Protective effects of Wuwei Xiaodu Drink against chronic osteomyelitis through Foxp3+CD25+CD4+ Treg cells via the IL-2/STAT5 signaling pathway / 中国天然药物

To explore the effectiveness and safety of a Chinese medicinal decoction Wuwei Xiaodu Drink (WWXDD) in inhibiting chronic osteomyelitis via regulatory T cells signaling. The effective constitutes of WWXDD and osteomyelitis related genes were screened. Target proteins were cross-validated using the Venny database. GO function and KEGG pathway analysis were performed for target proteins, while pharmacological network was constructed. The bone properties were analyzed by HE staining and the concentrations of immune factors were measured by ELISA. The expression of CTLA-4 and Foxp3 mRNA and STAT5, p-STAT5, CTLA-4 and Foxp3 protein were detected using Real-time PCR and Western blot, respectively. FACS was used to analyze the percentages of cells. A total of 117 genes overlapped between 785 target genes of the active compounds of WWXDD and 912 osteomyelitis related genes. Inflammation-related genes, including IL-6, TNFα, IL-1β and IL-2 showed high connection degree in the drug-compound-disease-target network. GO function and KEGG pathway analysis revealed that 117 intersection genes mainly enriched in virus infection related pathways, immune related pathways and chemokine signaling pathway. Furthermore, the development of chronic osteomyelitis was suppressed in model rats after treatment with WWXDD. Meanwhile, the concentrations of IL-2 and CD4+CD25+Foxp3 Treg percentages together with the levels of p-STAT5, CTLA-4 and Foxp3 were also down-regulated. Furthermore, IL-2 and WWXDD drug-containing serum exhibited opposite effects on regulating IL-2, IL-10, TGF-β1, Foxp3, CTLA4 and STAT5. In addition, a STAT5 phosphorylation inhibitor suppressed the expression of Foxp3 and CTLA-4. WWXDD can treat chronic osteomyelitis through suppressing the main regulating factors of Tregs and interfere its immunodepression. Our results bring a new solution for chronic osteomyelitis.

Impact of Allium sativum ethanol extract on immuno-regulatory T cells and anti-inflammatory cytokine profile in murine schistosomiasis

@#Parasite immune response against schistosomal antigens involves both the innate and adaptive immune response. Tregs have a suppressive effect and play a role on the parasite’s immune evasion. This study aimed to evaluate active compounds of Allium sativum (AS) ethanol extract and the impact of AS extract alone or in combination with praziquantel on Tregs and anti-inflammatory cytokines TGF-β and IL-10 in mice infected with S. mansoni. Phytochemical screening of AS bulbs for various active constituents and qualitative and quantitative analysis of the flavonoids and phenolic acids were done using HPLC. Measurement of splenocytes Treg cell phenotypes and anti-inflammatory cytokines TGF-β and IL-10 was done by flow cytometric analysis. The data are expressed as mean ± SD. Statistical significance was determined by one-way ANOVA utilizing the statistical package (SPSS version 17.0). HPLC of AS ethanol extract revealed presence of 22 and 18 compounds of flavonoids and phenolic acids, respectively. S. mansoni infection upregulated the Treg cells subsets (CD4, CD25, Foxp3) frequencies and the levels of TGF-β and IL-10 anti-inflammatory cytokines when compared to healthy control. AS ethanol extract alone or combined with PZQ decreases the production of Treg cells from spleen in addition to the reduction in antiinflammatory cytokines IL-10 and TGF-β. This study recommends that the combination of AS ethanol extract and PZQ may play a role in maintaining the homeostasis of the immune system during schistosomiasis by decreasing Treg cells and anti-inflammatory cytokines IL10 and TGF-β production.

Effect of fasudil on experimental autoimmune neuritis and its mechanisms of action

This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.

Blockade of IL-27 signaling ameliorates herpes stromal keratitis with upregulated CD4+ Foxp3+ regulatory T cells influx in mice

Purpose: The purpose of this study was to investigate the production of IL-27 p28 and EBI3 in the ocular inflammatory sites, and the role of IL-27 signaling in a model of HSV-1 induced herpetic stromal keratitis (HSK). Methods: The BALB/c mice were injected intraperitoneally (24 h before infection) with anti-IL-27 antibody or IgG antibody as control, infected with HSV-1 via corneal scarification, and then injected intraperitoneally with anti-IL-27 antibody or IgG antibody at 1, 3, and 5 days postinfection. Slit lamp and histopathology were used to assess disease outcome. The levels of IL-27 p28 and EBI3 in corneas were determined by western blotting and immunofluorescence. Furthermore, viral titers were determined, and immune cell infiltrates were collected and analyzed by flow cytometry. Results: We found that the levels of IL-27 p28 and EBI3 in corneas were elevated significantly at the peak of HSK, and both of them were expressed simultaneously in the epithelium, stroma, and endothelium of corneas. In the group of anti-IL-27 treatment, the severity of the corneal lesion and CD4+ T cells infiltration were significantly decreased, and the percentage of CD4+ Foxp3+ Tregs was upregulated markedly in the spleen, DLNs and cornea of HSK mice compared to IgG treatment. Conclusion: These results provided evidence that IL-27 as a pathogenic pro-inflammatory cytokine controlled CD4+ Foxp3+ Tregs production in HSK, which ultimately resulted in promoting the progression of HSK and poor prognosis.

Regulatory T cells (CD4+CD25+FOXP3+) in lupus nephritis

Background: Systemic lupus erythromatosus (SLE) is an autoimmune disease with 20–65% of patients developing lupus nephritis (LN). Studies have reported 10% of LN patients will end up with end stage renal disease and their mortality rate is higher compared to patients without LN. Abnormality of regulatory T cells (Tregs) level is thought to be a potential factor for this LN development. The aim of study was to evaluate the percentage of Tregs in LN patients.Methods: This was a comparative cross sectional study involving LN patients and age and gender matched controls with a 2:1 ratio. The patients were grouped into active and inactive LN based on their lupus activity index; complement levels, ANA, dsDNA antibodies, ESR, SLE Disease Activity Index (SLEDAI2K) score and also urine PCI (uPCI>0.05 for active group). Disease history, demographic data, routine blood test, peripheral blood for differentials count were taken and recorded. Peripheral blood mononuclear cells were stained with CD4, CD25 and Foxp3 antibodies and percentage of Tregs was analysed using BD fluorescence-activated cell sorting (FACS) cytometer. We compared demographic and laboratory parameters between healthy controls and LN patients as well as active and inactive LN patients.Results: A total of 34 LN patients (32 females, 2 males) were recruited. Their mean age and disease duration were 37.97±11.14 years and 110.95±65.07 months respectively.  Thirteen matched controls with mean age 35.23±7.89 years were enrolled. There was no demographic difference between 2 groups of LN patients. Tregs were significantly lower in active LN compared to inactive LN and healthy control (0.44±0.37% vs. 1.89±0.46% vs. 3.12±0.56% of the CD4+, P<0.001). C3 and C4 complement fragments were significantly reduced in patients with active disease (C3; 50.92±28.43 vs. 76.31±25.63, P=0.011) and (C4; 11.17±8.41 vs. 16.70±6.50 P=0.044). Proteinuria was significantly higher while serum albumin levels were significantly lower in active patients compared to inactive patients and healthy control (urine PCI; 0.25(0.15-0.3) vs. 0.03(0.01-0.05) vs. 0.01, P<0.001) and (albumin; 29.89±6.87 vs. 36.87±3.58 vs. 40.62±1.89mmol/L, P<0.001). We found positive inversely correlation between Tregs with SLEDAI2K (r = -0.572, P=0.011) and proteinuria (r = -0.451, P=0.007).Conclusions: Tregs, C3 and C4 complements, and albumin were significantly lower while proteinuria was significantly higher in active LN. There was positive inversely correlation between the percentage of Tregs with SLEDAI2K score and proteinuria.

Altered Tregs and oxidative stress in pregnancy associated lupus

Abstract Aim SLE is a systemic autoimmune disease generally affecting woman in the reproductive age. It is associated with an altered level of Tregs and oxidative stress while an increase in Tregs, and different antioxidant mechanisms to combat oxidative stress are essential for successful pregnancy. Hence, this study aims to determine the level of CD4+ and CD8+ Tregs and oxidative stress in pregnant lupus patients. Methods Ten healthy and 10 pregnant lupus volunteers from the North Indian population, within the age group of 20-30 years were enrolled in the study. All the patients were non-smokers, non-alcoholics and were not associated or undergoing therapy for any other disease. They had a SLEDAI of 37.4 ± 7.32 with 5.2 ± 1.93 years of disease duration. Oxidative stress was determined by measuring the enzyme activity of anti-oxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) and the level of reduced glutathione and lipids peroxidised, spectrophotometrically. Flowcytometry was performed for immunophenotyping to determine CD8+ and CD4+ Tregs. Results Elevated CD8+ Tregs and diminished CD4+ Tregs were observed in pregnant lupus patients. Oxidative stress was significantly increased as the activities of anti-oxidant enzymes and level of reduced glutathione was considerably diminished. There was a substantial increase in the amount of lipids peroxidised. Conclusion Pregnant lupus patients undergo considerable level of oxidative stress in comparison to healthy pregnant woman. The decreased level of CD4+ Tregs and an increase in CD8+ Tregs might be another important factor responsible for pregnancy associated complications. Hence, lupus leads to alterations in the necessary conditions for a successful pregnancy, which might eventually cause higher mortality, morbidity and associated complications.

Effect of RNA interfering Kvl. 3 channel expression on regulatory T lymphocytes in rats / 中国药理学通报

To investigate the changes in activation and proliferation of Tregs after targeted RNA inter-ference of Kvl. 3 channel genes and in vitro administration of eplerenone (EPL). Methods After lenti-virus vector was transfected to regulatory T cells (Tregs) of rats, qPCR and whole-cell patch-clamp methods were used to detect gene knockout efficiency, and EOSA method was used to detect cytokine secretion IL-10 and TGF-p levels of Tregs group,Tregs + EPL group,RNAi-Tregs group,and RNAi-Tregs + EPL group. Results Lentivirus vector was successfully transfected into Tregs cells, and the mRNA level and current density of Kvl. 3 channel was 78% and 71.3% respectively; compared with Tregs group, extracellular and intracellular TGF-p levels in RNAi-Tregs group were significantly reduced (P < 0. 01), and extracellular and intracellular TGF-(3 levels in Tregs + EPL group were also reduced (P < 0. 05 ) ; compared with RNAi-Tregs group, extracellular and in-tracellular TGF-fJ levels in RNAi-Tregs + EPL group showed no change. However, IL-10 levels in Tregs group,Tregs + EPL group, RNAi-Tregs group, RNAi-Tregs + EPL group showed no significant change. Conclusions Kvl.3 channel mediates the activation and proliferation of Tregs cells, while EPL can reduce the activation and proliferation of Tregs cells by directly inhibiting Kvl.3 channel and reducing the secretion of TGF-fi levels, further indicating that EPL is a specific blocker of Kvl. 3 channel.

The research progress of soluble fibrinogen-like protein 2 / 国际检验医学杂志

Soluble fibrinogen-like protein 2 (sFGL2) , is an novel effector secreted by the CD4+CD25+CD127lowTregs cells and plays an important role in immunoregulation.It is critical for the maintenance of the activity and function of Tregs.It can induce the apoptosis of B cells, suppress the maturation of dendritic cells and suppress the activation and proliferation of T cells through the FcγRⅡB receptor.This review concluded the recent progress of the genes, structure, immunoregulatory effects of sFGL2, and discussed the clinical implications of sFGL2in hepatitis, autoimmunity, transplantation, tumors and atherosclerosis.

Polymorphisms in the TGFB1 and IL2RA genes are associated with clinical forms of leprosy in Brazilian population

BACKGROUND Leprosy is a chronic infectious disease caused by Mycobacterium leprae, and compromises the skin and peripheral nerves. This disease has been classified as multibacillary (MB) or paucibacillary (PB) depending on the host immune response. Genetic epidemiology studies in leprosy have shown the influence of human genetic components on the disease outcomes. OBJECTIVES We conducted an association study for IL2RA and TGFB1 genes with clinical forms of leprosy based on two case-control samples. These genes encode important molecules for the immunosuppressive activity of Treg cells and present differential expressions according to the clinical forms of leprosy. Furthermore, IL2RA is a positional candidate gene because it is located near the 10p13 chromosome region, presenting a linkage peak for PB leprosy. METHODS A total of 885 leprosy cases were included in the study; 406 cases from Rondonópolis County (start population), a hyperendemic region for leprosy in Brazil, and 479 cases from São Paulo state (replication population), which has lower epidemiological indexes for the disease. We tested 11 polymorphisms in the IL2RA gene and the missense variant rs1800470 in the TGFB1 gene. FINDINGS The AA genotype of rs2386841 in IL2RA was associated with the PB form in the start population. The AA genotype of rs1800470 in TGFB1 was associated with the MB form in the start population, and this association was confirmed for the replication population. MAIN CONCLUSIONS We demonstrated, for the first time, an association data with the PB form for a gene located on chromosome 10. In addition, we reported the association of TGFB1 gene with the MB form. Our results place these genes as candidates for validation and replication studies in leprosy polarisation.

Novel heterozygous mutation of the IL2RA gene in one Chinese girl with immunodeficiency 41 with lymphoproliferation and autoimmunity and diabetes mellitus, insulin-dependent 10: One case report and literature review / 中华内分泌代谢杂志

Objective To report a case of immunodeficiency 41 with lymphoproliferation and autoimmunity (IMD41) and type 10 insulin-dependent diabetes mellitus(IDDM10), caused by mutations of the interleukin 2 receptor α(IL2RA)gene.Methods Clinical symptoms were colleted,while IL2RA gene was sequenced.Results Here we reported a girl of 7 years and 6 months old who came to our hospital presented with lymphadenovarix for 5 years,debilitation for 2 months and alternation of hyperglycemia and hypoglycemia for 20 days. She was subsequently diagnosed with fungal pneumonia and ANCA-associated vasculitis. All exons of IL2RA gene were sequenced. c.340C>T(p.Q114X,paternal,novel mutation),c.64G>A(p.E22X,maternal) were detected. After treatments of dihydrocortisone,voriconazole combined with diabetic diet plus raw cornstarch, the pulmonary lesions reduced, autoantibodies disappeared and the blood glucose returned to normal. Literature review suggested that totally 5 IL2RA gene mutation patients were reported, the major clinical features were recurrent infection(infection of lung, skin, gastrointestinal tract) and immune abnormalities ( such as lymph node disease, autoimmune disease, hepatosplenomegaly,and diabetes mellitus). Conclusion In cases of atypical clinical symptoms, whole exon sequencing helps early diagnosis.

Psoralen inhibits RAW264.7 differentiation into osteoclasts and bone resorption by regulating CD4+T cell differentiation / 中国中药杂志

This paper aimed to investigate whether psoralen inhibits the differentiation and bone resorption by regulating CD4+T cell differentiation in RANKL-induced osteoclastogenesis in RAW264.7 cells, and elucidate its mechanism for osteoporosis. CD4+T cells were isolated from spleen cells of Balb/c mice by immunomagnetic separation method. The cells were divided into blank control group and psoralen group. The cells were cultured in 24-well plates and cultured for 3 days, and then they were collected for co-culture experiments after 4 days. Co-culture experiments were divided into RAW264.7 cell group, psoralen+RAW264.7 cell group, without psoralen treatment of CD4+T cells+RAW264.7 cell group, psoralen treatment of CD4+T cells+RAW264.7 cell group. After 5 days of co-culture, TRAP staining was used to detect the number of osteoclasts, and after 8 days of co-culture, bone resorption was evaluated by toluidine blue staining. The expressions of RORγt, Foxp3, IL-17, TNF-α, TGF-β and IL-10 in CD4+T cells and osteoclast differentiation-related genes MMP-9, TRAP and Cat-K were detected by Real-time polymerase chain reaction (RT-PCR); ELISA kit was used to detect IL-17, TNF-α, TGF-β and IL-10 and other cytokines levels. Our data confirmed that the psoralen significantly promoted the expression of Foxp3, TGF-β and IL-10 in CD4+T, and inhibited the expression of RORγt, IL-17 and TNF-α in CD4+T, the CD4+T cells without treatment by psoralen can significantly promote RANKL-induced differentiation of RAW264.7 to osteoclasts, and psoralen treatment of CD4+T can significantly inhibit RANKL-induced RAW264.7 osteoclast differentiation and bone resorption. Taken together, psoralen inhibits the differentiation and bone resorption of RAW264.7 into osteoclasts by promoting the development of CD4+ CD25+ Treg/Th17 balance in CD4+T cells to CD4+CD25+T.

Overexpression of indoleamine 2,3-dioxygenase correlates with regulatory T cell phenotype in acute myeloid leukemia patients with normal karyotype

BACKGROUND: Production of immunosuppressive enzymes such as indoleamine 2,3-dioxygenase (IDO) is one of the strategies employed by hematologic malignancies, including acute myeloid leukemia (AML), to circumvent immune surveillance. Moreover, IDO has the ability to convert CD4+CD25− conventional T cells into regulatory T cells (Tregs). In this study, we evaluated the expression of IDO in cytogenetically normal acute myeloid leukemia (CN-AML) patients and its correlation with the Treg marker, FOXP3, as well as clinical and laboratory parameters. METHODS: Thirty-seven newly diagnosed CN-AML patients were enrolled in our study along with 22 healthy individuals. The expression of the IDO and FOXP3 genes was analyzed by SYBR Green real-time PCR. RESULTS: Both IDO and FOXP3 were highly upregulated in CN-AML patients compared to control groups (P=0.004 and P=0.031, respectively). A positive correlation was observed between IDO and FOXP3 expression among AML patients (r=0.512, P=0.001). Expression of IDO and FOXP3 showed no significant correlation with laboratory parameters such as white blood cell and platelet counts, hemoglobin levels, bone marrow blast percentage, gender, and FLT3 mutation status (P>0.05). CONCLUSION: Higher IDO expression in CN-AML patients may be associated with an increased Treg phenotype which may promote disease progression and lead to poor prognosis of CN-AML patients.

The role of regulatory T cells, interleukin-10 and in vivo scintigraphy in autoimmune and idiopathic diseases - Therapeutic perspectives and prognosis / O papel de células T regulatórias, da interleucina 10 e da cintilografica in vivo em doenças autoimunes e idiopáticas - Perspectivas terapêuticas e prognóstico

Summary Previous studies have demonstrated the expression of the CD25 marker on the surface of naturally occurring T cells (Tregs) of mice, which have a self-reactive cellular profile. Recently, expression of other markers that aid in the identification of these cells has been detected in lymphocyte subtypes of individuals suffering of autoimmune and idiopathic diseases, including: CD25, CTLA-4 (cytotoxic T-lymphocyte antigen 4), HLA-DR (human leukocyte antigen) and Interleukin 10 (IL-10), opening new perspectives for a better understanding of an association between such receptors present on the cell surface and the prognosis of autoimmune diseases. The role of these molecules has already been described in the literature for the modulation of the inflammatory response in infectious and parasitic diseases. Thus, the function, phenotype and frequency of expression of the a-chain receptor of IL-2 (CD25) and IL-10 in lymphocyte subtypes were investigated. Murine models have been used to demonstrate a possible correlation between the expression of the CD25 marker (on the surface of CD4 lymphocytes) and the control of self-tolerance mechanisms. These studies provided support for the presentation of a review of the role of cells expressing IL-2, IL-10, HLA-DR and CTLA-4 receptors in the monitoring of immunosuppression in diseases classified as autoimmune, providing perspectives for understanding peripheral regulation mechanisms and the pathophysiology of these diseases in humans. In addition, a therapeutic approach based on the manipulation of the phenotype of these cells and ways of scintigraphically monitoring the manifestations of these diseases by labeling their receptors is discussed as a perspective. In this paper, we have included the description of experiments in ex vivo regulation of IL-10 and synthesis of thio-sugars and poly-sugars to produce radiopharmaceuticals for monitoring inflammation. These experiments may yield benefits for the treatment and prognosis of autoimmune diseases.

Resumo Estudos anteriores já haviam demonstrado a expressão do marcador CD25 na superfície de células T de ocorrência natural (Tregs) de camundongos, que apresentam perfil celular autorreativo. Recentemente, foi detectada, em subtipos de linfócitos de indivíduos acometidos por doenças autoimunes e de causa idiopática, a expressão de outros marcadores, que auxiliam na identificação dessas células, entre os quais: CD25, CTLA-4 (cytotoxic T-lymphocyte antigen 4), HLA-DR (human leucocyte antigen) e Interleucina 10 (IL-10), abrindo novas perspectivas para a melhor compreensão de uma associação entre esses receptores presentes na superfície celular e o prognóstico de doenças autoimunes. O papel dessas moléculas já havia sido descrito na literatura na modulação da resposta inflamatória em doenças infectoparasitárias. Dessa forma, foram investigados a função, o fenótipo e a frequência de expressão, do receptor de cadeia a da IL-2 (CD25) e de IL-10 em subtipos de linfócitos. O modelo murino tem sido utilizado para demonstrar uma possível correlação entre a expressão do marcador CD25 (na superfície de linfócitos CD4) e o controle dos mecanismos de autotolerância. Essas pesquisas forneceram suporte para apresentação de uma revisão sobre o papel das células que expressam os receptores de IL-2, IL-10, HLA-DR e CTLA-4 no monitoramento da imunossupressão, em doenças de classificação autoimune, abrindo perspectivas para o entendimento dos mecanismos de regulação periférica e sobre a fisiopatologia dessas doenças no ser humano. Além disso, é discutida como perspectiva uma abordagem terapêutica fundamentada na manipulação do fenótipo dessas células, bem como de modos de monitoramento cintilográfico das manifestações dessas doenças, por meio da marcação de seus receptores. Nestes, foram incluídas descrições das experiências em regulação ex-vivo de IL-10; de síntese de tioaçúcares e de poliaçúcares para produção de radiofármacos para monitoramento de inflamações. Essas experiências podem trazer benefícios na terapia e no prognóstico de doenças autoimunes.

Antagonization of eplerenone on proliferation of cardiac fibroblasts induced by co-cultured Treg cells based on inhibition of Kv1.3 channel of Tregs / 中国药理学通报

Aim To establish a co-incubation system in cardiac fibroblasts of SD neonatal rats and spleen CD4+ CD25 + regulatory T lymphocytes (Tregs) of normal adult SD rats,and to investigate the effects of eplerenone(EPL) on the interaction of two cells and the relationship with the Kvl.3 channel on Tregs cell membrane.Methods The spleen Tregs of normal adult SD rats were sorted by immunomagnetic bead sorting,and the myocardial fibroblasts of SD rats were isolated by differential adherence method.The experiment was conducted in the following groups:CFs,CFs + Tregs,CFs + Tregs + EPL,Tregs.The proliferation of CFs was detected by CCK-8 method.The expression levels of type Ⅰ collagen,type m collagen and matrix metalloproteinase 2 (MMP-2) secreted by CFs were detected by ELISA.The mRNA expression levels of Kv1.3,KCa3.1 on Tregs cell membrane and intracellular CRAC channel were detected by RT-qPCR technique.Tregs cell membrane Kvl.3 channel protein expression levels were determined by In-Cell Western blot.Results After 48 h incubation of the co-culture system,the cell proliferation was stable.CFs proliferation was marked(P ＜0.01),which could be inhibited by EPL(P ＜0.01).The type Ⅰ,type Ⅲ collagen and MMP-2 secreted by CFs increased (P ＜ 0.01).The expression levels of Kv1.3,KCa3.1 and CRAC channel mRNA in Tregs increased by 6.95,1.99 and 1.53 fold (CFs + Tregs vs Tregs,P ＜0.01),EPL decreased the mRNA level of each channel (CFs +Tregs + EPLvs CFs + Tregs,P＜0.01),and the decrease of Kv1.3 channel was the most significant (P ＜ 0.01).The Kv1.3 channel protein of Tregs increased by 67.9％ (CFs + Tregs vs Tregs,P ＜0.01),which could be inhibited by EPL(P ＜ 0.01).Conclusions Tregs cultured with CFs after 48 h can significantly promote the proliferation of CFs,and EPL can down-regulate the Kv1.3 channel expression on the Tregs membrane and inhibit the activation/proliferation of Tregs,indirectly inhibiting myocardial fibrosis.

Change and Clinical Significance of Peripheral Blood DN Tregs in Patients with Systemic Lupus Erythematosus / 现代检验医学杂志

Objective To study the expression and function of TCRαβ+ CD3+ CD4-CD8-T (Double negative regulatory T cells,DN Tregs) in peripheral blood of Systemic lupus erythematosus (SLE) patients,and investigate their function in pathogenesis of SLE.Methods TCRαβ+ CD4-CD8 T cells were quantified as percentage of total CD3+ T cells in peripheral blood from 20 SLE patients and 20 healthy controls by flow cytometry.Used ELISA to test the cytokine levels (IFN-γ,TNF-α,IL-6,IL-17A) in the plasma of SLE patients.And analyzed the relationship between the percentage of DN Tregs and cytokines levels.Results The ratio of DN Tregs in TCRαβ+ T cells was significantly increased in SLE patients compared to healthy donors (t=3.54,P＜0.01).The levels of IFN-γ,TNF-α and IL-6 in plasma of SLE patients were higher than healthy donors (t=2.824,2.085,2.304,P＜0.05).The frequency of DN Tregs was found to correlate with IFN-γ (r=0.52,P=0.02) but not TNF-α (r=0.17,P=0.16) and IL-6 (r=0.47,P=0.49).Conclusion This study reveals frequency of DN Tregs in peripheral blood of SLE patients was higher than healthy controls,the frequency of DN Tregs was also found to correlate with IFN-γ levels,which means that DN Tregs may play an important role in pathogenesis of SLE.

Spectrum drift characteristics of CD4+ CD25+ Tregs TCR CDR3 in hepatitis B patients before and after treatment / 中国免疫学杂志

Objective:To determine the spectrum drift characteristics of CI4+CD25+Tregs TCR β chain CDR3 in patients with different phases of acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients before and after the entecavir treatment.Methods:Anticoagulation venous blood was collected from 4 normal control subjects,3 AHB patients with acute phase and convalescent phase,and 4 CHB patients before and after the entecavir treatment;and peripheral blood mononuclear cells were isolated;CD4+ CD25+ Tregs were separated by using the magnetic beads,and total RNAs were extracted from CD4+ CD25+ Tregs and used for reverse transcription.The TRBV CDR3 was amplified by polymerase chain reaction (PCR) with forward primers specific for 24 TRBV families and one fluorescence-labeled common reverse primer specific for the BC region.The PCR products were sent out for Genescan,and results were analyzed for the TRBV family CDR3 spectrum characteristics by using the Peak Scanner Software vl.0.Data were analyzed with the comparative t-test to perform the statistical analysis.Results:The CDR3 spectral types of the TRBV family showed drift characteristics in 3 cases of AHB patients with acute and convalescent phases;single/oligo peak spectral type family was observed in most of patients with acute phase;multiple peak spectral type was seen in patients with convalescent phase;and the common spectrum shift of TRBV4,10,14,16,19 families seen in patients with acute phase was changed to multiple peak spectral type.The clonal expansion of TRBV family in the CD4+CD25+Tregs in PBMC from AHB patients with convalescent phase was significantly lower than AHB patients with acute phase (t =9.456,P =0.011).The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in C HB patients before treatment may interfere the virus removal through down-regulating the body's immune response;and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1,5.2,6,12,14,24 family may help body induce immune tolerance and result in the HBV persistence.The clonal expansion of TRBV family in the CI4+CD25+Tregs in PBMC from of CHB patients after antiviral treatment was increased (t =-0.666,P =0.553).Conclusion:TRBV4,10,14,16,19 family of spectrum shift seen in AHB patients with acute phase was changed to multiple peak spectral type in patients with convalescent phase,suggesting this transition may be associated with HBsAg and HBeAg turning to negative.The clonal expansion of Tregs TRBV13.2,15,16,18,20 family seen in CHB patients before treatment may interfere the virus removal through down-regulating the body's immune response;and with the decline of viral load in serum after the antiviral treatment,the clonal expansion of Tregs TRBV1,5.2,6,12,14,24 family may help body induce immune tolerance and result in the HBV persistence.

Effect of Compound Danshen Dropping Pills combined with simvastatin treatment on level of regulatory T cells in patients with carotid atherosclerotic cerebral infarction / 中草药

Objective: To study the correlation of regulatory T cells (Tregs) and carotid atherosclerosis (CAS) in patients with acute cerebral infarction, and the effects of Compound Danshen Dropping Pills and simvastatin on Tregs level in CAS patients. Methods: Flow cytometry (FCM) was used to detect the percentage of Tregs in the peripheral blood of treatment group and control group before and after treatment. Plasma levels of interleukin 10 (IL-10), and transforming growth factor β1 (TGF-β1) were measured by enzyme linked immunosorbent assay (ELISA) method to detect IL-10 and TGF-β1 of peripheral blood plasma. Results: With CAS aggravation, percentage of Tregs, IL-10, and TGF-β1 levels reduced. After two weeks of treatment, the levels of Tregs, IL-10, and TGF-β1 were higher than those before treatment. In severe CAS patients, Tregs and IL-10 increased significantly in treatment group than those in control group. Conclusion: The proportion of Tregs in peripheral blood CD4+T cells can indirectly reflect the degree of CAS lesions. The combination of simvastatin and Compound Danshen Dropping Pills has lipid-regulation, anti-inflammatory, and anti-oxidant effect, and also up-regulates the levels of Tregs and IL-10, which can achieve the role of anti-atherosclerosis.

Role of Regulatory Cells in Oral Tolerance

The immune system is continuously exposed to great amounts of different antigens from both food and intestinal microbes. Immune tolerance to these antigens is very important for intestinal and systemic immune homeostasis. Oral tolerance is a specific type of peripheral tolerance induced by exposure to antigen via the oral route. Investigations on the role of intestinal immune system in preventing hypersensitivity reactions to innocuous dietary and microbial antigens have been intensively performed during the last 2 decades. In this review article, we discuss how food allergens are recognized by the intestinal immune system and draw attention to the role of regulatory T (Treg) and B (Breg) cells in the establishment of oral tolerance and tolerogenic features of intestinal dendritic cells. We also emphasize the potential role of tonsils in oral tolerance induction because of their anatomical location, cellular composition, and possible usage to develop novel ways of specific immunotherapy for the treatment of allergic diseases.

High-dose heat shock protein gp96 immunization prevents type 1 diabetes via inducing regulatory T cells / 生物工程学报

Type 1 diabetes (T1D), the most prevalent human autoimmune disease, occurs in genetically susceptible individuals. Regulatory T cells (Tregs) are defective in T1D setting. Therefore, efforts to repair or restore Tregs in T1D may prevent or reverse this autoimmune disease. Here, we studied the potential role of rgp96 in preventing T1D, using non-obese diabetic (NOD) mice as an animal model. High-dose rgp96 immunization elicited efficient protection of mice against T1D, as evidenced by stable blood glucose, decreased disease incidence. Significantly increased CD4⁺ CD25⁺ Foxp3⁺ Tregs were observed in immunized mice. In vitro co-culture experiments demonstrated that rgp96 stimulation enhanced Treg proliferation and suppressive function by up-regulation of Foxp3 and IL-10. Our work shows that activation of Tregs by high-dose rgp96 immunization protects against T1D via inducing regulatory T cells and provides preventive and therapeutic potential for the development of an rgp96-based vaccine against T1D.

Correlation of peripheral blood bilirubin level with Tregs in patients with bile duct stones / 中华肝胆外科杂志

Objective To study and correlate serum bilirubin and regulatory T cell (Treg) levels in patients with bile duct stone.Methods Flow-cytometry and Enzyme-linked immunosorbent assay (Elisa) were used to study the peripheral blood expression level of Tregs and the bilirubin level in 27 patients with bile duct stones and jaundice.The changes in the expression level of Tregs and the bilirubin level were studied and correlated before and after treatment.Results After treatment,both the peripheral blood bilirubin level,the Tregs expression level and the cell cytokines decreased significantly.The total bilirubin level decreased from (102.8 ± 33.1) mmol/L to (15.3 ± 5.7) mmol/L (P ＜ 0.05),the direct bilirubin level decreased from (38.1 ± 12.8) mmol/L to (5.0 ± 1.6) mmol/L (P ＜0.05);the percentages of CD4+ CD25 +Foxp3 + T cells in CD4+ T decreased from (4.2 ± 2.0) ％ to (2.4 ± 1.0) ％ (P ＜ 0.05).Before treatment,the levels of IL-10 and TGF-β were 171.4 ± 13.7 and 2016 ±657 pg/ml but after treatment,the two cytokines decreased to 92.1 ± 7.4 and 1 686 ± 168 pg/ml,respectively (P ＜ 0.05).Conclusions Patients with bile duct stones and jaundice presented with high expressions of bilirubin and Tregs level.These expressions returned to normal after effective treatment.The Tregs expression level was positively correlated with the bilirubin level.