| Aplastic anemia(AA)is a hematological disorder characterized by reduced bone marrow hematopoietic tissue,reduced hematopoietic function,or failure of the hematopoietic stem cells resulting in complete cytopenia.Compared with Europe and the United States,Asian countries have a higher incidence of approximately(3.9-5.0)/106.The clinical manifestations of AA mainly include varied degrees of anemia,hemorrhage and infection.Most patients do not have hepatomegaly,splenomegaly and lymphadenopathy.The traditional mechanism of pathogenesis is believed to be related to hematopoietic stem/progenitor cell abnormalities,dysfunction of hematopoietic microenvironment and unbalance of the immune system.At present,most scholars believe that abnormal immune functions play an important role in the development of AA.Hematopoietic microenvironment and hematopoietic stem and progenitor cell damage are caused by abnormal immune functions.And in clinical practice,immunosuppressive agents have good effects on AA,providing more powerful evidence for this argument.The immune abnormalities of AA are manifested in many ways,including changes in the number and proportion of T subpopulations and cytokines,abnormalities of dendritic cells and natural killer cells.CD4+T lymphocytes and related cytokines are the key factors of immune response,which play important roles in the initiation and effect phase of the immune response.Studies have shown that CD4+CD25+CD127low Tregs have an important role in maintaining normal function of the immune system.CD4+CD25+CD127low Tregs are a subset of CD4+T cells and have the function of immunosuppression and induction of immune tolerance.CD4+CD25+CD127low Tregs can secrete cytokines with inhibitory functions,such as IL-10,TGF-? and other types of membrane surface inhibitory molecules.These inhibitory molecules eventually inhibit the proliferation and activation of cytotoxic T lymphocytes(CTL)and helper T cells(Th).Therefore,CD4+CD25+CD127low Tregs play an important role in regulating the immune response of T cells,preventing the occurrence of autoimmune diseases,tumor immunity,maintenancing of immune tolerance and other aspects.In light of this,more and more studies focused on the pathogenesis of AA are aimed at the abnormalities of the number and proportion of Tregs.Some studies have shown that the number and proportion of Tregs in peripheral blood and bone marrow of AA patients are all decreased compared with those in healthy populations.The number of Tregs in patients with effective immunosuppressive therapy(IST)can be significantly increased,whereas Tregs of those ineffective patients are not significantly changed.Due to the decrease of Tregs and the negative regulators(IL-10,IL-35 and TGF-?)of Tregs,the ability to maintain immune tolerance is impaired,so that the self-reactive T lymphocytes proliferate in a large amount and the cellular immune function is enhanced,which lead to the occurrence of AA.At present,among AA patients receiving immunosuppressive therapy()IST based on anti-thymocyte immunoglobulin(ATG)or anti-lymphocyte immunoglobulin(ALG),only 2/3 have varied degrees of hematopoietic recovery,while more than 40% of AA patients who don't respond to IST died in 5 years because of bleeding or infection.Furthermore,in terms of affordability alone,ATG-or ALG-based IST also overwhelmed a significant proportion of patients with AA.Therefore,it is undoubtedly an urgent thing to explore appropriate,effective and inexpensive treatment for these patients with AA.ATO is currently recognized as the first-line treatment for acute promyelocytic leukemia because of its ability to promote differentiation and induce apoptosis.At the same time,as a ubiquitous environmental toxicant,arsenic including ATO had complex effects on the immune system.And many scholars believe that the damage of arsenic to the immune system may be one of the important causes of these diseases.Studies have found that arsenic exposure can lead to immune damage,and CD4+T lymphocytes may be the early targets.The team of academician Shusen Zheng from China found that ATO could control acute graft-versus-host disease(GVHD)by inhibiting cellular immunity by the heart transplantation model of mouse.In this process,the levels of CD4+T cells,CD8+T cells,IFN-? and TNF-? m RNA decreased and the Treg and related cytokine TGF-? m RNA increased.There are also studies confirmed that low-dose and long-term exposure to arsenic could up-regulate Tregs levels in the spleen and inhibit T cell immunity of mice.Abnormalities of the proportion,number,and function of Tregs play an important role in the pathogenesis of AA.And ATO could inhibit the immune system function by increasing the proportion and/or number of Tregs according to previous studies.However,there is no study focus on whether ATO can increase the proportion of peripheral Tregs in AA patients and suppress T cell immunity to promote hematopoietic recovery currently.ObjectiveTo investigate the immunomodulatory effects of ATO on the proportions of CD4+CD25+CD127low Tregs in peripheral blood of patients with SAA,flow cytometry was used to detect the effect of different concentrations of ATO on the proportions of Tregs,and the results were verified by RT-PCR and ELISA.And we hope this research can be helpful to explore more economical and effective treatment methods for patients with AA who could not receive hematopoietic stem cell transplantation and with poor efficacy of conventional immunosuppressive drugs.Materials and Method1.General informationTwenty newly diagnosed SAA patients were collected from Henan Tumor Hospital and Henan Provincial peoples' Hospital.All patients signed informed consents as required.Among the 20 patients,13 were females and 7 were males.The median age was 34(13-56)years.All patients meet SAA diagnostic criteria and all the details can be found in the appendix table.2.Experimental methodThe proportions of CD4+CD25+CD127low Tregs in peripheral blood PBMC of SAA patients treated with different concentrations of ATO were measured by flow cytometry.The expression of Fox P3 m RNA was detected by RT-PCR and the concentrations of IFN-?,IL-4,IL-17 and TGF-?1 were detected by ELISA.3.Statistical analysisWe used SPSS.22 software for statistical analysis.Spearman rank correlation tests were used to analyze the correlations between ATO concentrations and Tregs ratio,concentrations of cytokines and Tregs ratio.Person correlation tests were used to analyze the correlations between expression levels of Fox P3 m RNA and Tregs ratio.The data were presented as mean±standard deviation(?X±S).Significant statistical differences were considered when P<0.05.Results1.Effects of ATO on Tregs ratio of PBMC in peripheral blood of patients with SAAAt the concentration of 2.5?mol/L and 5?mol/L,ATO could significantly up-regulate the ratio of Tregs(P<0.001).Treg ratio is positively correlated with ATO concentration(r=0.616,P<0.001).When the ATO concentration was 1 ?mol/L,the results was not statistically significant(P=0.052).2.The effects of ATO on the expression levels of Fox P3 m RNA in peripheral blood PBMC of patients with SAAAt 1?mol/L(P=0.032),2.5?mol/L(P<0.001)and 5?mol/L(P<0.001),ATO could up-regulate the expression levels of Foxp3 m RNA,and the expression levels of Foxp3 m RNA were positively correlated with Tregs ratio(r=0.523,P<0.001).3.Effects of ATO on cytokines in culture supernatant of PBMC of patients with SAAATO significantly reduced concentrations of IFN-? at 1?mol/L,2.5 ?mol/L and 5?mol/L,P<0.001.ATO significantly reduced concentrations of IL-4(2.5 ?mol/L,P=0.009;5 ?mol/L,P<0.001)and IL-17(2.5 ?mol/L,P=0.016;5 ?mol/L,P<0.001).ATO had no significant effects on the levels of TGF-?1 at 1 ?mol/L(P=0.458),2.5?mol/L(P=0.352),but significantly reduced the levels of TGF-?1 at 5 ?mol/L(P=0.032).Correlation analysis showed significant negative correlations between Tregs ratio and concentrations of IFN-?(r=-0.546,P<0.001),IL-4(r=-0.271,P=0.015)?IL-17(r=-0.293,P=0.008).ConclusionATO up-regulated Tregs ratio and expression levels of Foxp3 m RNA in PBMC of SAA patients,and reduced the levels of cytokines IFN-?,IL-4,IL-17 to varied degrees. |
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