| Background:Fc gamma receptor(Fc?),a member of the immunoglobulin superfamily,is one kind of glycoprotein expressed on the surface of a variety of cells.Fc? receptors are very important immune regulatory molecules by specifically binding to the Fc portion of IgG and causing a variety of immune responses.FcyRs not only maintain the body's normal physiological balance,but also play a very important regulatory role in immune injury,inflammatory diseases and the incidence and progression of tumors.Human FcyRs are divided into three categories,which is FcyRI/CD64,Fc?R?/CD32 and FcyR?/CD16.According to their different immune response regulation,FcyR includes activating receptor(FcyRI,Fc?RIIa,FcyR?)and inhibitory receptor(FcyR?b).FcyRI is the only high-affinity Fc? receptor distributed on the cell surface of monocytes,dendritic cells,macrophages and neutrophils.FcyRII,widely expressed on monocytes,part of dendritic cells,neutrophils,B cells,natural killer cells and platelets,consists of two subtypes FcyR?a and FcyRIIb and has low to moderate affinity,mainly combining to polymeric IgG.FcyR?,primarily expressed on surface of macrophages,neutrophils,NK cells,has weak binding capacity to monomeric IgG,and low to moderate binding force to polymerization IgG.The intracellular segment of Fc?R contains immunoreceptor tyrosine-based activating motif(ITAM)and immunoreceptor tyrosine-based inhibitory motif(ITIM),which decides activation or inhibition properties of Fc?R.Activating receptor combines to corresponding IgG-Fc ligand,next activation signals transmits through intracellular ITAM sequence,which induces phosphorylation of Src family tyrosine kinases and causes signaling cascades.Thus a series of immune activation responses generates,involving monocyte/macrophage phagocytosis,antibody-dependent cell-mediated cytotoxicity(ADCC),release of cytokines and inflammatory mediator and so on.FcyRIIb is the only inhibitory receptor of FcyRs.Intracellular ITIM signal is activated when immune complex binding with FcyRIIb and causes Src family tyrosine kinases dephosphorylation,thereby suppresses the immune response.In addition,ITIM can block activation of ITAM signals by combining inositol phosphatase SH2(SHIP)and inhibite persistent immune activation in vivo.Activating and inhibitory receptor often co-expressed on the cell surface,so cellular reaction result is determined by the ratio of the two receptors,which can be influenced by many factors.This will lead to abnormal immune response and cause various autoimmune diseases.Currently a large number of studies confirm that a variety of autoimmune diseases have imbalance of FcyR expression including systemic lupus erythematosus(SLE),multiple sclerosis(MS),rheumatoid arthritis(RA),primary immune thrombocytopenia(ITP),etc,which specifically appears in increased expression of activating receptor Fc?R?,Fc?R?a,FcyRIII and reduced expression of inhibitory receptor FcyRIIb.The imbalance of activating and inhibitory FcyR plays a crucial role in the development of autoimmune diseases.Therefore adjusting the balance of the two receptors is an effective way of maintaining immune tolerance and treating autoimmune diseases.ITP was formerly known as idiopathic thrombocytopenic purpura,and now renamed as primary immune thrombocytopenia according to unknown etiology,the pathogenesis of immune-related and vary bleeding manifestations.It is the most common bleeding disorder,accounting for 1/3 of clinical bleeding disorders,whose common clinical manifestations are bleeding of skin and mucous membrane,epistaxis,gingival bleeding and women menorrhagia,severe fatal bleeding including organ bleeding,intracranial hemorrhage,etc.Bleeding severity is negatively correlated with the number of platelets and certain other factors also associated with bleeding risk such as age,body condition,living environment,etc.According to the length of the disease,ITP is divided into three categories:new diagnosis,persistent(3-12 months)and chronic ITP(? 12 months).Studies have shown that ITP is an autoimmune disease.That is,immune effector cells of the body overreact,produce specific IgG against platelet glycoprotein GP?b/?a and GP?b/?X,which binds to platelets and acts on FcyR of monocyte surface.Finally the immune complex is engulfed by megakaryocytes through reticuloendothelial system to cause platelet destruction.New research shows the decrease of platelet production caused by abnormal megakaryocytes quantity and quality also plays a very important role in the pathogenesis of ITP.In addition,a variety of other immune imbalance is likely involved in the pathogenesis of ITP such as balance change of Th1/Th25 abnormal Th17,direct destroy of cytotoxic T cells,changes in gene expression of T cell antigen receptor(TCR)and so on.The exact pathogenesis remains to be confirmed by further studies.Currently the treatments of ITP includes two ways:preventing excessive platelet destruction and promoting platelet formation.Specific measures involve corticosteroids,immunoglobulins,splenectomy,rituximab,other immunosuppressive therapies and thrombopoietin treatment.ITP patients are usually effective for the above treatments,but usual relapse requires multiple medications to control,which leads to many side effects including osteoporosis,hypertension,immunocompromised,severe infection,etc.There are still about one-third of patients with delayed healing to become chronic ITP.Thrombopoietin(TPO),synthesized in the liver,is a kind of cytokines promoting platelet formation through stimulating bone marrow megakaryocyte formation,accelerating its differentiation and maturation to increase the number of platelets.In recent years,two kinds of new thrombopoietin receptor agonists(TPO-RAs),eltrombopag and romiplostim,have been used clinically and opened up new avenues for the treatment of chronic ITP.ITP is one of the most classic autoimmune diseases mediated by Fc? receptor.Studies have shown that upregulation of activating receptor and downregulation of inhibitory receptor is the basis for the onset of ITP.Accompanying with effective treatments of ITP,Fc? receptor balance tends to recover.Samuelsson et al.used intravenous immunoglobulin(IVIg)to treat mouse ITP and found that with the recovery of platelets the expression of Fc?R?b in mouse spleen macrophages increased.Ashi discovered that the expression of activating receptors Fc?R? and Fc?R?a decreased and inhibitory receptor FcyRIIb increased in ITP patients effective for the treatment of removing Helicobacter pylori,while patients with ineffective treatment did not respond similarly,which indicats that the removal of Helicobacter pylori is beneficial to restore Fc?R balance to treat ITP.Liu Xin-guang studied the expression of Fc?Rs in patients with ITP before and after treatment with high dose dexamethasone to confirm that dexamethasone affected the expression of Fc?Rs,especially inhibitory Fc?R?b.So far there has been no report on the effect of TPO-RAs on Fc?R expression in chronic ITP patients.Objective:To investigate the expression of Fc?R?,?,and ? on monocytes of patients with chronic ITP before and after treatment with eltrombopag and healthy controls,as well as changes in Fc?R?a,?b mRNA;To observe in vitro the ability changes of monocytes in phagocytosing autologous platelets before and after treatment;To detect the changes of Fc?R?,?,and ? expression on monocytes before and after administration of romiplostim in ITP mice;To explore the regulation mechanism of TPO-RAs on Fc?Rs system.Methods:1.Preparation of the study object 21 patients with chronic ITP and 20 healthy controls are enrolled.Blood samples were taken before and after taking eltrombopag for 6 weeks,followed by isolation of monocytes and plasma.2.Flow cytometry and real-time RT-PCR detections of monocyte Fc?Rs The expressions of Fc?R?/CD64,Fc?R?/CD32,Fc?R?b/CD32b and Fc?R?/CD16 on CD14+ monocytes were detected by flow cytometry.CD14+MCs were sorted by immunomagnetic beads and real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was used to determine the expressions of Fc?R?a and ?b mRNA.The levels of TGF-?1 in ITP patients and normal controls were measured by enzyme-linked immunosorbent assay(ELISA).3.In vitro phagocytosis assays Monocytes from ITP patients before and after treatment and healthy controls were solated and stimulated,then co-cultured with CMFDA-labeled sensitized autologous platelets.The monocytes/macrophages phagocytosis of autologous platelets was detected by flow cytometry.4.The expression of Fc?R?,?,? on monocytes/macrophages of ITP mouse Active ITP mouse model was induced and romiplostim was intraperitoneally injected.The spleens were taken with or without romiplostim treatment obtained and the expression of Fc?R? and ?/? on monocytes/macrophages was determined by flow cytometry.The expression of Fc?R? and ? mRNA was detected by real-time RT-PCR.Results:1.Efficacy of eltrombopag After twenty-one patients with chronic ITP were treated with eltrombopag for 6 weeks,15 patients were effective(71.43%),of which 6 were complete remission(28.57%),and 6 people were ineffective(28.57%).2.Flow expression of Fc?R?,Fc?R?,Fc?R?b and Fc?R? on monocyte surface in ITP patients before and after eltrombopag treatment(Expressed by mean fluorescence intensity,MFI)After 6 weeks of eltrombopag treatment,the expression of Fc?R? on monocytes of patients with ITP was significantly decreased,while Fc?R?b expression was significantly increased;There was no significant difference in the expression of Fc?R? and Fc?R? before and after treatment;In responders,the expression of Fc?R?b was significantly higher than that before treatment and the expression of Fc?R? was significantly reduced;In non-responders,there was no significant change in the expression of Fc?R?,Fc?R? and Fc?R?b before and after treatment;The expression of Fc?R? did not change significantly both in responders and non-responders.3.Genes expression of Fc?R?a and FcyRIIb in monocytes before and after treatment with eltrombopag detected by real-time reverse transcription polymerase chain reaction(real-time RT-PCR)After 6 weeks of eltrombopag treatment,the ratio of Fc?R?a/?b mRNA in monocytes was significantly reduced.Among them,the ratio decreased significantly in responders and did not change significantly in non-responders.4.The changes of phagocytic activity in monocytes/macrophages of ITP patients before and after eltrombopag treatment After 6 weeks of eltrombopag treatment,the phagocytic activity of macrophages from patients with effective treatment greatly reduced,while that from ineffective patients didn't show noticeable changes before and after treatment.Moreover,the phagocytic ability of macrophages from eltrombopag non-responders was significantly higher than that from responders.5.The changes of plasma TGF-?1 levels in ITP patients before and after eltrombopag treatment For eltrombopag responders,the plasma concentrations of TGF-?1 significantly increased compared with those before treatment;For non-responders,there was no significant change in TGF-?1 concentration.6.The expression of FcyR in monocytes/macrophages before and after romiplostim treatment with ITP mice Flow detection displayed that after the injection of romiplostim,the expression of Fc?R? in spleen macrophages of mice was significantly lower than that of normal controls,and the expression of FcyR?/? showed no obvious change compared with normal controls.Real-time RT-PCR test demonstrated the ratio of FcyR?/? mRNA in spleen macrophages of mice after romiplostim injection was significantly higher than that of normal controls;Romiplostim up-regulated the expression of FcyR? mRNA,but did not obviously alter the expression of FcyR? mRNA;The expression of FcyR? was negatively correlated with platelet count,while that of FcyRII was positively correlated with platelet count.Conclusions:1.Eltrombopag treatment with chronic ITP has a high rate of response and is well tolerated.2.After 6 weeks of eltrombopag treatment with chronic ITP,the expression of monocyte activating receptor Fc?R? decreased and that of inhibitory receptor Fc?R?b increased.At the same time,the ratio of Fc?R?a/?b mRNA decreased significantly.3.After effective treatment of eltrombopag,the activity of monocyte/macrophage phagocytosis of their own sensitized platelets decreased accompanied by an recovery of inhibitory receptor Fc?R?b expression in ITP patients.4.The concentration of plasma anti-inflammatory cytokine TGF-?1 increased in eltrombopag responders along with balance recovery of monocyte Fc?Rs.5.After treatment of ITP mice with romiplostim,the recovery of platelet count was associated with decreased expression of activating receptor Fc?R? and increased expression of inhibitory receptor Fc?R? on monocyte/macrophage.Primary immune thrombocytopenia(ITP)is an immune-mediated disease characterized by decreased count of platelet and increased risk of bleeding,accounting for about one-third of clinical bleeding disorders.The main clinical manifestations include bleeding of skin and mucous membrane,increased menstruation,severe organ bleeding and intracranial hemorrhage.Although the current immunosuppressive therapy and platelet promoting treatment in ITP have achieved great curative effect,there are still some patients with delayed healing and the development of chronic ITP.The underlying cause of immune tolerance in ITP is unclear,while autoantibody-mediated platelet clearance is still the current mechanism of platelet destruction in ITP.The body produces an autoantibody against specific glycoprotein(GP)on membrane surface of platelet.This autoantibody is primarily an IgG that recognizes platelet GPIIb/IIIa or Ib/IX.The formed antigen-antibody complex binds to the FcyRs on monocyte/macrophage surface and is cleared by the reticular endothelial system.Recently,more and more studies have found that changes in cellular immune regulation in patients with ITP,particularly T cell dysfunction,play an important role in the pathogenesis of ITP.Such dysfunction mainly includes the following aspects:(1)The ratio of Thl/Th2 increased.So the levels of IL-2 and IFN-? secreted by Thl were elevated.IL-2 can stimulate B lymphocyte proliferation and differentiation to increase antibody production.IFN-? promotes the transformation of B lymphocytes into plasma cells that produce autoantibodies.While the secretion of cytokines IL-4 and IL-10 from Th2 decreased,so that the inhibition of IFN-y effect weakened,which led to further destruction of platelets.(2)CD4+ CD25+Fox3 + regulatory T cells(Treg cells)decreased.Treg cells account for 5-10%of CD4+T lymphocytes and play a key role in maintaining the balance of autoimmune tolerance.Decreased peripheral Tregs in ITP patients broke the balance of autoimmune tolerance and caused abnormal activation of antigen-presenting cells(APC),thus stimulated their own reactive lymphocytes to produce platelet antibodies.(3)The levels of CD8+ T lymphocyte increased,so platelet damage mediated by Cytotoxic T lymphocyte increased.(4)The expression of Thl7and Th22 cells increased,suggesting its correlation with incidence of ITP.In addition to immune damage,megakaryocyte count and quality abnormalities is also an important risk factor for ITP.Anti-platelet autoantibodies can also bind to megakaryocytes,inhibiting megakaryocyte maturation and triggering its programmed death.All studies have shown that the incidence of ITP is caused by multiple factors.The combination of humoral immunity and cellular immunity causes increased platelet destruction and reduced production.Therefore,in-depth study of the pathogenesis of ITP is important to provide a new therapeutic strategy for ITP treatment.The initial CD4+ T cells express corresponding transcription factors under specific cytokines and produce helper T cells(Th)and regulatory T cells(Tregs)with different structures and functions,which play important roles in immune response and immune regulation.Th17 cells,belonging to CD4+ Th cells,were first discovered by Harrington in 2005.Th17 cells have been proposed to break the traditional Thl/Th2 equilibrium theory.Its differentiation process requires a variety of factors to participate including the initiating factor TGF-? and IL-6,promoting factors IL-21 and IL-23,inhibitory factor IL-4 and IL-27.Studies have shown that Th17 cells are the first to participate in the body's anti-infective immune response and its secretion of IL-17 is a key cytokine that is resistant to pathogens.IL-17-deficient mice were found to be highly susceptible to pathogens such as Candida and Klebsiella pneumoniae.At the same time Th17 can cause different degrees of inflammation injury of the body.Studies have shown that during the course of Hp infectious gastritis,IL-23 promoted Thl7 to release IL-17,which induced the secretion of IL-6,IL-8,TNF and other inflammatory factors,thus inducing neutrophils to raise in the inflammatory site.Moreover,IL-17 could further promote fibroblast to release matrix metalloproteinases and commonly caused inflammation of the gastric mucosa.In recent years,studies on the relationship between Th17 cells and autoimmune diseases are growing.Th 17 cells have been shown to play an important role in the development of various autoimmune diseases including SLE,MS,RA,etc.As a classic autoimmune disease,ITP also exists Thl 7 functional abnormalities.Th17 cells in patients with active ITP are significantly higher than those in normal controls.Th17 cells can promote the production of a series of proinflammatory molecules such as IL-6,IL-8 and granulocyte colony stimulating factor(G-CSF)by releasing IL-17A and IL-17F regulated by IL-23,thereby ameliorating the immune disorder in ITP.IL-17 is a major effector of Th17 cells,with a variety of biological functions including the promotion of T cell activation,induced secretion of a variety of cytokines,stimulation of neutrophil chemotactic factor secretion to cause neutrophil recruitment,and eventually leading to a series of inflammatory reactions.IL-17 cytokines are divided into six groups:IL-17A,IL-17B,IL-17C,IL-17D,IL-17E and IL-17F,from which IL-17A and IL-17F have the best biological characterization and regulatory functions.IL-17A and IL-17F are highly homologous.Both human and mouse genes that encode the two cytokines are located in adjacent positions of the same chromosome,so both have similar biological functions.There is growing evidence that IL-17A and IL-17F are key cytokines secreted by Thl7 cells.Further studies have found that in addition to Thl7 cells,innate immune cells including invariant natural killer T cells(iNKT),y8T cells and myeloid cells can also secrete IL-17.Recent studies have shown that certain activated B cells also express IL-17A and IL-17F.IL-17A and IL-17F gene polymorphisms have been shown to be associated with many diseases.Single nucleotide polymorphism(SNP)refers to the difference between individuals of the same species due to the deletion,insertion,or substitution of single nucleotide on the same DNA allele.There is an SNP site about every 100-300 bases in the human genome.SNP is a key factor of individuals in different susceptibility to a particular disease.The IL-17A G197A gene locus is located in the promoter region,and the IL-17F A7488G gene locus is located in the exon region,which is a hot spot in recent years.Studies have found that IL-17F gene SNP is associated with the degree of chronic inflammation in gastrointestinal tract,such as Helicobacter pylori(Hp)infectious gastritis,ulcerative colitis(UC)and so on.IL-17 gene polymorphism plays an vital role in the progression of many autoimmune diseases.Nordang reported that the IL-17A G197A locus was closely related to the susceptibility of rheumatoid arthritis in the Norwegian population.Another study showed that the site SNP was also correlated with the pathogenesis of childhood asthma.Studies of the Han population in Sichuan have shown that IL-17F A7488G gene locus was associated with the incidence of MS and A allele may be a susceptible factor for MS.IL-17 gene polymorphism and the incidence of certain tumors also have a certain relationship.Alireza et al.found that population of IL-17F A7488G locus AA homozygous had a significantly higher risk of developing gastric cancer than other genes.Shibata's study of Japanese population also showed that IL-17F A7488G site A allele increased the risk of atrophic gastritis and gastric cancer.Other studies found that IL-17 gene polymorphism was associated with the pathogenesis of cardiovascular disease.IL-17F A7488G locus SNP was closely related to family history of hypertension and A7488G locus AA genotype had a significant increase in coronary heart disease risk.Recent studies have also found that IL-17 gene polymorphisms can predict the severity of GVHD after allo-HSCT.Previous studies have shown that a variety of cytokines,including tumor necrosis factor-?(TNF-?),IL-1?,IL-4 and IL-10,were associated with the immunopathogenic mechanisms of ITP.However,so far there has been little research about IL-17A and IL-17F gene polymorphisms and the susceptibility of ITP.In this study,we used PCR-RFLP to detect the IL-17A G197A and IL-17F A7488G gene polymorphisms of chronic ITP patients in Chinese population,then analyzed the association between IL-17A and-17F SNP and susceptibility of ITP,explored possible genetic mechanisms of ITP,thus provided new ideas for treatment of chronic ITP.Objective:To investigate the association between IL-17A and-17F gene polymorphisms and susceptibility of chronic ITP in Chinese population,and explore the possible pathogenesis of chronic ITP.Methods:1.DNA extractionA total of 146 adult patients with chronic ITP and 137 healthy controls were enrolled.Blood samples were taken to extract DNA by following operating instructions of QIAamp DNA Mini Kit(Qiagen,Germany).2.Detection of IL-17A and IL-17F genotypesThe IL-17A G197A and IL-17F A7488G loci were genotyped by PCR-RFLP.Firstly the genomic DNA was amplified by PCR,and the specific primer sequence was shown in Table 1;Then the PCR products of IL-17A G197A and IL-17F A7488G were digested with restriction endonuclease Xag?(Fermentas)and Nla?(New England BioLabs);Next the digested product was separated by 3%agarose gel electrophoresis.3.Statistical analysisWhether the healthy control group is consistent with the Hardy-Weinberg equilibrium law was evaluated;The differences in clinicopathological features and genotype distribution between ITP patients and normal controls were examined;Logistic regression analysis was used to calculate the odds ratios(ORs)and 95%confidence intervals(CIs).Results:1.There was no difference in age and gender distribution between the ITP group and the normal control group.2.The genotype distribution of IL-17A and IL-17F SNP in the normal control group was consistent with Hardy-Weinberg equilibrium;There was no significant difference in genotype and allele frequencies of IL-17A G197A between ITP patients and healthy controls;The frequency of IL-17F A7488G genotype AA and AG in ITP patients was higher than that in healthy controls,also the frequency of allele A was significantly higher than in healthy controls;AA and AG genotypes of IL-17F A7488G significantly increased the susceptibility of ITP and A-allele carriers may be associated with increased risk of ITP.3.In patients with ITP,there was no significant difference in IL-17F A7488G genotype distribution between male and female ones;There was no obvious association between the frequency of IL-17F genotype and the severity of thrombocytopenia;Also there was no association between IL-17F gene polymorphism and bleeding tendency.Conclusions:1.IL-17F A7488G genotype AA and AG had certain association with the susceptibility of chronic ITP in Chinese population,and allele A may be a risk factor for ITP.2.There was no significant association between IL-17F gene polymorphism and clinical manifestation and severity of chronic ITP. |
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