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Objective:To investigate the changes in IL-35 expression in patients with type 1 diabetes mellitus (T1DM) and to analyze the role of IL-35 in regulating Th9 cells.Methods:Thirty-one T1DM patients and 13 controls were enrolled. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated. The levels of IL-35 and IL-9 in plasma were measured by ELISA. The expression of IL-35 subunits, EBI3 and IL-12p35, as well as Th9 transcription factor PU.1 at mRNA level was detected by real-time PCR. The percentages of Th9 cells were measured by flow cytometry. Changes in cell proliferation, the percentage of Th9 cells, PU.1 expression at mRNA level and IL-9 secretion were detected after stimulating PBMCs from T1DM patients and controls with recombinant human IL-35. CD4 + CCR4 -CCR6 -CXCR3 - cells and CD8 + T cells were isolated from PBMCs of 11 T1DM patients. CD4 + CCR4 -CCR6 -CXCR3 - cells were first stimulated with recombinant human IL-35 and then co-cultured with CD8 + T cells. IFN-γ and TNF-α in the culture supernatants were measured by ELISA. Perforin and granzyme B secretion was measured by enzyme-linked immunospot assay. Student′s t-test, paired t-test or LSD- t test was used for statistical analysis. Results:Plasma IL-35 level was lower in T1DM patients than in controls [(67.13±9.94) pg/ml vs (97.77±23.61) pg/ml, P<0.000 1]. Compared with controls, T1DM patients had decreased expression of EBI3 and IL-12p35 at mRNA level in PBMCs ( P<0.000 1). The percentage of Th9 cells, PU.1 expression at mRNA level and plasma IL-9 level were increased in T1DM patients as compared with those in controls [(3.47±0.99)% vs (2.76±0.75)%, P=0.029; P<0.000 1; (99.08±11.85) pg/ml vs (86.38±12.72) pg/ml, P=0.002 8]. IL-35 had no significant influence on the proliferation of PBMCs from both T1DM patients and controls ( P>0.05). The percentage of Th9 cells and PU.1 expression at mRNA level in PBMCs from T1DM patients were down-regulated in response to IL-35 stimulation ( P<0.01), while no significant difference was observed in the control group ( P>0.05). IL-9 secretion by PBMCs was down-regulated in response to IL-35 stimulation in both T1DM and control groups ( P<0.01). CD4 + CCR4 -CCR6 -CXCR3 - cells promoted the secretion of IFN-γ, TNF-α, perforin and granzyme B by CD8 + T cells from T1DM patients ( P<0.05), but the effects could be inhibited by IL-35 ( P<0.05). Conclusions:Decreased IL-35 in T1DM patients could not exert effective immunosuppressive activity, leading to the enhancement of Th9 cell activity and inflammatory injury.
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Objective To investigate the relationship of the expression of peripheral T helper type 9 (Th9) cells and their related cytokines with the severity and clinical course of alopecia areata,and to explore their significance in the occurrence of alopecia areata.Methods From May to December in 2017,74 outpatients with alopecia areata enrolled from Department of Dermatology,Huashan Hospital,Fudan University served as the alopecia areata group,and 57 health checkup examinees in Huashan Hospital served as the control group.Peripheral blood samples were obtained from the patients and controls.Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum levels of interleukin (IL)-9,IL-4,transforming growth factor (TGF)-β1 and interferon (IFN)-γ,flow cytometry to determine the proportion of Th9 cells (CD4+IL-9+ T helper cells) in peripheral blood mononuclear cells (PBMC),and real-time fluorescence-based quantitative PCR to measure the mRNA expression of IL-9 and PU.1 in the PBMCs.Data were recorded in Microsoft Excel 2017 software,and statistically analyzed with SPSS17.0 software using two-sample t test and Spearman rank correlation analysis.Statistical charts were drawn with Graphpad prism 6 software.Results Compared with the control group,the alopecia areata group showed significantly decreased serum level of IL-9 (190.40 ± 12.33 ng/L vs.288.10 ± 17.38 ng/L,t =4.71,P ＜ 0.01),but significantly increased serum levels of TGF-β1 (6 191.00 ± 355.50 ng/L vs.4 026.00 ± 258.00 ng/L,t =4.41,P ＜ 0.05) and IFN-γ(15.71 ± 3.00 ng/L vs.8.79 ± 0.60 ng/L,t =2.001,P ＜ 0.05).However,there was no significant difference in the serum level of IL-4 between the alopecia areata group and control group (P ＞ 0.05).The serum level of IFN-γ was significantly lower in the patients with severe alopecia areata than in the patients with mild alopecia areata (P =0.02),and the serum level of IFN-γ in the patients with alopecia areata was negatively correlated with the severity of alopecia tool (SALT) score (ru =-0.298,P =0.010).There were no significant differences in the serum levels of IL-9,IL-4 and TGF-β1 between the patients with severe alopecia areata and those with mild alopecia areata (all P ＞ 0.05).The serum levels of IL-9,IL-4,TGF-β1 and IFN-γdid not differ between the patients with active alopecia areata and those with stable alopecia areata,as well as between the patients with clinical course of ＜ 6 months and those with clinical course of ＞ 6 months (P ＞ 0.05).The alopecia areata group showed significantly decreased proportion of Th9 cells in the PBMCs (t =2.04,P =0.045) and mRNA expression of IL-9 and PU.1 (t =2.12,2.178,both P ＜ 0.05) compared with the control group.Condusion The serum level of IL-9 and proportion of peripheral blood Th9 cells both decrease in patients with alopecia areata,and Th9 cells and their related cytokines may be involved in the occurrence of alopecia areata.
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Objective: To investigate the role of T helper 9 (Th9) cells in liver cirrhosis (LC) patients and whether chemokine receptor type 6 (CCR6)/chemokine ligand 20 (CCL20) axis is involving in the recruitment of Th9 cells into liver. Methods: Peripheral blood and liver tissue from 30 LC patients and 18 normal controls were recruited. The frequency of Th9 cells and CCR4, CCR6 in the peripheral blood was tested by flow cytometry. Serum interleukin (IL)-9 and CCL20 levels were tested by enzyme-linked immunosorbent assay. Immunohistochemical staining was used to detect α-smooth muscle actin, CCR6 and CCL20 expression in liver tissue. Results: The frequency of Th9 cells in LC patients was significantly increased compared with controls (P < 0.05). The serum IL-9 level and CCL20 level increased markedly in LC patients compared with controls (P < 0.05), and IL-9 was positively correlated to Th9 cells and CCL20. Furthermore, the frequency of Th9 cells was correlated to prothrombin time, total bilirubin level, hyaluronic acid and type IV collagen in LC patients. We also found that Th9 cells in LC patients expressed higher frequency of CCR4
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Objective To investigate the role of Th9 cells in primary biliary cirrhosis and its clinical significance.Methods The percentages of Th9 cells in patients with PBC (n=38)and healthy controls (n=38)were measured by flow cytometry. Patients clinical data were collected and Mayo risk scores were calculated.Determined the levels of IL-9,PU-1 and TGF-βmRNA expressions and the protein levels of IL-9 in plasma.Further analyzed the correlation between Th9 cells and clinical parameters.Results Compared to healthy controls,Th9 cells percentage was higher in PBC patients and increased with dis-ease stages (t=27.29;P<0.01).IL-9,PU-1 and TGF-βmRNA expressions were increased compared with healthy controls (t=14.69,23.92,10.48;all P<0.01)and the protein level of IL-9 was also up-regulated (t=11.66;P<0.01).The clinical parameters (ALP,AST,ALT,GGT and TBIL)were higher than healthy controls (t=10.94,12.95,10.56,14.92,27.70;all P<0.01).Moreover,the percentage of Th9 cells were positively correlated to Mayo risk,ALT,AST,GGT and TBIL (P<0.05).Conclusion Th9 cells may be involved in the pathogenesis of PBC and related to the disease stage,which provides new clues for future immunotherapy.
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Objective: To assess the role of Th9 and Th17 cells in malignant ascites (MA). Methods: MA from 30 hepatic carcinoma patients and benign ascites from 30 cirrhotic patients were collected. Corresponding peripheral blood samples from these hepatic carcinoma and cirrhotic patients as well as 30 healthy subjects were collected. The frequency of Th9 and Th17 cells was tested by flow cytometry. Serum levels of interleukin (IL)-9 and IL-17 were examined by ELISA. Results: The observed frequency of Th9 and Th17 cells, and the IL-9 and IL-17 serum levels were significantly higher in MA patients than those in cirrhotic patients and healthy control samples ( P<. 0.05). Moreover, the Th9 cells demonstrated positive correlation with Th17 cells as well as IL-9 in MA patients; however, this positive correlation was not observed in the cirrhotic patients or healthy control samples. The frequency of Th9 and Th17 cells was distinctly higher in MA patients presenting with stage III or IV malignancy and with lymph node or distant metastasis than those in patients in stage I or II and without distant metastasis ( P<. 0.05). Conclusions: The increased frequency of Th9 and Th17 cells in MA patients suggests that these two T cell subsets play a synergistic role in MA pathogenesis. This study also demonstrated that Th9 and Th17 cells may perform their biological functions in conjunction with IL-9 production.
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Objective To investigate the relationship of interleukin-9 (IL-9) and the transcription factor PU.1 with chronic urticaria.Methods Thirty patients with chronic urticaria (patient group) and 30 healthy volunteers (control group) were included in this study.Venous blood samples were collected from these subjects.Enzyme-linked immunosorbent assay (ELISA) was performed to detect serum levels of IgE,flow cytometry (FCM) to determine the percentage of CD4+IL-9+ T cells,real-time fluorescence-based quantitative PCR (RTFQ-PCR) to measure the mRNA expressions of IL-9 and PU.1 in peripheral blood mononuclear cells (PBMCs).Measurement data were compared between the two groups by independent-samples t test,and relationship among these parameters was assessed by Pearson correlation analysis.Results The expressions of IL-9 and PU.1 mRNAs in patients with chronic urticaria were significantly higher than those in healthy controls (4.44 ± 1.90 vs.3.20 ± 1.78,t =2.60,P＜ 0.05; 3.26 ± 1.59 vs.2.34 ± 1.47,t =2.34,P ＜ 0.05).Compared with the control group,the patient group showed increased percentage of CD4+IL-9+ T cells in peripheral blood (0.63％ ± 0.44％ vs.0.22％ ± 0.12％,t =5.04,P ＜ 0.01) and serum IgE levels ((82.04 ± 31.72) vs.(60.74 ± 28.26) IU/ml,t =2.75,P ＜ 0.01).The percentage of CD4+IL-9+ T cells and levels of IL-9 and PU.1 mRNAs in peripheral blood were all positively correlated with serum levels of IgE in these patients (r =0.596,0.767,0.746,respectively,all P ＜ 0.01).Conclusion T helper type 9 (Th9) cells and IL-9 may take part in the occurrence of chronic urticaria.
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Objective:To assess the role of Th9 and Th17 cells in malignant ascites (MA). Methods: MA from 30 hepatic carcinoma patients and benign ascites from 30 cirrhotic patients were collected. Corresponding peripheral blood samples from these hepatic carcinoma and cirrhotic patients as well as 30 healthy subjects were collected. The frequency of Th9 and Th17 cells was tested by flow cytometry. Serum levels of interleukin (IL)-9 and IL-17 were examined byELISA. Results: The observed frequency of Th9 and Th17 cells, and the IL-9 and IL-17 serum levels were significantly higher in MA patients than those in cirrhotic patients and healthy control samples (P Conclusions:The increased frequency of Th9 and Th17 cells in MA patients suggests that these two T cell subsets play a synergistic role in MA pathogenesis. This study also demonstrated that Th9 and Th17 cells may perform their biological functions in conjunction with IL-9 production.
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Objective To investigate the presence and significance of Th9 cells and the related transcription factor ( PU.1 ) and cytokine ( IL-9 ) in peripheral blood of patents with hashimoto thyroiditis ( HT) .Methods Thirty patients with HT and thirty age/gender matched healthy subjects were recruited in this study.The peripheral blood and serum samples were collected from each subject.The percentages of Th9 cells and the transcriptional levels of PU.1 in peripheral blood mononuclear cells ( PBMCs) were meas-ured by flow cytometry analysis and real-time RT-PCR.The concentrations of IL-9, the functions of thyroid and the titers of thyroid-specific autoantibodies ( TPOAb and TgAb) in serum samples were detected by using enzyme-linked immunosorbent assay ( ELISA ) and electrochemiluminescence immunoassay analysis ( ECLIA) .Results Compared with healthy subjects, the percentages of Th9 cells and the expression of PU.1 at mRNA level in PBMCs and the concentrations of IL-9 in serum samples were all significantly in-creased in patients with HT [(1.49±0.68)%vs (0.87±0.24)%], 4.91±2.14 vs 1.66±0.52, (26.90± 7.74) pg/ml vs (16.71±5.87) pg/ml, all P<0.01).Serum concentrations of IL-9 were positively correla-ted with the percentages of Th9 cells (r=0.419, P=0.021).Moreover, the percentages of Th9 cells were positively correlated with the titers of TPOAb and TgAb in serum samples (r=0.394, P=0.032;r=0.457, P=0.011) of patients with HT.Conclusion The levels of Th9 cells and the related cytokine IL-9 were in-creased in the peripheral blood of patients with HT.A positive correlation was found between the percentage of Th9 cells and the titers of thyroid-specific autoantibodies.This study indicated that Th9 cells might be in-volved in the pathogenesis of autoimmune damage in thyroid.
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Objective:To study the abnormal suppressive capacity of regulatory T cells ( Tregs) to effector cells,Th17 cells and Th9 cells in patients with mild to moderate asthma attack.Methods: Recruited asthmatic patients (n=30) and healthy control (n=30),collected peripheral venous blood from healthy control and asthmatic patients in mild to moderate attack stage respectively,and then isolated CD4+CD25+CD127-/low Tregs and CD4+CD25-T effect cells with immunomagnetic beads method ( purity was above 90%).The effect cells were cultured with PHA stimulation with or without Tregs.Measured the proliferation (3H-Thymidine), expression of RORC and PU.1 ( RT-PCR) genes,production of IL-17 and IL-9 ( LUMNEX) in effect cells with or without Tregs inter-vention.Analyzed the abnormality of Th17 and Th9 cells and the supressive capacity of Tregs on Th17 and Th9 cells in patients with asthma attack,comparing with control group.Results:Tregs of asthmatic patients could suppress the proliferation of effector cells,but less effectively than healthy control (P=0.03).In asthmatic patients,RORC expression and IL-17 production increased (P0.05).Conclusion:The suppressive capacity and amount of Tregs decreased,but the specific gene expression and cytokines production of Th17 cells and Th9 cells were upregulated in patients with mild to moderate asthma attack.This deficiency for the suppressive capacity of Tregs to Th17 but not Th9 cells may indicate some pathogenesis of asthma devel-opment.
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Objective To observe Th9 cells and IL-9 level in adult patients with primary immune thrombocytopenia (ITP), and to discuss their potential roles in the pathogenesis of ITP. Methods A total of 25 newly diagnosed ITP patients and 25 sex- and age-matched healthy controls were enrolled in the present study. The percentage of Th9 cells in the peripheral blood samples of the two groups were detected by flow cytometry, expressions of IL-9, TGF-ß, PU. 1 and IRF4 mRNA were analyzed by real time-RCR, and IL-9 protein level was examined by ELISA. The platelet count was recorded by sysmex XE-2100. Results Compared with the healthy controls, the ratio of Th9 cells was significantly increased in ITP patients(P30×109/L(P30 × 109/L(P<0.05). Compared with healthy controls, the mRNA expressions of IL-9, TGF-ß, PU.1 and IRF4 were raised significantly in ITP patients(P<0.05). The ratio of Th9 cells and IL-9 protein level were negatively correlated with PLT in ITP patients(r= -0.428 1, P = 0.032 8; r= -0.537 5, P = 0.005 6, respectively). Furthermore, follow-up study of 11 ITP patients found that both Th9 cells and IL-9 protein levels took a declining tendency after effective treatment(P<0.05). Conclusion Abnormal activation of Th9/IL-9 may participate in the occurrence and development of ITP disease, which provides new clues for further understanding of ITP pathogenesis and selecting potential therapeutic targets in immune therapy of ITP.