TREM-2 Drives Development of Multiple Sclerosis by Promoting Pathogenic Th17 Polarization / 神经科学通报·英文版

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease, mediated by pathogenic T helper 17 (Th17) cells. However, the therapeutic effect is accompanied by the fluctuation of the proportion and function of Th17 cells, which prompted us to find the key regulator of Th17 differentiation in MS. Here, we demonstrated that the triggering receptor expressed on myeloid cells 2 (TREM-2), a modulator of pattern recognition receptors on innate immune cells, was highly expressed on pathogenic CD4-positive T lymphocyte (CD4+ T) cells in both patients with MS and experimental autoimmune encephalomyelitis (EAE) mouse models. Conditional knockout of Trem-2 in CD4+ T cells significantly alleviated the disease activity and reduced Th17 cell infiltration, activation, differentiation, and inflammatory cytokine production and secretion in EAE mice. Furthermore, with Trem-2 knockout in vivo experiments and in vitro inhibitor assays, the TREM-2/zeta-chain associated protein kinase 70 (ZAP70)/signal transducer and activator of transcription 3 (STAT3) signal axis was essential for Th17 activation and differentiation in EAE progression. In conclusion, TREM-2 is a key regulator of pathogenic Th17 in EAE mice, and this sheds new light on the potential of this therapeutic target for MS.

Study on the mechanism of Yishen daluo decoction in alleviating nervous system inflammation in EAE model mice / 中国药房

OBJECTIVE To study the effects of Yishen daluo decoction on inflammatory factors and cyclic adenosine monophosphate（cAMP）/protein kinase A （PKA）/cAMP response element binding protein （CREB） signal pathway in experimental autoimmune encephalomyelitis （EAE） model mice by inhibiting the expressions of β-arrestin1， and to explore the mechanism of Yishen daluo decoction in the treatment of EAE. METHODS Sixty mice were randomly divided into normal group， model group， TCM group （Yishen daluo decoction 20 g/kg）， positive control group （prednisone acetate 3.9 mg/kg）， β-arrestin1 siRNA adeno- associated virus （AAV-β） group， AAV-β+TCM group， with 10 mice in each group. Except for normal group， EAE model was made in other groups. AAV-β group and AAV-β+TCM group were injected with AAV-β via tail vein to interfere with the expression of β -arrestin1 protein. Starting from the 8th day of modeling， they were given corresponding drug solution/normal saline intragastrically， once a day， for consecutive 14 days. The neurological function score of mice was detected； the pathological and morphological changes were observed in the brain and spinal cord tissues of mice； the serum levels of inflammatory factors [interleukin-2 （IL-2）， IL-23， interferon-γ （IFN-γ）] in mice were determined； the expressions of β-arrestin1， cAMP， PKA and CREB in brain and spinal cord were detected. RESULTS Compared with normal group， neurological function scores， serum levels of inflammatory factors， and protein expressions of β-arrestin1 in brain and spinal cord were significantly increased （P＜0.05 or P＜ 0.01）； protein expressions of PKA， CREB and cAMP in brain and spinal cord were decreased significantly（P＜0.05 or P＜0.01）. The deep staining of cellular shrinkage and aggregation of inflammatory cells were observed in most neurons of the brain and spinal cord， with varying degrees of demyelinating. Compared with model group， the neurological function scores， pathological changes in brain and spinal cord tissues， and most indicators （except for CREB and cAMP proteins in the brain tissue of AAV-β group） were significantly reversed （P＜0.05 or P＜0.01）.Compared with AAV- β group， the neurological function scores， the levels of IFN-γ in serum and β-arrestin1 in spinal cord were significantly decreased （P＜0.05 or P＜0.01）， PKA and cAMP in brain and spinal cord tissues were significantly increased in AAV- β +TCM group （P＜0.05 or P＜0.01）. CONCLUSIONS Yishen daluo decoction can inhibit the expression of β-arrestin1 in the central nervous system thus activating the cAMP/PKA/CREB signaling pathway， relieving nervous system inflammation， and ultimately alleviates the symptoms of EAE.

Improvement effect and mechanism of matrine on experimental autoimmune encephalomyelitis mice / 中国药房

OBJECTIVE To investigate whether matrine exerts improvement effect on experimental autoimmune encephalomyelitis （EAE） mice by regulating ferroptosis pathway. METHODS Totally 30 female C57BL/6 mice were randomly assigned into normal group， model group and matrine group， with 10 mice in each group. Model group and matrine group were given antigen emulsion containing inactivated Mycobacterium tuberculosis and MOG35-55 to induce EAE model. Matrine group was injected with Matrine injection （50 mg/kg） intraperitoneally since the 7th day after immunization； normal group and model group were given constant volume of normal saline intraperitoneally， once a day， since 18th day after immunization. The neurofunctional score of mice was recorded， and hematoxylin and eosin staining and Luxol fast blue staining were used to observe inflammatory cell infiltration and demyelination in spinal cord tissue. The quantitative reverse transcription PCR and Western blot assay were performed to determine the mRNA expressions of transferrin receptor 1 （TFR1）， nuclear receptor coactivator 4 （NCOA4） and hephaestin （Heph）， and the protein expressions of system Xc－ （xCT） and glutathione peroxidase 4 （GPx4）. RESULTS Compared with normal group， accumulative neurofunctional score was significantly increased in model group （P＜0.01）； inflammatory cell infiltration and demyelination were obvious in spinal cord tissue， and related scores were increased significantly （P＜0.01）. The mRNA expressions of TFR1 and NCOA4 in myelin tissue were up-regulated significantly， while the mRNA expression of Heph and the protein expressions of xCT and GPx4 were down-regulated significantly （P＜0.05 or P＜0.01）. Compared with model group， above indexes of matrine group were all improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Matrine can improve EAE mice， the mechanism of which may be associated with regulating iron metabolism pathway and xCT/GPx4 pathway in ferroptosis.

Progress in Mechanism of Astragalus membranaceus and Its Chemical Constituents on Multiple Sclerosis / 中国结合医学杂志

The primary chemical components of Astragalus membranaceus include polysaccharides, saponins, flavonoids, and amino acids. Recent studies have shown that Astragalus membranaceus has multiple functions, including improving immune function and exerting antioxidative, anti-radiation, anti-tumor, antibacterial, antiviral, and hormone-like effects. Astragalus membranaceus and its extracts are widely used in clinical practice because they have obvious therapeutic effects against various autoimmune diseases and relatively less adverse reaction. Multiple sclerosis (MS) is an autoimmune disease of central nervous system (CNS), which mainly caused by immune disorder that leads to inflammatory demyelination, inflammatory cell infiltration, and axonal degeneration in the CNS. In this review, the authors analyzed the clinical manifestations of MS and experimental autoimmune encephalomyelitis (EAE) and focused on the efficacy of Astragalus membranaceus and its chemical components in the treatment of MS/EAE.

Correlation of neutrophil extracellular traps with T cells differentiation in Hashimoto′s thyroiditis mice and vitamin D′s relief effect / 中华内分泌代谢杂志

Objective:To investigate correlation between neutrophil extracellular traps(NETs) formation and T cell subsets in mice with experimental autoimmune thyroiditis(EAT) and the impact of active vitamin D intervention.Methods:Six-week-old female BALB/c mice were randomly divided into Control group, EAT group and 1, 25 dihydroxy vitamin D 3[1, 25(OH) 2D 3] treatment group(VitD group; n=6/group). HE staining was used to observe thyroid pathology. Plasma thyroglobulin antibody(TGAb), thyroid peroxidase antibody(TPOAb), and 1, 25(OH) 2D 3 were measured by ELISA. Peripheral NETs formation, Th1, Th2, and Th17 cell ratio from spleen were measured by flow cytometry. Correlation between NETs formation rate and Th1, Th2, and Th17 cell ratio was analyzed. Results:Compared with Control group, mice in EAT group had significantly increased thyroid inflammation scores, thyroiditis morbidity, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + )and Th17 cell proportions( P were <0.001, 0.002, 0.007, <0.001, <0.001, 0.003, 0.001, and 0.002, respectively), and significant decreased 1, 25(OH) 2D 3, Th1 cell proportions, Th1/Th2(CD4 + IL-4 + ), Th1/Th2(CD4 + IL-13 + ), and Th1/Th17 ratios( P were 0.010, 0.018, 0.010, 0.005, and 0.007, respectively). Compared with the EAT group, the VitD group had lower thyroid inflammation scores, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportions( P were 0.044, 0.007, <0.001, 0.001, 0.014, 0.008, and 0.001, respectively), and significant higher Th1 cell ratio, Th1/Th2(CD4 + IL-13 + ) and Th1/Th17 ratio( P were 0.011, 0.009, and 0.003, respectively). The Th1/Th2(CD4 + IL-4 + ) was not significantly increased in VitD group compared with EAT group( P=0.174). NETs formation rate was positively correlated with Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportion( r were 0.65, 0.59, and 0.61; and P were 0.004, 0.010, and 0.007, respectively), but not with Th1 cell proportion( r=-0.47, P=0.051). Conclusion:EAT mice were more prone to NETs formation. Active vitamin D may relieve immune imbalance with increased Th2 and Th17 cell ratio and decreased Th1 cell ratio by reducing the formation of NETs in EAT mice. Vitamin D played the protective role in thyroid by reducing thyroid pathological damage and thyroid autoantibody levels, and relived overall lymphocyte imbalance.

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells / 中国结合医学杂志

OBJECTIVE@#To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action.@*METHODS@#This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively.@*RESULTS@#GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+ and CD45+CD4+ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05).@*CONCLUSION@#GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6 / STAT3 / ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice / 解剖学报

Objective To explore the effect of exogenous and endogenous erythrocyte membrane-associated protein (ERMAP) on helper T cell 17 (Th17) cell differentiation through interleukin 6 / signal transducers and activators of transcription 3 / retionoid-related orphan nuclear receptor-γt(IL-6 / STAT3 / ROR-γt) signal pathway in the mouse model of experimental autoimmune encephalomyelitis (EAE) . Methods Using flow cytometry to verify the function of ERMAP-Ig fusion protein at different concentrations; Agarose gel electrophoresis was performed to identify ERMAP knockout mice. Flow cytometry was performed to detect the effect of ERMAP-Ig fusion protein on Th17 cell differentiation in vitro. Forty 6-week-old normal C57BL / 6 mice were randomly divided into 2 groups to establish EAE models, control-Ig and ERMAP-Ig groups, with 20 mice in each group; Clinical scores were recorded; Flow cytometry was performed to detect Th17 cell differentiation in EAE mice in vivo. Forty 6-week-old identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models. Identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models, ERMAP

Study on protective effect of Shenqi Fuzheng injection on immune myocarditis injury induced by anti-PD-1 antibody / 中国药理学通报

Aim To investigate the effeot of Shenqi Fuzheng injection on the prevention of immune myocarditis induced by anti-PD-1 antibody by reducing the production of inflammatory factors and the expression of myocardial injury markers. Methods Thirty-two maie PD-1 humanized mice with C57BL/6 genetic background were randomly divided into control group, myocarditis model group, anti-PD-1 antibody group and Shenqi Fuzheng injection group (n = 8). Except the control group, mice in other groups were intraperitoneally injected with myocardial myosin heavy chain peptide (5 mg • kg

GLPS improves EAE demyelination through inhibition of TLR4/NF-KB pathway / 中国药理学通报

Aim To explore the protective effects of ganoderma lucidum polysaccharides (GLPS) on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS) and the underlying mechanism. Methods Thirty C57BL/6 mice were randomly divided into three groups: normal control group, EAE model group and GLPS group (5 mg • kg

JAK inhibition ameliorated experimental autoimmune encephalomyelitis by blocking GM-CSF-driven inflammatory signature of monocytes

Monocytes are key effectors in autoimmunity-related diseases in the central nervous system (CNS) due to the critical roles of these cells in the production of proinflammatory cytokines, differentiation of T-helper (Th) cells, and antigen presentation. The JAK-STAT signaling is crucial for initiating monocytes induced immune responses by relaying cytokines signaling. However, the role of this pathway in modulating the communication between monocytes and Th cells in the pathogenesis of multiple sclerosis (MS) is unclear. Here, we show that the JAK1/2/3 and STAT1/3/5/6 subtypes involved in the demyelination mediated by the differentiation of pathological Th1 and Th17 and the CNS-infiltrating inflammatory monocytes in experimental autoimmune encephalomyelitis (EAE), a model for MS. JAK inhibition prevented the CNS-infiltrating CCR2-dependent Ly6Chi monocytes and monocyte-derived dendritic cells in EAE mice. In parallel, the proportion of GM-CSF+CD4+ T cells and GM-CSF secretion were decreased in pathological Th17 cells by JAK inhibition, which in turns converted CNS-invading monocytes into antigen-presenting cells to mediate tissue damage. Together, our data highlight the therapeutic potential of JAK inhibition in treating EAE by blocking the GM-CSF-driven inflammatory signature of monocytes.

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.

Effect of Yishen Daluo Prescription on Rho/ROCK Signaling Pathway in EAE Mice Based on Silencing of β-arrestin1 / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Yishen Daluo prescription （YSDL） on Ras homolog（Rho）/Rho-associated coiled-coil containing protein kinase（ROCK）signaling pathway in mice with experimental autoimmune encephalomyelitis （EAE） based on the silencing of β-arrestin1 gene. MethodSixty C57BL/6 female mice were randomly divided into a blank group， a model group， a virus group， a YSDL group， a virus + YSDL group， and a prednisone acetate group （hormone group）. The EAE model was induced in mice except for those in the normal group. Adeno-associated virus（AAV）solution （150 μL， 1×1011 vg·mL-1） was injected into the tail vein of each mouse in the virus group and the virus + YSDL group on the 4th day of immunization. Drugs were administered on the 8th day of modeling. Specifically， normal saline was given to the mice in the normal group，the model group，and the virus group at 10 mL∙kg-1， prednisone acetate suspension to those in the hormone group at 3.9 g∙kg-1，and YSDL to those in other groups at 20 g∙kg-1 for 14 consecutive days. The mice were weighed and scored every day. The neurological function scores of mice in each group were recorded every day after immunization. Hematoxylin-eosin （HE） staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice. The protein expression of β-arrestin1，Ras homolog gene family member A（RhoA）， and Rho-associated coiled-coil forming protein kinase Ⅰ（ROCK Ⅰ） in spinal cord and brain tissues of EAE mice was determined by Western blot. ResultCompared with the model group， the virus group and the virus + YSDL group showed decreased neurological function scores （P<0.01），and the YSDL group also showed decreased neurological function scores（P<0.05）. HE results showed that there was obvious inflammatory reaction in the central nervous system （CNS） of the model group， which was alleviated to varying degrees in other groups compared with the model group. Western blot results showed that compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the spinal cord tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression levels of β-arrestin1， RhoA， and ROCKⅠ in the spinal cord tissues （P<0.01）. Compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the brain tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression level of β-arrestin1 in the brain tissues （P<0.01）， and the virus group and the YSDL group showed decreased protein expression levels of RhoA， and ROCKⅠ in the brain tissues （P<0.05）. Additionally， the virus + YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠ in the brain tissues （P<0.01）. ConclusionYSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS. The mechanism is presumably attributed to the fact that YSDL inhibits the expression of β-arrestin1 in CNS，thereby reducing the expression of Rho/ROCK signaling pathway. Furthermore， YSDL may have a synergistic effect with the inhibition of β-arrestin1 gene expression.

Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice / 西安交通大学学报(医学版)

【Objective】 To explore the effect and mechanism of Fasudil in the treatment of experimental autoimmune myocarditis (EAM) in mice so as to provide a theoretical basis for the clinical use of Fasudil in treating myocarditis. 【Methods】 Balb/c male mice were used as the research objects, and the EAM mice model was constructed using MyHC-α614-629 polypeptide. Mononuclear cells were isolated and cultured to detect the number of mononuclear cells in mouse spleen. Inflammation infiltration, fibrosis and IL-6 expression in mouse myocardial tissue were detected by HE staining, Masson staining and immunohistochemistry, respectively. The protein expressions of Notch1 and IL-6 were detected by Western blotting. qRT-PCR was used to detect the expressions of pro-inflammatory factors (IL-1α, IL-1β and IL-6) as well as key genes of TLRs and NOTCH signaling pathway. 【Results】 EAM mice showed increased HW, decreased BW, increased HW/e-BW, and increased inflammatory infiltration and fibrosis in myocardial tissue. The above-mentioned symptoms or pathological features were improved in EAM mice treated with Fasudil. The analysis showed that the pro-inflammatory factors IL-1α, IL-1β and IL-6 in the myocardial tissue of EAM mice were significantly increased, but only the expression of IL-6 was statistically different after Fasudil treatment compared with the control group. In addition, TLRs signaling pathway might also play an important role in the EAM mice treated with Fasudil. The expressions of IL-6 and Notch1 were consistent, and the expressions of the key genes of NOTCH signaling pathway (Notch1, Hes1 and Jag2) were down-regulated after Fasudil treatment. 【Conclusion】 Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice.

Cornuside ameliorated experimental autoimmune encephalomyelitis by limiting the recruitment of CD4+ T lymphocytes in the spinal cord

We conducted this study to determine whether cornuside could improve the neurological deficit symptoms of experimental autoimmune encephalomyelitis (EAE) rats, as well as determine the potential involvement of CD4+ T lymphocytes, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and tumor necrosis factor-α (TNF-α). Altogether, 32 Lewis rats were randomly divided into control, EAE, EAE/prednisolone, and EAE/cornuside, wherein their neurological function was assessed every day. CD4+ T lymphocyte recruitment into the spinal cord (SC) was evaluated using immunohistochemistry. The VCAM-1, ICAM-1 and TNF-α mRNA expressions in the SC were determined by real-time quantitative PCR, and the VCAM-1 and ICAM-1 proteins were determined by western blotting. Compared to the control group, the EAE group rats with neurological deficits had enhanced CD4+ T lymphocyte infiltration and higher expression levels of VCAM-1, ICAM-1, and TNF-α in the SC. Meanwhile, compared with the EAE group, the EAE/cornuside and EAE/prednisolone groups had lower neurological scores, less CD4+ T lymphocyte infiltrations, and lower expression levels of VCAM-1, ICAM-1, and TNF-α in the SC. Thus, cornuside ameliorated EAE, which could be owed to the inhibition of CD4+ T lymphocyte recruitment and VCAM-1, ICAM-1, and TNF-α expressions in the SC

Excessive iodine promotes the occurrence and development of experimental autoimmune thyroiditis in mice through p38 and intercellular adhesion molecule-1 / 中华地方病学杂志

Objective:To investigate the molecular mechanism of excessive iodine induced experimental autoimmune thyroiditis (EAT) in mice.Methods:Sixty female non-obese diabetic (NOD) mice were selected and divided into 5 groups according to body weight [(25 ± 3) g] via the random number table method, with 12 mice in each group: control group (group A), 10-fold high iodine group (group B), 100-fold high iodine group (group C), 1 000-fold high iodine group (group D) and 1 000-fold high iodine combined with polyinosinic acid-polycytidylic acid [Poly (I:C)] group (group E). The experiment period was 16 weeks. Mice in each group drank purified water with sodium iodine (NaI) content of 0.000, 0.005, 0.050, 0.500 and 0.500 mg/L, respectively; mice in group E were intraperitoneally injected with Poly (I:C) at week 7 and week 15, respectively. At the end of the 16th week, mice were dissected and blood samples and thyroid tissue were taken. The levels of serum thyroid function indexes [thyroid stimulating hormone (TSH), free triiodothyronine (FT 3), free thyroxine (FT 4), and thyroid peroxidase antibody (TPOAb)] were detected by enzyme-linked immunosorbent assay (ELISA); the pathological changes of thyroid tissue were observed after hematoxylin-eosin (HE) staining; differentially expressed genes in thyroid tissue were detected by RNA-sequencing (RNA-seq), and analyzed by KEGG pathway; mRNA and protein levels of p38, intercellular adhesion molecule-1 (ICAM-1) and chemokine 10 (CXCL10) in thyroid tissue were detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting, respectively. Results:There were statistically significant differences in serum levels of TSH (ng/ml: 6.53 ± 0.86, 6.61 ± 0.82, 7.68 ± 0.55, 7.93 ± 0.60, 8.73 ± 1.60), FT 3 (pg/ml: 59.35 ± 10.16, 53.73 ± 10.96, 46.19 ± 8.03, 41.01 ± 8.67, 34.21 ± 11.75), FT 4 (pg/ml: 136.74 ± 10.06, 124.33 ± 14.34, 101.80 ± 6.78, 91.37 ± 6.75, 73.29 ± 17.31), and TPOAb (U/ml: 130.81 ± 24.53, 145.47 ± 28.89, 166.52 ± 41.59, 199.78 ± 42.19, 201.99 ± 44.03) among the 5 groups of mice ( F = 4.77, 4.96, 23.12, 3.68, P < 0.05). Compared with group A, the serum TSH levels of mice in groups C, D and E were higher, the levels of FT 3 and FT 4 in groups B, C, D and E were lower, and the levels of TPOAb in groups D and E were higher, and the differences were statistically significant ( P < 0.05). HE staining showed that the thyroid follicle lesion in groups D and E was serious, and the EAT phenotype appeared in both groups. The differentially expressed genes were analyzed by KEGG pathway. Compared with group A, 8 metabolic pathways related to thyroid autoimmunity and inflammation were found in groups B, C, D and E. Further analysis found that 3 genes appeared in multiple pathways, namely p38, ICAM-1 and CXCL10. There were significant differences in the mRNA levels of p38, ICAM-1 and CXCL10 in thyroid tissue of the 5 groups of mice ( F = 14.77, 12.76, 16.39, P < 0.05); compared with group A, the mRNA levels of p38 in groups B, C, D and E were higher, and the mRNA levels of ICAM-1 and CXCL10 in groups C, D and E were higher ( P < 0.05). There were significant differences in the protein levels of p38, ICAM-1 and CXCL10 in thyroid tissue of the 5 groups of mice ( F = 7.97, 73.86, 18.02, P < 0.05); compared with group A, the protein levels of ICAM-1 and CXCL10 in groups B, C, D and E were higher ( P < 0.05). Conclusion:Excessive iodine promotes the occurrence and development of EAT in mice by up-regulating the expressions of p38 and ICAM-1 genes that are closely related to thyroid autoimmune and inflammatory responses.

Effect of interleukin-23R overexpression on Th17/Treg balance in experimental autoimmune uveitis mice / 中华眼底病杂志

Objective:To investigate the effect of interleukin (IL)-23 receptor (IL-23R) overexpression on the balance of T helper 17 (Th17 cells)/regulatory T cells (Treg cells) in experimental autoimmune uveitis (EAU) mice.Methods:Twelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group, with 6 mice in each group. Two groups of mice were injected with LV-Ctrl and LV-IL-23R lentiviruses through the tail vein, respectively; 7 days after injection, the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors. Starting from 13 days after immunization, the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed; 30 days after immunization, hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina. The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay; the proportion of Th17 cells and Treg cells was detected by flow cytometry. The relative mRNA expression of IL-23R, IL-17, retinoic acid-related orphan receptor γt (RORγt), IL-10 and forkhead transcripyion factor p3 (Foxp3) were detected by real-time quantitative polymerase chain reaction. Comparisons between groups were performed using repeated measures analysis of variance, independent samples Mann-Whitney U test, and independent samples t test. Results:Compared with the LV-Ctrl group, the retinal inflammatory reaction of the LV-IL-23R group was more severe. At 13 days after immunization, there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group ( t=-2.001, P=0.058); 15-29 days after immunization. The fundus inflammation scores of LV-IL-23R group were higher than those of LV-Ctrl group, and the difference was statistically significant ( t=-4.429,-6.578, -7.768, -10.183, -6.325, -7.304, -4.841, -6.872; P<0.001). Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased, the retinal structure was damaged more seriously, and the histopathological score was significantly increased, and the difference was statistically significant ( t=-4.339, P=0.001). Compared with the LV-Ctrl group, the relative expression of IL-23R mRNA in the spleen of the LV-IL-23R group was significantly increased, and the difference was statistically significant ( Z=2.087, P=0.037). The relative expression of IL-17 and RORγt mRNA increased, while the relative expression of IL-10 and Foxp3 mRNA decreased, and the differences were statistically significant ( t=-6.313,-5.922, 4.844, 7.572; P=0.003, 0.004, 0.008, 0.002). Compared with the LV-Ctrl group, the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased, and the difference was statistically significant ( t=-5.423, P=0.002); the proportion of Th17 cells in the spleen and lymph nodes was significantly increased, whereas, the proportion of Treg cells was significantly reduced, and the difference was statistically significant ( t=-4.290, 3.700; P=0.002, 0.006). Conclusion:IL-23R overexpression can promote Th17/Treg imbalance in EAU mice, and aggravate the clinical and pathological manifestations of EAU.

LncRNA Gm13568 regulates the activation of A1 astrocytes and affects the EAE process in mice / 中华微生物学和免疫学杂志

Objective:To investigate the effects of long non-coding RNA (lncRNA) Gm13568 on the activation of A1 astrocytes and the progress of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:A recombinant lentiviral vector (LV-Inhibit-Gm13568) carrying astrocyte-specific promoter of glial fibrillary acidic protein (GFAP) was established to inhibit the function of endogenous Gm13568. A control vector (LV-ctrl) was established as well. The recombinant vectors were packaged. C57BL/6 mice were injected with 1×10 7 transforming units of viral suspension via the tail vein and 7 d after the injection, myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) was used to establish the mouse model of EAE. Four groups, PBS group, EAE group, LV-ctrl+ EAE group and LV-Inhibit-Gm13568+ EAE group, were included in this study. Clinical signs of the mice were monitored daily in a double-blinded manner. The mice were sacrificed 23 d after the EAE model was established and the spinal cord tissues were collected. The expression of Serping 1, C3, Srgn and H2-T23 at mRNA level was detected by real-time PCR. Changes in the expression of IL-6, TNF-α, macrophage chemotactic protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) were measured. Western blot was used to investigate the expression of GFAP and Notch1 in spinal cord tissues and the phosphorylation of signal transduction and transcription activator 3 (STAT3). The expression of Notch1 intracellular domain (NICD) and GFAP in spinal cord tissues was detected by immunofluorescence. Furthermore, the infiltration of inflammatory cells and the demyelination of spinal cord were observed using HE and Luxol fast blue (LFB) staining methods. Results:Compared with PBS group, A1 astrocytes were activated and Notch1 expression was significantly up-regulated in EAE group and LV-ctrl+ EAE group. The clinical score of mice in LV-Inhibit-Gm13568+ EAE group was decreased from an average score of 3.5 to less than 1 on 23 d after antigen induction and the clinical symptoms were alleviated as compared with the mice in LV-ctrl+ EAE group. Meanwhile, the activation of A1 astrocytes was down-regulated, and the production of inflammatory cytokines and chemokines was also reduced. The expression of Notch1, GFAP and NICD at protein level and the phosphorylation of STAT3 were significantly reduced. Moreover, the infiltration of inflammatory cells and demyelination of spinal cord tissues were alleviated significantly.Conclusions:LncRNA Gm13568 might regulate the activation of A1 astrocytes via the Notch1/STAT3 pathway, thus affecting the production of inflammatory cytokines and chemokines and participating in the process of EAE.

Study on the effect of miR-223-3p regulating transcription factor Rbpj on the differentiation of Th1 and Th17 cells in rats with experimental autoimmune uveitis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the regulatory effect of miR-223-3p on the expression of transcription factor Rbpj and on the differentiation of Th1 and Th17 cells in experimental autoimmune uveitis(EAU)rats.<p>METHODS: The regulatory role of miR-223-3p in Rbpj gene expression was investigated by a dual luciferase expression reporter system. In the present study, 24 female Lewis rats were randomly divided into EAU model group, normal control(NC)group and blank control(BC)group, and each group included 8 rats. The EAU model group was injected with interphotoreceptor retinoid-binding protein(IRBP)emulsion containing Mycobacterium tuberculin H37RA and complete Freund's adjuvant to induce uveitis, while the NC group was injected with an equal volume of emulsion without IRBP peptide. The rats in the BC group received the same volume of sterile saline solution. At 12d after immunization, the spleen, lymph node and eye tissues in both groups were aseptically isolated, and the expression levels of miR-223-3p and Rbpj RNAs were detected by real-time quantitative PCR(Q-PCR); Meanwhile, the expression levels of Rbpj, IFN-γ and IL-17 proteins were detected by ELISA, and the levels of Th1 and Th17 cell lineages in each tissue from each groups were detected by flow cytometry. <p>RESULTS: The results of dual fluorescein assay indicated that Rbpj was the target gene which regulated by miR-223-3p. At 12d after immunization, compared with NC group, the relative expression levels of miR-223-3p in spleen, lymph node and eye tissues from EAU model rats were 0.33±0.29, 0.11±0.12 and 0.18±0.11, respectively, accompanied by the down-regulated expression, and the differences were statistically significant(all <i>P</i><0.05); Rbpj mRNA levels were 3.00±0.06, 1.52±0.12 and 3.01±0.34, respectively, and were all up-regulated, while the differences were statistically significant(all <i>P</i><0.05). Moreover, the differences in miR-223-3p and Rbpj mRNA levels in spleen, lymph node and eye tissues of rats in the blank control group were not statistically significant compared with those in the NC group(<i>P</i>>0.05); ELISA results revealed that the expression levels of RBPJ, IFN-γ and IL-17 proteins in all tissues from EAU rats at 12d after immunization were significantly higher than those in the NC group( all <i>P</i><0.05), and there was no statistically significant difference in the expression levels of Rbpj, IFN-γ and IL-17 protein in all tissues of rats in the blank control group compared with the NC group(<i>P</i>>0.05); Meanwhile, flow cytometry results showed that the proportions of Th1 and Th17 cell lineages in all tissues from EAU model group were significantly higher than those from the NC group at 12d after immunization, and the differences were statistically significant(all <i>P</i><0.05). Furthermore,there was no significant change in the proportion of Th1 and Th17 cells in each tissue in the BC and NC groups(all <i>P</i> >0.05). <p>CONCLUSION: The miR-223-3p can negatively regulate the expression of the transcription factor Rbpj of Notch signaling pathway. The down-regulated miR-223-3p expression in EAU rats can increase the expression levels of Rbpj gene and protein, and aggravate the differentiation of Th1 and Th17 cells and the expression levels of related molecules IFN-γ and IL-17, which in turn affect the development of uveitis.

Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice

Aging-induced changes in the immune system are associated with a higher incidence of infection and vaccination failure. Lymph nodes, which filter the lymph to identify and fight infections, play a central role in this process. However, careful characterization of the impact of aging on lymph nodes and associated autoimmune diseases is lacking. We combined single-cell RNA sequencing (scRNA-seq) with flow cytometry to delineate the immune cell atlas of cervical draining lymph nodes (CDLNs) of both young and old mice with or without experimental autoimmune uveitis (EAU). We found extensive and complicated changes in the cellular constituents of CDLNs during aging. When confronted with autoimmune challenges, old mice developed milder EAU compared to young mice. Within this EAU process, we highlighted that the pathogenicity of T helper 17 cells (Th17) was dampened, as shown by reduced GM-CSF secretion in old mice. The mitigated secretion of GM-CSF contributed to alleviation of IL-23 secretion by antigen-presenting cells (APCs) and may, in turn, weaken APCs' effects on facilitating the pathogenicity of Th17 cells. Meanwhile, our study further unveiled that aging downregulated GM-CSF secretion through reducing both the transcript and protein levels of IL-23R in Th17 cells from CDLNs. Overall, aging altered immune cell responses, especially through toning down Th17 cells, counteracting EAU challenge in old mice.

Research progress and application of animal models for drug development in multiple sclerosis / 中国药理学通报

Multiple sclerosis ( MS) is an immune-mediated chro¬nic inflammatory disease of the central nervous system (CNS) , which is regulated by multiple pathophysiological mechanisms.There are four clinical phenotypes of MS, including relapsing-re- mitting MS ( RRMS) , primary progressive MS ( PPMS) , sec- ondary-pmgressive MS ( SPMS) , and progressive relapsing MS ( PRMS) , among which RRMS is the main type.'Hie pathogen¬esis of MS is not clear and it could not he cured, so long-term drug treatment is needed for the MS patients.Nowadays, animal models play an important role in the preclinical research of MS drugs.'Hie MS animal models are mainly divided into experi¬mental autoimmune encephalomyelitis (EAE) model, toxin in¬ duced demyelination model, and vims induced demyelination model, among which EAE model is most widely used.'Hie three types of MS animal models demonstrate specific characteristics due to the different induction methods and animal species, and they correspond to specific clinical types of MS.According to the different clinical types of MS, the use of appropriate animal models for drug research and development will help us develop more targeted and potential therapeutic dnrgs, making it possible to cure MS.