The Role Of Aberrant Macrophage Polarization And HLA-G In The Pathogenesis Of Immune Thrombocytopenia

Part I High-dose dexamethasone or all-trans retinoic acid restores the balance of macrophage toward M2 in immune thrombocytopeniaImmune thrombocytopenia(ITP)is a common autoimmune hemorrhagic disease characterized by decreased platelet counts and an increased bleeding risk.The main cause of platelet destruction is autoantibody-mediated phagocytosis by monocytes/macrophages,and/or CD8+T cell mediated platelet lysis.Immune-mediated impaired maturation of megakaryocytes results in insufficient platelet production.The imbalance of Thl/Th2,increased ratio of mDCs/1DCs(myeloid/lymphoid dendritic cells),and the deficiency of tolerogenic DCs are involved in the pathogenesis of ITP,Macrophages play a key role in both innate and adaptive immunity,however,more functions of macrophage in ITP need to be explained.Macrophages can be divided into two subsets:classically activated macrophage(M1)and alternatively activated macrophage(M2).M1 macrophages are induced by lipopolysaccharide(LPS)and/or interferon(IFN)-y and characterized by elevated expression of co-stimulatory molecules(CD80,CD86),enhanced efficiency in antigen presentation,and ability to produce copious amounts of proinflammatory cytokines such as Interleukin(IL)-12,and show Th1 immunity.By contrast,M2 macrophages are activated by IL-4 or IL-13 and immune complexes,and mediate immunoregulatory Th2 functions through production of anti-inflammatory cytokines(including IL-10),higher levels of scavenger and mannose receptors and expansion of regulatory T cells(Tregs).The Ml/M2 polarization status can disturb the balance of lymphocyte subset differentiation or change their tolerogenic function,hence contributing to the pathogenesis of autoimmune diseases,such as rheumatoid arthritis and multiple sclerosis(MS).However,whether aberrant macrophage polarization are involved in the pathogenesis of ITP remains unclear.High-dose dexamethasone(HD-DXM)has been used as first choice for treatment of ITP patients.However,the mechanism of HD-DXM therapy in ITP was not fully understood,and 1/3 of patients didn’t response for a long time treatment.Recently,new therapies such as thrombopoietin receptor agonist(TPO-RA)and anti-CD20 antibody had been reported,however,some patients still failed to response.All-trans retinoic acid(ATRA)is an active inducer of differentiation and immunomodulator,and being used in multiple autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus(SLE).Dai and colleagues found that ATRA combined with prednisone was effective in 54.3%of chronic refractory ITP patients.Studies indicated that ATRA directly modulated the differentiation of T cells and induced tolerogenic DCs.Moreover,ATRA in combination with IL-4 dramatically enhanced the expression of Arginase-1,one of the most relevant markers of murine M2 activation.Nevertheless,whether DXM or ATRA affects macrophage polarization in ITP patients and contributes to disease remission is unknown.Objective:To explore the macrophage polarization and function in ITP and investigate whether HD-DXM or ATRA could correct this imbalance,and contribute to the responses towards therapies for ITP.Methods:1.Spleen specimens from 5 patients with ITP and 5 patients with traumatic spleen rapture(non-ITP control patients)were analyzed by immunofluorescence.Slides were incubated with primary anti-human CD68 antibody,anti-human CD 163 antibody,or anti-human iNOS antibody.The number of CD68+iNOS+(Ml)and CD68+CD163+(M2)was detected using confocal laser scanning microscopy.2.Twenty treatment-naive ITP patients was recruited and 10ml of peripheral blood was taken.CD14+ cells were separated with anti-CD14 microbeads and induced for M0,Ml or M2.3.The expression of CD163,CD206,CD209,CCR7,CD86 and CD80 of M1 or M2 was assessed by flow cytometry.4.The concentration of IL-12,TNF-a and IL-10 in the culture supernatant was assayed by enzyme-linked immunosorbent assay(ELISA).5.CFSE labeled CD4+T or CD8+ T cells were co-cultured with macrophages.The proliferation of T cells and the proportion of CD4+CD49b+LAG-3+ Tr1 cells were analyzed by flow cytometry.6.The co-culture supernatants were collected and cytokines were detected using ProcartaPlex Human Thl/Th2 Cytokine Panel.7.The 20 treatment-naive ITP patients received HD-DXM therapy,while the 23 steroid-resistant ITP patients received ATRA in combination with prednisone therapy.Peripheral blood was taken and M1/M2 were induced for the detection of phenotypes and functions.Results:1.The number of M1 in the spleens was greater in ITP patients than in non-ITP control patients(5.4 ± 0.9 vs 2.8 ± 0.8,P<0.05)and the number of M2 was not significantly different between ITP and control patients(3.0 ± 0.7 vs 3.2 ± 1.3,P>0.05).2.Macrophages induced from ITP patients showed higher expression of Ml surface markers(CD80,CD86)than those of healthy controls.Either HD-DXM or ATRA altered macrophage phenotype towards M2 by reducing CD80,CD86 and CCR7.HD-DXM increased the expression of CD 163 while ATRA increased CD209.3.M1-polarized macrophages from ITP patients secreted more IL-12p70 and TNF-a than those from healthy controls.Both DXM and ATRA reduced production of IL-12p70 and TNF-α,and increased IL-10 in M1 and M2 macrophages.4.Both M1 and M2 induced from ITP patients showed enhanced stimulation of T cells proliferation compared with those from healthy controls.Either DXM-or ATRA-modulated Ml or M2 dramatically suppressed the CD4+ and CD8+T cells proliferation in comparison to unmodulated ones.Either HD-DXM or ATRA improved the ability of M2 macrophages,not M1,to induce Tr1 cells in ITP patients.5.DXM-modulated M1 and M2 from ITP patients caused a marked increase in IL-4,IL-5,and IL-13 and decrease in IFN-y,IL-2,IL-12p70 produced by CD4+ T cells.ATRA-treated M1 and M2 from ITP also increased IL-4,IL-5,and IL-13 secretion and reduced IL-2 and IL-12p70 secretion.Only ATRA-modulated M2 significantly inhibited IFN-y production.6.In the HD-DXM arm,85%patients responded to the initial course of medication while in the ATRA arm,the overall response(OR)rate is 69.6%.Responders to either therapy showed suppressed expression of CD86,CD80 and CCR7,and alleviated CD4+ T cells proliferation.Responders to HD-DXM increased the expression of CD 163 while CD209 expression increased in responders to ATRA treatment.No significant changes were observed from non-responders in our study.Conclusion:In conclusion,our findings demonstrated an imbalance of macrophage polarization towards Ml in patients with ITP,with a loss of immune tolerance.Either HD-DXM or ATRA could correct this imbalance.The study may shed lights on new mechanisms of ITP pathogenesis and provide basis for the therapeutic potential of ATRA in ITP patients.Part II The effect of HLA-G and ILTs in immune thrombocytopeniaImmune thrombocytopenia(ITP)is a common autoimmune disease characterized by loss of immune tolerance.Several cellular defects,including activation of auto-reactive T and B cells,deficiency and dysfunction of regulatory T cells(Tregs),regulatory B cells(Bregs),tolerogenic dendritic cells(DCs)and myeloid-derived suppressor cells(MDSCs)have been reported in ITP patients.Such autoimmune disorders results in accelerated platelet destruction as well as insufficient platelet production in ITP patients.Human leukocyte antigen-G(HLA-G)is a group of non-classical HLA class-I molecule,including membrane-bound(mHLA-G)and soluble(sHLA-G)isoforms.Most cells could secret HLA-G and HLA-G exerts its inhibitory functions via interacting with three inhibitory receptors:immunoglobulin-like transcript 2,(ILT2/CD85j),immunoglobulin-like transcript 4(ILT4/CD85d),and killer cell immunoglobulin-like receptor(KIR2DL4/CD158d),which were expressed differentially on NK,T,and antigen presenting cells(APCs).The relevance of HLA-G in human pathological contexts has been intensively investigated.Previous literatures showed that insufficient and dysfunction of HLA-G was involved in many autoimmnue diseases such as rheumatoid arthritis(RA)and systemic lupus erythematosus(SLE).But no data was referred to the relationship of HLA-G and ITP patients.Objective:(1)To explore whether the expression of HLA-G and ILT2 in CD4+,CD8+,CD19+lymphocytes,and the expression of HLA-G and ILT4 in CD14+ cells were abnormal in ITP patients.(2)To investigate whether the rhHLA-G protein could correct the aberrant expression and function of HLA-G and ILTs in lymphocytes and dendritic cells in ITP patients,and explore the new therapeutic target for ITP.Methods:(1)Thirty ITP patients and 15 healthy controls were recruited and whole blood was collected.Plasma,platelets and peripheral blood mononuclear cells were isolated and CD14+ monocytes were separated with magnetic activated cell sorting(MACS).(2)The concentration of sHLA-G in platelet poor plasma was detected by enzyme-linked immunosorbent assay(ELISA).(3)The expression of mHLA-G and ILTs in PBMCs,the effect of rhHLA-G on the expression of ILTs in PBMCs,and the frequency of regulatory T cells in PBMCs after treatment with rhHLA-G were analyzed by flow cytometry.(4)The rhHLA-G-modulated PBMCs were co-cultured with autologous or allogeneic platelets for 4 hours and the apoptosis of platelets was assayed with JC-1.(5)CD14+ monocytes were cultured with the presence of granulocyte-macrophage colony stimulating factor(GM-CSF)and IL-6 for 5 days.To activate DCs,L.PS was added with or without the presence of rhHLA-G for another 48 hours.The expression of CD86 and CD80 was evaluated by flow cytometry.Results:(1)A significantly lower level of sHLA-G in the plasma from ITP patients positive for anti-glycoprotein(GP)Ⅱb/Ⅲa and/or anti-GPIb/IX autoantibodies was found,but no difference was observed between the autoantibody-negative patients and healthy controls.(2)The expression of HLA-G and ILT2 were significantly lower in CD4+,CD19+cells in ITP patients,the same as HLA-G and ILT4 in CD14+ cells.The rhHLA-G protein could up-regulate the expression of ILT2 in CD4+,CD19+ and ILT4 in CD14+cells.(3)The apoptosis of autologous or allogeneic platelets co-cultured with PBMCs from ITP patients was significantly lower in comparison with PBMCs from controls.And rhHLA-G could attenuate platelet destruction by PBMCs obtained from ITP patients.(4)The expression of CD86 and CD80 was found to be down-regulated on DCs with the presence of rhHLA-G.(5)There was no significant difference in the proportion of CD4+CD25+Foxp3+regulatory T cells in PBMCs pre-or post-modulated with rhHLA-G.Conclusions:(1)The expression of HLA-G and ILTs was decreased in ITP patients.(2)The rhHLA-G could increase the expression of ILTs,alleviate the cytotoxicity of PBMCs and inhibit the maturation of dendritic cells in ITP patients.(3)The novel discovery of inhibitory effect of HLA-G may play an important role in the treatment of ITP patients.