Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression

Accumulating evidence indicates an immunosuppressive role for CD4(+)CD25(+) regulatory T cells (Tregs) in autoimmune diseases. Although an impaired Treg function in patients with relapsing-remitting multiple sclerosis (RR-MS) has been reported recently, no information is available so far about Treg function in the progressive stage of the disease. In the present study, the phenotypic and functional characteristics of CD4(+)CD25(+) T cells isolated from the peripheral blood of patients with RR-MS and secondary progressive multiple sclerosis (SP-MS) were investigated. No significant quantitative or phenotypic abnormalities in CD4(+)CD25(+) T cells from RR- and SP-MS patients were detected. However, whereas a reduced suppressor function of CD4(+)CD25(+) T cells toward proliferation and interferon-gamma production of CD4(+)CD25(-) responder T cells was found in RR-MS patients, SP-MS patients showed a normal Treg function. The suppressive capacity of MS-derived CD4(+)CD25(+) T cells was correlated with disease duration but not with age, indicating that Treg function is more affected in the early phase of the disease process. Consistently with the suppressive capacity, CD4(+)CD25(+) T cells from SP-MS patients showed normal levels of FOXP3 mRNA in contrast to RR-MS patients that had a reduced FOXP3 expression. These data are the first to demonstrate differences in function and FOXP3 expression of CD4(+)CD25(+) T cells from patients with RR- and SP-MS.     

多发性硬化患者外周血CD4+CD25+ T细胞变化及其机制探讨

目的探讨多发性硬化(MS)患者外周血CD4 CD25 T细胞数量及叉头样转录因子(FOXP3)表达水平与MS病情的关系。方法选择温州地区MS患者44例(男12例、女32例),均按Poser诊断标准诊断,结合头颅MRI增强扫描排除合并其他神经系统和免疫系统疾病,并统一行EDSS评分;对照组43例(男13例、女30例)为健康查体者。具体方法:流式细胞仪检测外周血CD4 CD25 T细胞数量;免疫磁珠法分离CD4 CD25 T细胞;RT-PCR法检测CD4 CD25 T细胞FOXP3 mRNA表达并进行半定量分析。结果MS患者外周血中CD4 CD25 调节性T细胞数量与对照组比较无明显变化(P>0.05);活化的效应性T细胞数量增加(P<0.05)且活动期增加更为显著(P<0.01)。同一个体疾病活动期外周血CD4 CD25 调节性T细胞数量较非活动期减少(P<0.05)。MS患者外周血中CD4 CD25 T细胞的FOXP3 mRNA表达降低(P<0.05),且活动期降低更明显(P<0.01)。结论此组MS患者外周血CD4 CD25 调节性T细胞抑制活性降低,FOXP3 mRNA表达减少,活化的效应性T细胞数量增加,且与MS疾病活动性有关。     

Nerve growth factor content is increased in the rectal mucosa of children with diarrhea-predominant irritable bowel syndrome

Background The enteric nervous system is a complex network that includes, in the digestive mucosa, neuronal bodies and fibers interacting with the immune system and mucosal mast cells (MC). These interactions involve the secretion of messengers, such as the neurotrophin nerve growth factor (NGF), which influence colonic motility and sensitivity, both affected in irritable bowel syndrome (IBS). This study was designed to test the hypothesis that, in children with IBS, colonic mucosal innervation, NGF content, and MC infiltration are altered. We aimed to measure MC infiltration, number of neuronal bodies, distance from MC to nerve fibers, inflammation, and NGF content in rectal mucosa of pediatric patients with IBS as compared with controls. Methods Rectal biopsies from children (median age: 14 years) with diarrhea‐predominant IBS (n = 11) and controls (n = 14) were studied. MC and neuronal mucosal structures were identified by tryptase, CD117 and PGP9.5 immunoreactivity. Inflammatory cells (neutrophils, eosinophils, and lymphocytes) were counted. NGF was quantified in situ by ELISA. Key Results No mucosal inflammation was detected in IBS. MC infiltration and number of neuronal bodies were not significantly different between IBS and controls. The distance between MC and nerve fibers was not different in IBS compared with controls (5.2 ± 0.3 vs 5.0 ± 0.3 μm). Number of MC in close proximity to nerve fibers (<5 μm) was not different in the two groups. However, in IBS, NGF content was higher than controls (0.93 ± 0.3 vs 0.62 ± 0.3 pg mg^?1 protein, P < 0.05) and significantly correlated with MC number. Conclusions & Inferences Regardless of inflammation, NGF content is increased in rectal mucosa of diarrhea‐predominant IBS children.     

Frequency of naturally-occurring regulatory T cells is reduced in patients with ST-segment elevation myocardial infarction

BACKGROUND: Naturally-occurring regulatory T cells (Treg) constitute a mature T-cell population characterized phenotypically by co-expression of CD4 and CD25(high) surface molecules. We investigated here the frequency of circulating Treg in patients presenting with STEMI in comparison with subjects without coronary artery disease (CAD). The effect of primary percutaneous coronary intervention (PCI) with implantation of a bare (BS) or paclitaxel-eluting stent (PES) on peripheral Treg distribution was also examined. METHODS: Peripheral blood mononuclear cells were isolated from 30 consecutive patients presenting with STEMI and from 30 age-matched control subjects with angiographically normal coronary arteries. Treg were detected by flow cytometry according to their characteristic CD4+ CD25(high) membrane phenotype, and their frequency was assessed before PCI and at 48 h and at 6 days after PCI. CD27 expression identifying a highly suppressive Treg subset was also analysed. RESULTS: The percentages of both (CD27+)Treg and (CD27-)Treg were significantly lower in patients with STEMI in comparison with controls. In addition, the (CD27+)Treg/(CD27-)Treg ratio was skewed toward the CD27- population. The frequency of both Treg subsets significantly increased 48 h after either BS or PES implantation, remaining elevated for up to at least 6 days after PCI. CONCLUSIONS: Our data suggest that the percentage of circulating Treg is significantly reduced in patients with STEMI, suggesting that this immunosuppressive T-cell subset is compartmentalized within the acutely ischemic myocardium to limit the ongoing inflammation associated with this condition, and that coronary revascularization is associated with partial reconstitution of peripheral Treg pool.     

Paralysis of CD4(+)CD25(+) regulatory T cell response in chronic autoimmune encephalomyelitis

Increasing evidence strongly suggest that CD4(+)CD25(+) regulatory T (Treg) cells play a pivotal role in suppressing the development of autoimmune diseases. However, it remains poorly understood how these cells are involved in the persistence of, or recovery from, the diseases. In the present study, we examined the role of CD4(+)CD25(+) Treg cells in chronic EAE and compared the results with those obtained in acute EAE. In EAE lesions, CD25(+) cells decreased rapidly at the beginning of chronic EAE, whereas these cells were maintained at high levels during the recovery from acute EAE. The number of Foxp3(+)CD4(+)CD25(+) Treg and levels of Foxp3 mRNA in the lymphoid organ were significantly lower in chronic EAE. Importantly, the regulatory function of individual CD4(+)CD25(+) Treg cells was maintained in animals with chronic EAE. Furthermore, adoptive transfer of activated CD4(+)CD25(+) Treg cells suppressed the development of chronic EAE. These findings suggest that impairment of the CD4(+)CD25(+) Treg response is critical for development of chronic autoimmune diseases, and can be adjustable by autologous Treg transplantation.     

Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura

Immunoregulatory CD4(+)CD25(+) T cells play an important role in the induction and maintenance of peripheral self-tolerance. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. Therefore, CD4(+)CD25(+) T cells are believed to possibly play an important role in pathogenic autoimmune diseases. We measured the count of CD4(+)CD25(+) T cells in 44 patients with idiopathic thrombocytopenic purpura (ITP), and the number of CD4(+)CD25(+) T cells and clinical features were then analyzed. By using a flow cytometric analysis, the number of CD4(+)CD25(+) T cells in the patients with ITP showed a very wide distribution in comparison to healthy volunteers. The number of CD4(+)CD25(+) T cells was significantly lower in the ITP patients in the severe phase, and in patients positive for anti-glycoprotein IIb-IIIa antibody. However, the number of those cells increased in the patients at the complete remission phase, especially after a splenectomy. The Foxp3 mRNA levels of peripheral blood mononuclear cells (PBMC) of ITP patients were higher with an improved platelet count than in those with a low platelet count. In addition, the Foxp3 mRNA levels closely correlated with the number of CD4(+)CD25(+) cells. These mechanisms remain to be fully elucidated, however, the count of CD4(+)CD25(+) T cells is considered to possibly be related to the severity of ITP.     

Opioid maintenance therapy restores CD4+ T cell function by normalizing CD4+CD25(high) regulatory T cell frequencies in heroin user

There is an increasing body of evidence that heroin addiction is associated with severe alterations in immune function, which might contribute to an increased risk to contract infectious diseases like hepatitis B and C or HIV. However, the impact of heroin consumption on the CD4(+) T cell compartment is not well understood. Therefore, we analyzed the frequency and functional phenotype of CD4(+) T cells as well as immune-suppressive CD4(+)CD25(high) regulatory T cells (Tregs) isolated from the peripheral blood of opiate addicts currently abusing heroin (n=27) in comparison to healthy controls (n=25) and opiate addicts currently in opioid maintenance treatment (OMT; n=27). Interestingly, we detected a significant increase in the percentage of CD4(+)CD25(high) Tregs in the peripheral blood of heroin addicted patients in contrast to patients in OMT. The proliferative response of CD4(+) T cells upon stimulation with anti-CD3 and anti-CD28 antibodies was significantly decreased in heroin users, but could be restored by depletion of CD25(high) regulatory T cells from CD4(+) T cells to similar values as observed from healthy controls and patients in OMT. These results suggest that impaired immune responses observed in heroin users are related to the expansion of CD4(+)CD25(high) Tregs and more importantly, can be restored by OMT.     

Synapsin peptide fused to E. coli heat-labile toxin B subunit induces regulatory T cells and modulates cytokine balance in experimental autoimmune encephalomyelitis

We previously found that the preventive oral administration of a hybrid consisting of the C domain of synapsin and the B subunit of E. coli heat-labile enterotoxin (LTBSC) efficiently suppresses experimental autoimmune encephalomyelitis (EAE) development in rats. We investigated the effect of LTBSC on cytokine expression and on regulatory T (Treg) cells in rats with myelin induced EAE. LTBSC treatment increased the frequency of CD4(+)FoxP3(+) Treg cells in lymph nodes prior to challenge and in the EAE acute stage. LTBSC also up-regulated the expression of anti-inflammatory Th2/Th3 cytokines and diminished myelin basic protein-specific Th1 and Th17 cell responses in lymph nodes. CD4(+)CD25(+) Treg cells from LTBSC treated rats showed stronger suppressive properties than Treg cells from controls in vitro. Our observations indicate that LTBSC is a useful agent for modulating the autoimmune responses in EAE.     

Regulatory T cells in paraneoplastic neurological syndromes

Focusing on CD4(+)CD25(+) regulatory T lymphocytes (T(reg)), we studied the gene expression of T(reg) functional molecules in peripheral blood lymphocytes of patients with paraneoplastic neurological syndrome (PNS), including Lambert-Eaton myasthenic syndrome (LEMS) with small cell lung carcinoma (SCLC) and anti-Hu- or anti-Yo-antibody-positive PNS. T(reg)-rich subsets were sorted from the patients' peripheral blood mononuclear cells, and the mRNA expression levels of their functional genes were measured. The expression levels of FOXP3, TGF-beta and CTLA4 mRNA in T(reg)-rich subsets of PNS patients were down-regulated compared with that of SCLC patients without PNS. These results suggest that T(reg) dysfunction plays a role in PNS development.     

Specificity of regulatory CD4+CD25+ T cells for self-T cell receptor determinants

Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.     

Immunotherapy of rat glioma without accumulation of CD4~+CD25~+FOXP3~+ regulatory T cells

Immunotherapy may be used for the treatment of glioblastoma multiforme;however,the induced immune response is inadequate when either T cells or dendritic cells are used alone.In this study,we established a novel vaccine procedure in rats,using dendritic cells pulsed with C6 tumor cell lysates in combination with adoptive transfer of T lymphocytes from syngenic donors.On day 21 after tumor inoculation,all the rats were sacrificed,the brains were harvested for calculation of glioma volume,cytolytic T lymphocyte responses were measured by cytotoxic assay,and the frequency of regulatory T lymphocytes(CD4+CD25+FOXP3+) in the peripheral blood was investigated by flow cytometric analysis.The survival rate of rats bearing C6 glioma was observed.Results showed that the co-immunization strategy had significant anti-tumor potential against the pre-established C6 glioma,and induced a strong cytolytic T lymphocyte response in rats.The frequency of peripheral blood CD4+CD25+FOXP3+ regulatory T lymphocytes was significantly decreased following the combination therapy,and the rats survived for a longer period.Experimental findings indicate that the combined immunotherapy of glioma cell lysate-pulsed dendritic cell vaccination following adoptive transfer of T cells can effectively inhibit the growth of gliomas in rats,boost anti-tumor immunity and produce a sustained immune response while avoiding the accumulation of CD4+CD25+FOXP3+ regulatory T lymphocytes.     

B-cell activation in patients with irritable bowel syndrome (IBS)

Abstract  Patients with irritable bowel syndrome (IBS) may have a low grade immune activation. However, little is known about the properties of B cells of IBS patients. We therefore investigated activation level and antigen presenting phenotype of blood B cells of IBS patients. We also examined B-cell responses to lipopolysaccharide (LPS) and probiotic bacteria. Blood samples were obtained from 74 IBS patients and 30 healthy subjects. Peripheral blood mononuclear cells were isolated and stimulated with LPS or an UV-light inactivated bacterial cocktail consisting of the probiotic Gram-positive strains; Lactobacillus paracasei ssp. paracasei 19, Lactobacillus acidophilus La5, Bifidobacterium lactis B612. The phenotype of CD19⁺ B cells was investigated by flow cytometry before and after 72 h cell culture. Furthermore, IBS symptom severity was assessed. B cells isolated from blood of IBS patients displayed an amplified activation level as demonstrated by increased cell surface expression of IgG, and also the costimulatory molecules CD80 and CD86. Expression of antigen presenting HLA-DR and costimulatory molecule CD40 on B cells was, however comparable in IBS patients and controls. B cells of IBS patients displayed an impaired ability to increase expression of CD80, but not CD86, in response to both LPS as well as probiotic bacteria stimulations. To conclude, blood B cells of IBS patients have an increased activation level. Bacterial component induced expression of the costimulatory molecule CD80, regarded as important for tolerance induction, is impaired. These data suggest that B-cell antigen presentation in IBS patients is associated with altered capacity of providing costimulation to T cells.     

Tight junctions and IBS ‐ the link between epithelial permeability,low‐grade inflammation,and symptom generation?

In this issue of Neurogastroenterology and Motility, Dr Ewa Wilcz‐Villega and colleagues report low expression of E‐cadherin, a tight junction protein involved in the regulation of paracellular permeability, in the colonic mucosa of patients with the irritable bowel syndrome (IBS) with predominance of diarrhea (IBS‐D) or alternating symptoms (IBS‐A). These findings constitute an improvement in our knowledge of epithelial barrier disruption associated with IBS. There is mounting evidence to indicate that a compromised epithelial barrier is associated with low‐grade immune activation and intestinal dysfunction in at least a proportion of IBS patients. During the last 10 years of research, much interest has focused on the increase in the number of different types of immune cells in the gut mucosa of IBS patients including: mast cells, T lymphocytes, and other local cells such as enteroendocrine cells. The inflammatory mediators released by these cells or other luminal factors could be at the origin of altered epithelial barrier functions and enteric nervous system signaling, which lead to gut hypersensitivity. A current conceptual framework states that clinical symptoms of IBS could be associated with structural and functional abnormalities of the mucosal barrier, highlighting the crucial importance of elucidating the contributory role of epithelial barrier defects in the pathogenesis of IBS. More importantly, disruption of the epithelial barrier could also participate in the generation of persistent abdominal pain and discomfort mimicking IBS in patients with inflammatory bowel diseases considered in remission. This mini review gives a brief summary of clinical and experimental evidence concerning the mechanisms underlying epithelial barrier defects in IBS.     

IFN-β induces the proliferation of CD4+CD25+Foxp3+ regulatory T cells through upregulation of GITRL on dendritic cells in the treatment of multiple sclerosis

IFN-β is a major disease-modifying agent used for the treatment of multiple sclerosis (MS). Its mechanisms are complex and it has broad immunomodulatory effects on many types of immune cells. It was observed clinically that the quantity of CD4(+)CD25(+)Foxp3(+) regulatory T cells increases in some MS patients treated with IFN-β. In this study, we show that IFNAR engagement by IFN-β expands naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cell population through the modulation of dendritic cells (DCs). IFN-β has no effect on the conversion of CD4(+)CD25(-) T cells to adaptive Treg cells. The IFN-β-induced upregulation of GITRL on DC and downregulation of CTLA-4 on Treg cell work together to facilitate the proliferation of anergic Treg cells. In MS patients treated with Avonex or Rebif (IFN-β), it was found that GITRL expression is markedly upregulated on peripheral CD14(+) cells. Our findings help the better understanding of the complex effects of IFN-β in the treatment of MS.     

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in irritable bowel syndrome

Abstract  Enhanced stress responsiveness has been implicated as a potential mechanism contributing to the pathophysiology of irritable bowel syndrome (IBS), and should be reflected in altered function of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Both of these systems can modulate mucosal immune function. The aims of this study were: (i) to characterize the basal circadian rhythm of adrenocorticotropin hormone (ACTH) and cortisol in IBS vs healthy controls; (ii) to compare stimulated ACTH, cortisol and noradrenaline responses to a pelvic visceral stressor (sigmoidoscopy) in IBS and controls; and (iii) to correlate neuroendocrine responses with colonic mucosal cytokine expression and symptoms in IBS. Two separate studies were conducted in women. In Study 1, basal cortisol levels were analysed in 41 IBS and 25 controls using 24-h collections of plasma ACTH and cortisol (q10 min sampling). In Study 2, 10 IBS patients with diarrhoea (IBS-D) and 10 controls underwent sigmoidoscopy with measurements of stimulated neuroendocrine responses and cytokine mRNA expression in colonic tissue. Basal ACTH levels were significantly blunted ( P  <   0.05), while basal and stimulated plasma cortisol levels were higher in patients. Basal cortisol levels prior to an experimental visceral stressor positively correlated with anxiety symptoms ( P  <   0.004), but not IBS symptoms. Irritable bowel syndrome patients with diarrhoea had significantly decreased mRNA expression of mucosal cytokines [interleukin (IL)-2, IL-6] in the sigmoid colon vs controls ( P  <   0.05). Although dysregulations in stress-responsive systems such as the HPA axis and mucosal immune function are demonstrated in IBS, they do not appear to have a primary role in modulating IBS severity and abdominal pain.     

Immune derived opioidergic inhibition of viscerosensory afferents is decreased in Irritable Bowel Syndrome patients

Alterations in the neuro-immune axis contribute toward viscerosensory nerve sensitivity and symptoms in Irritable Bowel Syndrome (IBS). Inhibitory factors secreted from immune cells inhibit colo-rectal afferents in health, and loss of this inhibition may lead to hypersensitivity and symptoms. We aimed to determine the immune cell type(s) responsible for opioid secretion in humans and whether this is altered in patients with IBS. The β-endorphin content of specific immune cell lineages in peripheral blood and colonic mucosal biopsies were compared between healthy subjects (HS) and IBS patients. Peripheral blood mononuclear cell (PBMC) supernatants from HS and IBS patients were applied to colo-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). β-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived β-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on colo-rectal afferent mechanosensitivity than those from IBS patients. The inhibitory effects of PBMC supernatants were more prominent in CVH mice compared to healthy mice due to an increase in μ-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for β-endorphin secretion in humans. IBS patients have lower monocyte derived β-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS.