Mean Fluorescence Intensity Of Foxp3 Research Articles

ADENOSINE INFLUENCES FOXP3 EXPRESSION OF T REGS VIA THE A2AR/CREB PATHWAY IN A MOUSE MODEL OF SEPSIS.

The adenosine concentration and forkhead box protein (Foxp3) expression in T regulatory cells (T regs ) are increased during sepsis. However, the mechanism by which adenosine induces Foxp3 expression is incompletely understood. A cecal ligation and puncture (CLP) model was constructed using C57BL/J mice. The plasma adenosine concentration and Foxp3 expression in splenic T regs were increased consistently for 15 days after sepsis onset. Analysis of the mean fluorescence intensity of Foxp3 and adenosine concentration in the same mice revealed a linear correlation. In the CLP model, adenosine 2a receptor (A2aR) blockade inhibited Foxp3 expression in T regs . In vitro activation of A2aR promoted Foxp3 expression in T regs and facilitated secretion of extracellular vesicles. Transcriptome sequencing revealed that A2aR blockade led to changes in cyclic adenosine monophosphate response element-binding protein (CREB) transcription in T regs in our sepsis model. Use of adenosine or A2aR agonists promoted CREB expression, CREB phosphorylation at S133, T reg expression of Foxp3, and enhanced inhibition of proliferation of cluster of differentiation (CD)4+ lymphocytes. A2aR blockade or inhibition of CREB expression inhibited Foxp3 expression in T regs . In the CLP model, use of CREB inhibitors could inhibit Foxp3 expression and reduce the bacterial load. In summary, adenosine in sepsis promotes CREB phosphorylation via A2aR which, in turn, upregulates Foxp3 expression in T regs .

Increased circulating monocyte MDSCs positively correlate with serum Interleukin-10 in metastatic melanoma patients.

Numerous immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and inhibitory cytokines identified in melanoma microenvironment have the important role in immune escape. Therefore, in this study we analyzed monocytic (m)MDSCs in peripheral blood of metastatic melanoma (MM) patients. In peripheral blood of 35 MM patients and 30 healthy controls we analyzed percentage of CD14 + HLA-DR- mMDSCs in monocyte gate and the mean fluorescence intensity of Foxp3 in CD25 + CD4 + regulatory T cells by Flow cytometry. Serum levels of transforming growth factor beta, interferon-gamma, interleukin (IL)-6, IL-8, IL-10 are measured by ELISA assays. In this study MM patients have significantly higher percentage of CD14 + HLA-DR- mMDSCs, as well as increased the baseline mMDSC/PBMC subset (NK, T, B cells, monocytes) ratio. Although there is no significant difference in the percentage of mMDSCs between groups of MM patients with different localization of distant metastasis, patients with elevated serum lactate dehydrogenase (LDH) have statistically significant higher percentage of these cells compared to LDH negative patients. Furthermore, in MM patients there is statistically significant positive correlation between values of IL-10 and the percentage of mMDSCs, only. Therefore, therapeutics that target circulating mMDSCs and IL-10 may have a big importance in the improvement of antitumor immunity in MM patients.

The effects of miR-10a on the immune function of splenic CD4 + CD25 + Treg cells in septic mice

Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis. Methods Sepsis mouse model was established by cecal ligation and puncture(CLP). Balb/c mice of clean grade were sacrificed 1, 3, 5, and 7 days after operation. Blood as well as spleen samples were harvested at given intervals. The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads. Cells were cultured, and phenotypes were analyzed by flow cytometry. The miR-10a expressed in Treg cells were detected by Real-time PCR. After administration of LV-mmu-miR-10a-5p-inhibition, the immunosuppressive function have been detected. Statistical analyses were performed using one-way analysis of variance (SPSS19.0, Chicago, USA) test followed by Dunnett-t test to compare among three or more groups or by Student’s t-test to compare between two groups. Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)% in normal group, and the increase in percentage of Tregs in spleen has been observed in septic mice (P<0.05). The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P<0.05). The expression of miR-10a was significantly elevated on CLP 1-7 day (P<0.05). After down-regulation of miR-10a in septic mice, the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P<0.05), the MFI of Foxp3+Treg was increased in septic mice compared with control group (P<0.05). The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P<0.05).After down-regulation of miR-10a in septic mice, the CD4+T cell proliferative activity was significantly suppressed compared with control group (P<0.05). Conclusions Treg plays a critical role in immunosuppression in septic mice. Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg. Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy . Key words: Regulatory T cells; Sepsis; MicroRNA-10a; Forkhead/winged helix transcription factor p3; Immune function; Effector T cell; Proliferative activity; Mean fluorescence intensity

Functional defects in CD4+ CD25high FoxP3+ regulatory cells in ankylosing spondylitis.

Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) play a pivotal role in the preservation of self-tolerance, and Treg dysfunction has been implicated in many autoimmune diseases. Whether and how Tregs participate in the pathogenesis of ankylosing spondylitis (AS) has not been fully elucidated. Here, we investigated Treg function and found that Tregs in peripheral blood (PB) from patients with active AS had lower FoxP3 mean fluorescence intensity (MFI) than those from healthy controls and could not fully suppress naïve T cell (Tn) proliferation. We also studied the mechanisms underlying PB Treg dysfunction in this context and found that PB Tregs failed to effectively utilize IL-2 and had relatively little STAT5 phosphorylation in active AS. Moreover, PB Tregs from patients with active AS exhibited greater CpG island methylation in the CNS2 region of the FOXP3 gene. Therefore, our findings indicate that functional defects in Tregs are present in AS. Abnormal IL-2 signalling and aberrant CNS2 epigenetic control induced functional defects in PB Tregs and represents a potential new mechanism for AS pathogenesis. These findings may aid the design of new treatment approaches for AS.

Epstein–Barr virus and regulatory T cells in Egyptian paediatric patients with acute B lymphoblastic leukaemia

ObjectiveAcute B lymphoblastic leukaemia (B-ALL) is the most common type of childhood malignancy worldwide but little is known of its origin. Recently, many studies showed both a high incidence of...

Intrauterine growth restriction and prematurity influence regulatory T cell development in newborns

PurposeThe aim of this study was to determine the relationship of birth weight and gestational age with regulatory T cells (Tregs) in cord blood of human newborns. MethodsCord blood mononuclear cells (CBMCs) of 210 newborns were analyzed using flow cytometry to identify Tregs (CD3+, CD4+, CD25high, FoxP3high) and measure FoxP3 mean fluorescence intensity (MFI). Suppressive index (SI) was calculated as FoxP3 MFI per Treg. ResultsMode of delivery had no significant effect on Tregs at birth. Term babies with growth restriction had fewer Tregs than their appropriate weight counterparts but equivalent SI. Preterm babies had higher percentages of Tregs, but lower SI than term controls. SI steadily increased through gestation. ConclusionsIntrauterine growth restriction is correlated with fewer circulating Tregs and prematurity with decreased functionality of Tregs compared to term appropriate weight infants. This may have implications in diseases such as necrotizing enterocolitis that disproportionately affect premature and lower birth weight infants.

Bifidobacterium breve and Lactobacillus rhamnosus treatment is as effective as budesonide at reducing inflammation in a murine model for chronic asthma.

BackgroundAsthma is estimated to affect as many as 300 million people worldwide and its incidence and prevalence are rapidly increasing throughout the world, especially in children and within developing countries. Recently, there has been a growing interest in the use of potentially beneficial bacteria for allergic diseases. This study is aimed at exploring the therapeutic effects of long-term treatment with two different beneficial bacterial strains (Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1) and a glucocorticoid (budesonide), as a reference treatment, on inflammatory response in a murine model for chronic allergic asthma.MethodsTo mimic the chronic disease in asthmatic patients, we used the murine ovalbumin-induced asthma model combined with prolonged allergen exposure. Airway function; pulmonary airway inflammation; airway remodelling, mRNA expression of pattern recognition receptors, Th-specific cytokines and transcription factors in lung tissue; mast cell degranulation; in vitro T cell activation; and expression of Foxp3 in blood Th cells were examined.ResultsLactobacillus rhamnosus reduced lung resistance to a similar extent as budesonide treatment in chronically asthmatic mice. Pulmonary airway inflammation, mast cell degranulation, T cell activation and airway remodelling were suppressed by all treatments. Beneficial bacteria and budesonide differentially modulated the expression of toll-like receptors (TLRs), nod-like receptors (NLRs), cytokines and T cell transcription factors. Bifidobacterium breve induced regulatory T cell responses in the airways by increasing Il10 and Foxp3 transcription in lung tissue as well as systemic by augmenting the mean fluorescence intensity of Foxp3 in blood CD4+ T cells.ConclusionThese findings show that Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1 have strong anti-inflammatory properties that are comparable to budesonide and therefore may be beneficial in the treatment of chronic asthma.

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

BackgroundLeprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4+/CD8+CD25highFOXP3+ and CD4+/CD8+CD25highFOXP3high cells in leprosy patients and household contacts, in both cases under 15 years old.Methodology/Principal FindingsPBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4+/CD8+ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4+FOXP3+ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4+FOXP3high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8+FOXP3+ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8+FOXP3high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.Conclusions/SignificanceWe have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4+ and CD8+ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.

Vitamin D as an adjunct to subcutaneous allergen immunotherapy in asthmatic children sensitized to house dust mite

We aimed to investigate the efficacy, safety, and T regulatory cell response of vitamin D as an adjunct to allergen-specific immunotherapy (IT). Fifty children with asthma and receiving pharmacotherapy were randomized into three groups as: subcutaneous IT (SCIT) along with vitamin D supplementation (650U/day; n: 17), SCIT alone (n: 15), and pharmacotherapy alone (n: 18). All patients were evaluated at baseline, 6th and 12th months for scorings of symptoms and medication, skin prick testing, total IgE, specific IgE, and Der p 1-specific IgG4. In addition, D.pteronyssinus-induced CD4(+) CD25(+) FOXP3(+) T regulatory cell percentage, intracellular Foxp3 expression, and peripheral blood mononuclear cell IL-10 and TGF-β responses were assessed. In the SCIT+vitamin D and SCIT alone groups, total asthma symptom score (TASS), total symptom score (TSS), and total medication scores (TMS) were significantly lower than pharmacotherapy group at the end of 1year. While the comparison of delta values (Δ 6th and Δ 12th month-baseline) of those scores revealed no significant differences between the two IT groups, TASS at the 6th month was lower in the SCIT+vitamin D group compared with others. There was a significant and positive trend in the levels of Der p 1-specific IgG4 in both IT groups throughout the study period. Whereas the levels of Der p 1-induced IL-10 and TGF-β were similar between IT groups, the mean fluorescence intensity of Foxp3 was highest in the SCIT+vitamin D group compared with others at the 12th month. The rate of discontinuation of inhaled corticosteroid (ICS) was 6/17 in SCIT+vitamin D, 3/15 in SCIT, and 0/18 in the pharmacotherapy group (P=0.02). Both SCIT groups fared better than pharmacotherapy alone at the end of 1year. Although the clinical and immunologic outcomes were mostly similar between the two IT groups, some favorable outcomes of vitamin D warrant further investigation in more selected populations with varying doses as adjunct to IT.

Changes of Treg-Associated Molecules on CD4+CD25+Treg Cells in Myasthenia Gravis and Effects of Immunosuppressants

Myasthenia gravis (MG) is a CD4(+) T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4(+)CD25(+)Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them. We adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs. The number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4(+)CD25(+)FOXP3(+)Helios(+)T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4(+)CD25(+)FOXP3(+)Helios(+)T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG. The significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.

P101 Regulatory T cells exhibit reduced phenotypic stability upon pro-inflammatory challenge in autoimmune hepatitis

IntroductionIn autoimmune hepatitis (AIH) CD4+CD25high regulatory T-cells (Tregs) are numerically defective and fail to suppress T-cell-mediated immune responses. Expression by Tregs of the ectoenzyme CD39 contributes to their ability to...

Skewed X-chromosomal inactivation impacts T regulatory cell function in systemic sclerosis

OBJECTIVES: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). METHODS: 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. RESULTS: Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012). CONCLUSIONS: Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

Skewed X chromosomal inactivation impacts T regulatory cell function in systemic sclerosis

ObjectivesTo investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs).Methods217 women with SSc...

Regulatory T cell dysfunction in subjects with common variable immunodeficiency complicated by autoimmune disease

Approximately 25% of subjects with common variable immunodeficiency (CVID) develop autoimmune disease. We analyzed T cell subsets, specifically regulatory T cells along with B cell subsets to determine whether there were changes in regulatory T cells which would correlate with the autoimmune disease clinical phenotype in CVID subjects. We hypothesized that regulatory T cell (CD4 + CD25hiCD127lo) suppressive function would be impaired in CVID subjects with autoimmune disease. Using purified, sorted Treg from CVID subjects ( n = 14) and from healthy controls (HC, n = 5) in standard suppression assays, we found the suppressive function of Treg from CVID subjects with autoimmune disease (CVID w/ AI, n = 8) to be significantly attenuated compared to CVID subjects with no autoimmune disease (CVID w/o AI, n = 6) and to HC ( n = 5). A number of proteins associated with Treg function were decreased in expression as detected through immunofluorescent antibody via flow cytometry (mean fluorescence intensity (MFI) of FoxP3, Granzyme A, XCL1, pSTAT5, and GITR in Treg was significantly lower (by up to 3 fold) in CVID w/ AI compared to CVID w/o AI and HC. Furthermore, a statistically significant correlation was found between intracellular MFI of FoxP3, Granzyme A, and pSTAT5 in Treg and the degree of Treg dysfunction. These results suggest that attenuation of Treg function is associated with autoimmune disease in CVID subjects and may contribute to autoimmune pathogenesis.