Experimental Autoimmune Encephalomyelitis Model Research Articles

Integrated Analysis of Immune Infiltration and Hub Pyroptosis-Related Genes for Multiple Sclerosis.

Studies on overall immune infiltration and pyroptosis in patients with multiple sclerosis (MS) are limited. This study explored immune cell infiltration and pyroptosis in MS using bioinformatics and experimental validation. The GSE131282 and GSE135511 microarray datasets including brain autopsy tissues from controls and MS patients were downloaded for bioinformatic analysis. The gene expression-based deconvolution method, CIBERSORT, was used to determine immune infiltration. Differentially expressed genes (DEGs) and functional enrichments were analyzed. We then extracted pyroptosis-related genes (PRGs) from the DEGs by using machine learning strategies. Their diagnostic ability for MS was evaluated in both the training set (GSE131282 dataset) and validation set (GSE135511 dataset). In addition, messenger RNA (mRNA) expression of PRGs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in cortical tissue from an experimental autoimmune encephalomyelitis (EAE) model of MS. Moreover, the functional enrichment pathways of each hub PRG were estimated. Finally, co-expressed competitive endogenous RNA (ceRNA) networks of PRGs in MS were constructed. Among the infiltrating cells, naive CD4+ T cells (P=0.006), resting NK cells (P=0.002), activated mast cells (P=0.022), and neutrophils (P=0.002) were significantly higher in patients with MS than in controls. The DEGs of MS were screened. Analysis of enrichment pathways showed that the pathways of transcriptional regulatory mechanisms and ion channels associating with pyroptosis. Four PRGs genes CASP4, PLCG1, CASP9 and NLRC4 were identified. They were validated in both the GSE135511 dataset and the EAE model by using qRT-PCR. CASP4 and NLRC4 were ultimately identified as stable hub PRGs for MS. Single-gene Gene Set Enrichment Analysis showed that they mainly participated in biosynthesis, metabolism, and organism resistance. ceRNA networks containing CASP4 and NLRC4 were constructed. MS was associated with immune infiltration. CASP4 and NLRC4 were key biomarkers of pyroptosis in MS.

Aureane-type sesquiterpene tetraketides as a novel class of immunomodulators with interleukin-17A inhibitory activity.

Interleukin (IL)-17A, a pro-inflammatory cytokine, is a fundamental function in the onset and advancement of multiple immune diseases. To uncover the primary compounds with IL-17A inhibitory activity, a large-scale screening of the library of traditional Chinese medicine constituents and microbial secondary metabolites was conducted using splenic cells from IL-17A-GFP reporter mice cultured under Th17-priming conditions. Our results indicated that some aureane-type sesquiterpene tetraketides isolated from a wetland mud-derived fungus, Myrothecium gramineum, showed remarkable IL-17A inhibitory activity. Nine new aureane-type sesquiterpene tetraketides, myrogramins A-I (1, 4-11), and two known ones (2 and 3) were isolated and identified from the strain. Compounds 1, 3, 4, 10, and 11 exhibited significant IL-17A inhibitory activity. Among them, compound 3, with a high fermentation yield dose-dependently inhibited the generation of IL-17A and suppressed glycolysis in splenic cells under Th17-priming conditions. Strikingly, compound 3 suppressed immunopathology in both IL-17A-mediated animal models of experimental autoimmune encephalomyelitis and pulmonary hypertension. Our results revealed that aureane-type sesquiterpene tetraketides are a novel class of immunomodulators with IL-17A inhibitory activity, and hold great promise applications in treating IL-17A-mediated immune diseases.

Electroacupuncture at ST36 acupoint regulates stem cells during experimental autoimmune encephalomyelitis

BackgroundElectroacupuncture (EA) is given to assist in the treatment of MS, which is an effective therapeutic method. However, the therapy mechanism of EA related to stem cells in the treatment of MS is not yet known. In this study, we used a classic animal model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) to evaluate the therapeutic effect of EA at Zusanli (ST36) acupoint in EAE and shed light on its potential roles in the effects of stem cells in vivo. MethodsThe EAE animal models were established. From the first day after immunization, EAE model mice received EA at ST36 acupoint, named the EA group. The weight and clinical score of the three groups were recorded for 28 days. The demyelination, inflammatory cell infiltration, and markers of neural stem cells (NSCs), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs) were compared. ResultsWe showed that EAE mice treated with EA at ST36 acupoint, were suppressed in demyelination and inflammatory cell infiltration, and thus decreased clinical score and weight loss and mitigated the development of EAE when compared with the EAE group. Moreover, our data revealed that the proportions of NSCs, HSCs, and MSCs increased in the EA group compared with the EAE group. ConclusionsOur study suggested that EA at ST36 acupoint was an effective nonpharmacological therapeutic protocol that not only reduced the CNS demyelination and inflammatory cell infiltration in EAE disease but also increased the proportions of various stem cells. Further study is necessary to better understand how EA at the ST36 acupoint affects EAE.

Remibrutinib (LOU064) inhibits neuroinflammation driven by B cells and myeloid cells in preclinical models of multiple sclerosis

BackgroundBruton’s tyrosine kinase (BTK) is a key signaling node in B cell receptor (BCR) and Fc receptor (FcR) signaling. BTK inhibitors (BTKi) are an emerging oral treatment option for patients suffering from multiple sclerosis (MS). Remibrutinib (LOU064) is a potent, highly selective covalent BTKi with a promising preclinical and clinical profile for MS and other autoimmune or autoallergic indications.MethodsThe efficacy and mechanism of action of remibrutinib was assessed in two different experimental autoimmune encephalomyelitis (EAE) mouse models for MS. The impact of remibrutinib on B cell-driven EAE pathology was determined after immunization with human myelin oligodendrocyte glycoprotein (HuMOG). The efficacy on myeloid cell and microglia driven neuroinflammation was determined in the RatMOG EAE. In addition, we assessed the relationship of efficacy to BTK occupancy in tissue, ex vivo T cell response, as well as single cell RNA-sequencing (scRNA-seq) in brain and spinal cord tissue.ResultsRemibrutinib inhibited B cell-dependent HuMOG EAE in dose-dependent manner and strongly reduced neurological symptoms. At the efficacious oral dose of 30 mg/kg, remibrutinib showed strong BTK occupancy in the peripheral immune organs and in the brain of EAE mice. Ex vivo MOG-specific T cell recall response was reduced, but not polyclonal T cell response, indicating absence of non-specific T cell inhibition. Remibrutinib also inhibited RatMOG EAE, suggesting that myeloid cell and microglia inhibition contribute to its efficacy in EAE. Remibrutinib did not reduce B cells, total Ig levels nor MOG-specific antibody response. In brain and spinal cord tissue a clear anti-inflammatory effect in microglia was detected by scRNA-seq. Finally, remibrutinib showed potent inhibition of in vitro immune complex-driven inflammatory response in human microglia.ConclusionRemibrutinib inhibited EAE models by a two-pronged mechanism based on inhibition of pathogenic B cell autoreactivity, as well as direct anti-inflammatory effects in microglia. Remibrutinib showed efficacy in both models in absence of direct B cell depletion, broad T cell inhibition or reduction of total Ig levels. These findings support the view that remibrutinib may represent a novel treatment option for patients with MS.

Nose to brain delivery of Astragaloside IV by β-Asarone modified chitosan nanoparticles for multiple sclerosis therapy

Multiple sclerosis (MS), an autoimmune disease, has been considered an inflammatory disorder of the central nervous system (CNS) with demyelination and axonal damage. Although there are certain first-line therapies to treat MS, their unsatisfactory efficacy is partly due to the limited CNS access after systemic administration. Besides, there is an urgent need to treat MS by enhancing remyelination or neuroprotection, or dampen the activity of microglia. Astragaloside IV (ASI) bears anti-inflammatory, antioxidant, remyelination and neuroprotective activity. While its poor permeability, relatively high molecular weight and low lipophilicity restrict it to reach the brain. Therefore, β-asarone modified ASI loaded chitosan nanoparticles (ASI-βCS-NP) were prepared to enhance the nose-to-brain delivery and therapeutic effects of ASI on EAE mice. The prepared ASI-βCS-NP showed mean size of about 120 nm, and zeta potential from +19 to +25 mV. DiR-βCS-NP was confirmed with good nose-to-brain targeting ability. After intranasal administration, the ASI-βCS-NP significantly reduced behavioral scores, decreased weight loss, suppressed inflammatory infiltration and astrocyte/microglial activation, reduced demyelination and increased remyelination on a mice EAE model. Our findings indicate that ASI-βCS-NP may be a potent treatment for MS after nose-to-brain drug delivery.

Involvement of NRON and TUG1 long noncoding RNAs in inflammation and the pathogenesis of EAE

There is growing evidence that the long noncoding RNAs (lncRNAs) contribute to the pathogenesis of various neurodegenerative diseases such as multiple sclerosis (MS). The role of lncRNAs nuclear repressor of NFAT (NRON) and Taurine up-regulated 1 (TUG1) in the inflammatory processes occurring in the experimental autoimmune encephalomyelitis (EAE) model of MS is yet to be investigated. Transcript levels of NRON and TUG1 in acute and chronic phases of EAE and cultured macrophages as well as the correlation between NRON and TUG1 expression with inflammatory cytokines, were evaluated in this study. EAE experimental model was induced in female C57BL/6 mice with subcutaneous injection of MOG35–55/CFA. Mice were scored for 28 days and then sacrificed. The expression of lncRNAs TUG1 and NRON in lumbar spinal cords, activated and controlled macrophages as well as the expression of IL-1, IL-6, and CDe-3 inflammatory cytokines, were assayed by real-time RT-PCR. The lncRNAs TUG1 and NRON were significantly down-regulated in lumbar spinal cords tissues in the acute phase of EAE compared to the control group. TUG1 and NRON were significantly down-regulated in macrophages treated with 10 ng lipopolysaccharide (LPS) compared to the control macrophages. A negative correlation was identified between NRON and TUG1 expression and IL-1, IL-6, and CDe-3 inflammatory cytokines. The present study demonstrates the dysregulation of lncRNAs TUG1 and NRON in spinal cord tissue lesions of EAE and activated macrophages, pointing to their potential role in the pathogenesis of EAE.

Anti-Glycolipid Antibody Examination in Five EAE Models and Theiler's Virus Model of Multiple Sclerosis: Detection of Anti-GM1, GM3, GM4, and Sulfatide Antibodies in Relapsing-Remitting EAE.

Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG)35-55, MOG92-106, or myelin proteolipid protein (PLP)139-151, with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler's murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP139-151-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.

Loss of the Novel Myelin Protein CMTM5 in Multiple Sclerosis Lesions and Its Involvement in Oligodendroglial Stress Responses.

This study comprehensively addresses the involvement of the protein CKLF-like Marvel transmembrane domain-containing family member 5 (CMTM5) in the context of demyelination and cytodegenerative autoimmune diseases, particularly multiple Sclerosis (MS). An observed reduction in CMTM5 expression in post-mortem MS lesions prompted further investigations in both in vitro and in vivo animal models. In the cuprizone animal model, we detected a decrease in CMTM5 expression in oligodendrocytes that is absent in other members of the CMTM protein family. Our findings also confirm these results in the experimental autoimmune encephalomyelitis (EAE) model with decreased CMTM5 expression in both cerebellum and spinal cord white matter. We also examined the effects of a Cmtm5 knockdown in vitro in the oligodendroglial Oli-neu mouse cell line using the CRISPR interference technique. Interestingly, we found no effects on cell response to thapsigargin-induced endoplasmic reticulum (ER) stress as determined by Atf4 activity, an indicator of cellular stress responses. Overall, these results substantiate previous findings suggesting that CMTM5, rather than contributing to myelin biogenesis, is involved in maintaining axonal integrity. Our study further demonstrates that the knockdown of Cmtm5 in vitro does not modulate oligodendroglial responses to ER stress. These results warrant further investigation into the functional role of CMTM5 during axonal degeneration in the context of demyelinating conditions.

The novel small molecule TPN10518 alleviates EAE pathogenesis by inhibiting AP1 to depress Th1/Th17 cell differentiation

Multiple sclerosis (MS) is one of the most common autoimmune diseases of central nervous system (CNS) demyelination. Experimental autoimmune encephalomyelitis (EAE) is the most classic animal model for simulating the onset of clinical symptoms in MS. Previous research has reported the anti-inflammatory effects of artemisinin on autoimmune diseases. In our study, we identified a novel small molecule, TPN10518, an artemisinin derivative, which plays a protective role on the EAE model. We found that TPN10518 reduced CNS inflammatory cell infiltration and alleviated clinical symptoms of EAE. In addition, TPN10518 downregulated the production of Th1 and Th17 cells in vivo and in vitro, and decrease the levels of related chemokines. RNA-seq assay combined with the experimental results demonstrated that TPN10518 lowered the mRNA and protein levels of the AP1 subunits c-Fos and c-Jun in EAE mice. It was further confirmed that TPN10518 was dependent on AP1 to inhibit the differentiation of Th1 and Th17 cells. The results suggest that TPN10518 reduces the production of Th1 and Th17 cells through inhibition of AP1 to alleviate the severity of EAE disease. It is expected to be a potential drug for the treatment of MS.

Novel nano-carriers with N-formylmethionyl-leucyl-phenylalanine-modified liposomes improve effects of C16-angiopoietin 1 in acute animal model of multiple sclerosis

Gradual loss of neuronal structure and function due to impaired blood–brain barrier (BBB) and neuroinflammation are important factors in multiple sclerosis (MS) progression. Our previous studies demonstrated that the C16 peptide and angiopoietin 1 (Ang-1) compound (C + A) could modulate inflammation and vascular protection in many models of MS. In this study, nanotechnology and a novel nanovector of the leukocyte chemotactic peptide N-formyl-methionyl-leucyl–phenylalanine (fMLP) were used to examine the effects of C + A on MS. The acute experimental autoimmune encephalomyelitis (EAE) model of MS was established in Lewis rats. The C + A compounds were conjugated to control nano-carriers and fMLP-nano-carriers and administered to animals by intravenous injection. The neuropathological changes in the brain cortex and spinal cord were examined using multiple approaches. The stimulation of vascular injection sites was examined using rabbits. The results showed that all C + A compounds (C + A alone, nano-carrier C + A, and fMLP-nano-carrier C + A) reduced neuronal inflammation, axonal demyelination, gliosis, neuronal apoptosis, vascular leakage, and BBB impairment induced by EAE. In addition, the C + A compounds had minimal side effects on liver and kidney functions. Furthermore, the fMLP-nano-carrier C + A compound had better effects compared to C + A alone and the nano-carrier C + A. This study indicated that the fMLP-nano-carrier C + A could attenuate inflammation-related pathological changes in EAE and may be a potential therapeutic strategy for the treatment of MS and EAE.

1-Oleoyl-lysophosphatidylethanolamine stimulates RORγt activity in TH17 cells.

Metabolic fluxes involving fatty acid biosynthesis play essential roles in controlling the differentiation of T helper 17 (TH17) cells. However, the exact enzymes and lipid metabolites involved, as well as their link to promoting the core gene transcriptional signature required for the differentiation of TH17 cells, remain largely unknown. From a pooled CRISPR-based screen and unbiased lipidomics analyses, we identified that 1-oleoyl-lysophosphatidylethanolamine could act as a lipid modulator of retinoid-related orphan receptor gamma t (RORγt) activity in TH17 cells. In addition, we specified five enzymes, including Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2, suggestive of the requirement of glycerophospholipids with monounsaturated fatty acids being required for the transcription of Il17a. 1-Oleoyl-lysophosphatidylethanolamine was reduced in Pla2g12a-deficient TH17 cells, leading to the abolition of interleukin-17 (IL-17) production and disruption to the core transcriptional program required for the differentiation of TH17 cells. Furthermore, mice with T cell-specific deficiency of Pla2g12a failed to develop disease in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus, our data indicate that 1-oleoyl-lysophosphatidylethanolamine is a lipid metabolite that promotes RORγt-induced TH17 cell differentiation and the pathogenicity of TH17 cells.

Early urinary candidate biomarkers and clinical outcomes of intervention in a rat model of experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system and is difficult to diagnose in early stages. Without homeostatic control, urine was reported to have the ability to accumulate early changes in the body. We expect that urinary proteome can reflect early changes in the nervous system. The early urinary proteome changes in a most employed multiple sclerosis rat model (experimental autoimmune encephalomyelitis) were analysed to explore early urinary candidate biomarkers, and early treatment of methylprednisolone was used to evaluate the therapeutic effect. Twenty-five urinary proteins were altered at day 7 when there were no clinical symptoms and obvious histological changes. Fourteen were reported to be differently expressed in the serum/cerebrospinal fluid/brain tissues of multiple sclerosis patients or animals such as angiotensinogen and matrix metallopeptidase 8. Functional analysis showed that the dysregulated proteins were associated with asparagine degradation, neuroinflammation and lipid metabolism. After the early treatment of methylprednisolone, the incidence of encephalomyelitis in the intervention group was only 1/13. This study demonstrates that urine may be a good source of biomarkers for the early detection of multiple sclerosis. These findings may provide important information for early diagnosis and intervention of multiple sclerosis in the future.

Microglial expression of CD83 governs cellular activation and restrains neuroinflammation in experimental autoimmune encephalomyelitis

Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.

Lipocalin-2 promotes adipose–macrophage interactions to shape peripheral and central inflammatory responses in experimental autoimmune encephalomyelitis

ObjectiveAccumulating evidence suggests that dysfunctional adipose tissue (AT) plays a major role in the risk of developing multiple sclerosis (MS), the most common immune-mediated and demyelinating disease of the central nervous system. However, the contribution of adipose tissue to the etiology and progression of MS is still obscure. This study aimed at deciphering the responses of AT in experimental autoimmune encephalomyelitis (EAE), the best characterized animal model of MS. Results and MethodsWe observed a significant AT loss in EAE mice at the onset of disease, with a significant infiltration of M1-like macrophages and fibrosis in the AT, resembling a cachectic phenotype. Through an integrative and multilayered approach, we identified lipocalin2 (LCN2) as the key molecule released by dysfunctional adipocytes through redox-dependent mechanism. Adipose-derived LCN2 shapes the pro-inflammatory macrophage phenotype, and the genetic deficiency of LCN2 specifically in AT reduced weight loss as well as inflammatory macrophage infiltration in spinal cord in EAE mice. Mature adipocytes downregulating LCN2 reduced lipolytic response to inflammatory stimuli (e.g. TNFα) through an ATGL-mediated mechanism. ConclusionsOverall data highlighted a role LCN2 in exacerbating inflammatory phenotype in EAE model, suggesting a pathogenic role of dysfunctional AT in MS.

Pparα knockout in mice increases the Th17 development by facilitating the IKKα/RORγt and IKKα/Foxp3 complexes

The helper CD4+ T cell-type 17 (Th17) cells and regulatory CD4+ T cells (Tregs) are balanced through numerous molecular regulators, particularly metabolic factors, and their alteration causes immune dysregulation. Herein, we report that peroxisome proliferator of activated receptor-alpha (Pparα), a lipid metabolism regulator, suppresses Th17 differentiation. We demonstrated that Pparα ablation improves Th17 and pro-Th17 factor HIF-1α by enhancing the expression and nuclear localization of NFκB-activator IκB kinase-alpha (IKKα). Unexpectedly, we found that IKKα directly interacts with RORγt and enhances the expression of Il17a gene. Meanwhile, IKKα also interacts with Foxp3, leading to the post-translational regulation of Foxp3 by elevating its proteasomal degradation, and influencing Th17 development. Pparα deficiency leads to enhanced Th17 development in vivo and is associated with enhanced pathology in a murine experimental autoimmune encephalomyelitis (EAE) model. Overall, our data indicate that Pparα may serve as a potential therapeutic target for autoimmune and inflammatory diseases.

Genetic deletion of c-Jun amino-terminal kinase 3 (JNK3) modestly increases disease severity in a mouse model of multiple sclerosis

The c-Jun amino terminal kinases (JNKs) regulate transcription, and studies suggest they contribute to neuropathology in the EAE model of MS. To examine the role of the JNK3 isoform, we compared EAE in JNK3 null mice to wild type (WT) littermates. Although disease severity was similar in female mice, in male JNK3 null mice the day of onset and time to reach 100% incidence occurred sooner, and disease severity was increased. While glial activation in spinal cord was similar, white matter lesions were increased in JNK3 null mice. These results suggest JNK3 normally limits EAE disease in a sex-dependent manner.

MRNA expression profile reveals differentially expressed genes in splenocytes of experimental autoimmune encephalomyelitis model.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that can be used to investigate aetiology, pathogenesis, and treatment approaches for multiple sclerosis (MS). A novel integrated bioinformatics approach was used to understand the involvement of differentially expressed genes (DEGs) in the spleen of EAE mice through data mining of existing microarray and RNA-seq datasets. We screened differentially expressed mRNAs using mRNA expression profile data of EAE spleens takenfrom Gene Expression Omnibus (GEO). Functional and pathway enrichment analyses of DEGs were performed by Database for Annotation, Visualization, and Integrated Discovery (DAVID). Subsequently, the DEGs-encoded protein-protein interaction (PPI) network was constructed. The 784 DEGs in GSE99300 A.SW PP-EAE mice spleen mRNA profiles, 859 DEGs in GSE151701 EAE mice spleen mRNA profiles, and 646 DEGs in GSE99300 SJL/J PP-EAE mice spleen mRNA profiles were explored. Functional enrichment of 55 common DEGs among 3 sub-datasets revealed several immune-related terms, such as neutrophil extravasation, leucocyte migration, antimicrobial humoral immune response mediated by an antimicrobial peptide, toll-like receptor 4 bindings, IL-17 signalling pathway, and TGF-beta signalling pathway. In the screening of 10 hub genes, including MPO, ELANE, CTSG, LTF, LCN2, SELP, CAMP, S100A9, ITGA2B, and PRTN3, and in choosing and validating the 5 DEGs, including ANK1, MBOAT2, SLC25A21, SLC43A1, and SOX6, the results showed that SLC43A1 and SOX6 were significantly decreased in EAE mice spleen. Thus this study offers a list of genes expressed in the spleen that might play a key role in the pathogenesis of EAE.

Oral administration of procyanidin B2 3,3"-di-O-gallate ameliorates experimental autoimmune encephalomyelitis through immunosuppressive effects on CD4+ T cells by regulating glycolysis

Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system caused by the excessive activation of T cells. Procyanidins are polyphenols that exhibit anti-inflammatory activity. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3″-di-O-gallate (PCB2DG)] inhibits cytokine production in T cells by suppressing the acceleration of glycolysis. In this study, we determined the effect of PCB2DG on T cell-mediated autoimmune disease in vivo. We examined the immunosuppressive effects of PCB2DG using an experimental autoimmune encephalomyelitis (EAE) model, which is a classic animal model for MS. Our results indicated that the clinical score for EAE symptoms improved significantly following the oral administration of PCB2DG. This effect was associated with the suppression of T cell-mediated cytokines (e.g., IFN-γ, TNF-α, and IL-17) and infiltrating T cells into the spinal cord, which ameliorated spinal cord injury. In addition, spleen cell culture experiments revealed that the increase of T cell-mediated pro-inflammatory cytokines in EAE mice was significantly decreased following PCB2DG treatment. We further analyzed the glycolytic activity of spleen cells to identify the mechanism of the immunosuppressive effects of PCB2DG. The production of lactate and the expression of glycolytic enzymes and transporters were increased following EAE induction, but not in PCB2DG-treated EAE mice. Collectively, our results indicate that a dietary polyphenol, which has a unique structure, improves the onset of EAE symptoms and inhibits the excessive activation of T cells by influencing glycolysis.

Naringenin stimulates aromatase expression and alleviates the clinical and histopathological findings of experimental autoimmune encephalomyelitis in C57bl6 mice.

This study was conducted to demonstrate the possible protective and therapeutic effects of naringenin, an estrogenically effective flavonoid, in experimental autoimmune encephalomyelitis (EAE), which is the rodent model of multiple sclerosis. For this purpose, 50 12-week-old C57BL6 male mice were divided into five groups; control, naringenin, EAE, prophylactic naringenin + EAE, and EAE + therapeutic naringenin. The EAE model was induced withmyelin oligodendrocyte glycoprotein(35-55), and naringenin (50mg/kg) was administered by oral gavage. The prophylactic and therapeutic effects of naringenin were examined according to clinical, histopathological, immunohistochemical, electron microscopic, and RT-PCR (aromatase, 3βHSD, estrogen receptors, and progesterone receptor expression) parameters. The acute EAE model was successfully induced, along with its clinical and histopathological findings. RT-PCR showed that expression of aromatase, 3βHSD, estrogen receptor-β, and progesterone receptor gene decreased, while estrogen receptor-α increased after EAE induction. Electron microscopic analysis showed mitochondrial damage and degenerative changes in myelinated axons and neurons in EAE, which could be behind the downregulation in the expressions of neurosteroid enzymes. Aromatase immunopositivity rates also decreased in EAE, while estrogen receptor α and β, and progesterone receptor immunopositivity rates increased. Naringenin improved aromatase immunopositivity rates and gene expression in both prophylactic and therapeutic use. Clinical and histopathological findings revealed that EAE findings were alleviated in both prophylactic and therapeutic groups, along with significantly decreased inflammatory cell infiltrations in the white matter of the spinal cords. In conclusion, naringenin could provide long-term beneficial effects even in prophylactic use due to stimulating aromatase expression, but it could not prevent or eliminate the EAE model's lesions completely.

GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression.

T-bet-expressing Th17 (T-bet+RORγt+) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo. Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2 Cre and Tbx21 Cre, respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via Cre-ERT2 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis of Gata3-sufficient and Gata3-deficient CNS-infiltrating CD4+ effector T cells from mixed congenic co-transfer recipient mice revealed an important, cell-intrinsic, function of GATA3 in regulating the expression of Egr2, Bhlhe40, and Csf2. Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression.