Multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS), predominately affects females compared to males. Tumor necrosis factor (TNF), a pro-inflammatory cytokine, signaling through TNF receptor 1 contributes to inflammatory disease pathogenesis. In contrast, TNF receptor 2 signaling is neuroprotective. Current anti-TNF MS therapies are shown to be detrimental to patients due to pleiotropic effects on both pro- and anti-inflammatory functions. Using a non-pertussis toxin (nPTX) experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice, we systemically administered a TNFR2 agonist (p53-sc-mTNF_R2) to investigate behavioral and pathophysiological changes in both female and male mice. Our data shows that TNFR2 activation alleviates motor and sensory symptoms in females. However, in males, the agonist only alleviates sensory symptoms and not motor. nPTX EAE induction in TNFR2 global knockout mice caused exacerbated motor symptoms in females along with an earlier day of onset, but not in males. Our data demonstrates that TNFR2 agonist efficacy is sex-specific for alleviation of motor symptoms, however, it effectively reduces mechanical hypersensitivity in both females and males. Altogether, these data support the therapeutic promise TNFR2 agonism holds as an MS therapeutic and, more broadly, to treat central neuropathic pain.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder with approximately 80 % of patients suffering from pain and 50 % from paralysis. Using a rodent model for MS, experimental autoimmune encephalomyelitis (EAE), researchers have predominately investigated paralysis/motor disease as the clinical symptom of EAE with fewer studying MS/EAE pain. However, in EAE, all mice exhibit a pain like phenotype and only a subset progresses to paralysis. Despite extensive research characterizing the disease pathology, the etiology that contributes to the range of pain and motor symptom occurrence in MS remains understudied. This is the first study to dissect MS symptom pathophysiology, using the non-PTX EAE model, in mice that experience mechanical hypersensitivity (pain-like phenotype) with and without paralysis. We found that mechanical hypersensitivity experienced by mice with or without paralysis is comparable between the two groups, irrespective of sex. In addition, there is a significant increase in the activation and infiltration of immune cells, demyelination, and heightened protein expression of B cell chemoattractant CXCL13 within the spinal cord of mice exhibiting mechanical hypersensitivity and paralysis, compared to mice only experiencing mechanical hypersensitivity.

Experimental autoimmune encephalomyelitis (EAE) is a widely used rodent model of multiple sclerosis (MS), typically induced with pertussis toxin (PTX) to achieve robust disease onset. However, PTX has been shown to exert broad immunomodulatory effects that include disruption of G protein-coupled receptor (GPCR) signaling, altered T-cell response, and exogenous suppression of regulatory T cells, all of which are not present in human MS pathophysiology. Moreover, PTX also obscures the sex differences observed in MS, limiting the translational value of EAE models that rely on it. Given EAE’s widespread use in preclinical therapeutic testing, there is a critical need for a model that better recapitulates both clinical and immunological features of MS without PTX-induced confounds. Here, we demonstrate a non-pertussis toxin (non-PTX) EAE model in C57BL/6 mice, using optimized concentrations of complete Freund’s adjuvant (CFA), Mycobacterium tuberculosis, and myelin oligodendrocyte glycoprotein (MOG_35-55) peptide. This model recapitulates hallmark features of MS that include demyelination, neuroinflammation, motor deficits, and neuropathic pain. Importantly, it retains sex-specific differences in disease onset and pathology, providing a more physiologically and clinically relevant platform for mechanistic and translational MS research.