Role Of Th17 Cells In The Pathogenesis Of Myelodysplastic Syndromes

Objective: Illuminate roles of Th17 cells in the pathogenesis of myelodysplastic syndromes(MDS) patients with different risks through measuring the frequency of Th17 cells(CD3+CD4+IL-17+), detecting levels of related cytokines in patients with different risks of MDS,analyzing the correlation between Th17 cells frequency and hematological parameters of MDS patients, testing levels of cytotoxic molecule expression in CD3+CD8+ T cells before and after human recombinant IL-17(rh IL-17) stimulation,ultimately to explore the role of Th17 cells in the pathogenesis of MDS.Background: MDS are a diverse group of clonal hematopoietic malignancies characterized by ineffective hematopoiesis, progressive bone marrow(BM) failure, cytogenetic and molecular abnormalities, and unpredictable risk of further deteriorating into acute myeloid leukemia(AML). Data from vast clinical and experimental exploration have revealed that T-lymphocytes were involved in the pathogenesis process of MDS, and our research team had found the abnormalities in the frequency and(or) function of nature killer cells, T regulatory cells,Th1 and Th2 cells as well as their ratio in patients with MDS in our previous studies. With Retinoid-related Orphan Receptor γt(RORγt) as their transcription factor to conduct differentiation and secretion, Th17 cells, whose proliferation, differentiation and secretion were regulated by IL-23 、IL-6 and Signal Transducer and Activator of Transcription-3(STAT-3), are a newly found subset of distinct CD4+ Th effector cells family. Many researches have confirmed their significance in inducing inflammation and autoimmune tissue injury to defense against microbial infections. Although recent researches proved that Th17 cells could also take part in pathogenesis of tumor diseases,the actual role of Th17 cells acting in the origin and development of malignant diseases remains equivocal as the available data showed a contribution from Th17 cells either to the protective antitumor immunity or to tumor growth. What’s more, despite more and more research results had supported the antitumor effects of Th17 cells,the specific pathway that Th17 worked through was still unclear. MDS are a common malignant disease of hematological system, but there were few reports on the role that Th17 cells acted in the pathogenesis of MDS.Materials and Methods: A total of 42 patients with MDS(L-MDS, n=22; H-MDS, n=20) and 23 healthy controls(HC) were enrolled in this study. The quantity of Th17 cells in peripheral blood(PB) and bone marrow(BM) were measured by flow cytometry. Then peripheral blood mononuclear cell(PBMNC) and bone marrow mononuclear cell(BMMNC) m RNA expression levels of RORγt, IL-17 as well as STAT-3 of 42 MDS patients and 23 healthy individuals were determined by SYBRGreen II real-time quantitative polymerase chain reaction(RT-PCR). In addition, IL-23、IL-6 and IL-17 levels of PB and BM plasma were examined by quantitative enzyme-linked immunosorbent assay(ELISA). Correlations between Th17 cells and peripheral blood routine, morphologic blast count in bone marrow smear as well as karyotype were analyzed. Levels of cytotoxic molecules including perforin, granzyme and Fas L in CD3+CD8+ T cells(cytotoxic T lymphocyte, CTL) before and after rh IL-17 stimulation were detected using flow cytometry in vitro.Results:1. Compared with HC, percentage of PB Th17 cells were significantly elevated in L-MDS group(4.42±2.59% vs 2.73±1.32%,P=0.006;) while decreased in H-MDS group(1.42±0.79% vs 2.73±1.32%, p<0.001), similar tendency was observed with Th17 percentages in BM. Percentages of Th17 cells in good, intermediate and poor karyotype groups were 4.591 ± 2.605%、2.450 ± 1.217%、0.913 ± 0.363% in PB and 4.686 ± 2.661%、2.921 ± 0.933%、0.882 ± 0.441% in BM respectively, with significant differences between any two groups in PB or BM(P<0.05). Percentage of Th17 cells was positively correlated with peripheral absolute neutrophil count(ANC) and hemoglobin(Hb) concentration while negatively correlated with BM blast percentage(BM blast%, P<0.05).2. Levels of PB IL-17 in H-MDS(22.44±5.64pg/ml) were markedly lower than those in patients with L-MDS(30.29±5.97pg/ml, P<0.001) and HC group(28.11± 4.64pg/ml,P=0.01); Levels of BM IL-17 in H-MDS group(131.67±49.71pg/ml) were markedly lower than those in patients with L-MDS(199.71±61.49pg/ml, P<0.001) and HC group(173.59± 52.70pg/ml,P=0.015). Levels of PB IL-23 in H-MDS(42.77±13.35 pg/ml) were markedly lower than those in patients with L-MDS(66.21±20.54 pg/ml,P<0.001) and HC group(54.20±15.40 pg/ml,P=0.030); Levels of BM IL-23 in H-MDS(77.75± 30.01pg/ml) were markedly lower than those in patients with L-MDS(137.54±47.32 pg/ml, P<0.001) and HC group(106.96±36.69 pg/ml,P=0.011); Levels of PB IL-6 in H-MDS(4.152±2.497pg/ml) were markedly lower than those in patients with L-MDS(7.834±3.977pg/ml, P=0.001) and HC group(6.377±3.578pg/ml, P=0.038); Levels of BM IL-6 in H-MDS( 10.467±5.131pg/ml) were markedly lower than those in patients with L-MDS(17.659±5.874pg/ml,P<0.001) and HC group(14.550±4.483 pg/ml,P=0.012), there was also a significant difference between the latter two groups(P=0.049).3. Consistent with levels of IL-17, levels of IL-17 m RNA in L-MDS、HC、H-MDS group were 11.81 ± 4.77,7.46 ± 3.05,4.72 ± 2.91 in PB and 10.66 ± 3.18,8.78 ±2.84,5.00±2.76 in BM,P values were all less than 0.05 between any two groups. Also, similar expression patterns were seen with the m RNA of IL-17 and IL-23 in PB and BM.Relative expression levels of PB and BM IL-17, RORγt and STAT-3 m RNA were significantly decreased in L-MDS compared to L-MDS and HC groups, consistent with the percentage of Th17 cells.4. Expression rate of Per、GB and Fas L in BM CTL was positively correlated with levels of IL-17 but negatively correlated with BM blast%. Expression rate of Per、GB and Fas L in CTL was significantly increased after stimulation with rh IL-17 4.56±4.23% vs 3.52± 3.17%, P<0.01; 59.47±16.47% vs 40.61 ± 18.98%, P<0.01; 13.94±10.58% vs 12.11±8.69%, P<0.05).Conclusion:(1)Th17 cells mainly played a protective role against the malignant clone in the pathogenesis of MDS, in that they were elevated in L-MDS and negatively correlated with BM blast percentages in the pathogenesis of MDS;(2)Th17 cells may relieve the inhibition of malignant clone on normal hematopoiesis while selectively killing malignant clone, maintaining the relative advantage of normal hematopoiesis;(3)In PB and BM, percentage of Th17 cells in normal karyotype group was higher than that in abnormal karyotype group markedly, and the lower the frequency of Th17 was,the poorer the karyotype was, further indicating that Th17 cells could assist in eradicating the malignant clone in MDS;(4)As upstream molecules of Th17 cells,abnormal levels of IL- 6 and IL- 23 might be the the chief culprit that led to the abnormalities in both percentage and function of Th17 cells;(5)Th17 cells might play antitumor effects through IL-17/CTL pathway in MDS.