Bifunctional Effect Of Inflammatory Cytokine TNFα On Human Megakaryopoiesis And Platelet Production

PurposePlatelets are affected by many factors,such as inflammation,bleeding,tumors.The close relationship between inflammation and platelets has been reported by many studies.Severe infections or sepsis can lead to thrombocytopenia,but some inflammatory status can also cause thrombocytosis.Tumor necrosis factor α(TNFα)is one of the important inflammatory cytokines,which has a killing effect on tumor cells and also regulates the selfrenewal and differentiation of hematopoietic stem cells.However,there are few studies on the effect of TNFα on megakaryocytes(MK)and platelets,and the mechanism of action remains largely elusive.Therefore,the main objectives of this subject are as follows:1.Explore the level of TNFα in platelet abnormalities-related diseases;2.Clarify the effects of TNFα on megakaryocyte proliferation,differentiation,maturation,pro-platelet formation and platelet production in vitro;3.Reveal the downstream targeting molecules and signaling pathway mechanism that involved in megakaryocytes regulation triggered by TNFα;4.Validate the effect of TNFα on megakaryocytes and platelets in mice.Methods1.Twenty patients with essential thrombocythemia and 10 healthy donors,20 patients with prolonged thrombocytopenia after HSCT,and 20 patients with good platelet reconstitution after HSCT were enrolled in this study.Inflammatory factors level of the patients was detected by ELISA and quantitative PCR,and to explore the level of TNFα in these patients.2.A culture system of CD34+cells inducing toward MK in vitro was established firstly.Different concentrations of TNFα and LPS were added and cultured for 12 days.Then the expression level of CD41,CD42,and CD61 in different treatment groups were detected by flow cytometry,evaluating the effect of TNFα and LPS on megakaryocyte differentiation and maturation,and identifying the appropriate concentration.The effects of TNFα on megakaryotic polyploid formation,pro-platelet production,and platelet production were further studied by flow cytometry and cell immunofluorescence.The expression levels of TNFa receptors,TNFR1 and TNFR2 were detected in different treatment groups to clarify the role of receptors in regulating megakaryocytes.3.Transcriptome sequencing of the different concentrations TNFa treated MKs was performed,and the possible downstream targeting molecules and pathways were explored through GO,KEGG,and GSEA analysis.Western Blot and real-time quantitative PCR were used to verified the expression of adhesive and migrated molecules.By treating with blocking antibody,the role of JAMC in regulating the expression of CD41,CD42 and CD61+MK,polyploid distribution,pro-platelet formation and platelet production,as well as adhesion and migration abilities were determined,and further clarify the relationship between JAMC and TNFα.The activation status of MAPK-ERK1/2,PI3K-Akt and NF-κB signaling pathway were detected in different concentrations TNFa treated groups,and then the pathway inhibitors U1026,LY294002 and Bay11-7082 were added into the culture system to determine the role of these pathways in MK differentiation,maturation and platelet production.4.The effect of TNFa on the MK adhesion and migration ability was detected.In order to explore the potential mechanism of TNFa on the process of platelet production,the cytoskeletal molecules including RhoA,ROCK1,Cofilin and MLC2 in different concentrations TNFα treated group were detected by western blot.5.Stable transfected JAMC knockdown and overexpression MK cell lines were constructed,and cell proliferation,apoptosis and cycle were detected.The phosphorylation level of Cofilin and MAPK-ERK1/2,as well as mitochondrial metabolism were detected.6.The mitochondrial metabolism and oxidative phosphorylation status,including the fluorescence level of MTG,DiIC1(5)and ROS were detected in different concentrations of TNFa treated groups by flow cytometry.7.Different doses of TNFa were administered in mice,then the platelet count was monitored dynamically.In addition,the effect of different dose of TNFa on the MK and platelet reconstitution after bone marrow transplantation were monitored.Results1.The level of TNFa in patients with essential thrombocythemia was significantly higher than that in the healthy control group(p<0.001),in addition,the expression level of TNFR1 was also significantly higher than that of the control group(p<0.01),however,the level of TNFR2 was significantly lower than that of the control group(p=0.029).The expression level of TNFα,IFN-γ and IL-10 in patients with prolonged thrombocytopenia after transplantation was significantly lower than that of patients with normal platelet reconstitution(p=0.040,p=0.034,p=0.048).While the expression levels of IL-6 were comparable between the two groups(p=0.373).2.Both TNFα and LPS showed influence on the differentiation and development of megakaryocyte.As the concentrations increase,a low concentration of TNFα 0.5 ng/ml tended to promote megakaryocyte differentiation,maturation,polyploidy and proplatelets formation as well as platelets production,while a high concentration of TNFa 10 ng/ml or higher showed a significant inhibitory effect.The LPS showed an inhibitory effect on megakaryocyte as the concentration increased.3.There was no significantly difference of TNFR1 and TNFR2 expression level between the low-concentration of TNFα 0.5 ng/ml group and the control group,while a slightly increase was observed in the high-concentration of TNFα 10 ng/ml group.AntiTNFR1 Ab treatment significantly weakened the stimulating effect of TNFa 0.5 ng/ml on CD42+MK percent,which was close to that of the control group.Both anti-TNFR1 Ab and TNFR2 Ab significantly weakened the positive effect of TNFα 0.5 ng/ml on platelet,especially the anti-TNFR1 Ab.The two blocking antibodies showed no additive effect.4.The RNA sequencing data indicated that the expression of adhesion and migration molecules significantly changed upon TNFa stimulation.In vitro experiments validated that the expression of JAMC,which belonged to the junction adhesion molecule family,is regulated by TNFa.Low concentration of TNFa 0.5 ng/ml significantly upregulated the expression of JAMC,while high concentration of TNFa 10 ng/ml showed a significantly inhibitory effect.In addition,the expression of JAMC gradually increased as the MK development.When the JAMC was blocked by anti-JAMC Ab,the megakaryocyte maturation and platelet production was significantly inhibited.In addition,the stimulating effect of low concentration of TNFa on megakaryocyte was weakened in the co-presence of anti-JAMC Ab and TNFa 0.5 ng/ml.5.In the low concentration TNFα 0.5 ng/ml group,the phosphorylation level of ERK1/2 decreased significantly,the phospho-Akt level increased,while the NF-κB pathway showed no significant activation.The high concentration of TNFa 10 ng/ml treatment obviously promoted the phosphorylation activation of ERK1/2 and significantly inhibited the phosphorylation of NF-κB,while the activation status of Akt pathway showed no significant change.MAPK-ERK1/2 inhibitor U1026 increased the percent of CD41+,CD61+MK and platelets production.Akt inhibitor LY294002 significantly inhibited the percent of CD42+MK and platelets number.NF-κB pathway inhibitor Bay11-7082 significantly decreased the proportion of CD41,CD42,CD61 megakaryocyte and the number of platelets.In addition,the phosphorylation level of ERK1/2 in the anti-JAMC Ab treatment group was significantly increased,while,the phosphorylation level was close to that of the control group in the costimulation of anti-JAMC Ab and TNFα 0.5 ng/ml.Akt and NF-κB pathways showed no obvious response to anti-JAMC Ab treatment.6.The number of adhesive and migrated cell in the TNFα 0.5 ng/ml treatment group was significantly higher than that in the control group,while the TNFa 10 ng/ml treatment group showed an opposite effect.The treatment of anti-JAMC Ab and anti-MAC1Ab,which act as a counter-receptor of JAMC,led to an impaired adhesion and migration ability of MK.In terms of cytoskeleton molecules,the phosphorylation level of Cofilin significantly decreased,while the phospho-MLC2 significantly increased in the TNFα 0.5 ng/ml group,and the high concentration of TNFa 10 ng/ml treatment showed an opposite effect.The TNFα stimulation showed no significant impact on RhoA and ROCK1 expression.The effect of anti-JAMC Ab on these molecules are similar to high concentration TNFα 10 ng/ml.The level of phospho-Cofilin was significantly increased,and the phospho-MLC2 slightly reduced.For the co-stimulation of anti-JAMC Ab and TNFα 0.5 group,the level pf phosphoCofilin was higher than that of the TNFα 0.5 ng/ml group,and the level phospho-MLC2 level was higher than that of the anti-JAMC Ab treatment group.7.Knockdown of JAMC by shRNA and overexpression by PCDH-JAMC had no significant effect on the proliferation of megakaryocytic cell lines,and the cell viability showed no significant difference with the control group at different time points.In terms of cell apoptosis,the early apoptosis percent in JAMC knockdown group was significantly higher than that of the control group,and the late apoptosis proportion also slightly increased;there was no significant difference of the early apoptosis percent between the JAMC overexpression group and the control,while the late apoptosis percent significantly reduced in the JAMC overexpression group.In cell cycle detection,the proportion of G1 phase in the JAMC knockdown group was slightly lower than that in the control group,and the proportion in S phase and G2 phase showed no significant difference between the different groups.The proportion of cells in G1 phase in the JAMC overexpression group was slightly higher than that in the control group,and there was no significant difference in the percent of S phase and G2 phase compared with the control group.As for adhesion assay,the number of adhesive cells in the JAMC knockdown group was significantly lower than that of the control,while there was no significant difference between the JAMC overexpression group and the control.In the migration assay,the number of cells migrated to the low chamber membrane in the JAMC knockdown group was similar to the control group,while the migrated cell in the JAMC overexpression group was significantly higher than that in the control group.Regarding the expression of downstream molecules,the phosphorylation level of ERK1/2 in the JAMC knockdown group significantly increased,meanwhile the phosphorylation of Cofilin was upregulated remarkably.When JAMC is overexpressed,ERK1/2 phosphorylation was obviously inhibited,and Cofilin was dephosphorylated.8.There was no significant difference of the MTG fluorescence level between the different concentration of TNFα treatment groups.The DiICi(5)level significantly reduced in the TNFα 10 ng/ml treatment group,while no significant difference was observed between TNFα 0.5 ng/ml group and control group.The ROS level was significantly higher in the TNFα 10 ng/ml group than that of the control group,and showed no significant change in the TNFα 0.5 ng/ml treatment group.The DiIC1(5)fluorescence level in the JAMC knockdown group was significantly lower than that of control group,while the JAMC overexpression group was slightly higher than that of the control group.9.The administration of TNFα 0.5 μg can lead to a transient increase of platelets in wild type mice(Balb/c).In mice received syngeneic bone marrow transplantation,platelets continued to decline within 1 week after transplantation,and reached the lowest point on the 7th day.The platelet levels in the TNFα 0.5 μg and TNFα 2 μg groups were significantly higher than those of the control group within 14 days after transplantation,then returned to basic level and showed no significant difference with the control group.The platelet level in the TNFa 5 μg group was significantly lower than that of the control group within 14 days,and still slightly lower than the control group on the 21st day,then returned to the basic level on the 28th day,indicating a delayed platelet reconstitution.The number of MK in bone marrow and the distribution distance of MK from vascular endothelium also showed significantly change after TNFa administration.The distance between MK and the vascular endothelium in the low-dose TNFα group was shorter.The number of MK in the high dose group significantly reduced,and the distance from the vascular endothelium was farther than the control group.In mice that received allogeneic bone marrow transplantation,the TNFa 0.5 μg administration slightly promoted platelet recovery within one month after transplantation.While the platelet level of TNFα 2 μg group was significantly lower than that of the control group within 1 month after transplantation,indicating delayed platelet recovery.ConclusionsTherefore,we get the conclusions that 1.TNFa has a unique bifunctional effect on megakaryocyte maturation and platelet production,with a low concentration exerting a stimulating effect,and a high concentration showing a significant inhibitory effect.The effect of TNFa was mainly depended on TNFR1.2.JAMC was modulated by TNFa,and showed a directly regulation on megakaryocytes.JAMC may serve as a downstream target mediating the regulatory effect on megakaryocyte triggered by TNFa.3.The MAPK-ERK signaling pathway showed obvious response to TNFa treatment.JAMC also directly regulated the activation or inactivation of ERK1/2.Therefore,we speculate that the MAPK-ERK1/2 signaling pathway may act as a terminal target,which may regulate the differentiation and development of megakaryocytes.4.The activation status of Cofilin was regulated by both TNFa and JAMC.Cofilin may act as another terminal molecule of TNFα/JAMC,participating megakaryocyte skeleton reorganization and platelet production.5.In mice,low-dose TNFα showed a short-term promoting effect on platelet reconstitution after bone marrow transplantation,while high-dose delayed platelet reconstitution.