Center of Ningxia Medical University.

Materials

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

Referencias

Swanberg, K., et al. Multiple sclerosis diagnosis and phenotype identification by multivariate classification of in vivo frontal cortex metabolite profiles. Sci Rep. 12 (1), 13888 (2022).
Zahoor, I., et al. An emerging potential of metabolomics in multiple sclerosis: a comprehensive overview. Cell Mol Life Sci. 78 (7), 3181-3203 (2021).
Vitturi, B., et al. Spatial and temporal distribution of the prevalence of unemployment and early retirement in people with multiple sclerosis: A systematic review with meta-analysis. PloS one. 17 (7), 0272156 (2022).
Brambilla, R. The contribution of astrocytes to the neuroinflammatory response in multiple sclerosis and experimental autoimmune encephalomyelitis. Acta neuropathologica. 137 (5), 757-783 (2019).
Niedźwiedzka-Rystwej, P., Tokarz-Deptuła, B., Deptuła, W. Characteristics of T lymphocyte subpopulations. Postepy Hig Med Dosw. 67, 371-379 (2013).
Loos, J., et al. Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy. J neuroinflammation. 17 (1), 357 (2020).
Prajeeth, C., et al. Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties. J neuroinflammation. 14 (1), 204 (2017).
Nielsen Birgitte, R., et al. Characterization of naïve, memory and effector T cells in progressive multiple sclerosis. J Neuroimmunol. 310, 17-25 (2017).
Xie, L., et al. The role of CD4+ T cells in tumor and chronic viral immune responses. Med Comm. 4 (5), e390 (2023).
Sun, L., et al. T cells in health and disease. Signal Transduct Target Ther. 8 (1), 235 (2023).
Kawabe, T., et al. Memory-phenotype CD4+ T cells spontaneously generated under steady-state conditions exert innate TH1-like effector function. Sci Immunol. 2 (12), (2017).
Moon, J., et al. A study of experimental autoimmune encephalomyelitis in dogs as a disease model for canine necrotizing encephalitis. J Vet Sci. 16 (2), 203-211 (2015).
Pérez-Nievas, B., et al. Chronic immobilisation stress ameliorates clinical score and neuroinflammation in a MOG-induced EAE in Dark Agouti rats: mechanisms implicated. J neuroinflammation. 7, 60 (2010).
Zheng, J., et al. Prostaglandin D2 signaling in dendritic cells is critical for the development of EAE. J Autoimmun. 114, 102508 (2020).
Adamczyk, M., et al. The expression of activation markers CD25 and CD69 increases during biologic treatment of Psoriasis. J Clin Med. 12 (20), 6573 (2023).
Meng, R., et al. Echinococcus multilocularisIndoleamine 2,3-dioxygenase 1 signaling orchestrates immune tolerance in -infected mice. Front Immunol. 13, 1032280 (2022).
Sheinin, M., et al. Suppression of experimental autoimmune Encephalomyelitis in mice by β-Hydroxy β-Methylbutyrate, a body-building supplement in humans. J Immunol. 211 (2), 187-198 (2023).
Ghareghani, M., et al. Hormones in experimental autoimmune encephalomyelitis (EAE) animal models. Transl Neurosci. 12 (1), 164-189 (2021).
Pérez-Miralles, F., et al. Clinical impact of early brain atrophy in clinically isolated syndromes. Mult Scler. 19 (14), 1878-1886 (2013).
Nygaard, G., et al. Cortical thickness and surface area relate to specific symptoms in early relapsing-remitting multiple sclerosis. Mult Scler. 21 (4), 402-414 (2015).
Biberacher, V., et al. Atrophy and structural variability of the upper cervical cord in early multiple sclerosis. Mult Scler. 21 (7), 875-884 (2015).
Ma, Y., et al. Epsilon toxin-producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege. J Clin Invest. 133 (9), e163239 (2023).
Cibrián, D., Sánchez, M. CD69: from activation marker to metabolic gatekeeper. Eur J Immunol. 47 (6), 946-953 (2017).
Clarkson Benjamin, D., et al. Preservation of antigen-specific responses in cryopreserved CD4 and CD8 T cells expanded with IL-2 and IL-7. J Transl Autoimmun. 5, 100173 (2022).
Raeber Miro, E., et al. The role of cytokines in T-cell memory in health and disease. Immunol Rev. 283 (1), 176-193 (2018).
Ville, S., et al. Co-stimulatory blockade of the CD28/CD80-86/CTLA-4 balance in transplantation: Impact on memory T cells. Front Immunol. 6, 411 (2015).
Natalini, A., et al. OMIP-079: Cell cycle of CD4+ and CD8+ naïve/memory T cell subsets, and of Treg cells from mouse spleen. Cytometry A. 99 (12), 1171-1175 (2021).
Joffre, O., et al. Cross-presentation by dendritic cells. Nat Rev Immunol. 12 (8), 557-569 (2012).
Montes, M., et al. Oligoclonal myelin-reactive T-cell infiltrates derived from multiple sclerosis lesions are enriched in Th17 cells. Clin Immunol. 130 (2), 133-144 (2009).
Feruglio, S., Kvale, D., Dyrhol, R. T. Cell responses and regulation and the impact of in vitro IL-10 and TGF-β modulation during treatment of active tuberculosis. Scand J Immunol. 85 (2), 138-146 (2017).