Therapeutic Effect And Mechanism Study Of Kv1.3-targeting Peptide On Rat EAE Model

The voltage-gated Kvl.3K+channel in effector memory T cells (TEM) serves as a new therapeutic target for multiple sclerosis (MS). In our previous studies, the novel peptide ADWX-1was designed and synthesized as a specific Kvl.3blocker. This peptide has a high potency and high selectivity toward Kv1.3channel. However, it is unclear if and how ADWX-1alleviates experimental autoimmune encephalomyelitis (EAE), a model for MS.In this study, the administration of ADWX-1significantly ameliorated the rat EAE model by selectively inhibiting CD4+CCR7-phenotype TEM cell activation. In vivo the peptide can decrease the clinical score of the EAE rats, and reduce the inflammatory infiltration, demyelination and axonal loss in the central nervous system (CNS) by histological study including the H.E. stain, the LFB stain and the Transmission Electron Microscopy (TEM). This neuroprotective effect of the peptide might be attributed to its immunomodulation on CD4+T cells, Therefore series of inflammatory cytokines were analyzed by ELISA. Results showed that the peptide can suppress the serum Th1type cytokines productions in EAE model including IL-2, IFN-γ, and TNF-α The peptide also reduced IL-2secretion in the CNS of EAE rats. In vitro study showed that the peptide inhibited the activation of Jurkat T cells and EAE PBMCs by measuring the IL-2levels after activation. And the peptide further suppressed the proliferation of EAE PBMCs.Further more we elucidated the underlying mechanism of this afficacy. By the methods of ELISA combined with FCAS to detect the cytokines secretions after antigen activation, the peptide selectively inhibit CD4+CCR7" phenotype TEM cell activation and proliferation in vitro. In contrast, the Kvl.3-specific peptide had little effect on CD4+CCR7+cells, therefore limiting side effects. Furthermore, we determined that ADWX-1is involved in the regulation of NF-kB signaling through upstream protein kinase C-θ (PKCθ) in the IL-2pathway of CD4+CCR7-cells. The elevated expressions of Kvl.3mRNA and protein in activated CD4+CCR7-cells were reduced by ADWX-1engagement; however, an apparent alteration in CD4+CCR7+cells was not observed. Moreover, the selective regulation of the Kv1.3channel gene expression pattern by ADWX-1provided a further and sustained inhibition of the CD4+CCR7-phenotype, which depends on the activity of K.v1.3to modulate its activation signal. In addition, ADWX-1mediated the activation of differentiated Th17cells through the CCR7-phenotype, but having no obvious effect on the differentiation process induced by cytokines. Similarly we found the effect of the peptide on CD8+T cells; it selectively suppressed the GrB secretion from CD8+CCR7-cells rather than the CD8+CCR7+cells, in vivo the peptide prevented the GrB production in EAE, the alleviated the neural injury to oligodendrocytes by GrB, reducing the over-exhaust of myelin proteins.The efficacy of ADWX-1is supported by multiple functions, which are based on a Kv1.3high CD4+CCR7-T cell selectivity through two different pathways including the classic channel activity-associated IL-2pathway and the new Kvl.3channel gene expression pathway. This study makes the peptide a promising candidate drug for MS therapy.