Alteration of Th17 and Foxp3+ regulatory T cells in patients with unexplained recurrent spontaneous abortion before and after the therapy of hCG combined with immunoglobulin.
ABSTRACT: We conducted this study to investigate the effects of the combination therapy of human chorionic gonadotropin (hCG) plus immunoglobulin (IG) on the levels of T helper 17 (Th17) cells and Foxp3+ regulatory T cells (Treg) in patients with unexplained recurrent spontaneous abortion (URSA). Twenty pregnant women with URSA underwent combination therapy of hCG plus IG. Flow cytometry was performed to measure the proportions of Th17 and Treg cells before and after treatment. RT-PCR and ELISA were applied to detect the concentrations of interleukin (IL)-17, IL-6, IL-10, and transforming growth factor (TGF)-β1 in the peripheral blood. The therapy of hCG combined with IG may induce a decrease in the Th17/Treg ratio and the Treg bias, which may be beneficial for the maintenance of pregnancy. The levels of Th17 cells and related cytokines IL-17 and IL-6 decreased after the treatment (P<0.05). At the same time, levels of Treg cells and the related cytokines IL-10 and TGF-β1 increased (P<0.05). The Th17/Treg ratio decreased significantly after treatment. In conclusion, the occurrence of URSA may be associated with the Th17/Treg balance disorder, and correcting the disorder may be one of the mechanisms behind the efficacy of combination therapy for URSA.
Project description:BACKGROUND: Several types of T cells have been associated with the pathogenesis of unexplained recurrent spontaneous abortion (URSA), including Th1/Th2/Th17/Tregs cell. It has been appreciated that immunotherapy with paternal or third party lymphocytes is an effective method of treatment for URSA patients. The balance of Th1/Th2 cells could be maintained and an increase of Treg cells would be beneficial after immunotherapy; however, the mechanism by which the Th17/Treg balance affects URSA has not yet been fully elucidated. METHODS: Here, we used flow cytometry, liquid chip technology and quantitative real-time PCR (qPCR) methods to characterize Th17/Treg cell populations after immunotherapy. We found that after immunotherapy in URSA patients, the percentage of Th17 cells decreased and the percentage of Treg cells in peripheral blood mononuclear cells (PBMC) increased, as detected by flow cytometry. RESULTS: Immunotherapy may induce a decrease in the Th17/Treg ratio and the Treg bias, which may be beneficial for the maintenance of pregnancy. The expression level of ROR gamma t, a transcription factor found in Th17 cells, decreased and the expression of the Treg-specific transcription factor Foxp3 increased in peripheral blood as detected by qPCR. Immunotherapy may induce a decrease in the ratio of ROR gamma t to Foxp3 and a Treg cell bias, which would be beneficial for pregnancy maintenance. The secretion of the Treg-associated cytokine TGF-beta, as well as Th2 cytokines, was increased in serum, while the secretion of Th17-associated cytokine IL-17A and Th1 cytokine production was decreased. The Th1/Th2 cytokine ratio significantly decreased. Similarly, the Th17/Treg ratio significantly decreased in the total patient after immunotherapy. CONCLUSIONS: These results indicate that in patients with URSA, immunotherapy with mononuclear cells derived from the baby's father could affect both Th1/Th2 and Th17/Treg balance, and we found that the Th2 and Treg bias would be beneficial for pregnancy, which may lead to a balancing of the Th17/Treg ratio in URSA patients after immunotherapy.
Project description:This study aimed to investigate the role of aryl hydrocarbon receptor (AhR) and STAT3 gene during the differentiation of cluster of differentiation (CD)4 T cells into T helper (Th)17 and T regulatory (Treg) cells.First, CD4 T cells were isolated from the spleen of BALB/c mice. Then, stable CD4 T cells expressing STAT3 shRNA were constructed. CD4 T cells were assigned to one of the following treatments: Th17 group: antibodies against CD3 and CD28, 2.5 ng/mL transforming growth factor β (TGF-β)1, 30 ng/mL interleukin (IL)-6, and 30 ng/mL IL-23; 6-formylindolo[3,2-b]carbazole (FICZ) group: antibodies against CD3 and CD28, 2.5 ng/mL TGF-β1, 30 ng/mL IL-6, 30 ng/mL IL-23, and 100 nM FICZ; FICZ + STAT3 RNAi group (shSTAT3 group): antibodies against CD3 and CD28, 2.5 ng/mL TGF-β1, 30 ng/mL IL-6, 30 ng/mL IL-23, 100 nM FICZ, and STAT3 RNAi; naphthoflavone group: antibodies against CD3 and CD28, 2.5 ng/mL TGF-β1, 30 ng/mL IL-6, 30 ng/mL IL-23, and 3 μM naphthoflavone; 5) no antibodies were added in the control group. Later, the proportions of Th17 and Treg cells in each group were measured by flow cytometry; phospho-STAT3 and -STAT5 levels were measured by western blotting; and AhR, STAT3, STAT5, receptor-related orphan nuclear receptor γt (RORγt), FOXP3, T-cell receptor (TCR), CD25, IL-6R, IL-10, and IL-17 mRNA levels were also measured by real-time PCR.Th17 cells showed a rise and Treg cells showed a decrease in the FICZ group, but revised in the shSTAT3 group and the naphthoflavone group. Significant differences were observed in CD25, IL-6R, IL-10, and IL-17 mRNA levels among different groups.STAT3 may cooperate with AhR to regulate the differentiation of both Th17 and Treg cells.
Project description:BuShen HuoXue decoction (BSHXD) has been used to treat patients with unexplained recurrent spontaneous abortion (URSA). However, the chemical compounds and mechanism by which BSHXD exerts its therapeutic and systemic effects to promote the proliferation of decidual stromal cells (DSCs) has not been elucidated. This work sought to elucidate the cellular and molecular mechanism of BSHXD in terms of inflammatory factors IL-17A in DSCs in vitro because of the critical roles of inflammation, apoptosis, and immunity in the development and progression of pregnancy loss. Twelve migratory chemical compounds from BSHXD extract were qualitatively analyzed by high-performance liquid chromatography (HPLC). DSCs were collected from normal early pregnancy (NEP) and URSA to determine whether BSHXD affects IL-17A/IL17RA via the PI3K/AKT pathway. Abnormal apoptosis and activated p-AKT were observed in URSA DSCs. RhIL-17?A, LY294002 (a PI3K pathway inhibitor), and BSHXD were individually or simultaneously administered in NEP DSCs, suggesting that BSHXD restored cell proliferation without excessive stimulation and IL-17A promotes proliferation via the PI3K/AKT pathway. Using the same intervention in URSA DSCs, qRT-PCR measured the upregulated mRNA levels of IL-17?A/IL-17RA, PI3K, AKT, p-AKT, PTEN, Bcl-2, and Bcl-xL and downregulated mRNA levels of BAD and ACT1 after treatment with BSHXD. We demonstrated that BSHXD affected IL-17A/IL-17R via PI3K/AKT pathway to promote the proliferative activity of DSCs in URSA. These results provide a new insight to further clarify the relationship between inflammation and apoptosis and the mechanism of imbalance in the dynamic equilibrium between Th17/Treg immune cells at the maternal-fetal interface.
Project description:Objective: Cooking and heating with coal is the main source of household air pollution in acid rain-plagued areas of China and is a leading contributor to disease burden. In this study, we investigated the adverse effects of exposure to indoor fine particulate matter emission from coal combustion on the expression levels of inflammatory factors in ovalbumin (OVA)-induced mice. Methods: Forty BALB/c male mice were randomly divided into four groups (control group, PM2.5 group, OVA group, and OVA + PM2.5 group; n = 10) and treated with ovalbumin (OVA) or PM2.5, alone or together. Interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-17 (IL-17), transforming growth factor β1 (TGF-β1), and interferon γ (IFN-γ) protein expression levels were measured in bronchoalveolar lavage fluid (BALF). Additionally, serum immunoglobulin (Ig) IgE and IgG1 levels were measured. The mRNA expression levels of IL-7 and IFN-γ in pulmonary tissue were also analyzed. Bronchoalveolar lavage (BAL), inflammatory cell counts, and histopathological examinations were also performed. Results: Exposure to PM2.5 + OVA induced abnormal pathological changes and inflammatory responses in lungs compared to the control. The levels of IL-4, IL-7, IL-8 and IL-17 in BALF from the OVA + PM2.5 group were higher than those in BALF from the control group, OVA group, and PM2.5 group (P < 0.05). PM2.5 plus OVA significantly raised the serum IgE and IgG1 levels compared with the control group. An increasing IL-7 mRNA trend was found among the treatment groups (P < 0.05). The expression level of IFN-γ mRNA was significantly higher in the control group than in the other 3 groups (P < 0.05). Conclusion: Indoor coal PM2.5 was sufficient by itself to cause inflammatory cellular infiltration of pulmonary tissue, leading to organelle injury and physiological structure change. Additionally, it promoted the occurrence and development of asthma by influencing the expression levels of IL-7 and various relevant inflammatory factors (such as IL-4 and IL-8) and changing the equilibrium between Treg and Th17 cells.
Project description:The present study investigated the effects of electroacupuncture on blood pressure in spontaneously hypertensive rats (SHRs) by regulating the immune balance of T helper 17 cells (Th17 cells) and regulatory T cells (Treg cells). This study investigated the role of electroacupuncture in the immune balance of SHRs using Western blot, flow cytometry, and ELISA techniques. Electroacupuncture significantly improved blood pressure, downregulated the expression of ROR?t, and upregulated the expression of Foxp3, reduced the production of Th17 cells, promoted the production of Treg cells, reduced the secretion of IL-6 and IL-17, and increased the secretion of TGF-?1 and IL-10. These findings suggest that electroacupuncture therapy effectively improved the systolic blood pressure of SHRs, and its mechanism may be related to promotion of the immune balance between Th17 and Treg.
Project description:IgD-Fc-Ig fusion protein, a new biological agent, is constructed by linking a segment of human IgD-Fc with a segment of human IgG1-Fc, which specifically blocks the IgD-IgDR pathway and selectively inhibits the abnormal proliferation, activation, and differentiation of T cells. In this study we investigated whether IgD-Fc-Ig exerted therapeutic effects in collagen-induced arthritis (CIA) rats. CIA rats were treated with IgD-Fc-Ig (1, 3, and 9?mg/kg) or injected with biological agents etanercept (3?mg/kg) once every 3 days for 40 days. In the PBMCs and spleen lymphocytes of CIA rats, both T and B cells exhibited abnormal proliferation; the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1(CD4+IFN-?+), and Th17(CD4+IL-17+) were significantly increased, whereas the Treg (CD4+CD25+Foxp3+) cell percentage was decreased. IgD-Fc-Ig administration dose-dependently decreased the indicators of arthritis; alleviated the histopathology of spleen and joint; reduced serum inflammatory cytokines levels; decreased the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1 (CD4+IFN-?+), and Th17(CD4+IL-17+); increased Treg (CD4+CD25+Foxp3+) cell percentage; and down-regulated the expression of key molecules in IgD-IgDR-Lck-NF-?B signaling (p-Lck, p-ZAP70, p-P38, p-NF-?B65). Treatment of normal T cells with IgD (9??g/mL) in vitro promoted their proliferation. Co-treatment with IgD-Fc-Ig (0.1-10??g/mL) dose-dependently decreased IgD-stimulated T cell subsets percentages and down-regulated the IgD-IgDR-Lck-NF-?B signaling. In summary, this study demonstrates that IgD-Fc-Ig alleviates CIA and regulates the functions of T cells through inhibiting IgD-IgDR-Lck-NF-?B signaling.
Project description:INTRODUCTION: Although immune dysfunction plays a role in the pathogenesis of systemic sclerosis (SSc), involvement of T helper 17 (Th17) and T regulatory (Treg) cells remains unclear. We aimed to investigate the presence of Th17 and Treg cells in SSc patients and the role of Th17 cells in collagen production in SSc fibroblasts. METHODS: We analyzed inflammatory cell profiles in the skin of 13 SSc patients by immunohistochemistry, the percentage of Th17 and Treg cells in peripheral blood mononuclear cells (PBMCs) of 45 SSc patients and 24 healthy controls by flow cytometry, gene expression in PBMCs by real-time reverse transcription-polymerase chain reaction and interleukin-17 (IL-17) in sera and culture supernatants by enzyme-linked immunosorbent assay. We also investigated the effect of Th17 cell-derived IL-17 on fibroblast growth and collagen production. RESULTS: Infiltration of inflammatory cells including IL-17? and Foxp3? lymphocytes was detected in the skin of patients with early SSc. The percentages of circulating Th17 cells and IL-17 production were elevated in samples from patients with active SSc, whereas the percentage of circulating Treg cells was not affected. The number of Th17 cells was closely related to disease activity. IL-17 from SSc patients promoted fibroblast growth and collagen production, whereas IL-17 neutralizing antibody effectively blocked collagen production. CONCLUSION: SSc progression might be linked to expansion of circulating Th17 cells and increased infiltration of IL-17? cells in skin. Th17-derived IL-17 is involved in fibroblast growth and collagen production. IL-17 blocking antibody may be a useful tool for intervention in the fibrotic course of SSc.
Project description:Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.
Project description:A subset of human regulatory T cells (Tregs) secretes IL-17 and thus resembles Th17 effector cells. How IL-17(+) Tregs differentiate from naive precursors remains unclear. In this study, we show that IL-17-producing T cells can differentiate from CCR6(+) naive T cell precursors in the presence of IL-2, IL-1?, TGF-?, and IL-23. CCR6(+) naive T cells are present in adult peripheral and umbilical cord blood and in both conventional T naive and FOXP3(+) naive Treg subsets. IL-17(+) cells derived from CCR6(+) naive Tregs (referred to as IL-17(+) Tregs) express FOXP3 but not HELIOS, another Treg-associated transcription factor, and these cells display suppressor capacity and a surface phenotype resembling memory Tregs. Remarkably, the IL-17(+) Treg compartment was preferentially reduced relative to the canonical Th17 and Treg compartments in a subset of HIV(+) subjects, suggesting a specific perturbation of this subset during the course of disease. Our findings that CCR6(+) naive precursors contain a predetermined reservoir to replenish IL-17-secreting cells may have implications in balancing the Th17 and IL-17(+) Treg compartments that are perturbed during HIV infection and potentially in other inflammatory diseases.
Project description:The role of T-helper type 17 (Th17) responses in airway remodeling in asthma is currently unknown. We demonstrate that both parenteral and mucosal allergen sensitization, followed by allergen inhalation, leads to Th17-biased lung immune responses. Unlike Th17 cells generated in vitro, lung Th17 cells did not produce tumor necrosis factor-? or interleukin (IL)-22. Eosinophilia predominated in acute inflammation, while neutrophilia and IL-17 increased in chronic disease. Allergen-induced tolerance involved Foxp3-, Helios-, and glycoprotein-A repetitions predominant-expressing regulatory T cells (Treg) and IL-10/interferon-? priming. This Treg phenotype was altered in inflamed lungs and abrogated by inhalation of IL-17. Using Th17-deficient mice with genetic disruption of gp130 in T cells, we showed that Th17 cells induce airway remodeling independent of the Th2 response. All-trans retinoic acid administration ameliorated Th17-mediated disease and increased Treg activity, while dexamethasone inhibited eosinophilia but not neutrophilia, and enhanced Th17 development in vitro. Targeting the Th17/Treg axis might therefore be therapeutic in neutrophilic and glucocorticoid-refractory asthma.