Roles And Mechanisms Of Exosomes Derived From Macrophages On Experimental Autoimmune Neuritis

BackgroundGuillain-Barre syndrome(GBS)epitomizes immune-mediated disorders against peripheral nervous system.Nowadays,it is the most common and severe acute paralytic neuropathy.Despite the fact that many patients benefit from current available standard therapeutic regimens,GBS remains to be a potentially life-threatening and disabling condition.Acute inflammatory demyelinating polyneuropathy(AIDP)is the major subtype of GBS,for which experimental autoimmune neuritis(EAN)is the well-accepted animal model.Studies from EAN and human pathological data have illuminated the immune rationale underlying AIDP,in which CD4+T cells and macrophages play dominant rolesMacrophages are the key component of the innate immunity.They maintain high plasticity and exhibite distinct phenotypes and functions in response to environmental changes.M1 macrophages,activated by inflammatory insults and T helper 1(Th1)cell derived IFN-?,can in turn shape the magnitude of adaptive immunity.M1 macrophages have well-characterized roles in the progressive stage of AIDP.M2 macrophages can arise in Th2 settings and have been implicated in inflammation resolution of EAN and subsequent promotion of tissue repair.Considering the engagement in both inflammation initiation and resolution,macrophages are emerging as a promising therapeutic target for GBS and other inflammatory disorders.Since effective functioning of macrophages requires elaborate interactions between macrophages and other types of immune cells,there arise demands for comprehensive explorations of the crosstalk network under certain contextsExosomes,with size ranging from 50 nm to 150 nm,belong to extracellular vesicles(EVs).Exosomes contain nucleic acids,proteins and lipids,and their potentials for mediating intercellular exchange of these functional components are increasingly appreciated,which has greatly expanded our knowledge of intercellular communication.A growing body of evidence has highlighted the importance of exosomes for macrophage biology under both homeostatic and pathological circumstances.However,the roles of macrophage derived exosomes in inflammatory disorders and the mechanisms behind have been understudied and remain blank in EAN/GBS.Objectives1.In this thesis,we sought to evaluate the effects of M1 and M2 macrophage derived exosomes on the development of EAN and investigated the cellular mechanisms involved,which may provide novel therapeutic targets for both GBS and other inflammatory disorders2.Another aim of the thesis was to investigate the regulatory effects of macrophage derived exosomes on T cells,which may further extend our understanding of the interaction network between macrophages and T cells and advance the development of macrophage-based therapeuticsMethodsPart ?1.Bone marrow derived macrophages(BMDMs)preparationFemurs and tibias were harvested from 6-8 week old healthy Lewis rats.Bone marrow mononuclear cells(MNCs)were prepared.Bone marrow progenitor cells were differentiated into BMDMs by recombinant rat M-CSF2.BMDM polarizationBMDMs were treated with LPS+recombinant rat IFN-? for M1 polarization.Nitric oxide(NO)and IL-12 production were examined by Griess reagents and ELISA,respectively.Recombinant rat IL-4 was used for inducing M2 polarization,and surface expression of CD 163 and CD206 was detected by flow cytometry3.Exosome isolation and characterizationThe conditioned media were collected.M1 macrophage derived exosomes(M1 exosomes)and M2 macrophage derived exosomes(M2 exosomes)were isolated by differential centrifugation.Transmission electron microscopy was used to characterize the morphology of exosomes.Flow cytometry and immunoblotting analysis were performed to detect surface and internal exosomal protein markers.Size distribution was measured by nanoparticle tracking analysis(NTA).4.Establishment of EAN and macrophage derived exosome treatmentTo establish EAN,rats were immunized at the base of tail subcutaneously with BPM emulsified in incomplete Freund's adjuvant containing Mycobacterium tuberculosis.Rats were then randomized into three groups and intravenously administrated with M1 or M2 exosomes,or with an equal volume of vehicle on day 5,day 7,day 9 and day 11 post immunization.Neurogenic signs of EAN and body weight were evaluated daily.All rats were euthanized on day 13 post immunization.Inguinal lymph nodes,spleens and sciatic nerves were harvested for further experiments5.HistologyHistological analysis was performed to evaluate cell infiltration and demyelination Sciatic nerves were fixed,dehydrated and embedded in paraffin.Longitudinal sections were prepared and stained with hematoxylin and eosin(H&E)and Luxol fast blue.6.Immunoregulatory effects of macrophage derived exosomes in vivoLymph node and splenic MNCs were prepared.Flow cytometry analysis was performed to evaluate the effects of M1 and M2 exosomes on Th cell subtypes,regulatory T(Treg)cells,germinal center B(GCB)cells and certain aspects of innate immunity.7.Statistical AnalysisStatistical analyses were performed with SPSS statistics 22.0.Normality and test of homogeneity of variance were performed by Shapiro-Wilk test and Levene's test,respectively.For data with normal distribution,hypothesis testing was performed by Student's t-test or one-way ANOVA.Multiple comparisons were conducted by LSD test with equal variances or Dunnett's T3 test without equal variances.For data without normal distribution,non-parametric test was applied,followed by pairwise comparisonsPart ?1.Preparation of M1 exosomes and M2 exosomesBone marrow MNC suspension from femurs and tibias of healthy Lewis rats were prepared,and BMDMs were differentiated from bone marrow progenitor cells with M-CSF.M1 macrophages and M2 macrophages were polarized with LPS+IFN-? and IL-4,respectively.Cell culture media from polarized macrophages were collected and M1 or M2 exosomes were isolated by differential centrifugation approach.2.Co-culture of macrophage derived exosomes with splenic MNCsSplenic MNCs were prepared and co-cultured with M1 or M2 exosomes.Flow cytometry and ELISA were performed to evaluate the effects of exosomes on IFN-?production of T cells.Proliferation and cell apoptosis of T cells were also detected by flow cytometry.3.Co-culture of Ml exosomes with bone marrow derived dendritic cells(BMDCs)BMDCs were differentiated from bone marrow progenitor cells with GM-CSF and IL-4,activated by LPS and co-cultured with M1 exosomes.Flow cytometry was used to detect CD80 and MHC ? expression,indicating the maturation status of these cells.4.Direct effects of M1 exosomes on T cellsSplenic T cells were purified by magnetic separation,and co-cultured with PKH26-labeled M1 or M2 exosomes.Samples were then imaged using a fluorescence microscope to confirmed cellular uptake of exosomes.Splenic T cells were further co-cultured with M1 exosomes and evaluated for IFN-y production by flow cytometry5.Statistical AnalysisStatistical analyses were performed with SPSS statistics 22.0.Normality and test of homogeneity of variance were tested.For data with normal distribution,hypothesis testing was performed by Student's t-test or one-way ANOVA.Multiple comparisons were conducted by LSD test or Dunnett's T3 test.For data without normal distribution,non-parametric test was applied,followed by pairwise comparisonsResultsPart ?1.BMDM polarizationBMDM treated with LPS+IFN-? secreted NO and IL-12,indicating successful M1 polarization,while M2 polarization induced by IL-4 was supported by upregulated CD 163 and CD206 expression in BMDMs2.Exosome characterizationRecovered exosomes were examined by transmission electron microscopy,and intact exosomes with typical morphology and size range could be observed.Exosomes were then passively absorbed to 4 ?m aldehyde-sulfate latex beads,making them detectable by flow cytometers,and the expression of CD81,one of the surface molecules enriched in exosomes,was identified.The presence of another two exosome-enriched proteins,TSG101 and ALIX,was confirmed by immunoblotting.Size distribution of small EVs was displayed by NTA.3.M1 exosomes exacerbate EAN,while M2 exosomes possess potentials to mitigate disease severityM1 exosomes aggravated EAN disease severity,while M2 exosomes exhibited potentials to mitigate the development of EAN.Histological assessments were then conducted to confirm the infiltration of inflammatory cells and demyelination in sciatic nerves,and the results were consistent with the observed clinical manifestations.4.Regulatory effects of M1 and M2 exosomes on adaptive immunityWe found that M1 exosomes increased the proportions of Thl and Th17 subtypes.M2 exosomes,albeit causing a decrease of splenic CD4+T cell frequency,failed to restrict overall Thl and Th17 differentiation.Both M1 and M2 exosomes exerted no significant effects on Treg cell frequencies.We found an increase of GCB cell frequency by both M1 and M2 exosome treatment,and these effects might result from increased ICOS expression in T cells.5.Effects of M1 and M2 exosomes on splenic natural killer cells(NK cells)and macrophagesThe frequency of splenic NK cells and the expression of CD80 and CD86 on splenic macrophages were not affected by exosone treatment.Part ?1.Ml and M2 exosomes differ in their capacities to regulate IFN-y production of T cells in vitroWe co-cultured splenic MNCs with M1 or M2 exosomes.M1 exosomes promoted IFN-y production in both CD4+T cells and CD8+T cells,which exhibited a dose-effect relationship,whereas M2 exosomes did not affect IFN-y expression in T cells.2.The effects of M1 and M2 exosomes on the proliferation and apoptosis of T cellsIn co-culture assays in vitro,we found that M1 exosomes,but not M2 exosomes,promoted proliferation of CD4+T cells and CD8+T cells,while neither of them affect cell apoptosis of T cells.3.The effects of M1 exosomes on the maturation of BMDCsM1 exosomes did not further upregulate expression of MHC ? and CD80 induced by LPS stimulation.4.M1 exosomes exert direct regulatory effects on IFN-? production of T cells in vitroPurified splenic T cells were first co-cultured with fluorescently labeled exosomes,and cellular uptake of M1 exosomes and M2 exosomes by T cells was confirmed.Further co-culture of purified T cells with M1 exosomes showed that M1 exosomes increased IFN-? expression in CD4+and CD8+T cells,supporting a direct regulation of Ml exosomes on the differentiation and effector function T cells.Conclusions1.M1 exosomes promoted the development of EAN,possibly through regulating Th1 and Th17 responses.M2 exosomes showed potentials to alleviate EAN via a mechanism bypassing Thl and Th17 responses.2.M1 exosomes could modulate proliferation and IFN-y production in T cells.These effects could be exerted on T cells in a direct manner.