Sex differences in EAE reveal common and distinct cellular and molecular components
Introduction
Experimental autoimmune encephalomyelitis (EAE) is an ascending paralytic autoimmune disease of the Central Nervous System (CNS) in animals that predominantly involves myelin-specific T cells and myeloid cells that migrate from the periphery into the CNS. Upon reactivation in spinal cord, these cells attack and damage myelinated axons [1], [2], [3], [4], [5]. The EAE model may be useful for understanding the disease process in multiple sclerosis (MS) due to similarities in the formation of demyelinating perivascular white-matter lesions and induction of acute, chronic, and progressive motor disabilities [5], [6], [7], [8], [9], [10]. EAE induction involves activation of encephalitogenic T cells upon immunization of susceptible strains of rodents by strain-specific myelin peptides emulsified in complete Freud’s adjuvant (CFA), with an additional requirement in some strains for subsequent injections of Bordetella pertussis toxin (Ptx) [10], [11], [12], [13], [14], [15]. Although the cause of MS has defied intense investigation, the assumption is that EAE induction to some extent resembles a similar process in MS that involves induction of pro-inflammatory T and B cells and activation of myeloid cells, perhaps due to CNS damage by microbial infection and/or molecular mimicry of myelin peptides with microbial antigens. In both diseases, emergence of clinical signs likely depends upon the amplification of autoimmune recognition of CNS target antigens [15], [16], [17], [18], [19].
It is widely known that MS disproportionately affects females [20], [21], [22], [23], [24], [25], [26], [27], [28], although MS disabilities are often more severe in males with progressive disease [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. Yet, few EAE studies have systematically evaluated sex differences in disease induction or severity. Thus, NIH guidelines [39] indicate the need to study both sexes in various disease models.
The goal of this study was to evaluate sex differences as well as the contribution of myelin peptides emulsified in CFA with added Ptx injections to the induction of severe acute EAE in both male and female C57BL/6 mice, with reference in all assays to naïve, untreated control mice as well as mice injected with CFA + Ptx alone. We thus evaluated clinical and histological EAE disease severity, composition of immune cell subtypes in spleen, and mRNA expression levels of a panel of 84 cytokines, chemokines/receptors, and cell process factors in spinal cord tissue from female and male mice in each of the three treatment groups. We found that EAE disease severity is governed not only by the degree of immune cell infiltration into the CNS and demyelination, but also by the induction of pro-inflammatory vs. regulatory splenocyte cell subtypes and spinal cord expression levels of cytokines, chemokines, and receptors, many of which varied in expression in a sex-dependent manner.
Section snippets
Animals
C57BL/6 male [33] and female [32] mice were obtained from The Jackson Laboratory (Sacramento, CA) at 6–7 weeks of age and used in experiments between 8 and 12 weeks of age. Mice were maintained on a 12-hour light/dark cycle with access to food and water ad libitum in the Animal Resource Facility at the Portland Veterans Affairs Health Care System. This study was carried out in strict accordance with Federal, NIH, and Institutional guidelines using a protocol approved by the Portland VA Animal
Sex differences in EAE induction
To investigate the role of cell types and associated factors in the inflammatory process in EAE induction, we compared clinical and histological signs of disease in Naïve, CFA/Ptx-immunized (CFA) and CFA/Ptx/MOG-35-55 peptide-immunized (EAE) groups of C57BL/6 male and female mice that were 8–12 weeks old at immunization.
Conclusion
The results of this study provide new perspectives on two major issues regarding the underlying processes involved in the induction of EAE in C57BL/6 mice. First, regarding sex differences, the clinical course and severity of EAE were nearly identical in male and female mice despite increased spinal cord lesions and demyelination potentially enabled by fewer immunoregulatory mechanisms in females vs. males. Understanding such sex differences may allow further evaluation of which components are
Declarations
Ethics approval and consent to participate
All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.
Consent to publish
Not applicable
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Funding
This work was funded by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development Merit Review Award 2I01 BX000226 and Senior Research Career Scientist Award 1IK6BX004209 (AAV) and by the National Institute of Allergy and Infectious Diseases award 2R42AI122574 (AAV). The contents do not represent the views of the Department of Veterans Affairs or the US Government.
Author contributions
GG, HS, PC, GK and AM performed experiments, analyzed data, and reviewed the manuscript. AH analyzed data, reviewed and helped with manuscript preparation. JW provided biostatistician analysis, analyzed data, wrote and reviewed the manuscript. AAV, MR and HO contributed to experimental design, analyzed data and wrote and reviewed the manuscript. All authors read and approved the final manuscript.
Declaration of Competing Interest
Drs. Vandenbark, Offner, Meza-Romero, and OHSU have a significant financial interest in Artielle ImmunoTherapeutics, Inc., a company that may have a commercial interest in the results of this research and technology. This potential conflict of interest has been reviewed and managed by the OHSU and VA Portland Health Care System Conflict of Interest in Research Committees.
Acknowledgements
The authors would like to acknowledge both the OHSU Histopathology and Advanced Light Microscopy Core for their contributions to the spinal cord tissue embedding, staining, and image acquisition. They would also like to thank the VAPORHCS Veterinary staff for housing and maintaining mouse environments.
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2022, Cellular ImmunologyCitation Excerpt :The changes induced by DRα1-MOG-35-55 beyond genotype effects can be observed in Fig. 4 for females and Figure S4 for males. The distribution and overlap of numbers in subgroups of these factors and their sensitivity to interventions considered in this study (i.e. MIF-1 deletion and DRα1-MOG-35-55 treatment) are illustrated in a Venn diagram for females vs. males (Fig. 5), color-coded by association intersection region and intervention sufficient for suppression (DRα1-MOG-35-55 treatment in WT vs. MIF-1-KO genotype vs. both), listing total counts and names of EAE- and adjuvant-associated factors that were shown to be important for EAE in our previous study [38] and observed to be suppressed under one of these conditions in the current study. The top panel shows the divisions of counts along each axis, and the bottom panel provides annotation of gene names for each region of the diagram, organized by color. (
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Contributed equally to this study.