The Effects Of A2A Receptor Agonist And Parthenolide On The Immune Response Of Experimental Autoimmune Neuritis

Background:Guillain-Barre syndrome(GBS)is an acute inflammatory disease of peripheral nervous system,which is characterized by symmetrical ascending flaccid paralysis,with or without sensory disturbance.The pathological examination of nerves shows inflammatory cells infiltration,demyelination and axonal degeneration.The exact causes of GBS are not clear now,and there is no specific effective treatment.Currently,the predominant therapies are plasma exchange and intravenous immunoglobulin injection(IVIG).Plasma exchange requires special machines and the hospitals equipped with such facilities are not prevalent.Immunoglobulin is a blood product,which means that the resource is limited.Besides,both these two treatments are expensive.Thus,it is necessary to explore the pathogenesis of GBS and find other effective therapies.Adenosine is a naturally occurring nucleoside.The extracellular adenosine increased sharply in stress state.There are four types of adenosine receptors have been identified,A1,A2A,A2B,and A3 receptor.A2A receptor(A2AR)belongs to the G-protein-coupled receptor family.A2AR activation could inhibit the activity of macrophages,neutrophils,NK cells,NKT cells and lymphocytes.Its anti-inflammatory functions have been proved in a number of animal models,like ischemia-reperfusion injury and rheumatoid arthritis.Experimental autoimmune neuritis(EAN)is the animal for GBS,which is wildly used in studying the pathogenesis of GBS and screen new therapies.Although the anti-inflammatory functions of A2AR activation are shown in various studies,its role in EAN has not been reported.Objective:To explore the effects of A2AR activation in the immune response of EANMethods:1.EAN induction and clinical symptoms assessment Lewis rats were immunized with bovine peripheral myelin(BPM).The rats were observed,assessed,and weighted daily after immunization.The symptoms were graded as follows:0 ? no illness;1 = flaccid tail;2 = moderate paraparesis;3 = severe paraparesis;4 = tetraparesis;5 =death.2.Treatment with CGS21680The immunized rats were divided into two groups randomly:treatment group and control group.The two groups were injected intraperitoneally with A2AR specific agonist,CGS21680,(1mg/kg)and the same volume of saline respectively.Rats were injected daily from day 5 post immunization(p.i.)to the end of experiments.On day 17 p.i.,rats were sacrificed,and the sera,sciatic nerves and inguinal lymph nodes were collected.3.Flow cytometry analysis of cell surface molecules,intracellular cytokines and immune cell populationsLymph nodes were grained though strainers to get mononuclear cells(MNCs).MNCs were stained with fluorescein conjugated antibodies against cell surface molecules(CD3,CD4,CD8,CD161a,CD80,CD86,CD103,CD19 and ?? TCR)or intracellular cytokines(IL-10,IL-4,IL-17A,TNF-? and IFN-y).Regulatory T(Treg)cells and follicular helper T(Tfh)cells were marked as CD4+Foxp3+ cells and CD4+CXCR5+ cells respectively.After staining,cells were analyzed with a flow cytometer.4.Flow cytometry analysis of lymphocyte proliferationLymph node MNCs were stained with carboxy-fluorescein diacetate,succinimidyl ester(CFSE).Then MNCs were incubated in the presence of ConA or P0 peptide(180-199)for 3 days.Finally cells were collected and analyzed by a flow cytometer.5.ELISA analysis of the levels of IL-2 in seraMNCs were incubated in the presence of P0 peptide(180-199).3 days later,the culture supernatants were collected and the levels of IL-2 in the supernatants were analyzed with a rat IL-2 ELISA kit.5.ELISA analysis of the levels of IgG and IgG subtypes in sera96 well plates were coated with PO peptide(180-199).The levels of P0 peptide-specific IgG,IgG 1,IgG2a and IgG2b in sera were detected by indirect ELISA method.6.Histological analysis of inflammatory cell infiltration and demyelination of peripheral nerves After fixation and dehydration,the sciatic nerves of each rat were processed into paraffin sections.Then hematoxylin and eosin(HE)staining,immunohistochemistry staining,and fast blue staining were performed to detect infiltrated inflammatory cells,macrophages(CD68+ cells)and demyelination respectively.7.Statistical analysisData were analyzed with GraphPad Prism 6.Clinical scores and demyelination scores were analyzed using Mann-Whitney test.The other results were analyzed using student's t-test.Significance level was set at p<0.05.Results:1.CGS21680 intervention promotes the development of EAN Compared with the rats from control group,the rats treated with CGS21680 developed symptoms earlier,showed severer paralysis,recovered later and lost more weight.The clinical scores of CGS21680 treated group were higher on day 11,13 and 14 p.i.,and the weights were lower on day 10,12,13 and 14 p.i..2.CGS21680 intervention promotes inflammatory cell infiltration and demyelination of sciatic nervesCompared with control group,sciatic nerves from CGS21680 intervention group showed more macrophages infiltration and demyelination.3.CGS21680 intervention elevates the levels of PO peptide-specific antibodies in serum ELISA analysis showed that the levels of PO peptide-specific IgG,IgG1 and IgG2a were elevated in CGS21680 intervention group.4.CGS21680 intervention suppresses Thl and Th17 cytokines,and powerfully inhibits lymphocyte proliferation and IL-2 secretionFlow cytometry analysis showed that the percentages of TNF-a,IFN-y,and IL-17 positive cells among MNCs were decreased after CGS21680 intervention.Lymphocyte proliferation and IL-2 production were significantly suppressed after CGS21680 treatment.5.CGS21680 intervention reduces the proportions Treg cells while increases Tfh cells,B cells and dendritic cells in draining lymph nodesCompared with control group,the percentages of CD4+Foxp3+ Treg cells were decreased,while the percentages of CD4+CXCR5+ Tfh cells,B cells and dendritic cells were increased in CGS21680 intervention group.6.CGS21680 intervention increases the expressions of MHC class ? and CD86 Flow cytometry analysis showed that compared with control group,both the percentages of positive cells and the mean fluorescence intensities of MHC class ? and CD86 were increased after CGS21680 treatment.Conclusion:A2AR agonist CGS21680 intervention aggravated EAN in Lewis rats induced with BPM.It promoted the inflammatory cell infiltration and demyelination in peripheral nerves.Although CGS21680 inhibited pro-inflammatory cytokine production and lymphocyte proliferation,it decreased the proportions of Treg cells,increased Tfh cells and B cells,and promoted the formation of autoantibodies.Background:Parthenolide(PAR)is a sesquiterpene lactone extracted from feverfew(Tanacetum parthenium L.).PAR has been proved to have strong anti-inflammatory functions.Previous reports show that PAR could inhibit lymphocyte proliferation and proinflammatory cytokine production.The anti-inflammatory function of PAR is mostly attributed to the inhibition of NF-?B.PAR could target multiple molecules of NF-?B signaling pathway and PAR is often treated as a NF-?B inhibitor.NF-?B is an evolutional conservative transcription factor.It is first found to bind near the ? gene in B cells and thus named as nuclear factor binding near the Klight-chain gene in B cells.Further researches show that NF-?B is expressed in nearly all mammal cells and NF-?B participates in cell proliferation,differentiation,migration and survival.NF-?B plays a master role in immune response.A number of stimuli could induce the activation of NF-?B,such as infection,stress,cytokine,and chemical reagents.Activated NF-kB could be detected in the brain sample of both multiple sclerosis patients and its animal model,experimental autoimmune encephalitis(EAE).In animal models of GBS,experimental autoimmune neuritis(EAN),NF-?B is also found in peripheral nerves.Besides,PAR is shown to directly inhibit the activity of caspase-1 and the activation of inflammasome,which prevents the release of IL-1?.Its anti-inflammatory functions have been documented in various animal models such as cyclophosphamide-induced cystitis,sulfate sodium-induced colitis and collagen-induced arthritis.However,the effect of PAR in autoimmune diseases of nervous system has not been reported.Objective:To explore the effect of PAR in EAN.Methods:1.EAN induction and symptom assessment Lewis rats were immunized with bovine peripheral myelin(BPM).The rats were observed,assessed,and weighted daily after immunization.The symptoms were graded as follows:0 = no illness;1 = flaccid tail;2 = ataxia or mild paraparesis;3 = moderate paraparesis;4 = severe paraparesis;5 = tetraparesis;6 = moribund;7 = death.2.Treatment with PARThe rats were randomly divided into three groups:control group,low dose treatment group with 2 mg/kg PAR and high dose treatment group with 8mg/kg PAR.The treatments started on day 5 post immunization(p.i.).The rats in control group received the same volume of solvent in the same way.Rats were treated daily until the end of experiments.On day 10 and 14 p.i.,one set of rats were sacrificed.The sera,sciatic nerves and inguinal lymph nodes were harvested for further detection.3.Analysis of proinflammatory cytokine production and CD4+T lymphocyte populations of lymph node mononuclear cells(MNCs)by flow cytometryThe inguinal lymph nodes were grained through cell strainers to get mononuclear cells(MNCs).After staining with fluorescein-conjugated antibodies,MNCs were analyzed by flow cytometry to detect the percentages of intracellular cytokines TNF-a,IFN-y and IL-17,and the proportions of Thl(CD4+IFN+),Th17(CD4+IL-17+)and Treg(CD4+Foxp3+)cells.4.Detecting of apoptosis by flow cytometryLymph node MNCs were stained with apoptosis detection kit and then analyzed by flow cytometry.5.Analysis of the levels of IL-1? in sera by ELISA The levels of IL-1 ? in the sera collected from rats were analyzed with a rat IL-1? ELISA kit.6.Histological analysis of sciatic nervesAfter fixation and dehydration,the sciatic nerves of each rat were processed into paraffin sections.The sections were observed under a microscope after hematoxylin and eosin(HE)staining to examine the inflammatory cell infiltration.7.Lymphocyte proliferation assay in vitro Lymph node MNCs were harvested from rats on day 8 p.i..MNCs were fist stained with CFSE,and incubated in the presence of ConA or BPM.Different concentrations PAR were added into the culture media.3 days late,cells and culture supernatants were collected.Cells were analyzed by flow cytometry.The levels of IL-17 and TNF-a in supernatants were analyzed by ELISA.8.Statistical analysisStatistical analysis was performed with GraphPad Prism 6.Clinical scores were analyzed by nonparametric test.The other data were analyzed by ANOVA.The significance level was set at p<0.05.Results:1.PAR inhibits lymphocyte proliferation in vitro Results showed that PAR significantly inhibited both ConA induced antigen-nonspecific and BPM induced antigen-specific lymphocyte proliferations in a dose dependent manner.And the production of IL-17 and TNF-a were also suppressed.2.PAR treatment inhibits the initiation of EAN in early phase and impedes the recovery in late phaseIn the induction phase,the clinical scores of high dosed treated rats were lower than those of control group on day 11,12 and 13 p.i..The rats in low dose PAR treated group developed disease similarly with rats in control group.After day 14 p.i.,clinical scores were similar between high dose treated group and control group.The scores of low dose treated group were higher than control group on day 16,17 and 18 p.i..3.PAR treatment inhibits the production of TNF-?,IFN-?,IL-17 and IL-1? and decreases the proportions of Th1 and Th17 cells at early time point of EAN On day 10 p.i.,the results showed that high does PAR treatment inhibited the production of TNF-?,IFN-?,IL-17 and IL-1? and decreased the proportions of Thl and Th17 cells.Low dose PAR inhibited the production of IL-1?while it had little effect on other cytokines or cell populations.PAR treatment did not affect Treg cells.4.Neither high dose nor low dose PAR treatment inhibits proinflammatory cytokine production or modulates CD4+ T cell populations at late time point On day 14 p.i.,the examination of cytokines and T cell populations showed no significant difference among the three groups.5.PAR treatment inhibits the apoptosis of lymph node MNCs and inhibits the expression of Fas at late time pointOn day 10 and day 14 p.i.,the apoptosis and Fas/FasL expression of lymph node MNCs were examined.Results showed that at early time point there was no difference among three groups,while at late time point both high dose and low dose PAR treatment inhibited the apoptosis and the expression of Fas of MNCs.6.PAR treatment does not prevent the infiltration of inflammatory cells in peripheral nerve systemHistological examination showed lots of inflammatory cells infiltrated in all three groups and there was no difference among the three groups.Conclusion:PAR exhibited potent anti-inflammatory functions in vitro,but its effect in vivo is more complicated.PAR played dual roles in EAN.PAR treatment inhibited the development of EAN in initiation phase while impeded the recovery of EAN in late phase.PAR may not be a proper treatment for autoimmune diseases in nervous system.