Amlexanox Attenuates Experimental Autoimmune Encephalomyelitis By Inhibiting Dendritic Cell Maturation Through TBK1/Akt Signaling Pathways

Amlexanox(ALX),a TBK1 inhibitor,can modulate immune responses and has anti-inflammatory function.To investigate its role in regulating the development and progression of experimental autoimmune encephalomyelitis(EAE),we studied the effect of ALX on dendritic cell(DC)maturation and TBK1/Akt signaling in vitro and in vivo.Our data showed that ALX inhibits the maturation of bone marrow-derived dendritic cells(BMDCs),and BMDC-stimulated T cell proliferation in vitro,accompanied by decreased TBK1 activity and AKT phosphorylation.ALX inhibited the development and severity of inducible EAE in C57BL/6 mice by decreasing inflammation and demyelination in the central nervous system(CNS).Treatment with ALX reduced the frequency of splenic Th1 and Th17 cells and levels of IFN-? and IL-17 in serum,spleen and spinal cord.T-bet and ROR?t was also decreased in spleen and spinal cord particularly in the lumbar enlargement of mice.Frequency of CD11c+CD80+ and CD11c+CD86+ dendritic cells was decreased with significantly impaired maturation of dendritic cells via lowering CD80,CD86 and MHC II expression and serum and splenic IL-12 and IL-23 concentration in mice.Finally,treatment with ALX also mitigated AKT phosphorylation in the spleenic DC of mice.These data indicated that ALX ameliorates EAE at least partially through inhibiting DC maturation and pathogenic Th1 and Th17 responses by targeting TBK1/Akt Pathways in dendritic cells.Therefore,TBK1 may be an attractive therapeutic target and potentially ALX is valuable for intervention of multiple sclerosis.Part one Amlexanox inhibits BMDC phenotypic and functional maturation through the TBK1/Akt signaling pathway Objective: Bone marrow cells(BMC)were used to generate DCs in the presence of GM-CSF.Cells were stimulated with LPS with or without different concentrations(0 to 500 ?M)of ALX.We explored the influence of ALX on viatability,maturation and DC-stimulated proliferation of T cells.Methods:1 Bone marrow cells(BMC)were freshly isolated from the tibia and femur bones of C57BL/6 mice.The cells were cultured in the presence of GM-CSF to generated BMDCs.After 8-day culture,some cells were stimulated with LPS with or without different concentrations(0 to 500 ?M)of ALX for 48 h.Then the different groups of BMDCs were collected.2 The viability of BMDCs was determined by the Cell Counting Kit-8assay(CCK8).the different groups of BMDCs were co-cultured in triplicate with T cells at a ratio(DC:T)of 1:5,1:10,or 1:20,respectively in 96-well plates for 2 days.The proliferation in individual groups of T cells was determined by CCK-8 assay.3 The cells were stained with PE-anti-CD80,PE-anti-CD86,PE-anti-MHC II and analyzed by flow cytometry.4 The morphology of BMDCs from various group was examined by scanning electron microscope(SEM)and transmission electron microscope(TEM).5 The level of IL-6?IL-12 and IL-23 in cultured supernatants of BMDCs was detected by ELISA.6 The levels of TBK1?p-TBK1(Ser172)?Akt?p-Akt(Ser473,Thr308)in BMDCs were detected by western blot.7 SPSS 23.0 software was used for the statistical analysis.The data are presented as mean± standard error of mean(SEM).The statistical comparisons among two groups were examined using the Student's t-test or t'-test.The statistical comparisons among groups were examined using the Dunnet t multiple comparison tests.A value of P< 0.05 was considered significant.Results:1.Electron microscope was used to detect the GM-CSF induced BMDC.On day 8,the dendritic structure was short and scarce.After 2-days treatment of LPS,dendritic structure developed abundantly.2.The proliferation of BMDCs was determined by CCK-8 analysis.Treatment with ALX at high doses(20-100 ?M)significantly inhibited LPS-induced proliferation of BMDCs,while having no effect at low concentrations(2-10 ?M).3.Proliferation of CD4+ T cells was determined by CCK-8 assay and co-culture of LPS-stimulated BMDCs with CD4+ T cells promoted remarkable T cell proliferation in a dose-dependent manner.This BMDC-mediated allogeneic T cell proliferation was suppressed with the addition of ALX(2?M,10?M).4.Flow cytometric analysis showed that LPS substantially induced high levels of CD80,CD86 and MHC II;co-treatment with ALX did not change CD80,but significantly reduced the levels of CD86 and MHC II in LPS-activated BMDCs.5.The results showed that ALX addition reversed LPS-induced IL-12 and IL-23 elevation,but had no effect on IL-6 expression at the indicated doses.6.LPS increase the phosphorylation of all these proteins(p-Akt-T308,p-Art-S473,p-TBK1-S172).Addition of ALX further enhanced p-TBK1-S172,but drastically inhibited LPS-induced phosphorylation of Akt-T308,Akt-S473.Part two Amlexanox ameliorates EAE by inhibiting Th1 and Th17 cellullar responses in mice Objective: The experiment taken EAE mice as the animal model and ALX was administrated orally.EAE development and weight of mice in each group were evaluated.We explored the influence of ALX to the differentiation and function of Th1 and Th17 cells in spleen and CNS.Methods:1 Female C57BL/6 mice at 6-8 weeks old and weighing at 18-20 g C57BL/6 mice were immunized subcutaneously at the base of tail with 250 ?g myelin oligodendrocyte glycoprotein peptide MOG35-55 in 50% complete Freund's adjuvan,containing 4 mg/ml heat-inactivated Mycobacterium tuberculosis H37 Ra.On day 0 and 2,the mice were injected intraperitoneally with 500 ng pertussis toxin.2 The control group of mice receiving carboxymethylcellulose sodium(CMC)by gavage.The immunized mice were randomized and treated with CMC or ALX at 50 mg/kg twice daily by gavage.From day 1,the mice neurological symptom was evaluated by two observers twice a day and recorded the scores according to Knoz scoring method.3 The lumbar enlargement of spinal cord were taken and studied by HE staining,LFB staining and transmission electron microscopy to evaluate the inflammatory infiltration and demyelination respectively.4 Flow cytometry was used to detect the ratio of Th1 and Th17 cells in spleen.5 The level of IFN-? and IL-17 in cultured supernatants of spleen cells and serum was detected by ELISA.6 The m RNA transcriptional levels of IFN-?,IL-17,ROR?t and T-bet in spleen and spinal cord were detected by rt PCR.7 The levels of IFN-? and IL-17 in spinal cord were detected by western blot.8 SPSS23.0 software was used for the statistical analysis.The data are presented as mean ± standard error of mean(SEM).The difference in scores between two groups of mice was analyzed using the Mann–Whitney U test.All other statistical comparisons among two groups were examined using the Student's t-test or t'-test.A value of P< 0.05 was considered significant.Results:1.ALX treatment significantly retarded EAE development and reduced disease scores.ALX alleviated the weight loss during the progression of EAE.2.According to HE staining,LFB staining and TEM,inflammation and demyelination were significantly reduced in the white matter of the spinal cord from the ALX-treated mice versus Vehicle.3.The percentages of splenic Th1 and Th17 cells,as well as levels of IFN-? and IL-17 A in serum and supernatant of splenocyte in the ALX mice were significantly lower than in Vehicle.Splenic IFN-?,T-bet,ROR?t,and IL-17 A m RNAs in ALX-treated mice were significantly lower than in Vehicle.4.Less IFN-?+ and IL-17A+ cells from ALX-treated mice.Expression of IFN-? and IL-17 A protein and IFN-?,T-bet,ROR?t,and IL-17 A m RNAs in the lumbar regions from the ALX-treated mice were significantly reduced,as compared with the Vehicle group.Part three ALX inhibits maturation of splenic DCs though TBK1/Akt signaling pathway Objective: The experiment taken EAE mice as the animal model and ALX was administrated orally.We explored the influence of ALX to the maturation of DCs in spleen and TBK1/Akt signaling pathway.Methods:1 Female C57BL/6 mice at 6-8 weeks old and weighing at 18-20 g C57BL/6 mice were immunized as stated in part two.2 The control group of mice receiving carboxymethylcellulose sodium(CMC)by gavage.The immunized mice were randomized and treated with CMC or ALX at 50 mg/kg twice daily by gavage.3 Flow cytometry was used to detect the expression of MHC II,CD80 and CD86 in CD11c+ cells in spleen.4 The level of IL-6?IL-12 and IL-23 in cultured supernatants of spleen cells and serum was detected by ELISA.5 The splenic tissue sections from individual mice were stained with anti-CD11 c and intracellularly stained with anti-p AKT(S473)or anti-p AKT(T308),followed by staining with DAPI.The fluorescent signals were examined under a fluorescent microscope.6 SPSS23.0 software was used for the statistical analysis.The data are presented as mean±standard error of mean(SEM).All other statistical comparisons among two groups were examined using the Student's t-test or t'-test.A value of P< 0.05 was considered significant.Results:1.In comparison with the Vehicle,ALX significantly decreased the frequency of CD80+ and CD86+,but not MHC II+ cells,and potently reduced mean fluorescence intensity(MFI)of CD80,CD86 and MHC II in CD11c+cells.2.IL-12 and IL-23,but not IL-6,in cultured supernatants of splenocyte and serum of ALX-treated mice were significantly lower than in Vehicle mice.3.Immunofluorescent analysis revealed that the expression of p AKT(S473)and p AKT(T308)was decreased in the splenic CD11c+ cells of ALX-treated mice compared to Vehicle mice.Conclusions:Amlexanox might attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and Th1 and Th17 cells differentiation through the TBK1/Akt signaling pathways in DCs.