Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system.Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis,a traditional experimental model of multiple sclerosis.This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis.We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type,as shown by reduced expression of inducible nitric oxide synthase/nitric oxide,interleukin-12,and CD16/32 and increased expression of arginase-1,interleukin-10,CD14,and CD206,which was linked to inhibition of Rho kinase activity,decreased expression of toll-like receptors,nuclear factor-κB,and components of the mitogen-activated protein kinase signaling pathway,and generation of the pro-inflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6.Crucially,Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis,resulting in later onset of disease,lower symptom scores,less weight loss,and reduced demyelination compared with unmodified macrophages.In addition,Fasudil-modified macrophages decreased interleukin-17 expression on CD4^(+)T cells and CD16/32,inducible nitric oxide synthase,and interleukin-12 expression on F4/80^(+)macrophages,as well as increasing interleukin-10 expression on CD4^(+)T cells and arginase-1,CD206,and interleukin-10 expression on F4/80^(+)macrophages,which improved immune regulation and reduced inflammation.These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response,thereby providing new insight into cell immunotherapy for multiple sclerosis.

Emodin attenuates inflammation and demyelination in experimental autoimmune encephalomyelitis

Emodin,a substance extracted from herbs such as rhubarb,has a protective effect on the central nervous system.However,the potential therapeutic effect of emodin in the context of multiple sclerosis remains unknown.In this study,a rat model of experimental autoimmune encephalomyelitis was established by immune induction to simulate multiple sclerosis,and the rats were intraperitoneally injected with emodin(20 mg/kg/d)from the day of immune induction until they were sacrificed.In this model,the nucleotide-binding domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome and the microglia exacerbated neuroinflammation,playing an important role in the development of multiple sclerosis.In addition,silent information regulator of transcription 1(SIRT1)/peroxisome proliferator-activated receptor-alpha coactivator(PGC-1α)was found to inhibit activation of the NLRP3 inflammasome,and SIRT1 activation reduced disease severity in experimental autoimmune encephalomyelitis.Furthermore,treatment with emodin decreased body weight loss and neurobehavioral deficits,alleviated inflammatory cell infiltration and demyelination,reduced the expression of inflammatory cytokines,inhibited microglial aggregation and activation,decreased the levels of NLRP3 signaling pathway molecules,and increased the expression of SIRT1 and PGC-1α.These findings suggest that emodin improves the symptoms of experimental autoimmune encephalomyelitis,possibly through regulating the SIRT1/PGC-1α/NLRP3 signaling pathway and inhibiting microglial inflammation.These findings provide experimental evidence for treatment of multiple sclerosis with emodin,enlarging the scope of clinical application for emodin.

EAE （Experimental Autoimmune Encephalomyelitis）, Corticotropin-Releasing Factor and the Blood Brain Barrier

EAE （experimental autoimmune encephalomyelitis） is an established, inducible animal model employed in the study of MS （multiple sclerosis） characterized by inflammation, BBB （blood brain barrier） malfunction, demyelination and neuronal disruption. CRF （corticotropin releasing factor） is a neuropeptide critically associated with immune function, BBB permeability, and the hypothalamic-pituitary-adrenal axis. Potential CRF targets in the brain include astrocytes, as well as endothelial cells of cerebral microvessels, since they have been reported to express CRFR （CRF receptors）. Further, both of these cell types function critically in regulating BBB permeability. CRF-BP （CRF binding protein） is also expressed in both neurons and glial cells. Changes in the cortical CRF system could be a contributing factor to the BBB disruption associated with MS/EAE and has been suggested to play a protective role against cytokine-induced inflammation. The current study assessed alterations associated with the C57BL/6 mouse model of EAE in the cortical CRF system and correlated these events with changes to the microvascular unit. Immunohistochemical confocal microscopy was used to analyze the distribution of CRF, CRF-BP, and CRFR in the mouse cerebral cortex. The authors observed a reduction in detectable CRF immunofluorescence in the EAE motor cortex, an increase in CRFBP immunoreactivity in EAE astrocytes and a concurrent reduction in astrocytic CRFR immunofluorescence. Staining techniques were used to visualize astrocytes/microvessels to document alterations in BBB integrity. Changes in the CRF system were associated with a modification of the blood brain barrier as manifested by a poorly defined astrocytic barrier in EAE microvessels. Evidence suggests that manipulation of CRF signaling pathways offers an intriguing target for interventional therapies designed to modify BBB permeability that may be beneficial for treating disease states such as MS.

Role of nuclear factor κB in multiple sclerosis and experimental autoimmune encephalomyelitis

The transcription factor nuclear factor κB（NF-κB） plays major roles in inflammatory diseases through regulation of inflammation and cell viability.Multiple sclerosis（MS） is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system（CNS）.It has been shown that NF-κB is activated in multiple cell types in the CNS of MS patients,including T cells,microglia/macrophages,astrocytes,oligodendrocytes,and neurons.Interestingly,data from animal model studies,particularly studies of experimental autoimmune encephalomyelitis,have suggested that NF-κB activation in these individual cell types has distinct effects on the development of MS.In this review,we will cover the current literature on NF-κB and the evidence for its role in the development of MS and its animal model experimental autoimmune encephalomyelitis.

MicroRNAs as disease progression biomarkers and therapeutic targets in experimental autoimmune encephalomyelitis model of multiple sclerosis

Multiple sclerosis is an autoimmune neurodegenerative disease of the central nervous system characterized by pronounced inflammatory infiltrates entering the brain,spinal cord and optic nerve leading to demyelination.Focal demyelination is associated with relapsing-remitting multiple sclerosis,while progressive forms of the disease show axonal degeneration and neuronal loss.The tests currently used in the clinical diagnosis and management of multiple sclerosis have limitations due to specificity and sensitivity.MicroRNAs(miRNAs)are dysregulated in many diseases and disorders including demyelinating and neuroinflammatory diseases.A review of recent studies with the experimental autoimmune encephalomyelitis animal model(mostly female mice 6–12 weeks of age)has confirmed miRNAs as biomarkers of experimental autoimmune encephalomyelitis disease and importantly at the pre-onset(asymptomatic)stage when assessed in blood plasma and urine exosomes,and spinal cord tissue.The expression of certain miRNAs was also dysregulated at the onset and peak of disease in blood plasma and urine exosomes,brain and spinal cord tissue,and at the post-peak(chronic)stage of experimental autoimmune encephalomyelitis disease in spinal cord tissue.Therapies using miRNA mimics or inhibitors were found to delay the induction and alleviate the severity of experimental autoimmune encephalomyelitis disease.Interestingly,experimental autoimmune encephalomyelitis disease severity was reduced by overexpression of miR-146a,miR-23b,miR-497,miR-26a,and miR-20b,or by suppression of miR-182,miR-181c,miR-223,miR-155,and miR-873.Further studies are warranted on determining more fully miRNA profiles in blood plasma and urine exosomes of experimental autoimmune encephalomyelitis animals since they could serve as biomarkers of asymptomatic multiple sclerosis and disease course.Additionally,studies should be performed with male mice of a similar age,and with aged male and female mice.

Sinomenine reduces iNOS expressionvia inhibiting the T-bet IFN-γ pathway in experimental autoimmune encephalomyelitis in rats

Sinomenine is a bioactive alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating rheumatic and arthritic diseases. In our previous studies, we found that sinomenine reduced cellular infiltration within the spinal cord and alleviated experimental autoimmune encephalomyelitis （EAE） in rats. In this study, we further investigated the mechanisms of sinomenine treatment in EAE rats. In EAE rats, treatment with sinomenine exerted an anti-inducible NO synthase （anti-iNOS） effect, which is related to the reductions of Thl cytokine interferon-y （IFN-7） and its transcription factor, T-bet, in spinal cords. Moreover, sinomenine treatment of splenocytes stimulated with anti-CD3 antibody and recombinant rat in- terleukin 12 reduced the expression of T-bet and IFN-y in vitro and also reduced the capability of supernatants of splenocyte culture to induce iNOS expression by primary astrocytes. However, sinomenine had no direct inhibito- ry effect on iNOS produced by astrocytes cultured with IFN-y and tumor necrosis factor α in vitro. In conclusion, the anti-iNOS effect of sinomenine on EAE is mediated via the suppression of T-bet/IFN-y pathway.

Amyloid precursor protein and growth-associated protein 43 expression in brain white matter and spinal cord tissues in a rat model of experimental autoimmune encephalomyelitis

Studies have demonstrated that amyloid precursor protein （APP） expression increases in multiple sclerosis tissues during acutely and chronically active stages. To determine the relationship between axonal injury and regeneration in multiple sclerosis, an animal model of experimental autoimmune encephalomyelitis was induced using different doses of myelin basic protein peptide. APP and growth-associated protein 43 （GAP-43）, which is considered a specific marker of neural regeneration, were assessed by western blot analysis. Expression of APP and GAP-43, as well as the correlation between these two proteins, in brain white matter and spinal cord tissues of experimental autoimmune encephalomyelitis rats at different pathological stages was analyzed. Results showed that APP and GAP-43 expression increased during the acute stage and decreased during remission, with a positive correlation between APP and GAP-43 expression in brain white matter and spinal cord tissues. These results suggest that APP and GAP-43 could provide nutritional and protective effects on damaged neurons.

Nogo receptor expression in microglia/macrophages during experimental autoimmune encephalomyelitis progression

Myelin-associated inhibitory factors within the central nervous system（CNS） are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1（NgR1） has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis（MS）. In this study, the animal model of MS, experimental autoimmune encephalomyelitis（EAE） was induced in ngr1^＋/＋ and ngr1^–/– female mice following injection with the myelin oligodendrocyte glycoprotein（MOG_（35–55）） peptide. A fatemap analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively（increasing locomotor disability） from both genotypes, using the CD11 b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1^＋/＋ and ngr1^–/– mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1^＋/＋ and ngr1^–/– mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1^＋/＋ and ngr1^–/– mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of Ng R on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1^–/– mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1^–/– mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1^–/– mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.

Zuogui pills for myelinolysis in a rat model of experimental autoimmune encephalomyelitis

Zuogui pills have been shown to attenuate the inflammatory reaction in a rat model of experimental autoimmune encephalomyelitis （EAE）. The present study attempted to investigate the pathology underlying the influence of Zuogui pills on myelinolysis in EAE rats. Hematoxylin-eosin and Luxol fast blue staining showed that the myelinolysis foci in the cerebrum, cerebellum, brain stem, and the spinal cord of EAE rats were significantly decreased, along with serum myelin basic protein content following treatment with Zuogui pills.

Nogo-A and Nogo-A receptor expression in periventricular white matter of experimental autoimmune encephalomyelitis rats

BACKGROUND：Previous studies have focused on the correlation between Nogo-A expression and multiple sclerosis or between Nogo-A receptor （NgR） expression and multiple sclerosis in the central nervous system. Expression patterns of Nogo-A and NgR remain poorly understood in rat models of experimental autoimmune encephalomyelitis （EAE）.OBJECTIVE：To observe dynamic changes in Nogo-A and NgR protein expression, and to verify the correlation between Nogo-A and NgR protein, as well as expression patterns at various time points, in periventricular tissue of EAE rats.DESIGN, TIME AND SETrlNG：A neuroimmunological, randomized, controlled experiment was performed at the Clinical Institute of Hunan People＇s Hospital of China from September to November 2008.MATERIALS：Immunohistochemistry （streptavidin-biotin-peroxidase complex method） kit was purchased from Boster, China.METHODS：A total of 60 female, Wistar rats, aged 6-8 weeks, ware randomly assigned to EAE and control groups （n = 30, respectively）. Guinea pig spinal cord homogenate, self-made complete Freund＇s adjuvant （0.2 mL/100 g）, and pertussis vaccine （0.2 mL） were subcutaneously injected into the hindlimb foot pad of rats from the EAE group to create rat models of EAE. Complete Freund＇s adjuvant （0.2 mL） was infused into rats from the control group.MAIN OUTCOME MEASURES：Nogo-A and NgR protein expression was determined in periventricular white matter using immunohistochemical methods. Neurological scores ware determined in all rats.RESULTS：Rats from the EAE group developed acute-onset EAE following immunization. The pathogenetic symptoms reached a peak on day 15, and neurological scores ware also greatest at this time point. Neurological scores decreased with recovery of the illness. Nogo-A was shown to be expressed in neuronal cells and oligodendrocytes, and expression increased 11 days after immunization （P 〈 0.01）, decreased by day 13 （P 〈 0.01）, and then increased again by day 15. Nogo-A expression remained greater in the EAE group compared with the control group at day 30 （P 〈 0.01）. In the EAE group, NgR protein was primarily expressed on the surface of neuronal bodies and axons. NgR expression increased 13-18 days after immunization （P 〈 0.01 or P 〈 0.05）.CONCLUSION：Nogo-A and NgR protein expression altered with disease course in periventdcular white matter of EAE rats. Results suggested that Nogo-A and NgR were involved in EAE occurrence.

Effects of Yishendaluo decoction on blood-brain barrier integrity in mice with experimental autoimmune encephalomyelitis

This study investigated the effects of Yishendaluo decoction on the loss of blood-brain barrier integrity in mice exhibiting experimental autoimmune encephalomyelitis.To this end,we used real-time fluorescent quantitative PCR to measure the levels of mRNAs specific to the T cell markers CD4 and CD8,and the monocyte marker CD11b.In addition,we used Evans blue dye extravasation in the spinal cord and brain tissues to assess blood-brain barrier permeability.The results indicated that an increase in blood-brain barrier permeability was associated with an increase in CD4,CD8 and CD11b mRNA expression in experimental autoimmune encephalomyelitis mice.Yishendaluo decoction administration significantly reversed inflammatory cell accumulation in cerebral tissues of experimental autoimmune encephalomyelitis mice.

High doses of α-galactosylceramide potentiate experimental autoimmune encephalomyelitis by directly enhancing Th17 response

α-Galactosylceramide （u-GC） is widely known to activate invariant natural killer T （iNKT） cells to suppress my- elin antigen-specific Thl responses, protecting susceptible mice against experimental antoimmune encephalomyelitis 0EAE）. Here, we demonstrate an unexpected finding that high doses of α-GC exacerbated, rather than ameliorated, EAE. Similar results were observed when MOG35.ss-specific T cells treated with high-dose α-GC were transferred into naive syngeneic recipient mice. Further study showed that high doses of a-GC directly enhance the Thl7 and Thl re- sponse by activation of CD4＋CD44＋ memory T cells through phosphorylation of STAT3 and activation of NF-kB. Un- like the activation of iNKT cells by low doses of a-GC, high doses of a-GC directly interacted with CDld expressed on T ceils and activated Thl7 and Thl cells. Furthermore, antigen-presenting cells （APCs） predominantly express CDldl, whereas the majority of CD4~ T cells express CDld2. Knockdown of CDldl or CDld2 gene expression by RNAi interfered with the activation of iNKT or Thl7/Thl cells, respectively. Therefore, α-GC treatment could im- prove or worsen EAE by engaging either APCs or Thl7/Thl cells depending on the dose used.

Axonal damage in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in a C57BL/6 mouse model may be not secondary to inflammatory demyelination

The present study established a chronic experimental autoimmune encephalomyelitis model in C57BL/6 mice induced by myelin oligodendrocyte glycoprotein peptides and complete Freund＇s adjuvant. Onset latency was 12 days, with an incidence rate of 100%. Neuropathological characteristics included perivascular inflammatory cell infiltration, demyelination, neuronal degeneration, and axonal damage within cerebral and myelic white matter. Electron microscopy revealed swollen mitochondria, complete organ disappearance, and fused or broken myelin sheath structure, which were accompanied by myelin sheath reconstruction. Moreover, axonal damage was not consistent with demyelination distribution, and severity of axonal damage did not correlate with demyelination. Results suggested that axonal damage in an experimental autoimmune encephalomyelitis model is not secondary to inflammatory demyelination.

Variation in experimental autoimmune encephalomyelitis scores in a mouse model of multiple sclerosis

Multiple sclerosis(MS) is a common demyelinating central nervous system disease associated with progressive physical impairment. To study the mechanism underlying disease pathogenesis and develop potential treatments, experimental autoimmune encephalomyelitis(EAE) is often used as an animal model. EAE can be induced in various species by introducing specific antigens, which ultimately result in motor dysfunction. Although the severity of the paralysis is indicated using the EAE score, there is no standard scoring system for EAE signs, and there is variability between research groups with regard to the exact EAE scoring system utilized. Here, we describe the criteria used for EAE scoring systems in various laboratories and suggest combining EAE score with another quantitative index to evaluate paralysis, such as the traveled distance, with the goal of facilitating the study of the mechanisms and treatment of MS.

Eotaxin-2 blockade ameliorates experimental autoimmune encephalomyelitis

AIM： To study the effect of blocking the eo-2 pathwaon the development and severity of experimental autoimmune encephalomyelitis （EAE）. METHODS： We produced mAb directed against eo-2named D8. MOG35-55 induced-EAE mice were dailintravenously injected with either 25 μg or 100 μg D8or with vehicle control alone [phosphate-buffered saline（PBS）], starting from day 0 post immunization and weremonitored for EAE clinical score （n = 10 in each group）Mice were sacrifced on day 58 and their sera were assessed for the presence of anti-myelin oligodendrocyteglycoprotein （anti-MOG） antibodies autoantibodies, awell as for the profle of pro-infammatory cytokines andchemokines. Histological analysis of brain sections waperformed by hematoxylin and eosin staining.RESULTS： Daily treatment of EAE induced mice with D8 signifcantly decreased the severity of EAE symp-toms. Treatment with both concentrations of D8 ame-liorated EAE symptoms compared to PBS treated mice, starting from day 42 post immunization （0.89 ± 0.35 in D8 25 μg and D8 100 μg treated groups vs 2.11 ± 0.38 in the PBS treated group, P = 0.03）. A signifcant im-provement in EAE clinical score compared to total IgG treated mice was observed with the higher concentra-tion of D8 （0.81 ± 0.38 in D8 100 μg treated group vs 2.11 ± 0.31 in IgG1 treated group, on day 56 post immunization, P = 0.04）. D8 treated mice with EAE did not signifcantly exhibit lower sera levels of anti-MOG autoantibodies compared to IgG-treated mice. How-ever, they expressed lower sera levels of the pro-in-fammatory cytokines： tumor necrosis factor （7.8 ± 0.2 pg/mL in D8 100 μg treated mice vs 19.9 ± 3.4 pg/mL in IgG treated mice, P = 0.005） and interferon-gamma （1.4 ± 0.6 pg/mL in D8 100 μg treated mice vs 3.6 ± 0.4 pg/mL in IgG treated mice, P = 0.02）, as well as reduced levels of the chemokine macrophage che-moattractant protein-1 （27.2 ± 3.1 pg/mL in D8 100 μg treated mice vs 63.7 ± 12.3 pg/mL in IgG treated mice, P = 0.03）. These fndings indicate that blocking the eo-2 pathway in EAE may affect not only eosino-phil infltration into the central nervous system （CNS）, but also have an effect on monocytes and T cells, but not humoral, mediated responses. Histological analysis of the brains of D8 treated mice with EAE support that this treatment decreases immune cells infltrates in the CNS.CONCLUSION： Taken together, these fndings suggest a role for eo-2 in EAE pathogenesis and consequen-tially may support a therapeutic potential of anti-eo-2 neutralizing mAb in multiple sclerosis.

Effect of cornel iridoid glycoside on experimental autoimmune encephalomyelitis attenuation through JAK/STAT signaling pathway,at least partially

OBJECTIVE In order to investigate whether cornel iridoid glycoside(CIG),the main component extracted from Cornus officinalis,can treat demyelinating diseases of the central nervous system(CNS)such as multiple sclerosis(MS).METHODS CIG(30,60 and 120mg·kg-1)or vehicle was intragastrically administered once daily to rats,starting immediately after purified myelin basic protein(MBP)68-86 peptides immunization until day 20 post immunization(p.i.).Histopathological staining,enzyme-linked immunosorbent assay,biochemical methods and Western blotting approaches were used to evaluate the disease incidence and severity,neuroinflammatory and neurotrophic response in the CNS.RESULTS Neurological deficit and proportion of incidence seen in EAE rats were significantly reduced by CIG treatment in a dose-dependent manner.Histopathological staining showed that CIG treatment alleviated demyelination and inflammatory infiltration,increased the number of oligodendrocytes,enhanced the expression of brain-derived neurotrophic factor(BDNF).Production of proinflammatory molecules such as interleukin-1β(IL-1β),tumour necrosis factor-αand interferon-γwere also inhibited by CIG administration.CIG could ameliorate phosphorylation of STAT1,STAT3 and JAK1 as well as IL-6/IL-6 Rexpression,which involved in immune response and inflammation.CONCLUSION Our results demonstrated that CIG may ameliorate EAE rats through down-regulation of JAK/STAT signaling pathway.This study gave new insight into the novel regulatory mechanism of CIG and highlight novel therapeutic targets and a potential therapeutic agent for the treatment of MS.

Ameliorative effects of human adipose tissue-derived mesenchymal stem cells on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats

Mesenchymal stem cells have been previously shown to exert an immunomodulatory function. The present study sought to investigate the effects of multipotential human adipose tissue-derived mesenchymal stem cells （hAdMSCs） on disease progression and cytokine expression in Lewis rats with experimental autoimmune encephalomyelitis （EAE） induced by myelin basic protein. The duration of EAE paralysis in the group treated on day 7 posfimmunization with 5 × 10^6 hAdMSCs was significantly reduced compared with the vehicle-treated controls and the 1 x 106 hAdMSC- treated group. The duration of EAE paralysis in the groups treated with 5 × 10^6 hAdMSCs on both day 1 and day 7 postimmunization was significantly reduced compared with the vehicle-treated controls and the groups treated with 5 × 10^6 hAdMSCs on both day 7 and day 10 postimmunization. The mRNA expression of interleukin-10 and indoleamine 2, 3-dioxygenase was significantly decreased in the hAdMSC-treated group compared with the vehicle-treated group. These findings suggest that the ameliorative effects of hAdMSCs on EAE symptoms operate in a dose- and time-dependent manner and can be mediated in part by the ample production of anti-inflammatory cytokines.

Matrine ameliorates experimental autoimmune encephalomyelitis by regulating the differentiation of encephalitogenic Th1/Th17 and Th2/regulatory T cells

OBJECTIVE Experimental autoimmune encephalomyelitis(EAE),the classical animal model for multiple sclerosis(MS)is triggered by an impaired balance of T helper(Th)cells and regulatory T(Tregs)cells.Matrine(MAT),a quinolizidine alkaloid derived from the herb Radix Sophorae Flave,has been shown to ameliorate the clinical signs,inflammatory infiltration,demyelination in acute EAE rats.However,whether MAT protect from EAE by adjusting Th and Treg cells response in specific-cellular and molecular level is unknown.METHODS Herein,MAT was tested for its effects on Th1,Th2,Th17 and Treg cells in the spinal cord of EAE mice and splenocyte-extracted from EAE mice with MOG35-55-restimulated,respectively.RESULTS Our findings revealed that MAT significantly inhibit the proliferation of splenocyte,and remarkably down-regulate the differentiation of Th1/Th17 cells with decreased expressions of CD4+IFN-γ+cells and CD4+IL-17+cells in vivo and IL-17,IFN-γ,ROR-γt,T-bet in vitro,meanwhile it dramatically up-regulate the Th2/Treg cells response associated with increased levels of CD4+TGF-β+1cells and CD4+IL-10+cells in vivo and IL-4,IL-10,TGF-β1,Foxp3 and GATA3in vitro.CONCLUSION Considering the effective therapeutic effects of MAT on EAE,it′s worth to find its new values on other autoimmune diseases.

Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis —II

Therapeutic administration of fingolimod hydrochloride (FTY720), the functional antagonist at sphingosine 1-phosphate (S1P) receptor 1 (S1P1) shows a marked improving effect on experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice. However, this treatment showed an only partial inhibition of Th1/Th17 cell infiltration into the central nervous system (CNS), suggesting that down-regulation of lymphocytic S1P1 is insufficient to explain the therapeutic effect of FTY720 on EAE. On the other hand, the therapeutic administration of FTY720 reduced the mRNA expressions of IL-6, CCL2, and glial fibrillary acidic protein, an activation marker of astrocytes, in the CNS of EAE mice. In human astrocytic glyoma, U373MG cells, mRNA expression of S1P1 was higher as compared with those of the other S1P receptor subtypes and phosphorylation of Akt was induced by S1P, FTY720-phosphate (FTY720-P), or an S1P1-selective agonist, SEW2871. FTY720-P appeared to induce down-regulation of S1P1 in U373MG cells, implying a functional antagonism at S1P1 on astrocytes. S1P but not FTY720-P induced production of IL-6, IL-8, and CCL2 significantly and treatment with FTY720-P or SEW2871 inhibited production of these pro-inflammatory cytokines from U373MG cells stimulated with S1P. These results suggest that S1P-S1P1 axis induces production of pro-inflammatory cytokines by astrocytes. Consequently, it is highly probable that the therapeutic effects of FTY720 on EAE are caused by inhibiting not only egress of myelin-specific Th cells from the draining lymph nodes but also activation of astrocytes in the CNS.

Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis —I

Infiltration of myelin-specific helper T (Th) cells into the central nervous system (CNS) plays a key role in pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the involvement of sphingosine 1-phosphate (S1P)-S1P receptor 1 (S1P1) axis in lymphocytes for EAE development when C57BL/6 mice were immunized with myelin oliogodendrocyte glycoprotein (MOG). The expression of S1P1 mRNA and S1P responsiveness of lymphocytes in draining lymph nodes (DLN) were down-regulated markedly after MOG immunization until onset of EAE. Accompanying with reacquisition of down-regulated S1P1 transcript and S1P responsiveness in DLN lymphocytes, MOG-immunized mice developed EAE symptoms with significant infiltration of Th1 and Th17 cells into the CNS and a marked elevation of IFN-γ, T-bet, IL-17, and RORγt mRNA expressions. Prophylactic administration of an S1P1 functional antagonist, fingolimod hydrochloride (FTY720, 0.3 mg/kg, orally) significantly inhibited EAE development and almost completely prevented infiltration of Th1 and Th17 cells into the CNS with a marked reduction of IFN-γ, T-bet, IL-17, and RORγt mRNA expressions. Similar results were obtained by treatment with an S1P1-selective agonist, SEW2871 or an S1P lyase inhibitor, 2-acetyl-4-tetrahydroxybutylimidazole. Moreover, FTY720-phosphate and SEW2871 inhibited in vitro migration of Th1 and Th17 cells toward S1P but did not affect cytokine production or generation of Th1 or Th17 cells. These results suggest that reacquisition of S1P1 expression in DLN lymphocytes plays a major role in trafficking of myelin antigen-specific Th1/Th17 cells from DLN to the CNS in EAE and that prophylactic effect of FTY720 on EAE is predominantly caused by functional antagonism via lymphocytic S1P1.