Immune-regulation Of Sulfatides On Experimental Autoimmund Neuritis

BackgroundGuillain-Barre syndrome(GBS)is an autoimmune disease of peripheral nervous system,characterized by symmetrical limb weakness,limb areflexia,cranial nerve palsy,and respiratory failure.Based on clinical,electrophysiological and pathological characteristics,GBS can be divided into several variants:acute inflammatory demyelinating polyradiculoneuropathies(AIDP),acute motor axonal neuropathy,acute motor and sensory axonal neuropathy,Miller-Fisher sy drome,Pharyngeal-cervical-brachial(PCB),acute panautonomic neuropathy,and other rare variants.AIDP is the most common subtype.Experimental autoimmune neuritis(EAN)has many similarities with AIDP in immunology and clinical manifestations,and is an ideal animal model for studying the pathogenesis and treatment of GBS.Both AIDP and EAN is pathologically characterized by accumulation of autoreactive T cells and macrophages in the peripheral nervous system(PNS),and demyelination.The onset of AIDP and EAN is associated with Thl,Th2,Thl7,and Treg cells.The pathogenesis of AIDP and EAN is related to the expansion of Thl cells,which is characterized by secreting interferon-γ(IFN-γ)and tumor necrosis factora(TNF-a).Cytokines produced by Thl cells contribute to the disease development by recruiting inflammatory cells to the PNS and enhancing the release of other inflammatory products by other cells.Th2 cytokines include IL-4 and IL-10,which primarily promote B cell activation and inhibit macrophage activation.In EAN,Th1 cytokines mainly mediate inflammatory destruction of peripheral nerves,while Th2 cytokines are mainly involved in disease recovery.In addition,Thl7 cells also contribute to the development of EAN by secreting interleukin 17.Tregs are a T cell subset that negatively regulate immune responses and play an important role in the maintenance of self-tolerance.Previous studies have shown that Tregs can reduce inflammatory cell infiltration in the sciatic nerve and protect EAN.Sulfatides,also known as 3-O-galactosylsphingosine,are mainly distributed in the myelin of the nervous system,islet cells and kidneys.Sulfatides play an important role in maintaining myelin function and its stability.Mice lacking sulfatides will exhibit severe tremoring and even develop progressive hmdlimb paralysis.In addition,Sulfatides also have immunosuppressive effects mainly by activating type Ⅱ NKT cells.To date,studies have confirmed that sulfatides play preventive roles in the pathogenesis of experimental autoimmune encephalomyelitis(EAE),type 1 diabetes,and autoimmune liver disease,by suppressing Thl7 cells differentiation,type I natural killer T(NKT)cells,dentritic cells(DCs),IFN-γ and interleukin-4(IL-4)productions.Sulfatides can be presented to type Ⅱ NKT cells by CD 1d molecules expressed by antigen-presenting cells,such as DCs,macrophages,subsets of B cells.Sulfatide-mediated type Ⅱ NKT cell activation result in inhibition of type Ⅰ NKT cells,CD4+ and CD8+T cells.The pathogenesis of GBS is still inaccurate,and there is currently no effective specific treatment.At present,the mam treatment methods are intravenous immunoglobulin(IVIG)and plasma exchange(PE).However,these two methods are expensive and can only alleviate the condition of some patients.About 10%of patients still have severe physical disabilities,and even 3%-5%of patients die.Other patients still suffer from physical disabilities and even a small number of patients die.Therefore,it is necessary to explore new drugs for the treatment of GBS.Although many studies have demonstrated that sulfatides have protective effects against a variety of autoimmune diseases,there are currently no reports of sulfatides for the treatment of ENA.ObjectiveTo explore the effect of sulfatides on EAN and its immunological mechanism.Methods1.Induction of EAN animal model and evaluation of symptomsAnimal models were prepared by immunizing Lewis rats with bovine peripheral myelin(BPM).Rats were weighed and scored daily after immunization.Scoring criteria:O=no illness;1=paraparesis of the tail;2=paraparesis of the hind limbs;3=tetra-paresis;4=moribund;5=death.2.Grouping and treatment of EAN animal modelsENA rats were randomly divided into 2 groups:control group and treatment group.On days 5,8,and 11 after EAN induction,the rats in the treatment group were administered intraperitoneally(i.p.)with 140 μg sulfatides,and the rats in the control group were given the same dose of vehicle(0.5%polysorbate-20[Tween 20]and 0.9%saline).Rats were sacrificed 13 days after immunization at the peak of the disease.The lymph nodes and sciatic nerves of the rats were collected for later experiments.3.Flow cytometry analysisMononuclear cell suspensions of lymph nodes were obtained by grinding inguinal lymph nodes.CD4+IFN-γ+、CD4+IL4+、CD4+IL17A+、CD4+CD25+Foxp3+、CD3+CD161a and CD3-CD161a+cells were used to recognize Thl,Th2,Th17,Tregs,NKT and NK cells,respectively.Cell membrane surface molecules were labeled with fluorescein-labeled antibodies of CD3.CD4,CD161a,CD25,respectively.Tregs were labeled with CD4,CD25 and Foxp3 antibodies.Follicular helper T cells were labeled with fluorescein-labeled CD4,CXCR5,and ICOS antibodies.Intracellular cytokines were labeled with fluorescein-labeled IL-4,IL-10,TNF-α,IFN-γ,IL-17A antibodies.Cells were analyzed with a flow cytometer.4.Cell proliferation assayThe mononuclear cells(MNCs)of lymph nodes were stained with carboxy-fluorescein diacetate succinimidyl ester(CFSE).Then MNCs were cultured in a 37℃ incubator after adding ConA or BPM,and collected after 3 days.The cells were detected by flow cytometry5.Detection of IL-17 levels in cell proliferation supernatant by ELISAMNCs of Lymph nodes were cultured triplicates for 3 days in the presence of ConA or BPM at 37℃ in the incubator,and then the supernatants were collected.IL-17 levels were measured using a rat IL-17 ELISA kit6.Histopathology and immunohistochemistry of sciatic nerveThe sciatic nerves of rats were fixed with 4%paraformaldehyde,then dehydrated,embedded in paraffin and sliced into sections(3 μm).The inflammatory cell infiltration of the sciatic nerve was detected by HE staining.After paraffin section dewaxing and antigen reactivation,macrophages were labeled with rat CD68 antibody,and the infiltration of sciatic nerve macrophages was observed by immunohistochemistry7.Statistical analysisStatistical analysis of the data was performed using SPSS 22 and Prism 6 software.Data differences between the two groups were analyzed using the student’s t-test.Results were expressed as mean±SEM,and a level of p<0.05 was considered to be significantResults1.Sulfatides inhibits the development of EANRats in control group showed clinical symptoms on day 9 p.i.,while rats injected with sulfatides showed neurological deficits on day 10 p.i.The symptoms progressed rapidly and peaked around day 12 p.i..Clinical scores were lower in the sulfatides treatment group than those in control group on days 12 and I3p.i.(p<0.05).Clinical scores were 2.20±0.27 and 2.30±0.27 in the sulfatide treated group on days 12 and 13 p.i.,while clinical scores were 3.17±0.82 and 3.67± 1.25 respectively in control group.2.Sulfatides treatment inhibits Thl and Thl7 cellsImbalance of CD4+T cell subtypes plays a critical role in the pathogenesis of EAN.Thus,the differences of Thl,Th2,Th17,and Tregs in sulfatides-treated EAN rat lymph nodes were analyzed by flow cytometric analysis.Our data showed that the percentages of Th1 and Th17 cells in CD4+ cells are reduced compared with the control group.At the same time,tumor necrosis factor-α positive cells in CD4+ cells were also significantly reduced.However,there were no significant percentage differences of Th2 cells or interleukin-10 positive cells between the control group and sulfatides treatment group.Also,we did not observe any differences of Tregs between these two groups.As a result,sulfatides treatment could upregulate the ratios of Tregs to Thl and to Th17 cells.3.Sulfatides treatment inhibits Tfh cells and antigen-presenting cellsStudies have found that B cells participate in the pathogenesis of EAN.Tfh cells are the major T cell subset involved in helping B cell proliferation and differention.Thus we determined percentages of Tfh cells in different groups and found that sulfatides depressed the percentages of Tfh cells in lymph nodes.Previous studies demonstrated that sulfatides ameliorates the pathogenesis of EAE by suppressing dentritic cells,so the impacts of sulfatides on antigen presenting cells in lymph nodes were investigated.Our results showed that rats treated with sulfatides exhibited less CD80 and CD86 positive cells compared with control group.A trend but not statistical difference of decreases in MHC Ⅱ positive cells in lymph nodes were also observed after sulfatides treatment.4.Sulfatides treatment inhibits NK and NKT cellsNK and NKT cells are involved in the pathogenesis of autoimmune diseases.By activating type Ⅱ NKT cells,sulfatides inhibit type Ⅰ NKT cells and alleviate various autoimmune diseases.Therefore,we investigated the effects of sulfatides treatment on NK cells and NKT cells.We found that the percentages of NK and NKT cells among lymph node MNCs were decreased after sulfatides treatment.However,we did not observe any difference in IFN-γ-secreting NK or NKT cells.5.Sulfatides inhibits T cell proliferation and IL-17 secretion in vitroTo evaluate the effects of sulfatides on lymphocyte proliferation,lymphocytes from both groups were stained by CFSE and cultured in vitro in the presence of Con A or BPM for 72 h.Compared with control group,CD4+T cell proliferations were inhibited after sulfatides treatment both in Con A and BPM stimulation conditions The levels of IL-17 in MNCs’ culture supernatants in the presence of Con A or BPM were also suppressed after sulfatides treatment.6.Sulfatides treatment did not reduce inflammatory cell infiltration of peripheral nerves.To investigate whether sulfatides treatment could prevent inflammatory cell infiltration in peripheral nerve system,the sciatic nerves of rats were harvested and stained with HE and immunohistochemistry.HE examination showed lots of inflammatory cells infiltrated in both groups,but there was no difference between them.We also did not find any difference of macrophage cell infiltrations,which is shown by immunohistochemical stain of macrophage cell biomarker,CD68 molecule.ConclusionSulfatides delayed the onset and inhibited the development of EAN by inhibition of Thl,Thl7 cells,Tfh and antigen presenting cells.Since type Ⅱ NKT cells are present in humans,sulfatides may have a therapeutic potential in human Guillain-Barre syndrome by activating type Ⅱ NKT cells.