Sex differences in EAE reveal common and distinct cellular and molecular components

Highlights

• Female and male mice use partially overlapping mechanisms to develop EAE.

• Females have more spinal cord infiltrating cells and demyelination than males.

• Males have increased inflammatory but more regulatory cell types than females.

• EAE disease process induces a wide spectrum of inflammatory factors.

• Advanced analyses reveal major sex-dependent adjuvant and EAE-associated chemokines.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is commonly used as an animal model for evaluating clinical, histological and immunological processes potentially relevant to the human disease multiple sclerosis (MS), for which the mode of disease induction remains largely unknown. An important caveat for interpreting EAE processes in mice is the inflammatory effect of immunization with myelin peptides emulsified in Complete Freund’s Adjuvant (CFA), often followed by additional injections of pertussis toxin (Ptx) in some strains to induce EAE. The current study evaluated clinical, histological, cellular (spleen), and chemokine-driven processes in spinal cords of male vs. female C57BL/6 mice that were immunized with mouse (m)MOG-35-55/CFA/Ptx to induce EAE; immunized with saline/CFA/Ptx only (CFA, no EAE); or were untreated (Naïve, no EAE). Analysis of response curves utilized a rigorous and sophisticated methodology to parse and characterize the effects of EAE and adjuvant alone vs. the Naive baseline responses. The results demonstrated stronger pro-inflammatory responses of immune cells and their associated cytokines, chemokines, and receptors in male vs. female CFA and EAE mice that appeared to be offset partially by increased percentages of male anti-inflammatory, regulatory and checkpoint T cell, B cell, and monocyte/macrophage subsets. These sex differences in peripheral immune responses may explain the reduced cellular infiltration and differing chemokine profiles in the Central Nervous System (CNS) of male vs. female CFA immunized mice and the reduced CNS infiltration and demyelination observed in male vs. female EAE groups of mice that ultimately resulted in the same clinical EAE disease severity in both sexes. Our findings suggest EAE disease severity is governed not only by the degree of CNS infiltration and demyelination, but also by the balance of pro-inflammatory vs. regulatory cell types and their secreted cytokines and chemokines.

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.