Daphnetin Ameliorates Experimental Autoimmune Encephalomyelitis Through Regulating Heme Oxygenase-1

Neurochemical Research - To assess the potential role of daphnetin, a clinically used anti-inflammatory agent, on the development of the inflammatory and neurodegenerative disease, we investigated...     

Rapamycin Augments Immunomodulatory Properties of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis

The immunomodulatory and anti-inflammatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) have been considered as an appropriate candidate for treatment of autoimmune diseases. Previous studies have revealed that treatment with BM-MSCs may modulate immune responses and alleviate the symptoms in experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis. Therefore, the present study was designed to examine immunomodulatory effects of BM-MSCs in the treatment of myelin oligodendrocyte glycoprotein (MOG) 35-55-induced EAE in C57BL/6 mice. MSCs were obtained from the bone marrow of C57BL mice, cultured with DMEM/F12, and characterized with flow cytometry for the presence of cell surface markers for BM-MSCs. Following three passages, BM-MSCs were injected intraperitoneally into EAE mice alone or in combination with rapamycin. Immunological and histopathological effects of BM-MSCs and addition of rapamycin to BM-MSCs were evaluated. The results demonstrated that adding rapamycin to BM-MSCs transplantation in EAE mice significantly reduced inflammation infiltration and demyelination, enhanced the immunomodulatory functions, and inhibited progress of neurological impairments compared to BM-MSC transplantation and control groups. The immunological effects of rapamycin and BM-MSC treatments were associated with the inhibition of the Ag-specific lymphocyte proliferation, CD8+ cytolytic activity, and the Th1-type cytokine (gamma-interferon (IFN-γ)) and the increase of Th-2 cytokine (interleukin-4 (IL-4) and IL-10) production. Addition of rapamycin to BM-MSCs was able to ameliorate neurological deficits and provide neuroprotective effects in EAE. This suggests the potential of rapamycin and BM-MSC combined therapy to play neuroprotective roles in the treatment of neuroinflammatory disorders.     

Neuroprotection by Ulinastatin in Experimental Autoimmune Encephalomyelitis

Ulinastatin has previously been used as a drug for patients with acute inflammatory disorders. The goal of the present study was to investigate the protective effects of ulinastatin on myelin sheaths and oligodendrocytes in experimental autoimmune encephalomyelitis (EAE), and to explore the possible underlying mechanism. Mice were divided into an ulinastatin treatment group, a normal saline treatment group, and a normal control group. EAE was induced in the mice with and without ulinastatin treatment. Demyelination was evaluated, as was the number of oligodendrocytes. The ulinastatin treatment group had a significantly lower clinical score, demyelinating score, and large numbers of oligodendrocytes compared with the group without ulinastatin treatment. Furthermore, ulinastatin treatment increased the expression of nerve growth factor and brain-derived neurotrophic factor, and protected against oligodendrocyte apoptosis. Thus, ulinastatin is shown to have a protective effect against EAE.     

Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is mediated by myelin-specific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. The molecular bases for expansion and activation of these cells, plus trafficking to the CNS for peripheral cells, are not fully understood. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca²⁺ binding adapter-1 [Iba-1]) is induced in leukocytes in MS and EAE; here we provide the first assessment of Aif-1 function in this setting. After myelin oligodendrocyte glycoprotein peptide (MOG_35–55) immunization, Aif-1–deficient mice were less likely than controls to develop EAE and had less CNS leukocyte infiltration and demyelination; their spinal cords contained fewer CD4 T cells and microglia and more CD8 T cells. These mice also showed significantly less splenic CD4 T-cell expansion and activation, plus decreased proinflammatory cytokine expression. These findings identify Aif-1 as a potent molecule that promotes expansion and activation of CD4 T cells, plus elaboration of a proinflammatory cytokine milieu, in MOG_35–55-induced EAE and as a potential therapeutic target in MS.     

Gene-Expression Profiling of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that serves as an experimental tool for studying the etiology, pathogenesis, as well as new therapeutic approaches of multiple sclerosis (MS). EAE is a polygenic chronic inflammatory demyelinating disease of the nervous system that involves the interaction between genetic and environmental factors. Previous studies have identified multiple quantitative trait loci (QTL) controlling different aspects of disease pathogenesis. However, progress in identifying new susceptibility genes outside the MHC locus has been slow. With the advent of new global methods for genetic analysis such as large-scale sequencing, gene expression profiling combined with classic linkage analysis and congenic and physical mapping progress is considerably accelerating. Here we review our preliminary work on the use of gene expression mapping to identify new putative genetic pathways contributing to the pathogenesis of EAE.     

Artemisinin Analogue SM934 Ameliorates Murine Experimental Autoimmune Encephalomyelitis through Enhancing the Expansion and Functions of Regulatory T Cell

Background

Artemisinin analogue SM934 was previously reported to possess immunosuppressive properties. The aim of this study was to determine the effects and the underlying mechanisms of SM934 in murine experimental autoimmune encephalomyelitis (EAE).

Methods

Female C57BL/6 mice immunized with MOG_35–55 were treated with or without SM934, then the clinical scores and other relevant parameters were assessed. Th1, Th17 and regulatory T (Treg) cell profiles were determined through ELISA, qRT-PCR, flow cytometry and BrdU incorporation assay. The effects of SM934 on Th1, Th17 and Treg cells differentiation were explored through intracellular staining and flow cytometry examination.

Results

In vivo, administration of SM934 significantly inhibited the development of EAE and suppressed the elevation of serum IL-17. Ex vivo, upon antigen-recall stimulation, IL-2, IFN-γ, IL-17 and IL-6 production were decreased, whereas IL-10 and TGF-β production were increased from the splenocytes isolated from SM934-treated mice. Consistently, both flow cytometry and qRT-PCR results showed that SM934 treatment significantly increased the Treg, while strongly suppressed the Th17 and Th1, responses in the peripheral. Furthermore, in the spinal lesion, SM934 treatment dramatically decreased the infiltration of CD4⁺ T cells, within which the Treg cells percentage was enlarged, whereas the Th17, but not Th1 percentage, was significantly decreased comparing with the vehicle-treated groups. Finally, both BrdU incorporation and in vitro Treg differentiation assays revealed that SM934 treatment could directly promote the expansion of Treg cells in vivo and in vitro.

Conclusion

Taken together, this study demonstrated that SM934 treatment could ameliorate the murine EAE disease, which might be mediated by inducing Treg differentiation and expansion.     

Spasticity Treatment Ameliorates the Efficacy of Melatonin Therapy in Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model of Multiple Sclerosis

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease in the central nervous system (CNS). Melatonin is an effective treatment in MS patients and experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Melatonin secretion peaks at 2 AM, concomitant with the time at which the muscles are resting and the body is exerting its antioxidant activity. The current study was designed to investigate combination treatment of baclofen, a muscle relaxant drug, and melatonin in EAE mice. Results showed that melatonin (Mel) alone or in combination with baclofen (Bac + Mel) reduced clinical scores and demyelination by significantly increasing myelin oligodendrocyte glycoprotein (MOG) levels, a marker for mature oligodendrocytes, compared to EAE mice. Moreover, Mel or Bac + Mel therapy caused a significant increase in IL-4 serum levels, an anti-inflammatory cytokine, whereas IFN-γ serum levels, a pro-inflammatory cytokine, were significantly reduced. On the other hand, Mel or Bac + Mel caused a significant reduction in malondialdehyde (MDA) levels, a marker of oxidative stress, in comparison to EAE mice. In contrast, the activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) was significantly increased in Mel and Bac + Mel groups. In summary, combination therapy improved clinical scores and tend to enhance the efficiency of melatonin treatment by further promoting remyelination, decreasing inflammation, and stimulating the activity of antioxidant enzymes, which suggests that prior spasticity treatment increases the efficacy of melatonin therapy in EAE mouse model of MS. Further experimental and clinical studies are needed to ensure the beneficial role of this combination strategy.     

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.     

Rapamycin Enhances the Anti-Cancer Effect of Dasatinib by Suppressing Src/PI3K/mTOR Pathway in NSCLC Cells

Src and the mammalian target of rapamycin (mTOR) signaling are commonly activated in non-small cell lung cancer (NSCLC) and hence potential targets for chemotherapy. Although the combined use of Src inhibitor Dasatinib with other chemotherapeutic agents has shown superior efficacy for cancer treatment, the mechanisms that lead to enhanced sensitivity of Dasatinib are not completely understood. In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis. The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation. Mechanistic investigations demonstrated that FoxO1/FoxO3a and p70S6K/4E-BP1, the molecules at downstream of Src-PI3K-Akt and mTOR signaling, were significantly suppressed by the combined use of Dasatinib and Rapamycin. Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib. In addition, this finding was also validated by a series of assays using another two NSCLC cell lines, NCI-H1706 and NCI-H460. Conclusively, our results suggested that the combinatory application of Src and mTOR inhibitors might be a promising therapeutic strategy for NSCLC treatment.     

N-type Calcium Channel in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca²⁺ channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca²⁺ channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca²⁺ channel α_1B-deficient (α_1B^−/−) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35–55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α_1B^−/− mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α_1B^−/− mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α_1B^−/− mice and was significantly inhibited by a selective N-type Ca²⁺ channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca²⁺. These results suggest that the N-type Ca²⁺ channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.     

Reduced Severity of Experimental Autoimmune Encephalomyelitis in GMF-Deficient Mice

Glia maturation factor (GMF), a highly conserved brain-specific protein, isolated, sequenced and cloned in our laboratory. Overexpression of GMF in astrocytes induces the production and secretion of granulocyte-macrophage-colony stimulating factor (GM-CSF), and subsequent immune activation of microglia, expression of several proinflammatory genes including major histocompatibility complex proteins, IL-1β, and MIP-1β, all associated with the development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Based on GMF’s ability to activate microglia and induce well-established proinflammatory mediators, including GM-CSF, we hypothesize that GMF is involved in the pathogenesis of inflammatory disease EAE. In this present investigation, using GMF-deficient mice, we study the role of GMF and how the lack of GMF affects the EAE disease. Our results show a significant decrease in incidence, delay in onset, and reduced severity of EAE in GMF-deficient mice, and support the hypothesis that GMF plays a major role in the pathogenesis of disease.     

Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis

The cysteine cathepsins B, S, and L are functionally linked to antigen processing, and hence to autoimmune disorders such as multiple sclerosis. Stemming from several studies that demonstrate that mice can be protected from experimental autoimmune encephalomyelitis (EAE) through the pharmacologic inhibition of cysteine cathepsins, it has been suggested that targeting these enzymes in multiple sclerosis may be of therapeutic benefit. Utilizing mice deficient in cysteine cathepsins both individually and in combination, we found that the myelin-associated antigen myelin oligodendrocyte glycoprotein (MOG) was efficiently processed and presented by macrophages to CD4+ T cells in the individual absence of cathepsin B, S or L. Similarly, mice deficient in cathepsin B or S were susceptible to MOG-induced EAE and displayed clinical progression and immune infiltration into the CNS, similar to their wild-type counterparts. Owing to a previously described CD4+ T cell deficiency in mice deficient in cathepsin L, such mice were protected from EAE. When multiple cysteine cathepsins were simultaneously inhibited via genetic deletion of both cathepsins B and S, or by a cathepsin inhibitor (LHVS), MHC-II surface expression, MOG antigen presentation and EAE were attenuated or prevented. This study demonstrates the functional redundancy between cathepsin B, S and L in EAE, and suggests that the inhibition of multiple cysteine cathepsins may be needed to modulate autoimmune disorders such as multiple sclerosis.     

Hsp70 Regulates Immune Response in Experimental Autoimmune Encephalomyelitis

Heat shock protein (Hsp)70 is one of the most important stress-inducible proteins. Intracellular Hsp70 not only mediates chaperone-cytoprotective functions but can also block multiple steps in the apoptosis pathway. In addition, Hsp70 is actively released into the extracellular milieu, thereby promoting innate and adaptive immune responses. Thus, Hsp70 may be a critical molecule in multiple sclerosis (MS) pathogenesis and a potential target in this disease due to its immunological and cytoprotective functions. To investigate the role of Hsp70 in MS pathogenesis, we examined its immune and cytoprotective roles using both in vitro and in vivo experimental procedures. We found that Hsp70.1-deficient mice were more resistant to developing experimental autoimmune encephalomyelitis (EAE) compared with their wild-type (WT) littermates, suggesting that Hsp70.1 plays a critical role in promoting an effective myelin oligodendrocyte glycoprotein (MOG)-specific T cell response. Conversely, Hsp70.1-deficient mice that developed EAE showed an increased level of autoreactive T cells to achieve the same production of cytokines compared with the WT mice. Although a neuroprotective role of HSP70 has been suggested, Hsp70.1-deficient mice that developed EAE did not exhibit increased demyelination compared with the control mice. Accordingly, Hsp70 deficiency did not influence the vulnerability to apoptosis of oligodendrocyte precursor cells (OPCs) in culture. Thus, the immunological role of Hsp70 may be relevant in EAE, and specific therapies down-regulating Hsp70 expression may be a promising approach to reduce the early autoimmune response in MS patients.     

Cytokines as Mediators of Neuroinflammation in Experimental Autoimmune Encephalomyelitis

Cytokines play a pivotal role in maintaining homeostasis of the immune system and in regulation of the immune response. Cytokine dysregulation is often associated with development of various pathological conditions, including autoimmunity. Recent studies have provided insights into the cytokine signaling pathways that are involved not only in pathogenesis of autoimmune neuroinflammatory disorders, such as multiple sclerosis, but also in neurodegenerative states, for example, Alzheimer’s disease. Understanding the exact molecular mechanisms of disease pathogenesis and evaluation of relevant experimental animal models are necessary for development of effective therapeutic approaches.     

依达拉奉对大鼠实验性自身免疫性脑脊髓炎的保护作用

目的:研究依达拉奉对实验性自身免疫性脑脊髓炎(Experimental Autoimmune Encephalomyelitis,EAE)的影响。方法:72只健康成年雌性Wistar大鼠随机分为:正常对照组、EAE组、EAE+小剂量依达拉奉组、EAE+大剂量依达拉奉组(n=18)。正常对照组注射生理盐水,其它组采用自制完全抗原诱导EAE模型。EAE组建模后不做任何处理,EAE+小剂量依达拉奉组、EAE+大剂量依达拉奉组分别在建模后给予依达拉奉4mg/k·d、10mg/k·d。比较各组发病率并行神经功能评分,取脊髓组织行HE染色、iNOS、OPN免疫组织化学染色观察。结果:依达拉奉干预组大鼠较EAE组发病率、神经功能缺损评分均明显降低(P<0.05)。HE结果显示依达拉奉干预组较EAE组炎症反应减少,损伤程度减轻。依达拉奉组iNOS、OPN表达均明显小于EAE组(P<0.05)。大剂量依达拉奉iNOS、OPN表达均低于小剂量依达拉奉组(P<0.05)。结论:依达拉奉对EAE具有保护作用,可能与抑制小神经胶质细胞活化,减轻炎症反应,降低iNOS和OPN表达有关。     

Erythropoietin Ameliorates Rat Experimental Autoimmune Neuritis by Inducing Transforming Growth Factor-Beta in Macrophages

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. In addition, in various preclinical models EPO exhibited protective activity against tissue injury. There is an urgent need for potent treatments of autoimmune driven disorders of the peripheral nervous system (PNS), such as the Guillain-Barré syndrome (GBS), a disabling autoimmune disease associated with relevant morbidity and mortality. To test the therapeutic potential of EPO in experimental autoimmune neuritis (EAN) - an animal model of human GBS – immunological and clinical effects were investigated in a preventive and a therapeutic paradigm. Treatment with EPO reduced clinical disease severity and if given therapeutically also shortened the recovery phase of EAN. Clinical findings were mirrored by decreased inflammation within the peripheral nerve, and myelin was well maintained in treated animals. In contrast, EPO increased the number of macrophages especially in later stages of the experimental disease phase. Furthermore, the anti-inflammatory cytokine transforming growth factor (TGF)-beta was upregulated in the treated cohorts. In vitro experiments revealed less proliferation of T cells in the presence of EPO and TGF-beta was moderately induced, while the secretion of other cytokines was almost not altered by EPO. Our data suggest that EPO revealed its beneficial properties by the induction of beneficial macrophages and the modulation of the immune system towards anti-inflammatory responses in the PNS. Further studies are warranted to elaborate the clinical usefulness of EPO for treating immune-mediated neuropathies in affected patients.     

Induced Pluripotent Stem Cell Therapy Ameliorates Hyperoxia-Augmented Ventilator-Induced Lung Injury through Suppressing the Src Pathway

Background

High tidal volume (V_T) mechanical ventilation (MV) can induce the recruitment of neutrophils, release of inflammatory cytokines and free radicals, and disruption of alveolar epithelial and endothelial barriers. It is proposed to be the triggering factor that initiates ventilator-induced lung injury (VILI) and concomitant hyperoxia further aggravates the progression of VILI. The Src protein tyrosine kinase (PTK) family is one of the most critical families to intracellular signal transduction related to acute inflammatory responses. The anti-inflammatory abilities of induced pluripotent stem cells (iPSCs) have been shown to improve acute lung injuries (ALIs); however, the mechanisms regulating the interactions between MV, hyperoxia, and iPSCs have not been fully elucidated. In this study, we hypothesize that Src PTK plays a critical role in the regulation of oxidants and inflammation-induced VILI during hyperoxia. iPSC therapy can ameliorate acute hyperoxic VILI by suppressing the Src pathway.

Methods

Male C57BL/6 mice, either wild-type or Src-deficient, aged between 2 and 3 months were exposed to high V_T (30 mL/kg) ventilation with or without hyperoxia for 1 to 4 h after the administration of Oct4/Sox2/Parp1 iPSCs at a dose of 5×10⁷ cells/kg of mouse. Nonventilated mice were used for the control groups.

Results

High V_T ventilation during hyperoxia further aggravated VILI, as demonstrated by the increases in microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 (MIP-2) and plasminogen activator inhibitor-1 (PAI-1) production, Src activation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and malaldehyde (MDA) level. Administering iPSCs attenuated ALI induced by MV during hyperoxia, which benefited from the suppression of Src activation, oxidative stress, acute inflammation, and apoptosis, as indicated by the Src-deficient mice.

Conclusion

The data suggest that iPSC-based therapy is capable of partially suppressing acute inflammatory and oxidant responses that occur during hyperoxia-augmented VILI through the inhibition of Src-dependent signaling pathway.     

昆明小鼠实验性自身免疫性脑脊髓炎模型的建立

用豚鼠脊髓匀浆液和不完全弗氏佐剂制备免疫原,联合腹腔注射百日咳毒素,建立昆明小鼠实验性自身免疫性脑脊髓炎动物模型.发病小鼠临床神经系统症状逐渐加重,脑组织经HE染色后,可见血管周围有大量炎性细胞浸润,呈袖套状改变;大脑白质可见胶质细胞增生形成胶质细胞结节以及神经细胞坏死等病理改变.正常小鼠中枢神经系统功能及病理检查均未见异常.此模型为进一步阐明多发性硬化的发病机制打下基础.