| Aplastic anemia(AA)is a bone marrow failure syndrome caused by a variety of factors.Decreased hematopoiesis in bone marrow results in a decrease in granulocytes,reticulocytes,monocytes and platelets,with varying degrees of anemia,bleeding,and infection as clinical manifestations.It is.currently recognized that AA is an autoimmune disease mediated by T cells.In patients with AA,there are immune abnormalities in which helper T lymphocyte 0(Th0)is polarized to helper T lymphocyte 1(Th1)and helper T lymphocyte 2 is relatively reduced,resulting in an imbalance of Thl/Th2 cells ratio and migration to Th1 cells,which in turn leads to cellular immune hyperactivity and breakdown of autoimmunity.Such a change may have a bearing on the inhibition of proliferation and differentiation of hematopoietic stem cells by cytokines secreted by abnormally activated T cells,such as interleukin 12(IL-12)and interferon-y(IFN-y).Th1 cells are characterized by the secretion of IFN-y which mediates cellular immunity,while Th2 cells are represented by the secretion of interleukin 4(IL-4)and participate in humoral immunity.In this study,the immune status of Th1 and Th2 cells in patients with AA was indirectly observed by examining the protein expression of IFN-y and IL-4.B cells,derived from hematopoietic stem cells,mature in the bone marrow and are activated in secondary lymphatic organs,synthesizing and secreting antibodies that mediate humoral immunity.Activated B cells express a variety of cytokine receptors(CKR),secrete a variety of cytokines,and participate in antigen presentations.If activated B cells and activated T cells are involved jointly in the immune regulation of diseases,immune disorders will occur.B cells can be divided into effector B cells and regulatory B(Breg)cells according to their functions.Breg cells are a special subset of B cells similar to regulatory T(Treg)cells with immunosuppressive function.Breg cells exert their immunomodulatory effects mainly by secreting cytokines interleukin-10(IL-10)and transforming growth factor-P(TGF-β).In this way,other immune cells will be affected,excessive immune responses will be suppressed,and immune tolerance will be maintained.The expression of IL-10 in Breg cells is associated with the changes in the phosphorylation of the STAT3 family.The deficiency of Breg cells in systemic sclerosis is closely linked to the impairment of STAT3,further demonstrating that Breg cells secrete IL-10 dependent phosphorylation of STAT3.IL-10,as an immunosuppressive factor with a variety of biological activities,is characterized by its immunomodulatory function of inhibiting the proliferation and differentiation of CD4+T cells and the secretion of IFN-y,inhibiting the polarization of Th1 and Th2 cells,inducing the differentiation of T cells into Treg cells and the secretion of IL-10.TGF-β plays an immunomodulatory role by inhibiting the proliferation of hematopoietic cells,T cells and B cells,inhibiting the differentiation of CD4+T cells and CD8+T cells,inhibiting the production of inflammatory cytokines such as IFN-γ,inducing the differentiation of Treg cells and secreting cytokines.On the other hand,in Breg cells,the immune regulation effect can be exerted to influence the number and function of Treg cells via intercellular interaction.The number and function of Breg cells are decreased in a variety of immune system diseases,such AS ankylosing spondylitis(AS),systemic lupus erythematosus(SLE),immune thrombocytopenia(ITP),chronic graft-versus-host disease(cGVHD),etc.What are the role and immune regulation mechanism of Breg cells in the occurrence and development of AA in the same autoimmune disease?How do Breg cells change in patients with AA and how are they related to other immune cells?In order to study the role of Breg cells in the occurrence and development of AA,in this study,the changes of IL-10+CD19+Breg cell subsets and TGF-β+CD19+Breg cell subsets in the bone marrow and peripheral blood of patients with AA were first Analyzed.By detecting the expression of IL-10,TGF-β gene and protein,the functional status of Breg cells in AA patients was analyzed.By analyzing the correlation between Breg cells and peripheral blood indicators(neutrophil count,reticulocyte count,platelet count)that reflect the degree of myeloid hyperplasia,the association between Breg cells and the severity of AA disease as well as the treatment prognosis was evaluated.In addition,the role of Breg cells in the pathogenesis of AA and its clinical significance were discussed,proposing a new viewpoint and direction for the immunotherapy of AA.Interleukin 9(IL-9),a single chain glycoprotein with a molecular weight of 14kDa,falls into a member of the y family receptor.It is principally secreted by T cells,such as Th2,Th9,Th17 and Treg cells.IL-9 plays an immunoregulatory role when it binds to the interleukin 9 receptor(IL-9R)on effector cells,and the reverse transcriptional inhibition of Foxp3+Treg cells is enhanced when IL-9 cooperatively induces differentiation of naive CD4+T cells into Th17 cells with TGF-β.When IL-9 binds to IL-9R,STAT phosphorylation,especially STAT3 phosphorylation,will be facilitated by activation of mutual phosphorylation of JAK1 and JAK3 in JAK.Subsequently,IL-9 and IL-9R are transferred to the nucleus to activate the expression of IL-9-induced genes,thus affecting cell proliferation and apoptosis.It has been suggested in a number of studies that IL-9 has a bidirectional regulation effect on the immune system,and its inhibitory or promoting effects on various diseases vary under different circumstances.In the experimental autoimmune encephalomyelitis(EAE)mouse model,IL-9 has been shown to inhibit the pathogenesis of EAE.IL-9 levels in patients with rheumatoid arthritis(RA)and systemic sclerosis(SSc)were significantly higher than those in healthy controls,suggesting that IL-9 could promote the occurrence and development of disease.IL-9 features a certain role in promoting the occurrence and development of the above-mentioned autoimmune diseases,so how about its effect on the occurrence and development of AA disease,an autoimmune disease?In this study,enzyme-linked immunosorbent assay(ELISA),real-time quantitative polymerase chain reaction(RT-PCR)and Western blot were utilized to detect the gene and protein levels of IL-9 and IL-9R in peripheral blood and bone marrow of patients with AA,and to analyze the correlation between IL-9,IL-9R and the clinical features reflecting the degree of bone marrow hyperplasia in patients with AA.The role and clinical significance of IL-9 and IL-9R in the pathogenesis of AA were explored,providing a theoretical basis for IL-9-based targeted therapy.Part I:Changes of Regulatory B Cells in Patients with Aplastic Anemia and its Clinical SignificanceObjective:In this part,differences between the distribution of Breg cell subsets in patients with AA and those in healthy people,as well as their correlation with clinical indicators reflecting the degree of bone marrow hyperplasia,were studied to investigate the function of Breg cell subsets in patients with AA,and to try to discover the role and clinical significance of Breg cells in the occurrence and development of AA.Materials and methods:1.Subject selection:A total of 40 newly diagnosed patients with AA were enrolled,including 16 cases of severe aplastic anemia(SAA),24 cases of non-severe aplastic anemia(NSAA),or 20 cases of remission(remission group)and 20 cases of healthy volunteers(control group)after treatment.5-10 ml each of bone marrow and peripheral blood of the enrolled subjects were collected.2.Extraction and cell culture of mononuclear cells:the bone marrow and the peripheral blood samples of the newly diagnosed patients with aplastic anemia,the patients with aplastic anemia after treatment and the healthy controls were collected to extract mononuclear cells,and the purity of the mononuclear cells was identified by flow cytometry after extraction of the mononuclear cells.Cell concentration was adjusted to 1-2×106/ml.The cells were cultured in an incubator with 5%CO2 at 37℃,supplemented with 25 ng/mL phorbol ester(MULTI SCIENCES Biology co,Ltd),0.5 μg/mL ionomycin(MULTI SCIENCES Biology co,Ltd),0.5 μL/mL Brefeldin A(MULTI SCIENCES Biology co,Ltd)(PIB),5 μg/mL CpG(ODN 7909 Invivogen)and 0.5 μg/mL CD40L(RD)for stimulation culture for 5 hours.3,Detection of regulatory B cell subsets:the expression of IL-10+CD19+Breg cell subsets and TGF-β+CD19+Breg cell subsets in the bone marrows and the peripheral blood mononuclear cells of the severe aplastic anemia group,the non-severe aplastic anemia group,the post-treatment aplastic anemia group and the healthy control group were detected by flow cytometry and cellular immunofluorescence technique,respectively.4.Gene level detection of IL-10 and TGF-β:the expression levels of IL-10 and TGF-β in the bone marrow and the peripheral blood mononuclear cells of the severe aplastic anemia group,the non-severe aplastic anemia group,the post-treatment aplastic anemia group and the healthy control group were detected by RT-PCR at mRNA level.5.Detection of cytokine protein level:the expression levels of cytokines of IL-10,TGF-β,INF-γ and IL-4 in the bone marrow supernatant and the serum of the severe aplastic anemia group,the non-severe aplastic anemia group,the post-treatment aplastic anemia group and the healthy control group were detected by ELISA method.6.Clinical evaluation:complete disease histories of the research subjects were collected and physical examinations were carried out.Neutrophil absolute value,hemoglobin value,platelet count and reticulocyte absolute value were detected using blood routine analyzer by collecting fasting peripheral blood in the morning from four groups of experimental patients.7.Statistical analysis:all the results were analyzed by SPSS 21.0 software package for conducting statistical analysis.Results1.Bone marrow:In terms of the percentage of CD19+B cells in mononuclear cells were 7.62±3.54%,6.95±2.61%,7.62±1.98%,7.11±2.13%in the SAA group,the NSAA group,the remission group,and the control group,respectively.Statistical analysis showed that there was no significant increase or decrease in the SAA group,the NSAA group and the remission group compared with the control group,respectively,and there was no significant increase or decrease in the SAA group and the NSAA group compared with the remission group.2.Peripheral blood:In terms of the percentage of CD19+B cells in mononuclear cells were 6.84±2.67%,7.32±2.49%,7.12±2.92%,8.17±3.35%in the SAA group,the NSAA group,the remission group,and the control group,respectively.Statistical analysis showed that there was no significant increase or decrease in the SAA group,the NSAA group and the remission group compared with the control group,respectively,and there was no significant increase or decrease in the SAA group and the NSAA group compared with the remission group.3.Bone marrow:The proportions of IL-10+CD19+Breg cell subsets of the mononuclear cells in the CD 19+B cells were 0.55±0.17%,1.39±0.41%,2.99 ±0.72%,3.28±1.04%in the SAA group,the NSAA group,the remission group,and the control group,respectively.The proportions of TGF-β+CD19+Breg cell subsets of the mononuclear cells in the CD19+B cells were 1.16±0.2 6%,2.49 ± 0.90%,4.80 ±1.04%,5.42±1.08%in the SAA group,the NSAA group,the remission group,and the control group,respectively.The data demonstrated that the proportions in the severe aplastic anemia group and the non-severe aplastic anemia group were significantly lower than that in the healthy control group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group and the non-severe aplastic anemia group were lower than that of the post-treatment aplastic anemia group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group was lower than that of the non-severe aplastic anemia group,and there were significant differences(P<0.05).4.Peripheral blood:The proportions of IL-10+CD19+Breg cell subsets of the mononuclear cells in the CD 19+B cells were 0.65±0.19%,1.54±0.40%,2.97±0.69%,3.33±0.85%in the SAA group,the NSAA group,the remission group,and the control group,respectively.The proportions of TGF-β+CD19+Breg cell subsets of the mononuclear cells in the CD19+B cells were 0.98±0.20%,2.71 ± 0.79%,4.16 ±1.27%,4.64 ± 1.27%in the SAA group,the NSAA group,the remission group,and the control group,respectively.The data demonstrated that the proportions in the severe aplastic anemia group and the non-severe aplastic anemia group were significantly lower than that in the healthy control group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group and the non-severe aplastic anemia group were lower than that of the post-treatment aplastic anemia group,and there were significant differences(P<0.001),The proportion in the severe aplastic anemia group was lower than that of the non-severe aplastic anemia group,and there were significant differences(P<0.05).5.Bone marrow:In terms of the mRNA expression of IL-10 in mononuclear cells were 0.54±0.12,0.57±0.14,0.92±0.11,1±0.15 in the SAA group,the NSAA group,the remission group,and the control group,respectively.In terms of the mRNA expression of TGF-β were 0.54±0.10,0.69±0.16,0.89±0.21,1 ±0.16 in the four groups,respectively.Peripheral blood:In terms of the mRNA expression of IL-10 in mononuclear cells were 0.54±0.12,0.57±0.14,0.92±0.11,1±0.15 in the SAA group,the NSAA group,the remission group,and the control group,respectively.In terms of the mRNA expression of TGF-β were 0.54±0.10,0.69±0.16,0.89±0.21,1±0.16 in the four groups,respectively.The data demonstrated that the proportions in the severe aplastic anemia group and the non-severe aplastic anemia group were significantly lower than that in the healthy control group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group and the non-severe aplastic anemia group were lower than that of the post-treatment aplastic anemia group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group was lower than that of the non-severe aplastic anemia group,and there were significant differences(P<0.05).6.Bone marrow:In terms of the protein expression of IL-10 in mononuclear cells were 19.77±6.23 ng/mL,23.69±4.48 ng/mL,23.69±4.48 ng/mL,61.12 ±9.67 ng/mL in the SAA group,the NS A A group,the remission group,and the control group,respectively.In terms of the protein expression of TGF-β were 34.99±6.35 ng/mL,42.40±12.00 ng/mL,77.71±9.79 ng/mL、85.01±14.00 ng/mL in the four groups,respectively.Peripheral blood:In terms of the protein expression of IL-10 in mononuclear cells were 22.27±5.86 ng/mL,26.45±5.19 ng/mL,63.30±11.01 ng/mL,63.30±11.01 ng/mL in the SAA group,the NSAA group,the remission group,and the control group,respectively.In terms of the protein expression of TGF-β were 36.13±6.40 ng/mL,41.88±8.55 ng/mL,68.23±12.40 ng/mL,71.96±14.93 ng/mL in the four groups,respectively.The data demonstrated that the proportions in the severe aplastic anemia group and the non-severe aplastic anemia group were significantly lower than that in the healthy control group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group and the non-severe aplastic anemia group were lower than that of the post-treatment aplastic anemia group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group was lower than that of the non-severe aplastic anemia group,and there were significant differences(P<0.05).7.Bone marrow:The expression levels of INF-γ/IL-4 in bone marrow supernatant were 3.03±0.62,2.39±0.53,1.40±0.21,1.28±0.15 in the SAA group,the NSAA group,the remission group,and the control group,respectively.Peripheral blood:The expression levels of INF-γ/IL-4 in serum were 3.03 ± 0.62,2.39±0.53,1.40 ± 0.21,1.28±0.15 in the SAA group,the NSAA group,the remission group,and the control group,respectively.The data demonstrated that the proportions in the severe aplastic anemia group and the non-severe aplastic anemia group were significantly lower than that in the healthy control group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group and the non-severe aplastic anemia group were lower than that of the post-treatment aplastic anemia group,and there were significant differences(P<0.001).The proportion in the severe aplastic anemia group was lower than that of the non-severe aplastic anemia group,and there were significant differences(P<0.05).8.There was a positive correlation between IL-10+CD19+Breg subsets and TGF-β+CD19+Breg subsets and the related clinical indexes of the degree of myelohyperplasia in aplastic anemia reaction of the severe aplastic anemia group and the non-severe aplastic anemia group,and there were significant differences(P<0.05).Conclusions:1.The proportion of CD19+B cells in mononuclear cells in the patients with aplastic anemia is not significantly different from that in the healthy controls.2.The proportion of Breg cells(IL-10+CD19+Breg cell subsets and TGF-β+CD 19+Breg cell subsets)to CD 19+B cells in the bone marrow and peripheral blood in the SAA group and the NSAA group is significantly lower than that in the remission group and the control group,and that in the SAA group is lower than that in the NSAA group,which is positively correlated with the clinical indicators reflecting the degree of bone marrow hyperplasia in peripheral blood.The gene and protein levels of IL-10 and TGF-β are significantly lower in the SAA group and the NSAA group than that in the remission group and the control group,while those in the SAA group are significantly lower than those in the NSAA group,suggesting that Breg cells may play a certain role in the pathogenesis,severity of disease and prognosis of AA.3.The levels of INF-γ/IL-4 in the bone marrow and the peripheral blood of the patients with aplastic anemia are significantly higher those that of the healthy control group.Part Ⅱ:Changes of IL-9/IL-9R in Patients with Aplastic Anemia and its Clinical SignificanceObjective:In this part,the expression of IL-9 and IL-9R in the bone marrow and peripheral blood of patients with AA at gene and protein levels and their correlation with clinical indicators reflecting the degree of bone marrow hyperplasia(neutrophil count,reticulocyte count,platelet count)were studied.The role and clinical significance of IL-9 and IL-9R in the pathogenesis of AA were explored,providing a theoretical basis for IL-9-based immunotherapy.Materials and methods:1.Subject selection:A total of 40 newly diagnosed patients with AA were enrolled,including 16 cases of severe aplastic anemia(SAA),24 cases of non-severe aplastic anemia(NSAA),or 20 cases of remission(remission group)and 20 cases of healthy volunteers(control group)after treatment.5-10 ml each of bone marrow and peripheral blood of the enrolled subjects were collected.2.Mononuclear cell extraction:the bone marrow and the peripheral blood samples from the newly diagnosed aplastic anemia patients and the healthy controls were collected to extract mononuclear cells,and extract cDNA and protein from mononuclear cells.The bone marrow supernatant and serum were collected at the same time and freezed at-80℃.3.The expression levels of IL-9 in the bone marrow supernatant and the serum of the patients with aplastic anemia were detected by ELISA method,and the correlation between IL-9 expression and the related clinical indexes reflecting the degree of myelohyperplasia was analyzed.4.The gene and protein expression levels of IL-9 and IL-9R in the bone marrow and the peripheral blood mononuclear cells of the patients with aplastic anemia were analyzed by RT-PCR and Western blot,and the correlation with the clinical indexes reflecting the degree of myelohyperplasia was also analyzed.5.Statistical analysis:all the results were analyzed by SPSS 21.0 software package for conducting statistical analysis.Results:1.The protein expression of IL-9 in bone marrow supernatant of the SAA group,the NSAA group and the control group were 2.48±0.56 ng/mL,2.25±0.32 ng/mL,1.28±0.40 ng/mL,respectively.Statistical analysis showed that the protein expression of IL-9 in the SAA group was higher than that in the control group(P<0.05),that in the NSAA group was higher than that in the control group(P<0.05),and that in the SAA group was higher than that in the NSAA group(P>0.05),which was negatively correlated with the indicators reflecting the degree of bone marrow hyperplasia(P<0.05,r<0).2.The protein expression of IL-9 in the serum of the SAA group,the NSAA group and the control group were 3.15±0.39 ng/mL,3.01 ±0.46 ng/mL and 1.18±0.50 ng/mL,respectively.Statistical analysis showed that the protein expression of IL-9 in the SAA group was higher than that in the control group(P<0.05),that in the NSAA group was higher than that in the control group(P<0.05),and that in the SAA group was higher than that in the NSAA group(P>0.05),which was negatively correlated with the indicators reflecting the degree of bone marrow hyperplasia(P<0.05,r<0).3.The mRNA expression of IL-9 in bone marrow mononuclear cells of the SAA group,the NSAA group and the control group were 1.88±0.21,1.81±0.28,1.0±0.13,respectively.The mRNA expression of IL-9R in bone marrow mononuclear cells of the SAA group,the NSAA group and the control group were 1.73±0.19,1.62±0.26,1.0±0.17,respectively.The protein expression of IL-9 in bone marrow mononuclear cells of the SAA group,the NSAA group and the control group were 2.38±0.27,1.84 ± 0.02,1.0±0.16,respectively.The protein expression of IL-9R in bone marrow mononuclear cells of the SAA group,the NSAA group and the control group were 2.96±0.22,2.13±0.10,1.0±0.04,respectively.Statistical analysis showed that the protein expression of IL-9 in the SAA group was higher than that in the control group(P<0.05),that in the NSAA group was higher than that in the control group(P<0.05),and that in the SAA group was higher than that in the NSAA group(P>0.05),which was negatively correlated with the indicators reflecting the degree of bone marrow hyperplasia(P<0.05,r<0).4.The mRNA expression of IL-9 in peripheral blood mononuclear cells of the SAA group,the NSAA group and the control group were 1.52±0.33,1.44±0.35 and 1.0±0.14,respectively.The mRNA expression of IL-9R in peripheral blood mononuclear cells of the SAA group,the NSAA group and the control group were 1.66 ± 0.18,1.55 ± 0.22,1.0±0.15,respectively.The protein expression of IL-9 in peripheral blood mononuclear cells of the SAA group,the NSAA group and the control group were 3.23±0.08,2.12±0.25,1.0±0.16,respectively.The protein expression of IL-9R in peripheral blood mononuclear cells of the SAA group,the NSAA group and the control group were 3.63±0.18,2.38±0.20,1.0±0.03,respectively.Statistical analysis showed that the protein expression of IL-9 in the SAA group was higher than that in the control group(P<0.05),that in the NSAA group was higher than that in the control group(P<0.05),and that in the SAA group was higher than that in the NSAA group(P>0.05),which was negatively correlated with the indicators reflecting the degree of bone marrow hyperplasia(P<0.05,r<0).Conclusions:1.The level of IL-9 protein in bone marrow supernatant and peripheral serum of patients with AA is increased,especially in the SAA group,which is negatively correlated with the clinical indicators reflecting the degree of bone marrow hyperplasia.2.The gene and protein levels of IL-9 and IL-9R in the bone marrow and peripheral blood mononuclear cells of patients with AA are significantly higher than those of the healthy control group,especially in the SAA group,which is negatively correlated with the clinical indicators reflecting the degree of bone marrow hyperplasia.3.A certain correlation is observed between the abnormal expression of IL-9 and IL-9R and the occurrence and severity of AA.Supplement:As described in this study,the secretion of IL-10 by Breg cells has a close bearing on the phosphorylation of STAT3,and the corresponding biological role of IL-9 binding to IL-9R is also related to the phosphorylation of STAT3.In this study,the correlation analysis between Breg cell subsets and IL-9 and IL-9R was carried out,and no obvious correlation was found,which was considered to be linked to the small sample size.In view of this,positive countermeasures will be taken to increase the sample size to more accurately study the association between Breg cells and IL-9/IL-9R. |
PDF Full Text Request