Autoimmune Encephalomyelitis Mouse Model Research Articles

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

Cladribine treatment improves cortical network functionality in a mouse model of autoimmune encephalomyelitis

BackgroundCladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood–brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice.MethodsIn the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo.ResultsOral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient.ConclusionsOur results indicate that cladribine exerts a neuroprotective effect after crossing the blood–brain barrier independently of its peripheral immunosuppressant action.

C[sbnd]C chemokine receptor 5 antagonist alleviates inflammation by regulating IFN-γ/IL-10 and STAT4/Smad3 signaling in a mouse model of autoimmune encephalomyelitis

Multiple sclerosis (MS) is an immunopathological disease that causes demyelination and recurrent episodes of T cell-mediated immune attack in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model of MS. The roles of T cells in MS/EAE have been well investigated, but little is known about the role of CCR5+ cells. In the present study, we investigated whether treatment with DAPTA, a selective CCR5 antagonist, could modulate the progression of EAE in the SJL/J mice. EAE mice were treated with DAPTA (0.01 mg/kg) intraperitoneally daily from day 14 to day 42, and the clinical scores were evaluated. We further investigated the effects of DAPTA on IFN-γ-, TGF-β-, IL-10-, IL-17A-, IL-22-, T-bet, STAT4-, RORγT-, AhR-, Smad3-, and Foxp3-expressing CCR5+ spleen cells using flow cytometry analysis. We further explored the effects of DAPTA on mRNA/protein expression of IFN-γ, IL-10, IL-17A, IL-22, TGF-β, T-bet, STAT4, RORγT, AhR, Foxp3, and NF-H in the brain tissue. The severity of clinical scores decreased in DAPTA-treated EAE mice as compared to that in the EAE control mice. Moreover, the percentage of CCR5+IFN-γ+, CCR5+T-bet+, CCR5+STAT4+, CCR5+IL-17A+, CCR5+RORγt+, CCR5+IL-22+, and CCR5+AhR+ cells decreased while CCR5+TGF-β+, CCR5+IL-10+, CCR5+Smad3+, and CCR5+Foxp3+ increased in DAPTA-treated EAE mice. Furthermore, DAPTA treatment significantly mitigated the EAE-induced expression of T-bet, STAT4, IL-17A, RORγT, IL-22, and AhR but upregulated Foxp3, IL-10, and NF-H expression in the brain tissue. Taken together, our data demonstrated that DAPTA could ameliorate EAE progression through the downregulation of the inflammation-related cytokines and transcription factors signaling, which may be useful for the clinical therapy of MS.

Abstract 4177: Th17 inhibition with interleukin 6 blockade decouples immunotoxicity from tumor immunity

Abstract Durable remission rates with immune checkpoint inhibitor (ICI) monotherapy remain low. Combination ICI therapies could overcome immune resistance but are associated with higher rates of immune-related adverse events (irAEs). To elucidate the underlying irAE immunobiology, we profiled human intestinal tissue, enterocolitis (irEC), and tumor tissue from ICI-treated patients using NanoString PanCancer Immune Panel (NanoPCIP) and multiplex immunohistochemistry (mIHC); hypotheses generated from these translational results for potential irAE treatments were tested in parallel preclinical mouse model studies. In 23 patients identified with both normal intestinal and irEC tissue, we found genes encoding interleukin-6 (IL-6), Th17 differentiating cytokine, and neutrophil and monocyte chemotactic molecules were the highest significantly upregulated in irEC compared to patient-matched normal intestine. Using NanoPCIP cell scoring (23 patients) and mIHC (27 patients), Th17 cells were significantly higher in irEC than Th1 cells. T-cell subsets were similar between anti-CTLA-4 based regimen versus anti-PD-1monotherapy induced irEC, except Th17 memory cells were significantly higher CTLA-4i-induced irEC. We compared the degree of differential gene expression in irEC (normal intestine versus irEC) to the differential expression in tumors (baseline versus on-treatment tumors). The genes highest upregulated in irEC (including Th17 differentiating cytokines IL-6 and IL-1B) were not upregulated in responding tumors from patients receiving ICI. NanoPCIP Th17 cell score was significantly more upregulated than Th1 cells and neutrophils more upregulated than CD8 T cells in the irEC analysis, but not in the responding tumor analysis where IL-12 and CD8 cell score was significantly upregualted . Based on these discoveries, we used C57BL/6 and Balb/c mouse strains to address whether the antitumor effect of ICIs can be enhanced by IL-6 blockade while also improving irAEs. We found that IL-6 blockade + ICI was associated with improved tumor control and a higher density of CD4/CD8 effector T-cells, with reduced Th17, macrophages, and MDSCs compared to ICI alone. Furthermore, in the experimental autoimmune encephalomyelitis (EAE) mouse model, the pathogenesis of which is well documented to be Th17 mediated (Robinson et al., 2014), we demonstrated that combined IL-6 blockade and ICI enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICI alone. Based on the totality of the basic science, translational, and clinical data we hypothesize that IL-6 blockade combined with ICIs could de-couple autoimmunity from antitumor immunity. We are currently conducting a prospective phase 2 trials of combination ipilimumab (3mg/kg) + Nivolumab (1mg/kg) + Tocilizumab in patients with metastatic melanoma, EGFR mutant NSCLC, and urothelial carcinoma ( NCT04940299). Citation Format: Daniel H. Johnson, Yared Hailemichael, Salah-Eddine Bentebibel, Noha Abdel-Wahab, Sungnam Cho, Wai C. Foo, Khalida Wani, Stephanie S. Watowich, Suhendan Ekmekcioglu, Adi Diab. Th17 inhibition with interleukin 6 blockade decouples immunotoxicity from tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4177.

B cell-dependent EAE induces visual deficits in the mouse with similarities to human autoimmune demyelinating diseases

BackgroundIn the field of autoimmune demyelinating diseases, visual impairments have extensively been studied using the experimental autoimmune encephalomyelitis (EAE) mouse model, which is classically induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35–55). However, this model does not involve B cells like its human analogs. New antigens have thus been developed to induce a B cell-dependent form of EAE that better mimics human diseases.MethodsThe present study aimed to characterize the visual symptoms of EAE induced with such an antigen called bMOG. After the induction of EAE with bMOG in C57BL/6J mice, visual function changes were studied by electroretinography and optomotor acuity tests. Motor deficits were assessed in parallel with a standard clinical scoring method. Histological examinations and Western blot analyses allowed to follow retinal neuron survival, gliosis, microglia activation, opsin photopigment expression in photoreceptors and optic nerve demyelination. Disease effects on retinal gene expression were established by RNA sequencing.ResultsWe observed that bMOG EAE mice exhibited persistent loss of visual acuity, despite partial recovery of electroretinogram and motor functions. This loss was likely due to retinal inflammation, gliosis and synaptic impairments, as evidenced by histological and transcriptomic data. Further analysis suggests that the M-cone photoreceptor pathway was also affected.ConclusionTherefore, by documenting visual changes induced by bMOG and showing similarities to those seen in diseases such as multiple sclerosis and neuromyelitis optica, this study offers a new approach to test protective or restorative ophthalmic treatments.

Nobiletin attenuates inflammation via modulating proinflammatory and antiinflammatory cytokine expressions in an autoimmune encephalomyelitis mouse model

The aim of this study is to investigate the potential preventive and therapeutic effects of nobiletin by evaluating the expression of cytokines associated with inflammatory reactions in an autoimmune encephalomyelitis mouse model. A total of 60 male C57BL/6 mice aged between 8 and 10 weeks were used. Mice were divided into six groups (n = 10 mice per group): control, EAE, low-prophylaxis, high-prophylaxis, low-treatment and high-treatment. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin. Nobiletin was administered in low (25 mg/kg) and high (50 mg/kg) doses, intraperitoneally. The prophylactic and therapeutic effects of nobiletin on brain tissue and spinal cord were evaluated by expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), IL-6, IL-10 and transforming growth factor-beta (TGF-β) using immunohistochemistry and real-time polymerase chain reaction (RT-PCR). Prophylactic and therapeutic use of nobiletin inhibited EAE-induced increase of TNF-α, IL-1β and IL-6 activities to alleviate inflammatory response in brain and spinal cord. Moreover, nobiletin supplement dramatically increased the IL-10, TGF-β and IFNγ expressions in prophylaxis and treatment groups compared with the EAE group in the brain and spinal cord. The results obtained from this study show that prophylactic and therapeutic nobiletin modulates expressions of proinflammatory and antiinflammatory cytokines in brain and spinal cord dose-dependent manner in EAE model. These data demonstrates that nobiletin has a potential to attenuate inflammation in EAE mouse model. These experimental findings need to be supported by clinical studies.

Pericytes as mediators of infiltration of macrophages in multiple sclerosis

BackgroundMultiple sclerosis (MS) is a neurodegenerative condition of the central nervous system (CNS). It is associated with blood–brain barrier (BBB) breakdown and intravasation of leukocytes, particularly monocyte-derived macrophages, into the CNS. Pericytes are mural cells that are encased within the basement membrane of vasculature, and they contribute functionally to the neurovascular unit. These cells play an important role in maintaining BBB integrity and CNS homeostasis. However, the critical role of pericytes in mediating inflammation in MS or its models is unclear. Whether pericytes infiltrate into the CNS parenchyma in MS also needs clarification.MethodsCNS samples from the experimental autoimmune encephalomyelitis (EAE) mouse model of MS were collected at different time points for immunohistochemical analysis of pericytes along the inflamed vasculature. These findings were validated using MS brain specimens, and further analysis of pericyte involvement in inflammation was carried out by culturing primary pericytes and macrophages. Multiplex ELISA, transmigration assay and real-time PCR were used to study the inflammatory potential of pericytes in cultures.ResultsWe found that pericytes exhibit a heterogenous morphology, with notable elongation in the inflamed perivascular cuffs of EAE. This was manifested by a decrease in pericyte density but an increase in the coverage by pericytes along the vasculature. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix proteins enriched within inflamed perivascular cuffs, elevated levels of pro-inflammatory chemokines/cytokines in pericytes in culture. Importantly, pericytes stimulated with CSPGs enhanced macrophage migration. We did not detect pericytes in the CNS parenchyma during EAE, and this was corroborated in MS brain samples.ConclusionsOur data suggest that pericytes seek to restore the BBB through increased coverage, but that their exposure to CSPGs prompt their facilitation of macrophages to enter the CNS to elevate neuroinflammation in EAE and MS.

Prediction of combination therapies based on topological modeling of the immune signaling network in multiple sclerosis

BackgroundMultiple sclerosis (MS) is a major health problem, leading to a significant disability and patient suffering. Although chronic activation of the immune system is a hallmark of the disease, its pathogenesis is poorly understood, while current treatments only ameliorate the disease and may produce severe side effects.MethodsHere, we applied a network-based modeling approach based on phosphoproteomic data to uncover the differential activation in signaling wiring between healthy donors, untreated patients, and those under different treatments. Based in the patient-specific networks, we aimed to create a new approach to identify drug combinations that revert signaling to a healthy-like state. We performed ex vivo multiplexed phosphoproteomic assays upon perturbations with multiple drugs and ligands in primary immune cells from 169 subjects (MS patients, n=129 and matched healthy controls, n=40). Patients were either untreated or treated with fingolimod, natalizumab, interferon-β, glatiramer acetate, or the experimental therapy epigallocatechin gallate (EGCG). We generated for each donor a dynamic logic model by fitting a bespoke literature-derived network of MS-related pathways to the perturbation data. Last, we developed an approach based on network topology to identify deregulated interactions whose activity could be reverted to a “healthy-like” status by combination therapy. The experimental autoimmune encephalomyelitis (EAE) mouse model of MS was used to validate the prediction of combination therapies.ResultsAnalysis of the models uncovered features of healthy-, disease-, and drug-specific signaling networks. We predicted several combinations with approved MS drugs that could revert signaling to a healthy-like state. Specifically, TGF-β activated kinase 1 (TAK1) kinase, involved in Transforming growth factor β-1 proprotein (TGF-β), Toll-like receptor, B cell receptor, and response to inflammation pathways, was found to be highly deregulated and co-druggable with all MS drugs studied. One of these predicted combinations, fingolimod with a TAK1 inhibitor, was validated in an animal model of MS.ConclusionsOur approach based on donor-specific signaling networks enables prediction of targets for combination therapy for MS and other complex diseases.

Pathogenetic Mechanism and Construct of Neuromyelitis Optica Model by Immunization Activation

Objective: To obtain a mouse model that is clinically similar to neuromyelitis optica (NMO) via immunization.Methods: In this study, C57BL/6 mice were co-immunized by injecting human complement and aquaporin 4 (AQP4) antibody against NMO in the lateral ventricle, and the animals’ immune response was strengthened by injection of myelin glial cell glycoprotein and interleukin (IL)-17A and Bacilus Calmete-Guerin vaccine. To obtain a mouse model of autoimmune encephalomyelitis, after obtaining a model mouse with stable symptoms, we identified its behavioral performance, pathology, immunohistochemistry, molecular biology, and electrophysiology.Results: The behavioral observation showed that the model mice were similar to NMO patients in terms of limb symptoms and optic nerve performance. Regarding the pathology and immunohistochemistry, the spinal cords of model mice had obvious demyelination and AQP4 and glial fibrillary acid protein expression changes. Moreover, it was found that the inflammatory factors of the model mice, i.e., IL-1 and IL-17, were significantly expressed. In the detection of visual evoked potentials, we used the flash evoked potential detection method to demonstrate that the model and control groups had significantly longer S1 and T wave incubation periods, with significant differences.Conclusion: This research revealed that the mouse model of autoimmune encephalomyelitis prepared by this method is similar to the characteristics of NMO.

Pharmacological retraining for T cells

Immune Regulation Regulatory T cells (Tregs) expressing the Foxp3 transcription factor play a critical role in dampening overactive immune responses, including autoimmune diseases. Akamatsu et al. screened a library of small molecules and identified a compound that promotes Treg differentiation by inhibiting the cyclin-dependent kinases CDK8 and CDK19. The Treg-promoting activity of the CDK8/19 inhibitor reduced disease activity in mouse models of autoimmune diabetes and encephalomyelitis. CDK8/19 inhibitors are thus a new class of immunomodulatory drugs capable of generating Tregs with potential clinical applications in promoting tolerance and reducing autoimmunity. Sci. Immunol. 4 , eaaw2707 (2019).

Human umbilical cord blood derived cell therapy product, DUOC-01, ameliorates experimental autoimmune encephalomyelitis, a mouse model for multiple sclerosis

Background & Aim We have developed DUOC-01, a cord-blood derived, macrophage-based cell therapy product to treat demyelinating conditions of the central nervous system. Previously, we demonstrated that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the corpus callosum of immune-incompetent mice treated with cuprizone. To explore the mechanism of action and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in lysophosphatidylcholine (LPC) mediated demyelinated murine organotypic cerebellar brain slices and a mouse model of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). EAE has been recognized as the most widely used animal models to study the underlying molecular mechanisms of MS and the efficacy of potential therapeutics for treatment of disease. Methods, Results & Conclusion First, in the LPC-mediated demyelinated cerebellar brain slices, we found that DUOC-01 treatment increased remyelination, decreased gliosis, and promoted the proliferation of oligodendrocyte progenitor cells compared to the untreated control samples. EAE was induced in C57BL/6 mice by immunizing with myelin oligodendrocyte glycoprotein (MOG). After 10-12 days of EAE induction, when animals started showing early disease symptoms, we administered 3 × 105 DUOC-01 cells into the cerebrospinal fluid by a single intra-cisterna magna injection and clinical score was recorded for next several days. Compared to the vehicle injected control mice (n=9), in DUOC-01 treated mice (n=9), we observed significantly decreased severity of the disease as soon as the treatment started, as indicated by lower clinical scores based on motor dysfunction or severity of ascending paralysis. Histological analysis of lumber region of the spinal cord tissues from EAE mice revealed reduced inflammation and lower cellular infiltration in DUOC-01 treated samples compared to the vehicle injected controls. Presently we are analyzing single cell sequencing data to determine various sub-populations present in DUOC-01 cultures and to understand the functional pathways responsible for promoting remyelination and lowering neuroinflammation. In brief, our data suggest that DUOC-01 cell therapy product could be beneficial in treating MS and other diverse neurological conditions with demyelination.

Rapamycin alleviates inflammation by up-regulating TGF-β/Smad signaling in a mouse model of autoimmune encephalomyelitis

To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism. An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p- smad3 were investigated using Western blotting. High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 vs 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 vs 3.52 ± 0.32). In the in vitro experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.

Progress in the mechanism of neurogenic bladder in autoimmune encephalomyelitis

The autoimmune encephalomyelitis (EAE) mouse model is currently considered to be the preferred animal model for multiple sclerosis (MS). MS is a common demyelinating disease of central nervous system white matter, which often leads to neurogenic bladder. The mechanism of its occurrence has not been fully elucidated, and it may involve the abnormalities of multiple cell signal transduction pathways including bladder remodeling, nerve conduction, gap junction between detrusor and so on. We reviewed the molecular mechanism of neurogenic bladder in EAE mice for scientific research. Key words: Encephalomyelitis; Multiple sclerosis; Neurogenic bladder; Mechanism; Progress

CD93 regulates central nervous system inflammation in two mouse models of autoimmune encephalomyelitis.

Microglia and non-professional immune cells (endothelial cells, neurons) participate in the recognition and removal of pathogens and tissue debris in the injured central nervous system through major pro-inflammatory processes. However, the mechanisms involved in regulating these responses remain ill-characterized. We herein show that CD93, also known as complement C1qRp/AA4 stem cell marker, has an important role in the regulation of inflammatory processes. The role of CD93 was evaluated in two models of neuroinflammation. We used the MOG-experimental autoimmune encephalomyelitis (EAE) model and the antibody-dependent EAE (ADEAE), which were induced in wild-type and CD93 knockout mice. We found that CD93 was highly expressed by neurons, endothelial cells and microglia (ramified >> amoeboid). Astrocytes and oligodendrocytes did not to express CD93. We further observed that CD93-deficient (CD93-/- ) mice presented a more robust brain and spinal cord inflammation in EAE and ADEAE. Encephalitis in CD93-/- was characterized by increased numbers of infiltrating M1 macrophages (CD11c+ CD206- ) and amoeboid microglia exhibiting a more activated phenotype (Tomato Lectinhigh Cox2high ). Damage to and leakage through the blood-brain barrier was increased in CD93-/- animals and was associated with a more robust neuronal injury when compared with wild-type EAE mice. We propose that CD93 is an important neuro-immune regulator to control central nervous system inflammation.

Role of catalpol in ameliorating the pathogenesis of experimental autoimmune encephalomyelitis by increasing the level of noradrenaline in the locus coeruleus.

The endogenous neurotransmitter, noradrenaline, exerts anti-inflammatory and neuroprotective effects in vivo and in vitro. Reduced noradrenaline levels results in increased inflammation and neuronal damage. The primary source of noradrenaline in the central nervous system is tyrosine hydroxylase (TH)-positive neurons, located in the locus coeruleus (LC). TH is the rate-limiting enzyme for noradrenaline synthesis; therefore, regulation of TH protein expression and intrinsic enzyme activity represents the central means for controlling the synthesis of noradrenaline. Catalpol is an iridoid glycoside purified from Rehmannia glutinosa Libosch, which exerts a neuroprotective effect in multiple sclerosis (MS). The present study used an experimental mouse model of autoimmune encephalomyelitis to verify the neuroprotective effects of catalpol. Significant improvements in the clinical scores were observed in catalpol-treated mice. Furthermore, catalpol increased TH expression and increased noradrenaline levels in the spinal cord. In primary cultures, catalpol exerted a neuroprotective effect in rat LC neurons by increasing the noradrenaline output. These results suggested that drugs targeting LC survival and function, including catalpol, may be able to benefit patients with MS.

Therapeutic effects of walnut oil on the animal model of multiple sclerosis

Objectives: Therapeutic approaches for multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS), are accompanied by various undesirable side effects. Owing to the anti-inflammatory and antioxidant effects of walnut, we investigated its effects on the experimental autoimmune encephalomyelitis (EAE) mouse model of MS.Methods: After EAE induction in mice, the treated group was gavaged daily with walnut oil. The weights and clinical symptoms were monitored daily for 21 days following the onset of symptoms. The spleens and brains of the mouse were removed and used for ELISA and histological studies.Results: The average disease severity and plaque formation in the brains of the walnut oil-treated group were significantly lower (P < 0.05) than those of the untreated group. Stimulated splenocytes of the treated group expressed significantly less INF-γ and interleukin (IL)-17 than the untreated group with no significant differences in IL-10 or IL-5 production. In serum from the treated group, IL-17 expression was also significantly less than in the untreated group, while IL-10 was greater (P < 0.05).Conclusion: Walnut oil significantly reduced disease severity, inhibited plaque formation, and altered cytokine production. More studies are required to identify the mechanism of action of walnut oil as a valuable supplement in the treatment of MS.

Human chorionic gonadotropin (hCG) sub-chronic administration mediated MMP-9 activities and cytokine association deteriorate experimental autoimmune encephalomyelitis (EAE) condition in mice model

We investigated the role of sub-chronic administration of human chorionic gonadotropin (hCG) in an experimental autoimmune encephalomyelitis (EAE) mice model as a multiple sclerosis (MS) model. MS is a disease of malfunctioned immune system where infiltrating immune cells are stimulated by matrix metalloproteinase (MMP) activation and cause blood–brain barrier disruption followed by the destruction of myelin sheath by concurrent activation of cytokines. hCG is responsible for the expression and activation of MMPs and regulation of cytokines in different cells. Mice were implanted with the osmotic pump containing hCG and luteinizing hormone (LH) which maintain their release for 4 weeks with constant velocity. The activities of MMP-9 and NADPH oxidase and the levels of cytokines were measured in the plasma and the CNS tissues. The hCG treated EAE mice showed a decrease in body weights, increased mortality, accelerated onset stage and amplified EAE severity. MMP-9 activity was increased significantly in the EAE+hCG group compared to EAE group in both CNS and plasma. We also found increased activity of NADPH oxidase in plasma at onset stage and in brain tissues at day 38. IL-6 and IL-4 levels in brain tissue were found significantly higher in EAE+hCG group than in EAE group. EAE+LH group mice showed early onset patterns like EAE+hCG group and MMP-9 and NADPH oxidase levels were higher than EAE group, but cytokines levels were not significantly different from EAE group. We suggest that hCG treatment should be considered carefully as multiple or constant hCG treatment could exacerbate aggravation of MS induction and clinical symptoms.

Comment on: Dietary yeast reduce inflammation in central nerve system via microflora.

We recently read with great interest the article on the possible immunomodulatory effects of dietary yeast, specifically Candida kefir, on the central nervous system, notably the experimental autoimmune encephalomyelitis (EAE) mouse model by Takata et al.1 The role of yeast in inflammatory and autoimmune phenomena is nowadays controversial.2 The experiment from Takata et al. raises an important observation that not all yeast lead to the same effect on immunity. For instance, a significant reduction of proinflammatory cytokines such as IL‐17 and IFN‐γ was observed only in mice exposed to Candida kefir. Moreover, changes in microflora were different according to the microorganism used to feed EAE prone mice, with Saccharomyces cerevisiae being unable to reduce the ratio of Bacteroides or to ameliorate toluene‐2,4‐di‐isocyanate‐induced autoimmune dermatitis in mice. On the other hand, commensal microbiota which is not classically considered pathogenic can prompt and flare autoimmunity when fine regulation of immune tolerance does not work properly.2 This was indeed suggested for S. cerevisiae. Anti‐S. cerevisiae autoantibodies (ASCA) are associated with Crohn's disease and can be found even before clinical onset in ~30% of patients.3 Moreover, these antibodies can be found in several other autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome (APS), more frequently than in control subjects. Saccharomyces cerevisiae has immune‐modulating properties since cross‐reactive epitopes on β 2‐glycoprotein‐I and S. cerevisiae have been detected in ASCA‐positive APS patients.4 Several other overlaps in molecular sequence between yeast phosphopeptidomannan and conserved protein domains of specific autoantigens in humans have been analyzed and identified through the National Center for Biotechnology Information (NCBI) database.2 Saccharomyces cerevisiae proteins has been used as an adjuvant in vaccines such as hepatitis B vaccination and studies from our group led to the suspicion of an association with postvaccination autoimmune phenomena such as those described in the autoimmune/inflammatory syndrome induced by adjuvants (ASIA).5 Thus, the interesting experiment by Takata et al. may have revealed the importance of different dietary yeast in contribution to inflammation and autoimmunity. From one side, it is possible that some types (such as Saccharomyces boulardii) can be used as probiotics acting in a microbiome competition model leading to amelioration of autoimmune conditions.1 Nonetheless, it is fundamental to address the exact role of those types which, on the contrary, may even worsen an autoimmune/inflammatory condition.

Neuroprotective effects of a polyphenolic white grape juice extract in a mouse model of experimental autoimmune encephalomyelitis

In the last 20years, wine phenolic compounds have received increasing interest since several epidemiological studies have suggested associations between regular consumption of moderate amount of wine and prevention of certain chronic pathologies, such as neurodegenerative diseases.This study was aimed to investigate the possible neuroprotective role of a polyphenolic white grape juice extract (WGJe) in an experimental mice model of autoimmune encephalomyelitis (EAE), the most commonly used model for multiple sclerosis (MS) in vivo. EAE mimics the main features of MS, including paralysis, weight loss, demyelination, central nervous system (CNS) inflammation and blood–brain barrier (BBB) breakdown.Our study demonstrated that oral administration of WGJe (20 and 40mg/kg/day) may exert neuroprotective effects against MS, diminishing both clinical signs and histological score typical of disease (lymphocytic infiltration and demyelination). In particular, by western blot, histological evaluations and immunolocalization of the main markers of inflammation, oxidative stress and apoptosis (TNF-α, iNOS, Nitrotyrosine, PARP, Foxp3, Bcl-2, Caspase 3 and DNA fragmentation), we documented that WGJe counteracts the alteration of all these inflammatory and oxidative pathway, without any apparent sign of toxicity.On these bases, we propose this natural product as putative novel helpful tools for the prevention of autoimmune and neurodegenerative diseases such as MS. WGJe could have considerable implication for future therapies of MS, and this study may represents the starting point for further investigation on the role of WGJe in neuroinflammation.

The role of staphylococcal enterotoxin A in achieving oral tolerance to myelin basic protein in adult mice

Background: Oral tolerance is the biological process explaining the non-responsiveness of gut lymphoid tissue to intestinal content. Our study tested a new approach for the enhancement of oral tolerance to a multiple sclerosis-triggering auto-antigen–myelin basic protein, by its oral administration with the Staphylococcal enterotoxin A.Methods: Immune tolerance thus stimulated was assessed in adult BALB/c mice, by measuring different cytokines from the supernatant of mesenteric lymph nodes cells (IFN-γ, IL-4, IL-10, IL-17, and TGF-β), and in a SJL/E mouse model of experimental autoimmune encephalomyelitis, by evaluating the development of regulatory T cells in mesenteric lymph nodes and the clinical outcome of the intervention.Results: We obtained a significant rise in the levels of IL-10 and TGF-β compared with control and a significant decrease of IFN-γ, IL-4 (p < 0.05). Regulatory T cells were increased compared with control (p < 0.05). These results were attributable both to myelin basic protein and to Staphylococcal enterotoxin A. The clinical outcome of experimental autoimmune encephalomyelitis was influenced only by the administration of myelin basic protein.Conclusion: In our experiment, Staphylococcal enterotoxin A enhanced the immune tolerance to myelin basic protein in the gut mucosa, but had no impact on the clinical evolution of experimental autoimmune encephalomyelitis.