Interleukin 9 Prevents Immune Thrombocytopenia In Mice And Its Mechanism

Background and aimImmune thrombocytopenia(ITP)is a common autoimmune bleeding disorder characterized by persistent thrombocytopenia.ITP is caused by the production of auto-antibodies against platelet membrane glycoproteins,which mediate the destruction of platelets in the reticuloendothelial system.The incidence of ITP is 1.6?3.9/100 000 in adults and 3.0?5.3/100 000 in children per year.Clinically,it can be divided into acute type and chronic type.The former is predominant in children,and the latter is predominant in adults.The overall prognosis of ITP patients is good,but the quality of life of patients is seriously affected because of low platelet and high risk of bleeding.There are many different treatments for ITP including glucocorticoid,Intravenous immunoglobulin(IVIG),splenectomy,CD20 monoclonal antibodies,immunosuppressive medications,TPO and TPO-RA,but whose application was restricted due to the side effects,high costs,low efficiency,and poor efficacy.There is thus still an urgent need to find novel therapies to stimulate platelet production and prevent thrombocytopenia.Interleukin-9(IL-9)originally was identified as a growth factor for the human megakaryoblastic cell line MO7E,which showed significant homology with the murine T-cell growth factor P40.IL-9 is a pleiotropic cytokine produced mainly in T helper cell and generally functions through the IL-9R to activate JAK/STAT1,STAT3?STAT5 signaling in various biological processes.Many cytokines,such as IL-lb,IL-6,IL-la,and IL-11,have been demonstrated synergistic effects with TPO to influence the proliferation and/or differentiation of MK.Although IL-9 originally was identified as a hematopoietic cell regulatory factor,it has been reported promoting MK cell proliferation in culture in the presence of TPO and/or SCF.We previously showed that osteoblasts can secret IL-9 to support megakaryopoiesis and platelet formation.However,the effect of IL-9 in ITP has not been reported.In this study,we created a CD41-induced ITP mouse model to investigate the effect of IL-9 treatment,and explore its mechanisms.The results will provide further evidence to support the development of IL-9 as a promising therapeutic agent for the treatment of ITP.Materials and methods1.Rat anti-mouse CD41 monoclonal antibody(MWReg30)inducedthrombocytopenia mouse modelBALB/c male mice(6-8 weeks old)were divided into two groups randomly:ITP group and control group.ITP group were injected intraperitoneally with rat anti-mouse CD41 monoclonal antibody(MWReg30)for 8 consecutive days to induce ITP.Control mice were injected with the same dosage of IgGl.Briefly,mice were injected with 68 ?g/kg MWReg30 antibody on day 1 and day 2,102?g/kg on day 3,and 136 ?g/kg from day 4 to day 8 respectively.After injection,20-30 ?l peripheral blood was taken from the tail vein each day and diluted,to detect the numbers of white blood cells,red blood cells,and platelets.2.Flow cytometry analysisOn Day 8,bone marrow cells extracted from ITP group and control group were incubated with different antibodies to detect and calculate the percentage of CD41+Sca-1+,and CD41+CD42d+cells by FACScan flow cytometer3.Enzyme-linked immunosorbent assay(ELISA)On Day 8,blood was extracted from ITP mice and control mice.IL-9 concentrations in the serum was measured by ELISA,according to the manufacturer's protocol.4.Quantitative real time-polymerase chain reaction(qRT-PCR)On Day 8,bone marrow was obtained from ITP mice and control mice.Total RNA was extracted from tissues.The relative expression of IL-9 mRNA was analyzed by reverse transcription and polymerase chain reaction.5.The effects of IL-9 in ITP miceBALB/c male mice(8 weeks old)were divided into three groups:ITP+IL-9 group,ITP group and control group.ITP+IL-9 group mice were injected with MWReg30 and IL-9 for 8 consecutive days.Briefly mice were injected with IL-9(7.5 ?g/kg)on day 1 and day 2,11.25?g/kg on day 3,and 15 ?g/kg from day 4 to day 8.ITP group and control were same as previous(Method 1).The number of platelets was counted at different time points.On Day 8,bone marrow cells extracted from three groups were stained with different antibodies to detect the percentage of CD41+Sca-1+and CD41+CD42d+cells by FACScan flow cytometer.6.In vivo siRNA knockdown of IL-9 receptor(IL-9R)IL-9R siRNA or negative control siRNA was injected into the bilateral medullary cavity and mice were divided into three groups:negative control group,ITP+IL-9+IL-9rSiRNA group and ITP group.Mice were euthanized 7 days after the injection and BM was collected for MK counts by flow cytometry analysis.Whole blood was collected for quantitative analysis.7.JAK/STAT5 pathway analysis in bone marrow cells by western blottOn Day 8,bone marrow was extracted from ITP+IL-9 group,ITP group and control group.Tissues were lysed and the supernatants were separated by SDS-polyacrylamide 'gel electrophoresis and blotted onto a nitrocellulose membrane.The membrane was then incubated with antibodies against p-STAT5 and STAT5 and visualized using an enhanced chemiluminescence kit.8.Immunofluorescence analysis of tissue sectionsOn Day 8,Femur and tibia were obtained from ITP+IL-9 group,ITP group and control group.Femur and tibia tissues were dissected from the mice and fixed with 4%paraformaldehyde in PBS,and then decalcified in 15%EDTA.The tissues were embedded in paraffin,and 3-mm sagittal sections were prepared for immunofluorescence analysis,the sections were incubated with primary antibodies against CD42d and p-STAT5.9.Statistical analysisData are presented as the mean �standard deviation(SD)of three independent experiments.All statistical analyses were carried out using Student's t-test and ANOVA.A value of p<0.05 was considered as statistically significant.Results1.Successful establishment of ITP mouse modelBALB/c mice injected with the monoclonal antibody MWReg30 were developed into ITP.The basal numbers of peripheral blood platelets were(1250.13�36.02)�109/L and(1283.25�54.71)�109/L in the ITP group and control group,and there was no difference in platelet counts between two groups.The platelet counts(576.50�67.58)�109/L in ITP group was dropped to half of the initial values by 24 h.And then remained relatively constant from day 3 to day 8.Platelet counts remained stable throughout the study in the control mice injected with IgGl.The CD41+CD42d+cells in the BM were screened by flow cytometry and showed that the percentage of CD41+CD42d+cells was significantly lower in ITP mice((5.25 � 1.03)/104)compared with normal((15.13� 1.31)/104,n=8,p<0.001).2.The expression of IL-9 in ITP mice and control miceTo determine the circulating IL-9 in serum,ELISA assays were used in this study.We found a decreased IL-9 levels in ITP mice(13.1310.77pg/ml),compared to the normal(81.76�1.91pg/ml)(P<0.001).We tested the expression of IL-9mRNA in MK cells from both groups by q-PCR and found that the expression of IL-9mRNA were(0.12� 0.03)in ITP mice relative to the normal(p<0.001).3.IL-9 rescue in ITP miceWe tested the numbers of peripheral blood platelets on Day 1,There was no difference in platelet counts among ITP+IL-9group,ITP group and control group.On the second day the numbers of platelets in control group((1246.38�57.15)�109/L)were higher than that in ITP group((576.50�67.58)� 109/L)and ITP+IL-9group((950.00�61.35)�109/L,p<0.001),and platelet counts in ITP+IL-9 group were higher than ITP group(n=8,p<0.001).On Day 8,we detected the frequency of CD41+CD42d+cell from these three groups by flow cytometry and found that the percentage of CD41+CD42d+cells was significantly higher in ITP+IL-9 group((12.28 � 2.21)/104)compared with ITP group((5.25�1.03)/104,p<0.001),but lower than control group((15.13� 1.31)/104,p=0.086).4.IL-9 prevents CD41-induced thrombocytopenia via the IL-9RWe explored the mechanism of action of IL-9 in thrombocytopenia by injecting IL-9R siRNA into the BM in normal mice and ITP+IL-9 mice.IL-9R siRNA suppressed IL-9R expression and abrogated the therapeutic effect of IL-9 on ITP.5.IL-9 may prevent CD41-induced ITP by activating IL-9R/STAT5 pathwayWe further investigated the mechanism underlying the action of IL-9 in thrombocytopenia,especially its effects on downstream,by detecting p-STAT5 expression.The expression levels of p-STAT5 in the BM increased in ITP+IL-9 mice group compared with the ITP alone group.The proportion of mature MK(CD42d+)was also higher in the ITP+IL-9 group(2.69�0.34)%compared with the ITP group(0.65� 0.18)%,but still lower than in the control group(6.36� 1.13)%by flow cytometry.Similar results were obtained by immunofluorescence analysis of femur sections.Conclusion1.We reported that the expressions of IL-9 in CD41-induced ITP mice were lower than normal mice;2.IL-9 can stimulate the development and the mature of megakaryocytes and promote platelet formation in ITP mice.3.We further demonstrated that this effect is likely to be mediated via IL-9R with subsequent activation of the JAK2/STAT5 signaling in ITP mice.