Multiple Sclerosis (MS) pathology is marked by the massive infiltration of myelin-specific T cells into the central nervous system (CNS). Hallmarks of T helper (Th) cells during active disease are pro-inflammatory Th1/Th17 cells that predominate over immunoregulatory Th2/Treg cells. Neurodegeneration, a major factor in progressive MS, is often overlooked when considering drug prescription. Here, we show that oral dosing with SNJ-1945, a novel water soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis (EAE) clinical scores in vivo and has a two pronged effect via anti-inflammation and protection against neurodegeneration. We also show that SNJ-1945 treatment downregulates Th1/Th17 inflammatory responses, and promotes regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in vivo, which are known to have the capacity to suppress helper as well as cytotoxic T cell functions. Through analysis of spinal cord samples, we show a reduction of calpain expression, decreased infiltration of inflammatory cells and signs of inhibition of neurodegeneration. We also show a marked reduction of neuronal cell death in spinal cord (SC) sections. These results suggest that calpain inhibition attenuates EAE pathology by reducing both inflammation and neurodegeneration, and could be used in clinical settings to augment the efficacy of standard immunomodulatory agents used to treat MS.

The pathogenesis of multiple sclerosis (MS) is mediated by massive infiltration of myelin-specific T cells into the central nervous system (CNS). Self-reactive CD4+ T helper (Th) cells, specifically Th1 and Th17 cells, are hallmarks of active disease in progression, whereas Th2 cells are predominately in remission stages. Calpain has been shown to be upregulated in the CNS of MS patients and inhibition of calpain has been shown previously to decrease disease in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We investigated calpain involvement in Thcell bias. Here, we show that calpain inhibition in primary myelin basic protein (MBP) Ac1-11-specific T cells and MBP-specific T cell line cultures increase Th2 proliferation, cytokine profile, and transcription and signaling molecules. We also show a relative decrease in Th1 inflammatory factors in these same categories and a relative decrease in Th17 proliferation. These studies provide insight into the various roles that calpain plays in Th cell bias and proliferation and increases our understanding of the role that T cells play in the pathophysiology of EAE and MS. Results also indicate the mechanisms involved by which calpain inhibitor decreases the disease signs of EAE, suggesting that calpain inhibitor can be a possible therapeutic agent for the treatment of EAE and MS.

Myelin basic protein (MBP) is a major target of T cells in lesions of multiple sclerosis (MS) patients and its animal model, experimental autoimmune encephalomyelitis (EAE). Interactions between the major histocompatibility complex II containing antigenic peptides and the T cell receptor activate CD4+ T cells that perpetuate EAE and MS. Previously reported data has shown that treating with an altered peptide ligand (APL) in which the normal antigenic peptide sequence of MBP has been slightly changed at T cell contact positions is helpful in reducing disease in both rodents and humans. The use of natural peptides, which are susceptible to protease degradation, requires high concentrations that can create hypersensitivity reactions. Our hypothesis is that APL containing aza substitutions, CH(R)-N- > N(R)N, could lead to improved protease resistance, reduced clinical disease scores, and a shift in T cell profile. In this study, several aza-APLs and control peptides were synthesized and screened for the best aza-APL candidate (3aza-APL) based on dissociation half time from major histocompatibility complex (MHC) class II, induction of IL-2 response, and resistance to degradation by proteases. The efficacy was then tested in vivo. Results indicate that 3aza-APL is superior to currently available APLs in terms of protease resistance and disease suppression in EAE mice. The 3aza-APL induced anti-inflammatory immune responses by altering key transcription factors and cytokine genes which regulate T cell subpopulations. These data suggest that the novel 3aza-APL has increased protease resistance property and is effective in reducing clinical and physiological signs of disease in EAE animals.

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