The Role And Mechanism Of Long-lived Plasma Cells,NLRP3 Inflammasome And IL-36 In The Pathogenesis Of Primary Immune Thrombocytopenia

Part I:The effect of bortezomib on long-lived plasma cells in patients with steroid-resistant or relapsed ITPObjectives:To investigate the long-lived plasma cells in the bone marrow of patients with steroid-resistant or relapsed primary immune thrombocytopenia(ITP),and to explore the role of anti-platelet specific IgG secreting long-lived plasma cells in the pathogenesis of ITP by proteasome inhibition with bortezomib.Materials and Methods:1.We collected the bone marrow biopsy sections of ITP patients and healthy volunteers for immunohistochemistry analysis,and detected whether there is statistical difference in the percentage of CD138+plasma cells in bone marrow nucleated cells between the two groups.2.Bone marrow aspirates were collected and the percentage of CD38hiCD138+plasma cells in bone marrow nucleated cells of ITP patients and healthy volunteers was analyzed by flow cytometry after the erythrocytes were lysed,and the difference of average percentage of CD19-CD38hiCD138+total long-lived plasma cells between the two groups was assessed.3.The bone marrow tissues of ITP patients and healthy volunteers were collected and made into frozen sections.After CD 19 and CD 138 staining,immunofluorescence confocal analysis was carried out to observe the co-expression of CD 19 and CD 138.The negativity rate of CD19 in CD138+ cells and the percentage of CD19-CD138+long-lived plasma cells in nucleated cells were counted.4.Considering the proteasome inhibition effect of bortezomib,we studied whether bortezomib can reduce the number of long-lived plasma cells in bone marrow and further inhibit the production of anti-platelet autoantibodies.Bone marrow mononuclear cells isolated from ITP bone marrow aspirates were cultured with or without the presence of 0.25 ng/mL bortezomib for 5 days.Then the percentage of CD 19-CD38hiCD 13 8+long-lived plasma cells and the percentage of CD3+T cells,CD20+B cells and CD56+NK cells were analyzed by flow cytometry.5.In order to figure out the effect of bortezomib on the number of long-lived plasma cells secreting anti-GPⅡb/Ⅲa specific autoantibodies,GPIIb/IIIa specific ELISpot was performed on sorted CD19-CD138+long-lived plasma cells.Treatment group was supplemented with 25 ng/mL bortezomib and the same amount of normal saline was added to the control group for 20 hours.6.In order to determine the effect of bortezomib on the secretion of anti-platelet autoantibodies,we performed the modified MAIPA analysis using the supernatants of cultured CD19-CD138+long-lived plasma cells with or without 0.25 ng/mL bortezomib.7.Based on the mechanism of bortezomib as a proteasome inhibitor,we further investigated whether bortezomib could induce apoptosis of long-lived plasma cells using annexin V staining.At the same time,long-lived plasma cells were labeled with CD138-PE/cy7 and CD19-APC.Bone marrow mononuclear cells were divided into four groups and treated with four concentration gradients of bortezomib(0,0.25,25 and 2500 ng/mL).After 6 hours of culture,the proportion of annexin V positive apoptotic cells was determined by flow cytometry.8.In order to determine the mechanism of bortezomib reducing the number of long-lived plasma cells is through the inhibition of proteasome rather than through non-specific effects(such as cytotoxicity).We used the proteasome activity analysis kit to detect and analyze the proteasome activity between bortezomib group and control group.9.To investigate whether bortezomib can alleviate ITP in vivo,we established an active ITP mouse model to evaluate its effect.CD61 knockout mice were immunized with CD61+wild-type platelets.After immunization,spleen cells were collected and injected into SCID mice that had received whole-body irradiation.The model mice were divided into two groups:the bortezomib-treated group were injected with 20μg/kg bortezomib via tail vein twice a week from the beginning of the third week to the end of the fourth week,while the control group was injected with the same amount of normal saline.From the 19th to 28th day after irradiation,all mice received BrdU(0.8 mg/mL)through drinking water.Platelet count was recorded every week for 4 weeks.10.At the end of the 4th week,all mice were killed and bone marrow was collected for flow cytometry analysis.BrdU-CD138+plasma cells are long-lived plasma cells.The changes of CD3+T cells or CD20+B cells were also analyzed.Results:1.The results of immunohistochemistry showed that the average percentage of CD138+plasma cells in bone marrow of ITP patients was significantly higher than that of healthy controls.Consistent with this result,flow cytometry showed that the percentage of CD38hiCD138+plasma cells in bone marrow of ITP patients was significantly higher than that of healthy volunteers.However,there was no significant difference in the average percentage of total long-lived plasma cells of CD19-CD38hiCD13 8+between the two groups.2.The negativity rate of CD19 in CD138+cells of ITP patients was 30.96±16.07%,which was not significantly different from that of healthy controls(42.06 ± 23.08%).In addition,the percentage of CD19-CD138+long-lived plasma cells in nucleated cells was not significantly different between ITP patients and healthy volunteers.3.The percentage of CD19-CD38hiCD138+long-lived plasma cells in ITP bone marrow mononuclear cells of bortezomib-treated group was 0.02 ±0.01%,significantly lower than that of untreated control group(0.04 ± 0.02%).Bortezomib had no significant effect on the proportion of CD3+T cells,CD20+B cells and CD56+NK cells,suggesting a specific inhibition on plasma cells of bortezomib.4.GPⅡb/Ⅲa specific ELISpot analysis of CD19-CD138+long-lived plasma cells from patients with steroid-resistance or relapsed ITP showed that a certain number of anti GPⅡb/Ⅲa IgG secreting cells were observed,but almost none in healthy volunteers.In the ELISpot plate cultured for 20 hours,the number of spots in the bortezomib-treated group was significantly less than that in the untreated control group,which directly proved the significant effect of bortezomib on the clearance of long-lived plasma cells.5.The modified MAIPA analysis showed that the absorbance of the supernatant in the bortezomib-treated group was significantly lower than that in the untreated control group,which indicated that the concentration of anti-GPIIb/IIIa autoantibody in the bortezomib-treated group was significantly lower than that in the untreated control group.6.The apoptosis analysis showed that with the increase of bortezomib concentration,the percentage of annexin V positive apoptotic cells in the four concentration gradient groups also increased,indicating that bortezomib had a concentration dependent effect on the apoptosis of long-lived plasma cells.In contrast,the proportion of apoptotic B cells,T cells and NK cells in different groups remained stable,without statistical difference.7.The results of proteasome activity analysis showed that the relative fluorescence unit of bortezomib group was significantly lower than that of the control group,which suggested that bortezomib could inhibit the activity of proteasome in long-lived plasma cells.8.The results of active ITP mouse model experiments showed that the platelet count of mice treated with bortezomib was significantly higher than that of untreated mice at the end of the 4th week after irradiation,and the platelet count of bortezomib-treated mice increased faster over time.9.The percentage of plasma cells by flow cytometry in the bone marrow of bortezomib treated mice was 0.15±0.06%,significantly lower than that in control group(1.00 ± 0.30%).No significant changes in CD3+T cells or CD20+B cells were observed.The proportion of BrdU-CD138+long-lived plasma cells in bortezomib group was 0.06 ± 0.05%,significantly lower than that in the control group(0.69±0.11%).Conclusions:1.In the bone marrow of patients with steroid-resistant or relapsed ITP,there are anti-platelet specific long-lived plasma cells that continuously secrete antiplatelet autoantibodies.2.As a proteasome inhibitor,bortezomib can reduce the number of long-lived plasma cells which produces antiplatelet autoantibodies.3.Bortezomib can reduce the amount of anti-platelet autoantibodies in the supernatant of cultured long-lived plasma cells.4.In active ITP mouse model,bortezomib can effectively alleviate the severity of thrombocytopenia.5.This study supplements for the pathogenesis of ITP and provides a new feasible target and an alternative therapy for the clinical treatment of ITP.Part II:The involvement and mechanism of platelet NLRP3 inflammasome activation in ITPObjectives:To explore the alterations of intracellular antioxidant capacity and oxidative stress in ITP platelets,to investigate the activation of NLRP3 inflammasome in ITP platelets,and to improve the pathogenesis and pathophysiology of ITP.Materials and Methods:1.To investigate whether the expression of NLRP3 in the platelets of ITP patients is increased,the platelets of ITP patients and healthy volunteers were collected and stained with CD61 and NLRP3 to detect the expression of NLRP3 in CD61+ platelets.2.The total protein was extracted from the platelets of ITP patients and healthy volunteers.The protein expression level of NLRP3 inflammasome was further verified by western blotting analysis.The levels of NLRP3,ASC,cleaved caspase-1 and cleaved IL-1β were measured.3.Then we analyzed the transcription expression level by RT-qPCR.The platelets from ITP patients and healthy volunteers were collected,and the mRNA was extracted and reverse transcribed into cDNA.The mRNA expression of NLRP3,ASC,caspase-1 and IL-1β was detected by RT-qPCR.4.In order to further evaluate the activation of NLRP3 inflammasome,we also observed the co-localization of NLRP3 and ASC in platelets of ITP patients and healthy volunteers by immunofluorescence.The co-localization of NLRP3 and ASC in ITP platelets suggests that NLRP3 inflammasome has been assembled and activated.5.In addition,we also analyzed the activity of caspase-1 in ITP and healthy platelets with caspase-1 activity assay kit to further verify the activation of NLRP3 inflammasome.6.In order to find out the reason of NLRP3 inflammasome activation,we measured ROS level in ITP platelets with ROS detection kit,and detected the intracellular antioxidant capacity of platelets with DCFH-DA by flow cytometry under the stimulation of H2O2 for 30 min.7.Next,we used western blotting to detect the sensitivity of NLRP3 inflammasome activation to ROS response in ITP and healthy platelets,that is,the change of relative expression of NLRP3,ASC,cleaved caspase-1 and cleaved IL-1βbefore and after H2O2 treatment.8.We further elucidated the role of reduced antioxidant capacity in NLRP3 inflammasome activation in ITP platelets using reducing agent NAC.The expression of NLRP3,ASC,cleaved caspase-1 and cleaved IL-1(3 in platelets was assessed by western blotting after incubation with H2O2 for 30 minutes,and the activity of caspase-1 was detected by caspase-1 activity assay kit.9.Next,we evaluated the effect of NLRP3 inflammasome activation on platelet pyroptosis using NLRP3 inhibitor MCC950 and caspase-1 inhibitor Z-YVAD-FMK.Before exposure to H2O2,ITP platelets were pretreated with MCC950 or Z-YVAD-FMK for 30 min,then the percentage of annexin-V+/caspase-1+pyroptotic cells in platelets was analyzed by flow cytometry.Besides,the concentration of IL-1β secreted in the culture supernatant was detected by ELISA.Results:1.The results of flow cytometry showed that the mean fluorescence intensity of NLRP3 in ITP platelets was significantly higher than that in healthy platelets,suggesting that the expression of NLRP3 in ITP platelets increased.2.western blotting analysis of platelets showed that the levels of NLRP3,ASC,cleaved caspase-1 and cleaved IL-1β were significantly increased in ITP platelets,which further confirmed the enhanced expression of NLRP3 inflammasome at protein level.3.The mRNA expression of NLRP3,ASC,caspase-1 and IL-1β in ITP platelets was significantly higher than that in healthy platelets,suggesting that the transcription of NLRP3 inflammasome was also enhanced.4.The immunofluorescence analysis of platelets showed that NLRP3 and ASC co-located in ITP platelets,but not in healthy platelets,indicating that there was enhanced assembly and activation of NLRP3 inflammasome in ITP platelets.5.The caspase-1 activity assay of platelets showed that the activity of caspase-1 in the platelets of ITP patients was significantly higher than that of the healthy control group.Combined with the previous western blotting results of the increased level of cleaved IL-1β,these findings suggest that the NLRP3 inflammasome in ITP platelets was activated.6.There was no significant difference in ROS level between ITP platelets and healthy platelets,while the analysis of antioxidant capacity in platelets showed that the antioxidant capacity in ITP platelets was significantly reduced than that in healthy platelets.7.The relative expressions of NLRP3,ASC,cleaved caspase-1 and cleaved IL-1βin platelets after treatment with H2O2 were detected by western blotting,and the fold change of ITP platelets was significantly higher than that of healthy platelets,suggesting that the activation of NLRP3 inflammasome in ITP platelets was more sensitive to ROS than that of healthy platelets.8.The expression of NLRP3,ASC,cleaved caspase-1 and cleaved IL-1β in ITP platelets pretreated with NAC was significantly lower than that in the unpretreated control group.In accordance with this,the caspase-1 activity of ITP platelets pretreated with NAC after H2O2 treatment was also significantly decreased compared with that of the unpretreated control group.9.ITP platelets were pretreated with NLRP3 inhibitor MCC950 or caspase-1 inhibitor Z-YVAD-FMK,and then exposed to H2O2.The percentage of annexin-V+/caspase-1+ pyroptotic cells in pretreated ITP platelets was significantly lower than that in the pretreated control group.In addition,the concentration of IL-1β in ITP platelet culture supernatant pretreated with MCC950 or Z-YVAD-FMK was also lower than that of unpretreated control group.Conclusions:1.Both the protein and mRNA expression levels of NLRP3 inflammasome in ITP platelets was higher than that in healthy platelets.2.The assembly and activation of NLRP3 inflammasome in ITP platelets were stronger than that in healthy platelets.3.The antioxidant capacity of ITP platelets was significantly lower than that of healthy platelets.4.The sensitivity of NLRP3 inflammasome activation to ROS in ITP platelets was higher than that in healthy platelets.5.The decreased antioxidant capacity in ITP platelets leads to the increase of intracellular oxidative stress,which induces the activation of NLRP3 inflammasome.6.The activation of NLRP3 inflammasome induced the increase of platelet apoptosis and IL-1β secretion,leading to the enhancement of downstream inflammatory state.Part III:Analysis of expression profile of IL-36 cytokines and its role in ITPObjectives:To explore the expression profile of IL-36 cytokines(IL-36a,IL-36β,IL-36γ and IL-36Ra)in ITP,to preliminarily explore the correlation between IL-36 cytokines and ITP,and to further improve the mechanism or pathophysiology of ITP.Materials and Methods:1.Twenty-five active ITP patients and 13 ITP patients in remission were recruited.In addition,another 20 age and gender matched healthy volunteers were recruited as normal controls.2.We prepared platelet poor plasma from the collected peripheral blood,and detected the plasma levels of IL-36a,IL-36β,IL-36γ,IL-36Ra and IL-38.3.The peripheral blood mononuclear cells were isolated,and then RNA was extracted and reverse transcribed into cDNA.Real-time quantitative PCR was performed to detect the transcription level of IL-36 cytokines.4.Next,we analyzed the correlation between IL-36 cytokines in active ITP patients and platelet count which is characteristic in the disease.5.Considering that the presence of anti-platelet autoantibody is also characteristic in ITP.we also analyzed the relationship between IL-36 cytokines and anti-platelet autoantibody.The 17 active ITP patients who had autoantibody test were divided into two groups:autoantibody positive and autoantibody negative.Then the differences in IL-36 cytokines between the two groups were analyzed.6.In addition,we also analyzed the correlation between IL-36 mRNA level and IL-36R in peripheral blood mononuclear cells of patients with active ITP,and the correlation between IL-36 cytokines and IL-17 or IFN-y levels in plasma.7.Considering that IL-36 cytokines are divided into agonists and antagonists,we also analyzed the relationship between the ratio of IL-36 agonists/antagonists and platelet count or anti-platelet autoantibodies.The formula of IL-36 agonists/antagonists ratio is as follows:(IL-36α fold change+IL-36β fold change+IL-36γ fold change)/(IL-36Ra fold change+IL-38 fold change).Results:1.Compared with healthy control group and ITP in remission group,the plasma level of IL-36α in active ITP patients decreased significantly,and the plasma level of IL-36γ in active ITP patients also decreased significantly compared with healthy control group.However,there was no significant difference between ITP in remission group and healthy control group.In addition,there was no significant difference in the plasma levels of IL-36β,IL-36Rα and IL-38 among the three groups.2.The results of real-time quantitative PCR showed that the alteration of IL-36αmRNA level was consistent with that of plasma level,and the mRNA level of IL-36αin peripheral blood mononuclear cells of ITP patients was significantly decreased compared with healthy controls.There was no significant difference in the mRNA levels of IL-36γ,IL-36Ra and IL-38 among the three groups.The mRNA level of IL-36β is too low to detect.3.The correlation analysis of IL-36 cytokines and platelet count showed that IL-36α,IL-36β,IL-36γ,IL-36Ra and IL-38 had no significant correlation with platelet count.4.There was no significant difference in the levels of IL-36α,IL-36β,IL-36γ,IL-36Ra and IL-38 between the positive and negative anti-platelet autoantibody groups,primarily excluding the relationship between IL-36 and anti-platelet autoantibodies.5.There was no significant correlation between IL-36 mRNA level and IL-36R in peripheral blood mononuclear cells of active ITP patients,and there was no significant correlation between IL-36 and IL-17,IFN-γ in plasma.6.No significant correlation between IL-36 agonists/antagonists ratio and platelet count or anti-platelet autoantibody was noted.Conclusions:1.The levels of IL-36α and IL-36γ in the plasma of active ITP patients were lower than those in the healthy controls.2.The level of IL-36a mRNA in peripheral blood mononuclear cells of active ITP patients was lower than that of healthy controls.3.There was no significant correlation between IL-36 cytokines and platelet count,anti-platelet autoantibody,IL-36R,IL-17 or IFN-y.4.The changes of IL-36 cytokines in ITP are clear,but the specific mechanism of IL-36 cytokines involved in ITP still needs further screening and exploration in the future.