Helix Transcription Factor P3 Research Articles

Effect of Regulatory T Cells on Promoting Apoptosis of T Lymphocyte and Its Regulatory Mechanism in Sepsis.

With both in vivo and in vitro experiments, the present study was conducted to investigate the effect of regulatory T cell (Treg) on promoting T-lymphocyte apoptosis and its regulatory mechanism through transforming growth factor-beta (TGF-β1) signaling in mice. A murine model of polymicrobial sepsis was reproduced by cecal ligation and puncture (CLP); PC61 and anti-TGF-β antibodies were used to decrease counts of CD4(+)CD25(+) Tregs and inhibit TGF-β activity, respectively. Splenic CD4(+)CD25(+) Tregs and CD4(+)CD25(-) T cells were isolated. Phenotypes, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), forkhead/winged helix transcription factor p3 (Foxp3), and TGFβ1(m+), as well as the apoptotic rate of CD4(+)CD25(-) T cell, were analyzed by flow cytometry. Real-time reverse transcription-polymerase chain reaction was performed to determine mRNA expression of TGF-β1, and the expressions of Smad2/Smad3, Bcl-2 superfamily members of Bcl-2/Bim, cytochrome C, the mitochondrial membrane potential, and caspases in CD4(+)CD25(-) T cells were simultaneously determined. After treatment with PC61 or anti-TGF-β antibody, CTLA-4, Foxp3, and TGFβ1(m+) expressions of CD4(+)CD25(+) Tregs were markedly decreased in comparison to that of the CLP group and the apoptosis rate of CD4(+)CD25(-) T cells was significantly positively correlated with the expression of TGF-β1. Meanwhile, levels of P-Smad2/P-Smad3, proapoptotic protein Bim, cytochrome C, and activity of caspase-3, -8, -9 were downregulated, whereas the mitochondrial membrane potential and antiapoptotic protein Bcl-2 expression were restored. Taken together, our data indicated that the TGF-β1 signal could be partly involved in the apoptosis of CD4(+)CD25(-) T cells promoted by CD4(+)CD25(+) Tregs, therefore inhibition of TGF-β1 expression may provide a novel strategy for the improvement of host immunosuppression following sepsis.

Regulatory T cells in ankylosing spondylitis and the response after adalimumab treatment

ObjectivesThe aim of this study was to investigate the role of regulatory T cells (Tregs) in ankylosing spondylitis (AS). MethodsWe included 69 AS patients (15 of them received anti-tumor necrosis factor-apha agent-adalimumab) in the study and used a questionnaire to record the demographic data, disease activity index, functional index, human leukocyte antigen-B27, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Thirty healthy subjects were used as controls. The peripheral blood mononuclear cells (PMBCs) were stained with anti-CD4, anti-CD25 and anti-Forkhead/winged helix transcription factor P3 (anti-FoxP3) antibodies and flow-cytometry was used to determine cell populations. ResultsThe percentages of Tregs in PMBCs were significantly higher in AS patients than in healthy controls. In AS patients who had poor disease functional index with higher levels of ESR and CRP were positively and significantly correlated with Tregs percentages in PMBCs. After adalimumab treatment in 15 patients, the percentages of Tregs, the ESR/CRP levels and the Bath Ankylosing Spondylitis Disease Activity Index/Bath Ankylosing Spondylitis Functional Index were significantly and gradually decreased over time. ConclusionsThe high expression of FoxP3 and CD25 on CD4+ T cells in PBMCs in AS patients was noted, and could be reversed by adalimumab therapy. These findings suggest that Tregs may play a role in modulating the inflammatory process in AS. Whether Tregs can be taken as a predictor for disease activity or treatment outcome is unclear and requires further study.

Forkhead/winged helix transcription factor P3 gene methylation regulation of regulatory T Cells proliferation and function in murine autoimmune hepatitis

Objective To investingate whether T(Treg) cells forkhead/winged helix transcription factor P3(Foxp3)gene promoter CpG island methylation was participated in the Rhesus rotavirus(RRV) inhibiting regulatory Treg cells in BALB/c mice liver, to explore its role in autoimmune liver inflammation. Methods A total of 60 7-9-week-old BALB/c mice were divided randomly into 3 groups(20 each): injected intraperitoneally PFU= 106 RRV infection 24 h and 48 h,and normal control group injected the same volume of saline.Discontinuous Percoll gradient separation of monocytes, Using flow cytometry sorting liver CD4+ CD25+ Foxp3+ Treg cells.Application bisulfite sequencing method modified after sulphate (BSP) to detect BALB/c mouse spleen tissue carve DNA Foxp3 Treg cells selected gene promoter CpG island methylation status, Western blotting assay, spectrophotometer assay respectively. Results In the control group and after injection RRV 24 h or 48 h, accounted for the proportion of CD4+ cells was (5. 26±0. 21)%,(4. 30±0. 46)%,(3. 60±0. 19)% respectively, and the difference were statistically significance(P< 0. 05).Foxp3 gene CpG island methylation were(2. 78±0. 33)%,(26. 70±1. 87)%, (41. 18±3. 67)% respectively and the difference were statistically significance(P< 0. 01).Expression of Foxp3 Treg cells also decreased, with RRV infection time was positively correlated,and the differences were statistically significant(P< 0. 05). Conclusion RRV significantly promote the Treg cells Foxp3 gene promoter CpG island methylation in BALB/c mice liver,and Inhibit the proliferation of Treg cells and normal immune function.That may involved in autoimmune hepatitis in mice. Key words: Regulatory T Cells; Forkhead/winged helix transcription factor P3 gene; CpG island; Methylation; Autoimmune hepatitis

Significance of forkhead/winged helix transcription factor P3+ regulatory T cells infiltration in extrahepatic cholangiocarcinoma

Objective To explore the infiltration of regulatory T cells(Tregs)and its significance in extrahepatic cholangiocarcinoma(EHCC)and to elucidate the putative effects of cytochrome C oxidase- 2 (COX- 2)expression on the infiltration of Tregs. Methods The infiltration of forkhead/winged helix transcription factor P3(Foxp3)+ Tregs and the expression of COX- 2 were studied by immunohistochemistry in 94 and 77 cases of EHCC respectively.The correlation between the expression of COX- 2 and Foxp3+ Tregs infiltration with clinicopathological features and prognosis was analyzed. The influence of COX- 2 expression on infiltration of Foxp3+ Tregs was evaluated. Results Foxp3+ Tregs infiltration was seen in all the 94 cases of EHCC with the median Foxp3+ Tregs number being 2.35 per high- power field(HPF). There was no significant correlation between the number of infiltrated Foxp3+ Tregs in cancer tissue and histological grading, TNM stages and lymph node metastasis of EHCC.The patients with less number of infiltrated Foxp3+ Tregs showed significantly longer survival than those with greater number of infiltrated Foxp3+ Tregs(P<0.05).Cox analysis results showed that infiltration of Foxp3+ Tregs was an independent prognostic factor of EHCC(P< 0.05).The positive expression rate of COX- 2 in 77 cases of EHCC was 81.8%.COX- 2 expression was not significantly associatied with histological grading, TNM stages and lymph node metastasis of EHCC.COX- 2 expression was positively related with stromal Tregs infiltration(r= 4.89,P< 0.05). Conclusion The number of infiltrated Foxp3+ Tregs in EHCC tissue is negatively related with overall survival of the patients and is an independent prognosis factor of EHCC.COX-2 protein expression may contribute to Foxp3+ Tregs infiltration in cancer stroma. Key words: Extrahepatic cholangiocarcinoma; Cytochrome C oxidase-2; Forkhead/winged helix transcription factor P3; Regulatory T cells; Prognosis

Nicotine ameliorates experimental severe acute pancreatitis via enhancing immunoregulation of CD4+ CD25+ regulatory T cells.

Activation of "nicotinic anti-inflammatory pathway" could reduce severity of inflammation and injury induced by acute pancreatitis. However, the role of regulatory T (Treg) cells in this pathway is unclear. Severe acute pancreatitis (SAP) was induced in mice through retrograde injection of 50-μL 2% Na-taurocholate into the pancreatic duct of the mouse. In nicotine treatment group, nicotine (50, 100, and 300 μg/kg) was administered 1 hour before and after SAP operation through intraperitoneal injection. We compared the properties of Treg cell percentage and specific marker such as cytotoxic T-lymphocyte antigen 4 and forkhead box transcription factor forkhead/winged helix transcription factor p3 on Treg using quantitative reverse transcription polymerase chain reaction and flow cytometry. All experiment animal serum cytokines were measured using enzyme-linked immunosorbent assay. One-way analysis of variance was applied to evaluate the experimental data and for statistical comparisons. The survival rate data were analyzed using the log-rank test. Nicotine significantly protected mice from lethal SAP in a dose-dependent fashion by inhibiting tissue injury, digestive enzyme production, and proinflammatory cytokines production. Moreover, nicotine up-regulated the number and suppressive capacity of CD4 CD25 Treg via inducing the expression of immunoregulatory molecules and transforming growth factor β1 elevation. Modulating immunoregulation of CD4 CD25 Treg is a critical mechanism for nicotinic anti-inflammatory pathway and it may be feasible to use selective agonists as an immunotherapy for SAP.

Expression and function of forkhead box P3 in the tissue of rat gastric cancer model

Objective To study the expression and function of forkhead box P3(Foxp3)in the tissue of rat gastric cancer model. Methods Forty- six Wister rats were divided into two groups: group A(n= 23)given sarcosine ethyl ester hydrochloride and sodium nitrite carcinogen for 6 months to establish the rat gastric cancer model,and group B(n= 23)as the normal control group.The reverse transcriptase- polymerase chain reaction(RT- PCR), Western blotting,and immunohistochemistry were used to detect the expression and location of Foxp3 in the gastric tissues.Immunohistochemistry was used to detect the expression and localization of Foxp3 in gastric cancer tissues.The relationship between the expression of Foxp3 and gastric lesions was statistically analyzed. Results The expression of Foxp3 mRNA in the gastric tissue of group A existed to varying degrees, and increased with the grades, and there was no Foxp3 mRNA expression in the gastric tissues of group B.Immunohistochemistry revealed that the expression of Foxp3 protein was mainly distributed in the nuclei of gastric cancer cells.In 16 gastric cancer sections,4 cases of precancerous lesions,and 3 cases of proliferative lesions in group A, 10(62.50%), 2(50.00%) and 1 case(33.30%) was positive for the Foxp3 protein, respectively.In group B, no Foxp3 protein staining was detected all 23 cases.Statistical analysis showed a linear correlation of Foxp3 protein expression in gastric tissue and lesion classification(r= 4.26, P< 0.01). Conclusion The expression of Foxp3 universally exists in gastric cancer cells and tissues, suggesting Foxp3 may play important roles in the occurrence and development of gastric cancer. Key words: Gastric carcinoma; Forkhead/winged helix transcription factor P3

Immune killing effects of 1-methyltroptophan combined with peripheral blood mononuclear cells on human glioma cells

Objective To investigate the immune killing effects of 1- methyltroptophan(1- MT) combined with peripheral blood mononuclear cells(PBMC) on human A172 glioma cells and the relative mechanism. Methods PBMC were isolated from healthy donors by Ficoll density- gradient centrifugation. A172 cells were co- cultured with or without PBMCin the presence or absence of 1- MT. Cell counting kit-8(CCK- 8)assay was used to test the inhibitory effect of 1- MT(0,25,125,250,500μmol/L;1,2,4, 8,10 mmol/L)combined with PBMC on growth of A172 cells.Annexin V/propidium iodide(PI)staining was used to detect the effect of 1- MT(500μmol/L)combined with PBMC on apoptosis of A172 cells. The expression of indoleamine 2,3- dioxygenase(IDO)protein in A172 cells was detected by Western blotting in different groups.The expression changes of forkhead helix transcription factor p3(Foxp3) mRNA in PBMC were investigated by real- time quantitative polymerase chain reaction(RT- qPCR). Results 1- MT reduced the growth rate of A172 glioma cells in a dose- dependent manner under the condition of a constant PBMCamount.The apoptosis rate of A172 cells induced by combined 1- MT and PBMC(45.0%) was significantly higher than that induced by either 1- MT(33.7%)or PBMCs(20.0%).Western blotting analysis showed that the expression of IDO was considerably lower in 1- MT+ PBMC group(0.182±0.015)than in PBMC group(0.313±0.017).1- MT group and A172 blank group showed no significant IDO expression.The mRNA level of Foxp3 was significantly higher in PBMC group(2.764±0.180)than in 1- MT+ PBMC group (2.189±0.179)and PBMC blank group(1.000±0.028)by RT- qPCR. Conclusion 1- MT can significantly increase the immune killing effects of PBMC by inhibiting the expression of IDO and Foxp3 and the differentiation of Treg cells in A172 cells. Key words: Indoleamine 2,3-dioxygenase; Glioma cells; Peripheral blood mononuclear cells; Apoptosis

Role of Foxp3/Treg and RORγt/Th17 cell imbalance in rat model of chronic obstructive pulmonary disease

To observe the changes in forkhead/winged helix transcription factor p3(Foxp3), regulatory T cells (Treg), retinoid-related orphan receptor gamma (RORγt) in rat model of chronic obstructive pulmonary disease (COPD). Twenty Sprague-Dawley (SD) rats were randomly divided into normal control group and COPD model group, with 10 rats in each group. The COPD model was reproduced by smoke inhalation and tracheal instillation of lipopolysaccharide (LPS), and no such treatment was conducted in normal control group. Twenty-eight days after the model reproduction, the pulmonary function was determined, the pathological changes of lung tissue were observed with haematoxylin-eosin (HE) staining, interleukins (IL-6, IL-10) in serum were detected by enzyme-linked immunosorbent assay (ELISA), CD4⁺ CD25⁺ Foxp3⁺ Treg of peripheral blood was determined by flow cytometry, and the expressions of Foxp3, RORγt, IL-17 protein in lung tissue were assayed by Western Blot. Under light microscope, significal interstitial infiltration of inflammatory cells was found in alveoli and interstitial tissue of the lung, and destruction of alveolar tissue, alveolar wall thinning, and even rupture to fuse into bullae, and bleeding into alveoli in different degress could be observed. Compared with the normal control group, forced vital capacity (FVC), forced expiratory volume in 0.3 second (FEV0.3), FEV0.3/FVC, peak expiratory flow (PEF) in model group were significantly decreased [FVC (mL): 8.04 ± 2.03 vs. 9.97 ± 2.14, FEV0.3 (mL): 6.16 ± 2.23 vs. 8.84 ± 2.12, FEV0.3/FVC: 0.70 ± 0.09 vs. 0.85 ± 0.11, PEF (mL/s): 33.56 ± 4.76 vs. 40.14 ± 5.64, P<0.05 or P<0.01]. Serum IL-6 was obviously increased (ng/L: 93.17 ±20.96 vs. 76.28 ± 13.24, P<0.05), IL-10 was significantly decreased (ng/L: 78.62 ± 15.17 vs. 104.34 ± 19.46, P<0.01), and CD4⁺ CD25⁺ FoxP3(+)Treg was significantly diminished [(2.75 ± 0.83)% vs. (4.16 ± 1.14)%, P<0.01] in model group compared with those in the normal control group. The expression of Foxp3 protein in lung tissue in model group was significantly down-regulated compared with that in the normal control group (gray scale: 0.38 ± 0.15 vs. 0.63 ± 0.11, P<0.01), and RORγt and IL-17 protein expressions were significantly up-regulated [RORγt (gray scale): 0.96 ± 0.23 vs. 0.47 ± 0.11, IL-17 (gray scale): 1.02 ± 0.24 vs. 0.34 ± 0.08, both P<0.01]. Correlation analysis showed that FEV0.3 was positively correlated with Foxp3 (r=0.585, P<0.05), and FEV0.3/FVC was negatively correlated with IL-6 and RORγt (r=-0.655, r=-0.607, both P<0.05). PEF was positively correlated with Treg (r=0.573, P<0.05), and negatively correlated with IL-17 (r=-0.198, P<0.05). IL-6 was negatively correlated with Foxp3(r=-0.603, P<0.05),and positively correlated with RORγt (r=0.588, P<0.05). IL-10 was positively correlated with Treg (r=0.573, P<0.05). Treg was positively correlated with Foxp3 (r=0.607, P<0.05), and negatively correlated with IL-17 (r=-0.569, P<0.05). Foxp3 was negatively correlated with RORγt (r=-0.591, P<0.05). RORγt was positively correlated with IL-17 (r=0.578, P<0.05). There is a relationship among decreased pulmonary function, inflammation and imbalance of Foxp3/Treg and RORγt/Th17 in COPD.

Expression and Effects of High-Mobility Group Box 1 in Cervical Cancer

We investigated the significance of high- mobility group box1 (HMGB1) and T-cell-mediated immunity and prognostic value in cervical cancer. HMGB1, forkhead/winged helix transcription factor p3 (Foxp3), IL-2, and IL-10 protein expression was analyzed in 100 cervical tissue samples including cervical cancer, cervical intraepithelial neoplasia (CIN), and healthy control samples using immunohistochemistry. Serum squamous cell carcinoma antigen (SCC-Ag) was immunoradiometrically measured in 32 serum samples from 37 cases of squamous cervical cancer. HMGB1 and SCC-Ag were then correlated to clinicopathological characteristics. HMGB1 expression tends to increase as cervical cancer progresses and it was found to be significantly correlated to FIGO stage and lymph node metastasis. These findings suggest that HMGB1 may be a useful prognostic indicator of cervical carcinoma. In addition, there were significant positive relationships between HMGB1 and FOXP3 or IL-10 expression (both p < 0.05). In contrast, HMGB1 and IL-2 expression was negatively correlated (p < 0.05). HMGB1 expression may activate Tregs or facilitate Th2 polarization to promote immune evasion of cervical cancer. Elevated HMGB1 protein in cervical carcinoma samples was associated with a high recurrence of HPV infection in univariate analysis (p < 0.05). HMGB1 expression and levels of SCC-Ag were directly correlated in SCC (p < 0.05). Thus, HMGB1 may be a useful biomarker for patient prognosis and cervical cancer prediction and treatment.

Effect of iodine excess on Th1, Th2, Th17, and Treg cell subpopulations in the thyroid of NOD.H-2h4 mice.

Iodine is an indispensable micronutrient for thyroid hormone synthesis and metabolism. Iodine excess may trigger and exacerbate autoimmune thyroiditis (AIT). The pathogenetic mechanism of iodine excess-induced AIT is partly regarded as T helper type 1 (Th1) cell and/or T helper type 17 (Th17) cell dominant autoimmune disease. It is still unknown whether other cluster of differentiation 4+ T (CD4+T) cell subpopulations are involved. Therefore, we studied the profile of all the CD4+T cell subpopulations of the thyroid in iodine excess-induced nonobese diabetic-H2h4 (NOD.H-2h4) mice to explore the potential immunologic mechanism of iodine excess-induced AIT. A total of 40 healthy 8-week-old NOD.H-2h4 mice were randomly allocated into the normal group (NG, n=20) and the test group (TG, n=20), which were fed with double-distilled water and 0.05% sodium iodine (NaI) for 8 weeks, respectively. Compared to the NG, in the TG, the incidence of AIT was significantly higher, the expressions of interleukin-17 (IL-17), interleukin-23 (IL-23), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β) remarkably increased by immunohistochemistry, which were further verified by reverse transcription polymerase chain reaction (RT-PCR), while the protein and mRNA expressions of interleukin-4 (IL-4) and interferon-γ (INF-γ) decreased markedly. In the AIT mice, the expressions of retinoic acid-related orphan receptor gamma t (RORγt), retinoic acid-related orphan receptor alpha (RORα), and signal transducer and activator of transcription 3 (STAT3) were much higher, the expression of forkhead/winged helix transcription factor p3 (Foxp3) significantly lower by western blot, and the proportion of Th17 cells by flow cytometry method (FCM) much larger compared to those of the NG group. In conclusion, Th17 cells may promote an inflammatory reaction in the development of iodine-excess-induced AIT, which is negatively regulated by Th1, T helper type 2 (Th2), and regulatory T (Treg) cells.

Expression of tumor necrosis factor-α induced protein 8 like-2 contributes to the immunosuppressive property of CD4+CD25+ regulatory T cells in mice

Tumor necrosis factor-α induced protein 8 like-2 (TNFAIP8L2, TIPE2), a lately discovered negative regulator of innate immunity and cellular immunity, shares considerable sequence homology with members of the tumor necrosis factor-α induced protein 8 (TNFAIP8) family. It is preferentially expressed in lymphoid-derived and marrow-derived cells. However, it is unclear whether TIPE2 is expressed in the regulatory T cells (Tregs) to contribute to its negative regulatory property in immune response. The present study was designed to examine whether naturally occurring CD4+CD25+ Tregs isolated from murine spleens expressed TIPE2 by the use of Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Based on the expression of cell surface molecules, including cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on CD4+CD25+ Tregs, and cytokines including interleukin (IL)-10 as well as transforming growth factor (TGF)-β were analyzed to investigate the functional role of TIPE2 in controlling suppressive activity of CD4+CD25+ Tregs. Meanwhile, IL-2, the ratio of interferon (IFN)-γ/IL-4 in CD4+CD25+Treg/CD4+CD25− T cell coculture supernatant and nuclear factor of activated T cells (NF-AT) activation in T lymphocytes were determined to examine the effects of TIPE2 on the T-cell proliferation and differentiation induced by CD4+CD25+ Tregs. It was found that TIPE2 was a cytoplasmic protein expressed in CD4+CD25+ Tregs, and cell surface molecules as well as cytokines (IL-10, TGF-β) expressions were significantly down-regulated when TIPE2 gene silenced by siRNA. On the other hand, CD4+CD25+ Tregs treated with TIPE2 knock-down promoted T-cell proliferation as well as differentiation, and markedly upregulated IL-2 expression and intranuclear NF-AT activation. The results suggested that TIPE2 appeared to be a critical immunoregulatory molecule involved in immunosuppressive function of CD4+CD25+ Tregs.

Astragalus Polysaccharides Attenuate Postburn Sepsis via Inhibiting Negative Immunoregulation of CD4+CD25high T Cells

BackgroudAstragalus polysaccharides (APS) isolated from one of the Chinese herbs, Astragalus mongholicus, are known to have a variety of immunomodulatory activities. However, it is not yet clear whether APS can exert an effect on the immune functions of regulatory T cells (Tregs). This study was carried out to investigate the effect of APS on the immune function of peripheral blood Tregs in postburn sepsis.Methodology/Principal FindingsBalB/c mice were randomly divided into six groups as follows: sham burn group, burn control(burn without infection animals) group, burn plus P. aeruginosa group, burn plus P. aeruginosa with APS (50 mg/kg) treatment group, burn plus P. aeruginosa with APS (100 mg/kg) treatment group, and burn plus P. aeruginosa with APS (200 mg/kg) treatment group, and they were sacrificed on postburn day 1, 3, 5, and 7, respectively, with seven animals at each time point. Magnetic microbeads were used to isolate peripheral blood Tregs and CD4+ T cells. Phenotypes were analyzed by flow cytometry, and cytokine levels were determined with ELISA. In the burn plus P. aeruginosa group, forkhead/winged helix transcription factor p3 (Foxp3) expression on CD4+CD25+Tregs were strongly enhanced in comparison to the sham group, and the capacity of CD4+CD25+Tregs to produce interleukin (IL)-10 was markedly increased. Administration of APS to inhibit CD4+CD25+Tregs could significantly decrease expression of Foxp3 on CD4+CD25+Tregs, and IL-10 production in burned mice with P. aeruginosa infection. At the same time, proliferative activity and expression of IL-2 and IL-2Rα on CD4+ T cells were restored. In contrast, anti-Toll-like receptor 4 (TLR4) antibody could block the effect of APS on Tregs immune function.ConclusionAPS might suppress CD4+CD25+Treg activity, at least in part, via binding TLR4 on Tregs and trigger a shift of Th2 to Th1 with activation of CD4+ T cells in burned mice with P. aeruginosa infection.

High mobility group box-1 protein regulate immunosuppression of regulatory T cells through toll-like receptor 4

Introduction High mobility group box-1 protein (HMGB1), a recently recognized mediator of immune response might contribute to immune suppression when released extracellularly. The present study was performed to clarify effects of HMGB1 on regulatory T cells (Tregs) and the involvement of toll-like receptor (TLR) 4 signaling. Methods CD4 +CD25 +Tregs, isolated from spleens of normal mice and treated with HMGB1 in vitro, and those isolated from HMGB1-treated C3H/HeN (wild type) or C3H/HeJ (TLR4 mutant type) mice, were analyzed for expressions of cytotoxic T lymphocyte-associated antigen (CTLA)4, forkhead/winged helix transcription factor p3 (Foxp3) and interleukin (IL)-10 secretion. Results HMGB1-treatment was found to markedly decrease the expressions of CTLA4 and Foxp3, as well as IL-10 secretion. Administration of TLR4 neutralizing antibody abolished the phenotypic and functional changes in Tregs induced by HMGB1. Tregs from HMGB1-treated normal mice showed lower expression of CTLA4, Foxp3, and IL-10 secretion when compared with non-treated mice. Yet opposite results were observed in that of C3H/HeJ mice. Moreover, HMGB1 stimulation could down-regulate the expression of TLR4 on Tregs. Conclusion Our data suggest that HMGB1 has the ability to directly modulate the suppressive capacity of CD4 +CD25 +Tregs, and TLR4 might be a potential receptor essential for the negative effect of HMGB1 on CD4 +CD25 +Tregs activity.

The Potential Effect and Mechanism of High-Mobility Group Box 1 Protein on Regulatory T Cell-Mediated Immunosuppression

To investigate the effect of high-mobility group box 1 (HMGB1) protein on the regulatory T cells (Tregs) in vitro and its potential regulating mechanism in mice. Splenic CD4(+)CD25(+) Tregs and CD4(+)CD25(-) T cells were isolated. The time-dependent and dose-dependent responses between HMGB1 stimulation and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead/winged helix transcription factor p3 (Foxp3) expressions were analyzed. The secretion of various cytokines in the cell suspensions and the proliferation of CD4(+)CD25(-) T cells were determined. HMGB1 was found to be able to markedly down-regulate the expressions of CTLA-4 and Foxp3, especially at 72 h group and in 1,000 ng/mL group (both P < 0.01). The expression of Foxp3 mRNA showed a similar tendency as Foxp3 protein (P < 0.05 or P < 0.01). The secretion of interleukin (IL)-10 from Tregs was decreased when the concentration of HMGB1 was increased. The suppressive activity of proliferation of CD4(+)CD25(-) T cells exceeded 90% when the ratio of Tregs to CD4(+)CD25(-) T cells was 1:1; meanwhile, the proliferation of CD4(+)CD25(-) T cells was enhanced when cultured with HMGB1-stimulated Tregs. In the culture of HMGB1-stimulated Tregs, IL-2 and interferon-γ levels were elevated; whereas IL-4 and IL-10 decreased in CD4(+)CD25(-) T cells after the increased concentration of HMGB1 in comparison with unstimulated-Treg group. HMGB1 stimulation can result in markedly down-regulatory expressions of CTLA-4 as well as in Foxp3 expression and secretion of IL-10 from splenic Tregs in mice. HMGB1 appears to be involved in modulating cell-mediated immunity by influencing proliferation of effector T cells, secretion of IL-2, and cell polarization.

Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Increases Suppressive Capacity of Naturally Occurring CD4+CD25+Regulatory T Cells in Mice In Vitro

α7 Nicotinic acetylcholine receptor (α7 nAChR) has been found in several non-neuronal cells and is described as an important regulator of cellular function. Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. The present study investigated whether naturally occurring Tregs expressed α7 nAChR and investigated the functionary role of this receptor in controlling suppressive activity of these cells. We found that CD4(+)CD25(+) Tregs from naive C57BL/6J mice positively expressed α7 nAChR, and its activation by nicotine enhanced the suppressive capacity of Tregs. Nicotine stimulation up-regulated the expression of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on Tregs but had no effect on the production of interleukin (IL)-10 and transforming growth factor-β1 by Tregs. In the supernatants of CD4(+)CD25(+) Tregs/CD4(+)CD25(-) T-cell cocultures, we observed a decrease in the concentration of IL-2 in nicotine-stimulated groups, but nicotine stimulation had no effect on the ratio of IL-4/interferon (IFN)-γ, which partially represented T-cell polarization. The above-mentioned effects of nicotine were reversed by a selective α7 nAChR antagonist, α-bungarotoxin. In addition, the ratio of IL-4/IFN-γ was increased by treatment with α-bungarotoxin. We conclude that nicotine might increase Treg-mediated immune suppression of lymphocytes via α7 nAChR. The effect is related to the up-regulation of CTLA-4 as well as Foxp3 expression and decreased IL-2 secretion in CD4(+)CD25(+) Tregs/CD4(+)CD25(-) T-cell coculture supernatants. α7 nAChR seems to be a critical regulator for immunosuppressive function of CD4(+)CD25(+) Tregs.

Alteration of T helper cell subsets in the optic nerve of experimental autoimmune encephalomyelitis

The objective of this study was to detect interleukin-17 (IL-17), interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and forkhead/winged helix transcription factor p3 (Foxp3) protein and gene expression of the optic nerve and to further explore the role of T helper cell subsets such as Th1, Th2, Th17 and Treg in the pathogenesis of optic neuritis in experimental autoimmune encephalomyelitis (EAE). Mice in C57BL/6 background were randomly divided into control and EAE groups. At days 11, 15 and 19 post-immunization, optic nerves were dissected for morphological study to detect IL-17, IFN-gamma and IL-4. Protein analysis was done by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction for measuring the gene expression of IL-17, IFN-gamma, IL-4 and Foxp3. Concentrations of IL-17 protein in the optic nerve were significantly up-regulated at 11 days post-immunization, and IFN-gamma protein concentrations at 19 days. Concentrations of IL-4 protein in the optic nerve declined slightly in 19 days. mRNA expression of IL-17, IFN-gamma and IL-4 was consistent with their protein expression. Foxp3 mRNA transcription was down-regulated at 11-19 days post-immunization. Decreased expression of Foxp3 mRNA and Treg in the optic nerve may play a key role in the development of optic neuritis. IL-17 may mediate inflammatory pathogenicity at the early stage of optic neuritis, and IFN-gamma may aggravate inflammatory injury during the peak stage of optic neuritis.

Association between regulatory T cell activity and sepsis and outcome of severely burned patients: a prospective, observational study

IntroductionTo investigate the significance of changes in regulatory T cells (Tregs) activity and its relationship with sepsis, as well as outcome of patients with major burns.MethodsThe periphery blood samples of 106 patients were collected on post-burn days 1, 3, 7, 14, and 21. Tregs were isolated and their phenotypes (cytotoxic T-lymphocyte-associated antigen 4 and forkhead/winged helix transcription factor p3) were analyzed by flow cytometry, and the contents of cytokines (interleukin-10 and transforming growth factor-β1) released into supernatants by Tregs were also determined by enzyme-linked immunosorbent assay kits. Gene expressions of cytokines were assessed by real-time quantitative polymerase chain reaction.ResultsExpressions of Tregs phenotypes and gene/protein expression of cytokines were all elevated after burn, and there were obvious differences among patients with various burn sizes. They were also higher in septic patients than those without sepsis. Among septic patients, the expressions of Tregs phenotypes and the levels of cytokines were markedly lower in the survival group than those in patients with fatal outcome.ConclusionsSevere burn injury per se could lead to the changes in Tregs activities. Elevated levels of cytokines produced by Tregs and activation markers on Tregs surface might play an important role in the pathogenesis of sepsis and mortality in burned patients.

The Human “Treg MLR”: Immune Monitoring for FOXP3+ T Regulatory Cell Generation

Controversy exists about the conditions effecting the development of forkhead/winghead helix transcription factor P3 (FOXP3) expressing T cells and their relevance in transplant recipients. We generated carboxy-fluorescein diacetate succinimidyl ester-labeled CD4+CD25 high FOXP3+ cells in mixed lymphocyte reactions (MLRs) ("the Treg MLR"), with varying human leukocyte antigen (HLA) disparities and cell components. Five color flow cytometry and H-thymidine uptakes were the readouts. (1) Despite lower stimulation indices (SIs) than two DR-mismatched MLRs, 2 DR-matched MLRs generated more than twofold higher percentages when gating on proliferating CD4+CD25 high FOXP3+ cells; (2) Even with low numbers of proliferating cells, autologous and HLA identical MLRs generated the highest FOXP3+:FOXP3- cell ratios; (3) Elimination of either non-CD3+ responding cells (resulting in "direct presentation" only) or responding CD25+ (Treg generating) cells increased the SI but inhibited proliferating CD4+CD25 high FOXP3+ cell development; (4) MLR-generated CD4+CD25 high FOXP3+ cells added as third components specifically inhibited the same freshly set MLR SI and caused recruitment of new CD4+CD25 high FOXP3+ cells. As an example of the "Treg MLR" immune monitoring potential, addition of third component peripheral blood mononuclear cell containing high percentages of CD4+CD25 high FOXP3+ cells from an HLA identical kidney transplant recipient (in a tolerance protocol) caused donor-specific Treg MLR inhibition or recruitment. This was similar to the third component MLR Tregs generated entirely in vitro. In the Treg MLR, the generation of CD4+CD25 high FOXP3+ cells is more pronounced in the context of self-recognition (HLA matching, indirect presentation). These cells can be assayed for MLR inhibitory and Treg recruitment functions, so as to immunologically monitor the allospecific regulation after transplantation.

Effect of Xuebijing injection on lipopolysaccharide-induced apoptosis of CD4+ CD25+ regulatory T cells and immune function of effector T cells in vitro.

Objective To investigate the effect of Xuebijing injectiong on lipopolysaccharide(LPS)-induced apoptosis of CD4+CD25+regulatory T cells(Tregs)and immune function of effector T cells(Teff)in vitro.Method CD4+CD25+Tregs isolated from rat spleens were divided into the control group,anti-CD3/CD28 group,anti-CD3/CD28+LPS group,anti-CD3/CD28+Xuebijing injection group,and anti-CD3/CD28+LPS+Xuebijing injection group.The apoptosis rate and expression of forkhead/winged helix transcription factor p3 (Foxp3)and cytotoxic T-lymphocyte-associated antigen 4(CTLA-4)of CD4+CD25+Tregs were detected by flow cytometry(FCM),and the secretion of IL-10 of Tregs was measured by ELBA on day 3.CD4+CD25-T cells were co-cultured with CD4+CD25+Tregs(1:1)for 68 hours,proliferative activity of Teff was determined by MTT,and interleukin(IL)-2/sIL-2Rα levels were measured by ELISA.Results The apoptosis rate of CD4+CD25+Tregs in anti-CD3/CD28 group was 33.70± 3.06%,which was significantly higher than that in control group(12.84±0.84%).Also,apoptosis rate of CD4 CD25+Tregs in anti-CD3/CD28+LPS+Xuebijing injection group(45.13±2.70%)was much higher than that in anti-CD3/CD28+IPS group(29.41 ± 1.63%,P＜0.01).The expression of Foxp3 as well as CTLA-4,and the secretion of IL-10 were markedly decreased along with increases in the apoptosis rates.Compared with control group(54.48%),the mean inhibitory rate of Teff proliferative activity in response to Con A was significantly decreased in anti-CD3/CD28+Xuebijing injection group(31.26%,P＜0.05),and it was markedly decreased in anti-CD3/CD28+LPS+Xuebijing injection group comaped to anti-CD3/CD28+LPS group(P＜0.01).In addition,IL-2 levels in the supernatant of anti-CD3/CD28+Xuebijing injection group and anti-CD3/CD28+LPS+Xuebijing injection group were significantly higher than those of anti-CD3/CD28+IPS group(P＜0.01).Conclusions The inhibitory activity of CD4+CD25+Tregs on Teff appears to be upregulated by IPS stimulation in vitro,and Xuebijing injection could markedly enhance apoptosis of CD4+CD25+Tregs,thereby improving suppressive immune function of Teff. Key words: Sepsis; Regulatory T cells; Effector T cells; Apoptosis; Xuebijing injection

THE EFFECT OF HIGH-MOBILITY GROUP BOX 1 PROTEIN ON ACTIVITY OF REGULATORY T CELLS AFTER THERMAL INJURY IN RATS

The aim of the present study was to investigate in vivo the effect of high-mobility group box 1 protein (HMGB1) on activity of regulatory T cells (Tregs) and the influence on T-cell-mediated immunity after thermal injury. Male Wistar rats were randomly divided into four groups as follows: sham burn group, burn group, burn with ethyl pyruvate treatment group, and burn with antireceptor for advanced glycation end products (RAGE) antibody treatment group, and they were killed on postburn days 1, 3, 5, and 7, respectively, with eight animals at each time point. Magnetic cell sorting microbeads were used to isolate splenic Tregs and a column of nylon wool to obtain T cells. Phenotypes, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), forkhead/winged helix transcription factor p3 (Foxp3), RAGE, and IL-2Ralpha, were analyzed by flow cytometry. Levels of HMGB1, IL-10, IL-2, IL-4 and interferon gamma were determined by enzyme-linked immunosorbent assay kits, and real-time reverse transcription-polymerase chain reaction was performed to detect mRNA expression of IL-10, IL-2, and IL-2Ralpha. Serum HMGB1 levels were significantly elevated during postburn days 1 to 7. In the burn group, CTLA-4 and Foxp3 expression levels of Tregs were strongly enhanced in comparison to the sham-injured group, and the capacity of Tregs to produce IL-10 was markedly increased. Administration of ethyl pyruvate to inhibit HMGB1 or anti-RAGE antibody could significantly decrease expression levels of CTLA-4, Foxp3 on Tregs, and IL-10 production after burns. Simultaneously, proliferative activity and expression levels of IL-2 and IL-2Ralpha of T cell were restored. The excessively released HMGB1 might stimulate CD4+CD25+Treg activity via binding RAGE on the surface of Tregs and trigger a shift of T(H)1 to T(H)2 with suppression of T-lymphocyte immune function after burn injury.