Nannan Pang

TGF-β/Smad signaling pathway regulates Th17/Treg balance during Echinococcus multilocularis infection

Alveolar echinococcosis (AE) is a severe parasitic disease caused by the infection of Echinococcus multilocularis (Em). Very little is known on the relationship between TGF-β/Smad signaling pathway and Treg/Th17 balance in the infected liver at different periods after Em infection. Using qRT-PCR, immunohistochemistry, flow cytometry and CBA assay, we measured the expression levels of TGF-β, Smad2/3/7, ROR-γt, Foxp3, IL-17, IL-10 and percentages of Th17 cells and Treg cells in mouse AE model, from day 2 to day 270 after infection. In the early stage of infection (day 2 to day 30), Smad7 was up-regulated and the TGF-β pathway was inactivated. In the middle stage of infection (day 30 to day 90), TGF-β and Smad2/3 were up-regulated. And levels of Treg cells, Foxp3, Th17 cells, RORγt, IL-17, IL-10 and IL-6 were significantly increased. In the late stage of infection (day 90 to day 270), Treg cells, Foxp3, TGF-β and IL-10 maintained at high levels whereas Th17 cells and IL-17 decreased significantly. TGF-β/Smad signaling pathway was activated during the chronic infection. Our data suggest that there were Treg/Th17 imbalance in the middle and especially in the late stage of Em infection and that Treg/Th17 imbalance may be regulated by TGF-β/Smad signaling pathway. Treg and Th17 subsets may be involved in regulating immune tolerance and tissue inflammation, and facilitating the long-term survival of Em in the host.

The Th17/Treg balance and the expression of related cytokines in Uygur cervical cancer patients

BackgroundThe fine balance of Th17/Treg is crucial for maintenance of immune homeostasis. The objective of this study was to investigate the balance of Th17/Treg and the expression of related cytokines in Uighur cervical cancer patients.MethodsPeripheral blood was collected from 65 cases of cervical cancer patients, 42 cases of cervical CIN patients and 40 healthy people. Flow cytometry was used to detect the percentages of T cell subsets, including CD3+ T cells, CD4+ T cells, CD8+ T cells, Treg cells and Th17 cells. ELISA assay was conducted to detect expression levels of TGF-β, IL-6, IL-10, IL-17, IL-23 and IFN-γ.ResultsThere were no significant difference in the levels of CD3+ T cells, CD4+ T cells, CD8+ T cells, and the ratio of CD4+/CD8+ among the cervical cancer group, the CIN group and the healthy control group. However, compared with the healthy control group, the percentages of CD4+ CD25+ Treg, CD4+CD25+CD127- Treg, CD4+IL17+ Th17, CD4+CD25+Foxp3+, CD4+CD25- Foxp3+, CD8+CD25+CD127-Treg and CD8+CD25+Foxp3 were significantly higher in the cervical cancer group and the CIN group. Similar results were also found in the Th17/Treg ratio and the related cytokines. There was no significant difference between the cervical cancer group and the CIN group. Additionally, Th17 cell levels were positively correlated with IL-6, IL-23 and IL-17. Also, Treg cell levels were positively correlated with TGF-β, IL-10 and IL-6. Contrarily, Treg cell levels and IFN-γ were negatively correlated.ConclusionsOur data indicated that the Th17/Treg balance was broken in peripheral blood of cervical cancer patients. Analysis of Th17/Treg balance may have a significant implication in diagnosing cervical cancer.Virtual slidesThe virtual slide for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1813823795931511

Th9/IL-9 Profile in Human Echinococcosis: Their Involvement in Immune Response during Infection by Echinococcus granulosus

Th9 cells have been reported to contribute to immune responses; however, the role of Th9 cells in Echinococcus granulosus infection is unknown. This study is to determine whether Th9 cells and IL-9 are involved in human Echinococcus granulosus infection. Compared with healthy controls (HC group), the mRNA levels of PU.1, IL-9, and GATA-3 were significantly increased in patients before therapy (CE group), as revealed by qRT-PCR. Flow cytometry analysis showed that the percentages of Th9 and Th2 cells in CE group were significantly higher. The levels of IL-9, IL-4, IL-10, and TGF-β in CE group were also significantly increased, as detected by CBA assay. The percentages of Th9 and Th2 cells in CE group were positively correlated. After treatments of surgery in combination with albendazole, the PU.1 and GATA-3 mRNA levels were significantly decreased in patients after therapy (PCE group) compared with CE group. The numbers of Th9 and Th2 cells and levels of IL-9, IL-4, IL-10, and TGF-β were also significantly decreased in PCE group. In conclusion, the ratios of Th9 cells and IL-9 levels were significantly decreased after treatment, suggesting that Th9/IL-9 may be involved in immune response induced by Echinococcus granulosus infection.

Upregulation of PD-1 on CD4⁺CD25⁺ T cells is associated with immunosuppression in liver of mice infected with Echinococcus multilocularis.

Alveolar echinococcosis is a zoonotic disease caused by Echinococcus multilocularis (E. multilocularis) infection. The relationship between PD-1/PD-L1 pathway and Tregs at different stages of E. multilocularis infection has rarely been reported. This study aims to investigate the role of PD-1/PD-L1 in immunosuppression of Tregs in E. multilocularis infection. Hematoxylin-eosin staining, flow cytometry, immunohistochemistry, quantitative RT-PCR analysis, cytometric bead array and MTT assay were used to analyze liver pathological changes, percentages of PD-1(+) Tregs and PD-L1(+) dendritic cells (DCs), expression levels of PD-1, PD-L1 and Foxp3, levels of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) and proliferation of lymphocytes. During middle-late stage (day 30 to day 330) the percentages of PD-1(+) Tregs and PD-L1(+) DCs together with levels of Foxp3, IL-10 and TGF-β increased significantly and maintained at high level. The expression of PD-1 and PD-L1 was increased with the enlarging erosion of E. multilocularis, and was mainly distributed in hepatic sinus, fibrous wall of alveolar hydatid and germinal layer around foci of infection. PD-1/PD-L1 promoted the secretion of IL-10 and TGF-β. Our results indicate that engagement of the PD-1 and PD-L1 correlates with inhibition of T-cell effector function, cytokine secretion and proliferation. High expression of PD-1/PD-L1 may play an important role in stimulating CD4(+)CD25(+) T cells, and maintaining peripheral tolerance and immune evasion during chronic infection of E. multilocularis.

Elevated Expression of Programmed Death-1 and Programmed Death Ligand-1 Negatively Regulates Immune Response against Cervical Cancer Cells

The present study is to measure the expression of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), as well as its clinical significance in cervical cancer patients. Our results showed that different T cell subsets in patients with cervical cancer had high expression of PD-1, and DCs had high expression of PD-L1. High expression of PD-1 on Treg cells in cervical cancer patients facilitated the production of TGF-β and IL-10 but inhibited the production of IFN-γ. Cervical cancer elevated the expression of PD-1 and PD-L1 in mRNA level. PD-1 expression in peripheral blood of cervical cancer patients was related with tumor differentiation, lymph node metastasis, and invasiveness. PD-1/PD-L1 pathway inhibited lymphocyte proliferation but enhanced the secretion of IL-10 and TGF-β in vitro. In summary, our findings demonstrate that elevated levels of PD-1/PD-L1, TGF-β, and IL-10 in peripheral blood of cervical cancer patients may negatively regulate immune response against cervical cancer cells and contribute to the progression of cervical cancer. Therefore, PD-1/PD-L1 pathway may become an immunotherapy target in the future.

CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T cells are positively correlated with levels of IL-21 in active and transitional cystic echinococcosis patients.

BackgroundIn our study, we investigated whether circulating T follicular helper (Tfh) and the related cytokines are involved in human cystic echinococcosis (CE).MethodsA total of 64 patients with CE and 30 healthy controls were enrolled in this study. Percentages of CCR7loPD-1hi cells within CXCR5+ CD4+ T cells (circulating Tfh cells) were detected by flow cytometry. Levels of IL-21 and IL-4 in peripheral blood were detected by cytometric bead array. The mRNA expression of IL-21, IL-4, Bcl-6, and Blimp-1 in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR. Levels of IgG1, IgG2, IgG3, and IgG4 in the patients’ sera were measured using enzyme-linked immunosorbent assay.ResultsPercentages of circulating Tfh cells were significantly increased in the CE1, CE2, and CE3 groups (p < 0.05). The concentrations of IL-21 and IL-4 in the serum were significantly increased in CE1, CE2, and CE3 groups (p < 0.05). IL-21 was positively correlated with circulating Tfh cells in CE3 group (r = 0.779, p < 0.05). The mRNA levels of IL-21, IL-4, and Bcl-6 were increased in CE1, CE2, and CE3 groups. Levels of IgG1 and IgG4 in patients’ sera were increased in CE1, CE2, and CE3 groups. Levels of IgG2 and IgG3 were increased in CE4-5 group. Additionally, after stimulation with hydatid fluid in vitro, the levels of circulating Tfh cells, IL-21 and IL-4 in PBMCs isolated from CE patients were significantly increased (p < 0.05).ConclusionsThe levels of circulating Tfh and related cytokines were significantly increased in CE patients, suggesting that they are involved in human CE.

Percentages of PD-1+CD4+T cells and PD-L1+DCs are increased and sPD-1 level is elevated in patients with immune thrombocytopenia

ABSTRACT The present study is to measure the expression of programmed death (PD)-1 / programmed death ligand-1 (PD-L1) negative costimulatory molecules, soluble format sPD-1 in patients with immune thrombocytopenia (ITP), and to investigate their correlation with the secretion of cytokines. A total of 35 patients with ITP were included in the present study. Twenty healthy subjects who received physical examination at our hospital were included as control group. Peripheral blood was collected from all ITP patients and healthy subjects. Flow cytometry was performed to determine the percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients and healthy subjects. Enzyme-linked immunosorbent assay was performed to measure the concentrations of interferon (IFN)-γ, interleukin (IL)-17 and sPD-1 in peripheral blood from ITP patients and healthy subjects. Percentages of PD-1+CD4+T cells and PD-L1+DCs in peripheral blood from ITP patients before treatment were significantly higher than that from healthy subjects, but were not different from those after treatment. Serum concentrations of IFN-γ, IL-17 and sPD-1 in ITP patients before treatment were significantly higher than those in healthy subjects, and these concentrations were significantly reduced after treatment. The concentration of sPD-1 was positively correlated with the concentration of IFN-γ, and negatively correlated with platelet count. Percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients are higher than those in healthy subjects, but elevated sPD-1 concentration in the blood blocks PD-1/PD-L1 signaling pathway, leading to unaffected Th cell function. Elevated concentrations of IFN-γ and IL-17 in the blood may participate in the occurrence and development of ITP.

The effect of single course high dose dexamethasone on CD28/CTLA-4 balance in the treatment of patients with newly diagnosed primary immune thrombocytopenia

To evaluate the effect of a single course of high dose dexamethasone (HD-DXM) on CD28 and CTLA-4 expression in patients with newly-diagnosed primary immune thrombocytopenia (ITP). Twenty-8 ITP patients (18 females and 10 males, age range 18–65 years, median age 38.5 years) enrolled in this study and 26 healthy volunteers (19 women and 7 men, age range 16–66 years, median age 37 years) served as a control group. The patients were treated with HD-DXM (40 mg/day) for 4 consecutive days. CD28 and CTLA-4 expression was assessed by flow cytometry once-monthly for 6 months. Plasma levels of the cytokines IFN-γ and IL-10 were determined by enzyme-linked immunosorbent assay. One month after treatment, a platelet response was observed in 23 (82%) of the patients. The response rates over the next 5 months were 71%, 57%, 53%, 46%, and 39%, chronologically. We observed a significant decrease in CD28 expression after the first month (34.7 ± 4.8% vs. 44.5 ± 4.4% before treatment), after which the CD28 levels gradually increased. In contrast, CTLA-4 expression increased after the first month (3.2 ± 0.5% vs. 0.8 ± 0.4 before treatment), after which the CTLA-4 levels gradually decreased. Similar dynamic changes were seen in the levels of IFN-γ and IL-10. The dynamic changes of CD28 and CTLA-4 were consistent with those of IFN-γ and IL-10 and with the effectiveness of HD-DXM in the treatment of ITP. Our results suggest that a disturbed CD28/CTLA-4 balance may contribute to the immunopathogenesis of ITP.

Immunization of mice with egG1Y162-1/2 provides protection against Echinococcus granulosus infection in BALB/c mice

Aims: This study is to predict and purify the T‐B combined epitopes of egG1Y162 antigen in Echinococcus granulosus, and to evaluate their immunogenicity in mice. Methods: The bioinformatics software was used to predict the T‐B combined epitopes of egG1Y162 antigen. Recombinant egG1Y161/2 peptides were constructed, expressed and purified. Mice were immunized with egG1Y161/2 peptides. The serum and spleen cells were isolated. The isolated spleen cells were stimulated with egG1Y161/2 peptides in vitro and the culture supernatant was collected. The levels of IgG in serum and levels of IL‐4 and IFN‐&ggr; in the culture supernatant were measured by ELISA. The weight and number of the fresh hydatid cysts were evaluated. The serum ptotoscolicidal activity was measured by the complement dependent cytotoxicity assay. Results: Peptides of 6–19aa, 64–82aa, 106–119aa were predicted as T‐B combined epitopes of egG1Y162 antigen. And, recombinant protein egG1Y162–1 or egG1Y162–2, which contained T‐B combined epitope(s) of the 6–19aa, or the 64–82aa and the 106–119aa in egG1Y162 antigen, respectively, was successfully expressed and purified. Serum IgG levels of mice immunized with egG1Y162–1/2 were significantly increased during the immune response to Echinococcus granulosus. The levels of IFN‐&ggr;, IL‐4 and the ratio of IFN‐&ggr;/IL‐4 after egG1Y162–1/2 immunization were significantly higher. Weight and number of the fresh hydatid cysts in egG1Y162–1/2 immunized mice was significantly decreased. And, the serum protoscolicidal activity after egG1Y162–1/2 immunization was enhanced. Conclusions: The egG1Y162–1/2 induces production of serum IgG levels and Th1 cell immune response, which enhances the protective immunity in Echinococcus granulosus challenged mice and thus may be used as a potential vaccine candidate. HIGHLIGHTST‐B combined epitopes of egG1Y162 antigen are predicted.EgG1Y162–1/2 protein is expressed and purified in vitro.The IgG levels in serum are increased after EgG1Y162–1/2 immunization.egG1Y162–1/2 enhances protective immunity in mice.

TGF-β/Smad signaling pathway positively up-regulates the differentiation of Interleukin-9-producing CD4+ T cells in human Echinococcus granulosus infection

OBJECTIVES Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus (Eg) infection. Th9 cells are reported to be involved in the immune responses in CE patients. This study aims to investigate the role of TGF-β/Smad pathway in the regulation of Th9 cells in CE patients. METHODS Using Western blot analysis, flow cytometry, qPCR, immunohistochemistry, ELISA and MTT assay, we measured the expression levels of TGF-β/Smad, PU.1, IRF-4, and IL-9 in CE patients. RESULTS The levels of TGF-β, p-Samd3, PU.1 and IL-9 were elevated in the liver of CE patients. IL-9 and IL-9R expressions were also elevated in the infected liver tissue, and IL-9 level was positively correlated with the liver inflammation. The levels of IL-9, IL-4, TGF-β and IL-10 in the supernatant were also significantly increased after stimulating hepatic lymphocytes of CE patients with Eg antigen B. After blocking the TGF-β pathway signaling in vitro, PU.1 and IL-9 were obviously reduced. CONCLUSIONS IL-9 may aggravate the inflammatory response in the liver of CE patients. The TGF-β/Smad signaling pathway is activated, and the signaling pathway may promote the differentiation of Th9 cells and IL-9 expression in active CE patients.