The Role Of PGRN In Immune Thrombocytopenia

Part IPlasma PGRN levels in immune thrombocytopenia patientsBackgroundImmune thrombocytopenia(ITP)is a common clinical autoimmune hemorrhagic disease,which is caused by excessive platelet destruction and/or reduced platelet production.The increased risk of bleeding is its main clinical manifestation.And severe ITP patients may have intracranial hemorrhage,which would eventually affect survial of these patients.The pathophysiology of ITP is quite complicated and still unclear now.For instance,peripheral platelets in ITP patients can be opsonized by specific antibodies against platelet membrane glycoproteins.In addition,T cell abnormalities play a critical role,contributing to platelet reduction in ITP patients,through,for example,an imbalance of Th1/Th2,decreased and defective T regulatory cells(Tregs),enhanced activation of CD8+ T lymphocytes(CTLs).Large amounts of the immune regulatory factors are related to the genesis of ITP.To fully discover these immune regulatory factors and understand the pathogenesis of ITP may help improve levels of diagnosis and treatment.Progranulin(PGRN)is an autocrine glycoprotein comprising 593 amino acids.PGRN expression is widely observed in a variety of cell types,such as hematopoietic cells,chondrocyte,immunocyte,T cells.As a growth factor,PGRN promotes cellular proliferation,and is extensively involved in the regulation of neuropathology,cancer cell proliferation,infection and wound healing.Recent work has demonstrated that PGRN is also an important immune regulator in a variety of autoimmune diseases,including rheumatoid arthritis(RA),osteoarthritis(OA),and systemic lupus erythematous(SLE).At present,it is well accepted that the protective effects of PGRN toward autoimmune diseases are primarily exerted by competitively binding tumor necrosis factor-α(TNFα)receptors(TNFRs),including both TNFR1 and TNFR2.Beyond that,Wei Tang and colleagues reported that PGRN stimulated Treg proliferation and decreased the inhibitory effect of TNF α on them in vitro.Since ITP is found closely associated with the abnormal number and function of Tregs,which may lead to failure of immune tolerance,we tentatively surmise that PGRN may play a role in ITP,which has not been reported yet.In this study,we for the first time detected the plasma levels of PGRN in ITP patients over healthy controls or non-ITP patients with reduced platelet in peripheral blood.We conducted the correlation study of PGRN and ITP and provided a potential strategy for management of ITP.ObjectivePGRN is an important immune regulator in a variety of autoimmune diseases,and ITP is a common clinical autoimmune hemorrhagic disease.Our research is conducted to study the correlation of PGRN and ITP and provide a potential strategy for management of ITP.Methods1.Whole blood and plasma were obtained from 52 ITP patients(including 22 newly diagnosed and 30 relapsed patients),13 Non-autoimmune thrombocytopenia patients(Non-ITP)and 40 healthy controls.Platelet count was determined by complete blood count(CBC).2.PGRN levels in plasma were determined using an enzyme-linked immunosorbent assay(ELISA)kit.And PGRN levels in plasma of newly diagnosed(n = 9)and relapsed(n = 10)ITP patients were also measured after treatment.3.Plasma PGRN levels in ITP patients,Non-ITP patients and healthy controls were compared by the Mann-Whitney test.PGRN levels in plasma of ITP patients before and after treatment used the Wilcoxon matched-pairs test.P<0.05 means there were differences in statistics.The relationship between PGRN and platelet count of ITP patients was determined by Pearson correlation analysis.Results1.PGRN levels in plasma of ITP patients were significantly higher than non-ITP patients and normals(p<0.05).However,there was no significant difference between Non-ITP and healthy controls.There was no significant difference between newly diagnosed and relapsed patients,either(p>0.05).2.PGRN levels in plasma of ITP patients were markedly reduced after treatment(p<0.05).3.PGRN levels in ITP patient plasma were negatively correlated with their peripheral platelet count(p = 0.011,r =-0.350).Conclusion1.PGRN levels in plasma of ITP patients were significantly higher than non-ITP patients and normal,respectively,suggesting that PGRN may be involved in the mechanism of ITP,and plasma PGRN levels might used for differentiating ITP from other disorders causing reduction of peripheral platelet count.2.Plasma PGRN levels of ITP patients decreased after treatment,suggesting a role of PGRN in the disease development.3.The negative correlation between Plasma PGRN levels and platelet count of ITP patients also suggested a role of PGRN in the disease development.PartⅡStudy of the role and mechanism of PGRN in immune thrombocytopenia using murine modelsBackgroundImmune thrombocytopenia(ITP)is a common clinical autoimmune hemorrhagic disease characterized by decreased platelet counts and its diagnosis depends on exclusion.The pathogenesis of ITP is complicated and the main cause is autoantibody-mediated platelet destruction and reduced platelet production.Our study showed the correlation of PGRN and ITP by detecting the PGRN levels in plasma of ITP patients,non-ITP patients and healthy controls.We will study the mechanism of PGRN in ITP using two kinds of murine models.It is important to establish ideal animal models of ITP for pathogenesis investigation,new drugs research and development,et al.In earlier research,people established ITP murine model by X-ray radiation and bone marrow suppression drugs.But these murine models had side effects,such as aplastic anemia and bone marrow suppression after radiotherapy.There are two widely used murine models now:passive transfer ITP model and active ITP murine model.The passive transfer ITP model is an artificial immune model by injecting antigen or antibody,such as exogenous platelet,antiplatelet sera and monoclonal antibodies against platelet.Chow and colleagues proposed an ideal mouse model to mimic ITP pathophysiology that requires irradiation followed by intraperitoneal injection of platelet-immunized splenocytes into severe combined immunodeficient(SCID)mice.We investigated the effects and the mechanism of PGRN in ITP with two kinds of ITP murine models in this study.ObjectiveTo build two kinds of ITP murine models and investigate PGRN’s effect and mechanism in them.To investigate whether PGRN can become a potential agent for ITP treatment.Methods1.To build passive transfer ITP models:WT and weight-matched Grn-/-mice received an intraperitoneal injection of 5μg of the purified rat anti-mouse CD41 antibody(mAb,MWReg30).Their platelet counts were measured at 3 h,24 h,48 h,and 72 h after injection of anti-CD41 antibody.Statistically significant differences between two independent groups were detected by the Student’s t test,or the Mann-Whitney test if the data were not normally distributed.2.In passive transfer ITP model,Grn-/-mice received 50 μg of recombinant PGRN or the same volume of PBS by intraperitoneal injection.Platelet counts were measured 3 h,24 h,48 h,and 72 h after injection of anti-CD41 antibody.Statistically significant differences between two independent groups were detected by the Student’s t test,or the Mann-Whitney test if the data were not normally distributed.3.The Treg percentages of total CD4+ T cells from spleens of WT(n = 6)or Grn-/-(n =6)mice were determined by flow cytometry after staining with anti-CD4,anti-CD25,and anti-Foxp3 antibodies before or 24 h after treatment with anti-CD41 antibody.4.To build an active ITP mouse model:C57BL/6J CD61 knockout(KO)mice were transfused weekly with 108 platelets from WT mice for 3-4 weeks.Then the spleens were removed from the antiplatelet immune CD61 KO mice.The C57BL/6J severe combined immunodeficient(SCID)mice were subjected to 200 cGy total body irradiation to inhibit recipient innate immune responses.The SCID mice were injected intraperitoneally with the indicated splenocytes within 3 h of irradiation.SCID mice were randomly divided into two groups and their baseline platelet counts were determined.Mice in the experimental group then received an intraperitoneal injection of 50μg of PGRN,which was repeated once every three days.Meanwhile,mice in the control group were treated with the same volume of PBS.Platelet count was measured aweekly,until sacrifice 4 weeks after irradiation.Results1.In passive ITP murine model,Grn-/-mice were more sensitive to the anti-platelet effect of anti-mouse CD41 antibody at 3 h,24 h,48 h,and 72 h after injection compared to WT mice(p<0.05).2.Grn-/-mice pretreated with PGRN(n = 5)effectively protected against anti-mouse CD41-induced ITP,by significantly ameliorated platelet decline at 24h and enhanced platelet restoration at 48h after the induction of ITP compared with PBS-pretreated Grn-/-mice(n = 5,p<0.05).3.No significant difference was found in the percentage of matured Tregs between WT and Grn-/-mice without treatment of anti-CD41 antibody.However,the proportion of matured Tregs in Grn-/-mice decreased and was significantly lower than that in WT mice 24 h after injection of anti-CD41 antibody(p<0.05).4.In active ITP mouse model,Platelet count in the group treated with PGRN remained higher than in the PBS-treated mice 7,14,and 21 days after irradiation(p<0.05).ConclusionWe found the therapeutic efficacy of PGRN in both two kinds of ITP animal models.PGRN serves as a protective regulator in ITP animal models,providing evidence for its therapeutic potential in ITP.PGRN deficiency led to fewer Treg cells in ITP mouse spleens,suggesting that PGRN exerts an immunosuppressive effect in ITP.