Extracellular vesicles containing miR-181a-5p as a novel therapy for experimental autoimmune encephalomyelitis-induced demyelination.

Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system. Recent research has revealed that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs), containing specific miRNAs, possess immunomodulatory properties and have demonstrated therapeutic potential in the treatment of MS. This study aimed to investigate the role MSC-EVs, containing microRNA-181a-5p (miR-181a-5p) in both experimental autoimmune encephalomyelitis (EAE), an established animal model of MS, and lipopolysaccharide-stimulated BV2 microglia. We evaluated clinical symptoms and inflammatory responses in EAE mice following intrathecal injections of MSC-EVs. MSC-EVs containing miR-181a-5p were co-cultured with microglia to explore their impact on inflammation and cell pyroptosis. We validated the interaction between miR-181a-5p and its downstream regulators and conducted in vivo verification by injecting manipulated EVs containing miR-181a-5p into EAE mice. Our results demonstrated that MSC-EVs, containing miR-181a-5p reduced the clinical symptoms of EAE mice. Furthermore, we observed downregulation of miR-181a-5p in EAE model mice, and its expression was restored after treatment with MSC-EVs, which corresponded to suppressed microglial inflammation and pyroptosis. Additionally, EVs containing miR-181a-5p mitigated spinal cord injury and demyelination in EAE mice. Mechanistically, ubiquitin-specific protease 15 (USP15) exhibited high expression in EAE mice, and miR-181a-5p was specifically targeted and bound to USP15, thereby regulating the RelA/NEK7 axis. In conclusion, MSC-EVs containing miR-181a-5p inhibit microglial inflammation and pyroptosis through the USP15-mediated RelA/NEK7 axis, thus alleviating the clinical symptoms of EAE. These findings present a potential therapeutic approach for the treatment of MS.

Reduced serum IgG galactosylation is associated with increased inflammation during relapses of neuromyelitis optica spectrum disorders.

Background and Objective: Post-translational modifications of antibodies, with a specific focus on galactosylation, have garnered increasing attention in the context of understanding the pathogenesis and therapeutic implications of autoimmune diseases. However, the comprehensive scope and the clinical significance of antibody galactosylation in the context of Neuromyelitis Optica Spectrum Disorder (NMOSD) remain enigmatic.The primary aim of this research was to discern disparities in serum IgG galactosylation levels between individuals in the acute stage of NMOSD relapse and their age- and sex-matched healthy counterparts. Methods: A total of fourteen untreated NMOSD patients experiencing an acute relapse phase, along with thirteen patients under medication, were enrolled, and an additional twelve healthy controls of the same age and gender were recruited for this investigation. Western blot and lectin enzyme techniques were used to determine the level of IgG galactosylation in the serum samples from these subjects. The expression of CD45+, CD3+, CD3+CD4+, CD3+CD8+, CD19+, and CD16+CD56+ in peripheral blood leukocytes was measured by flow cytometry. The enzyme-linked immunosorbent assay (ELISA) was also used to quantify the amounts of IgG. Magnetic particle luminescence assays are used to detect cytokines. Robust statistical analysis was executed to ascertain the potential associations between IgG galactosylation and the aforementioned immune indices. Results: In the context of NMOSD relapses, serum IgG galactosylation exhibited a notable decrease in untreated patients (0.2482 ± 0.0261), while it remained comparatively stable in medicated patients when contrasted with healthy controls (0.3625 ± 0.0259) (p=0.0159). Furthermore, a noteworthy inverse correlation between serum IgG galactosylation levels and the Expanded Disability Status Scale (EDSS) score during NMOSD relapse was observed (r=-0.4142; p=0.0317). Notably, IgG galactosylation displayed an inverse correlation with NMOSD relapse among peripheral blood CD45+, CD3+, CD3+CD8+, CD19+ cells, as well as with IL-6 and IL-8. Nevertheless, it was not determined whether IgG galactosylation and CD3+CD4+ T cells or other cytokines are statistically significantly correlated. Conclusion: Our research identified reduced IgG galactosylation in the serum of NMOSD patients during relapses, significantly correlated with disease severity, thereby providing a novel target for the diagnosis and treatment of NMOSD in the realm of medical research.

A splicing site change between exon 5 and 6 of the nuclear-encoded chloroplast-localized HvYGL8 gene results in reduced chlorophyll content and plant height in barley.

The chloroplast is an important cellular organelle and metabolic hub, which is not only responsible for plant photosynthesis but is also involved in the de novo biosynthesis of pigments, fatty acids, and hormone metabolisms. Several genes that are responsible for rice leaf color variations have been reported to be directly or indirectly involved in chlorophyll biosynthesis and chloroplast development, whereas a few genes have been functionally confirmed to be responsible for leaf color changes in barley at the molecular level. In this study, we obtained a yellow leaf and dwarf ygl8 mutant from the progeny of Morex (a variety of barley) seeds treated with EMS. We performed bulked-segregant analysis (BSA) and RNA-seq analysis and targeted a UMP kinase encoding gene, YGL8, which generated a splicing site change between exon 5 and 6 of YGL8 due to a G to A single-nucleotide transition in the 5th exon/intron junction in the ygl8 mutant. The splicing site change between exon 5 and 6 of YGL8 had no effects on chloroplast subcellular localization but resulted in an additional loop in the UMP kinase domain, which might disturb the access of the substrates. On one hand, the splicing site change between exon 5 and 6 of YGL8 downregulated the transcriptional expression of chloroplast-encoded genes and chlorophyll-biosynthesis-related genes in a temperature-dependent manner in the ygl8 mutant. On the other hand, the downregulation of bioactive GA-biosynthesis-related GA20ox genes and cell-wall-cellulose-biosynthesis-related CesA genes was also observed in the ygl8 mutant, which led to a reduction in plant height. Our study will facilitate the understanding of the regulation of leaf color and plant height in barley.

Receptor-Interacting Protein 3/Calmodulin-Dependent Kinase II/Proline-Rich Tyrosine Kinase 2 Pathway is Involved in Programmed Cell Death in a Mouse Model of Brain Ischaemic Stroke.

Neuronal necroptosis and apoptosis are the most important pathways for programmed cell death after brain ischaemic stroke. Although apoptosis signalling pathways have been extensively studied, molecular mechanisms underlying necroptosis remain unclear. In this study, we found that receptor-interacting protein 3 (RIP3) deficiency reduced cerebral infarction volume, neurological deficits, and neuronal ultrastructural damage in a mouse model of brain ischaemic stroke by inhibiting programmed cell death. RIP3 deficiency inhibited the activation of both calmodulin-dependent kinase II (CaMKII) and proline-rich tyrosine kinase 2 (Pyk2) cascade, decreased the expression of classic necroptotic and apoptotic proteins, and ultimately decreased neuronal necroptosis and apoptosis. We further confirmed that RIP3 deficiency inhibited the decrease of mitochondrial membrane potential, the increase of calcium influx and reactive oxygen species (ROS) production. In addition, compared with WT primary cortical neurons, the decreased expression of CaMKII and Pyk2 was further verified in a Ripk3-/- primary cortical neurons underlying oxygen and glucose deprivation/reoxygenation (OGD/R) model. In conclusion, we first identified that the RIP3/CaMKII/Pyk2 pathway is involved in programmed cell death after brain ischaemic stroke, which suggests it is a promising therapeutic target in ischaemia-induced neuronal injury.

Maackiain Prevents Amyloid-Beta-Induced Cellular Injury via Priming PKC-Nrf2 Pathway.

Amyloid-beta (Aß) peptide induces neurotoxicity through oxidative stress and inflammatory response. Brain deposition of a large amount of amyloid-beta (Aß), in particular Aß 42, promotes the development of Alzheimer's disease (AD). Maackiain is extracted from traditional Chinese medicine peony root and possesses antioxidative, antiosteoporosis, antitumor, and immunoregulatory effects. Whether Maackiain can reduce neurotoxicity caused by Aß accumulation remains elusive. Herein, we found that Maackiain downregulated Aß 42-induced cell injury and apoptosis in PC12 cells. Moreover, Maackiain prevented Aß 42 stimulation-induced generation of oxidative stress and reduced Aß 42-caused impairment of mitochondrial membrane potential in PC12 cells. Maackiain increased the superoxide dismutase activity and decreased malondialdehyde content that was induced by Aß 42. Mechanistic studies showed that Maackiain increased intranuclear Nrf2 expression. Consistently, Nrf2 silencing by RNA interference weakened the protective role of Maackiain against Aß exposure. In addition, calphostin C, a specific antagonist of protein kinase C, attenuated the promoting effects of Maackiain on Nrf2 nuclear translocation. Moreover, calphostin C attenuated the antioxidant and anti-inflammatory capabilities of Maackiain in PC12 cells. Collectively, Maackiain promoted Nrf2 activation through the PKC signaling pathway, thus preventing PC12 cells from Aß-induced oxidative stress and cell injury, suggesting that Maackiain is a potential drug for AD treatment.

Ponesimod modulates the Th1/Th17/Treg cell balance and ameliorates disease in experimental autoimmune encephalomyelitis.

Sphingosine-1-phosphate receptor 1 (S1P1) plays an important role in autoimmune disease. Here, we evaluated whether ponesimod, an S1P1 modulator, affects inflammation in experimental autoimmune encephalomyelitis (EAE) and investigated Th1/Th2/Th17/Treg cell subsets. Ponesimod treatment ameliorated EAE and alleviated inflammatory infiltration. Compared with untreated EAE, ponesimod-treated mice had lower Th1 and Th17 cell numbers and higher Treg cell numbers; their IFN-γ, T-bet, IL-17, and RORγt levels as well as their pmTOR/mTOR ratio were diminished, while their TGF-ß and Foxp3 levels were enhanced. These results suggest that ponesimod modulates the Th1/Th17/Treg balance and regulates the mTOR pathway.

Amlexanox attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and reprogramming effector and regulatory T cell responses.

BACKGROUND: Amlexanox (ALX), a TBK1 inhibitor, can modulate immune responses and has anti-inflammatory properties. To investigate its role in regulating the progression of experimental autoimmune encephalomyelitis (EAE), we studied the effect of ALX on the maturation of dendritic cells (DCs) and the responses of effector and regulatory T cells (Tregs). METHODS: In vitro, bone marrow-derived DCs (BMDCs) were cultured and treated with ALX. Their proliferation, maturation, and their stimulatory function to induce T cells responses were detected. In vivo, the development of EAE from different groups was recorded. At the peak stage of disease, HE, LFB, and electronic microscope (EM) were used to evaluate inflammation and demyelination. Maturation of splenic DC and Th1/Th17/Treg response in the CNS and peripheral were also detected. To further explore the mechanism underlying the action of ALX in DC maturation, the activation of TBK1, IRF3, and AKT was analyzed. RESULTS: Our data indicated that ALX significantly inhibited the proliferation and maturation of BMDCs, characterized by the reduced MHCII, a co-stimulatory molecule, IL12, and IL-23 expression, along with morphological alterations. Co-culture of ALX-treated BMDCs inhibited allogeneic T cell proliferation and MOG-specific T cell response. In EAE mice, ALX significantly attenuated the EAE development by decreasing inflammatory infiltration and demyelination in the spinal cords, accompanied by reduced frequency of splenic pathogenic Th1 and Th17 cells and increased Tregs. Moreover, ALX treatment decreased Th1 and Th17 cytokines, but increased Treg cytokines in the CNS and spleen. Notably, ALX treatment reduced the frequency and expression of CD80 and CD86 on splenic DCs and lowered IL-12 and IL-23 secretion, further supporting an impaired maturation of splenic DCs. In addition, ALX potently reduced the phosphorylation of IRF3 and AKT in BMDC and splenic DCs, both of which are substrates of TBK1 and associated with DC maturation. CONCLUSIONS: ALX, a TBK1 inhibitor, mitigated EAE development by inhibiting DC maturation and subsequent pathogenic Th1 and Th17 responses while increasing Treg responses through attenuating the TBK1/AKT and TBK1/IRF3 signaling.

Rapamycin and fingolimod modulate Treg/Th17 cells in experimental autoimmune encephalomyelitis by regulating the Akt-mTOR and MAPK/ERK pathways.

Rapamycin prevents experimental autoimmune encephalomyelitis (EAE) and activates the MAPK/ERK pathway in EAE. Thus, we hypothesized combining rapamycin and fingolimod treatments would have synergistic effects in EAE. We show that combination therapy ameliorated EAE and regulated spinal cord IL-17 and TGF-ß levels in EAE mice. Combination therapy also modulated IL-17 and TGF-ß concentration, RoRγt and Foxp3 mRNA levels, and Th17 cell and Treg frequencies in the spleen. Moreover, rapamycin decreased ps6k and increased pAkt and pERK, while combination therapy downregulated pAkt, ps6 k and pERK in EAE mice. Our findings provide insight into using this drug combination to treat EAE.

Fingolimod ameliorates the development of experimental autoimmune encephalomyelitis by inhibiting Akt-mTOR axis in mice.

Fingolimod is a new immunosuppressive agent approved by Food and Drug Administration (FDA) for treating multiple sclerosis (MS). It acts as a functional antagonist to downregulate the S1P1 receptor, which is known to signal through the Akt-mTOR pathway. We investigated the mechanism of fingolimod action in the classical animal model of MS: experimental autoimmune encephalomyelitis (EAE). Fingolimod treatment significantly reduced clinical scores and histopathology in this model, even when treatment was begun after the onset of pathology. The Akt-mTOR signaling pathway was shown to be activated in the EAE model, by measuring the abundance of downstream activation markers, pAkt and ps6k. And this pathway was inhibited when EAE mice were treated with fingolimod. Mice with EAE exhibited an increased frequency of Th1 cells in the spleen, with concomitant increases in the mRNA levels of Tbet and Ifng and increased IFN-γ production by activated splenocytes; the frequency of Treg cells, as well as mRNA levels of Foxp3 and Tgfb, was reduced, as was TGF-ß production by activated splenocytes. After treatment with fingolimod, these parameters were reversed, suggesting that fingolimod treatment inhibits the Akt-mTOR axis in EAE, which affects the differentiation and function of Th1 and Treg cells. These results provide an insight into the mechanism of action of fingolimod treatment and may provide new ideas for treating EAE and MS.

[Anti-oxidative effects of dexamethasone on mice with experimental autoimmune encephalomyelitis].

OBJECTIVE: To investigate the effects of dexamethasone (DXM) on the level of oxidative stress and antioxidant enzymes in mice with experimental autoimmune encephalomyelitis (EAE). METHODS: C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) to induce EAE. The mice were randomly divided into control group, EAE group, DXM group, and their clinical symptoms were observed. On day 13, 20 and 30 post immunization, the content of malondialdehyde (MDA) in the brain was assayed by thiobarbituric acid method; the expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and NADP(H):quinine oxidoreductase 1 (NQO1) in the brain were detected by immunohistochemistry and Western blotting. RESULTS: The morbidity and neurological deficit score of DXM group were significantly lower than those of EAE group (P<0.05). On day 13, 20 and 30 post immunization, the content of MDA in the EAE group was obviously higher than that in the control group (P<0.05); the content of MDA in the DXM group was obviously lower than that in the EAE group (P<0.05). Compared with the control group, the expression levels of Nrf2 and NQO1 in the EAE and DXM groups significantly increased on day 13, 20 and 30 post immunization (P<0.05). Compared with the EAE group, the expression levels of Nrf2 and NQO1 in the DXM group notably increased on day 13, 20 and 30 post immunization (P<0.05). In addition, DXM promoted the nuclear translocation of Nrf2. CONCLUSION: DXM could mitigate oxidative insult by up-regulation of Nrf2 and antioxidant enzymes, which may be an important mechanism involved in its protective effects on EAE.

Sulforaphane ameliorates the development of experimental autoimmune encephalomyelitis by antagonizing oxidative stress and Th17-related inflammation in mice.

Sulforaphane (SFN) is an organosulfur compound present in vegetables and has potent anti-oxidant and anti-inflammatory activities. This study was aimed at investigating the effect of treatment with SFN on inflammation and oxidative stress, and the potential mechanisms underlying the action of SFN in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. Treatment with SFN significantly inhibited the development and severity of EAE in mice, accompanied by mitigating inflammatory infiltration and demyelination in the spinal cord of mice. The protective effect of SFN was associated with significantly improved distribution of claudin-5 and occludin, and decreased levels of MMP-9 expression, preserving the blood-brain barrier. Furthermore, the protection of SFN was also related to decreased levels of oxidative stress in the brains of mice by enhanced activation of the Nrf2/ARE pathway and increased levels of anti-oxidant HO-1 and NQO1 expression. In addition, treatment with SFN inhibited antigen-specific Th17 responses and enhanced IL-10 responses. Our data indicated that treatment with SFN inhibited EAE development and severity in mice by its anti-oxidant activity and antagonizing autoimmune inflammation. Our findings suggest that SFN and its analogues may be promising reagents for intervention of multiple sclerosis and other autoimmune diseases.