MSCs engineered with secreted Klotho alleviate blood–brain barrier disruption and reduce neuroinflammation more effectively than MSCs in experimental autoimmune encephalomyelitis

Abstract

Background The anti-aging protein, Klotho, has been shown to exert neuroprotective effects in neurodegenerative disorders. This study was designed to evaluate the effects of MSCs engineered with secreted Klotho (SKL-MSCs) on neuroinflammation in experimental autoimmune encephalomyelitis (EAE) mouse model and to investigate underlying molecular mechanisms. Methods EAE was induced in female C57BL/6 mice, and animals were then randomized to receive PBS, MSCs, or SKL-MSCs at the onset of disease. BBB permeability assay was performed. The mRNA and protein expression of inflammatory factors was detected in the brain of animals by real-time PCR and immunohistochemistry, respectively. The mRNA and protein expression of BBB-associated factors was detected in the brain of animals by real-time PCR and Western blotting, respectively. Results The results showed that SKL-MSCs slowed EAE progression and attenuated the disease severity more effectively than MSCs. SKL-MSCs also decreased the expression of TNF-α, IFN-γ, and IL-17 but increased the expression of IL-10 more potently than MSCs in the brain of EAE animals. Furthermore, SKL-MSCs reduced BBB permeability more significantly than MSCs, which was accompanied by decreased levels of BBB-associated factors, ICAM-1, VCAM-1, MMP-9, and CCL2, in the brain of EAE animals. However, in mice treated with MSCs, the reduction in the expression of BBB-associated factors was limited to ICAM-1 and MMP-9. Conclusions Our study highlighted the significantly greater therapeutic power of SKL-MSCs compared with MSCs in attenuating EAE disease severity and reducing neuroinflammation, which might be mediated through a more marked reduction in the BBB permeability and BBB-associated factors expression levels in the brain of animals.

1. Introduction

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) and the most common cause of neurological disability in young adults [1, 2]. This disease is characterized by infiltration of peripheral immune cells into the CNS through an impaired blood–brain barrier (BBB) in association with myelin sheath deterioration, which can ultimately culminate in severe relapsing or progressive neurocognitive and motor dysfunction [3, 4]. Experimental autoimmune encephalomyelitis (EAE) is one of the most commonly used animal models to study the pathogenesis and therapeutic approaches to MS, as it resembles the histopathological hallmarks of MS [5, 6].

Immunization of C57BL/6 mice with MOG35-55 peptide is one of the most widely distributed EAE models and can be considered as a reliable, replicable and well-to-use animal model. In both MS and EAE, activated auto-reactive immune cells cross the disrupted BBB and migrate into the CNS. Myelin-specific CD4+ T cells, especially the T helper (Th) 1 and Th17 lineages, is believed to play a vital role in the pathogenesis of EAE and MS [7–9]. Th1 cells are manifested by secretion of tumor necrosis factor (TNF-α) and interferon-γ (IFN-γ), whereas TNF-α and interleukin (IL)-17 are representative of the Th17 cell subset [10]. It has been demonstrated that systemic administration of TNF-α exacerbates the clinical outcomes of EAE, prolongs disease duration, and induces relapses [11]. In contrast, anti-TNF-α antibody attenuates the severity of EAE [12]. IFN-γ and IL-17 are found in CNS lesions and in the cerebrospinal fluid (CSF) of patients with MS, especially during the active phase of the disease [13]. In the CNS, studies have demonstrated that both IFN-γ and IL-17 could induce additional proinflammatory releasing and escalate immune activation [7, 14]. In contrast to effector Th cell lineages, Treg cells participate in the maintenance of immune homeostasis and self-tolerance. The protective effects of CD4+CD25+ Treg cells seem to be mediated by IL-10, an anti-inflammatory cytokine, since Treg cells for IL-10−/− mice failed to alleviate disease [8]. IL-10 knock-out mice develop more severe EAE symptoms, but IL-10 transgenic mice are less susceptible to EAE development.