Dexamethasone upregulates FOXP3 expression without increasing regulatory activity

Recent studies have shown the capacity of corticoids to increase forkhead box p3 (FOXP3) expression, which suggests that these drugs may be able to generate regulatory T cells (Treg). Therefore, corticoids may possibly be employed in protocols to generate or expand Treg cells with the aim of being used in cell transfer therapy. However, given that in humans FOXP3 is not necessarily associated with regulatory function, it is of great importance to ascertain whether FOXP3-expressing cells generated with corticoids are “truly” Treg cells. To this end, we studied the effect of dexamethasone on both human activated lymphocytes and in vitro generated Treg cells as well as regulatory activity of CD4⁺CD25^high cells from SLE patients users and non users of prednisone. Results show that dexamethasone markedly enhances FOXP3 expression and generates CD25^high cells with phenotypic characteristics attributable to natural Treg cells. Unexpectedly, in spite of their hyporesponsiveness and enhanced FOXP3 expression, these cells did not exert suppressive activity. Moreover, although dexamethasone was able to enhance FOXP3 expression in in vitro generated Treg cells, once again this effect was not correlated with increased regulatory activity. These results were supported by the fact that CD4⁺CD25^high cells from steroid-treated SLE patients did not show a higher antiproliferative function than those from non-steroid-treated patients. We conclude that the increment on FOXP3 expression caused by dexamethasone is not connected with regulatory function, supporting the fact that FOXP3 expression in humans is not an exclusive attribute of Treg cells. Subsequently, the use of FOXP3 as a Treg cell marker must be done cautiously, especially in patients with systemic inflammatory diseases or those under corticoid treatment.     

Decreased Expression of FOXP3 in Nasal Polyposis

Purpose

The pathogenesis of nasal polyposis (NP) is unclear. Eosinophils and mast cells are considered to play important roles in this process. In addition, the levels of Th2-type cells are increased, irrespective of the atopic status of the patient with NP. In this context, we and others have shown high levels of thymus and activation-related chemokine/CCL17, macrophage-derived chemokine, eotaxin, and RANTES in patients with NP. Forkhead box P3 (FOXP3) plays a key role in CD4+CD25+ regulatory T-cell function and represents a specific marker for regulatory T cells (Tregs). Decreased expression of FOXP3 has been reported in allergic diseases. The present study was designed to evaluate the presence and potential roles of Tregs, defined by the expression of FOXP3 protein, in NP.

Methods

Using immunohistochemistry, we estimated the numbers of FOXP3+ cells in the epithelium and lamina propria of the NPs of 17 patients with chronic rhinosinusitis with NP and the nasal mucosa of 15 patients with allergic rhinitis (AR). The number of FOXP3+ cells in NPs was compared with that in the nasal mucosa of patients with AR, and the numbers of FOXP3+ cells in atopic and non-atopic NP were also compared.

Results

The number of FOXP3+ cells in the lamina propria of patients with NP was significantly lower than that in the nasal mucosa of the AR patients (2.79 vs. 5.99, P=0.008). There was no statistically significant difference noted for the numbers of FOXP3+ cells between the epithelium of the NP and the nasal mucosa (3.60 vs. 2.39, P=0.180). Furthermore, the numbers of CD4+FOXP3+ cells were lower in NPs than in the allergic nasal mucosa. There was no difference in the number of FOXP3+ cells between the atopic and non-atopic NP patients.

Conclusions

Fewer Tregs (i.e., decreased FOXP3 expression) are found in NPs than in the nasal mucosa of AR patients. As the severity of eosinophilic, Th2-type inflammation and the levels of inflammatory mediators are much higher in NPs than in the nasal mucosa of AR patients, an inverse co-relationship may exist between these parameters and the number of Tregs. The deficiency of Tregs in NP may account for the more pronounced Th2-type inflammation seen in these patients.     

Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level

CD4+ CD25(high) regulatory T cells (Tregs) of patients with relapsing-remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single-cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25(high) FOXP3+ T cells and lower FOXP3 protein expression per cell in RR-MS patients than in SP-MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)-beta-treated RR-MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25(high) FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS patients, expressed CD103 and CD49d, adhesion molecules involved in T-cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25(high) CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS patients. Taken together, these data show aberrant FOXP3 expression at the single-cell level correlated with Treg dysfunction in RR-MS patients. Our results also suggest that Tregs accumulate in the CSF of RR-MS patients, in an attempt to down-regulate local inflammation in the central nervous system.     

Serum thymosin α 1 levels in patients with chronic inflammatory autoimmune diseases

Thymosin alpha 1 (Tα1) is a powerful modulator of immunity and inflammation. Despite years of studies, there are a few reports evaluating serum Tα1 in health and disease. We studied a cohort of healthy individuals in comparison with patients affected by chronic inflammatory autoimmune diseases. Sera from 120 blood donors (healthy controls, HC), 120 patients with psoriatic arthritis (PsA), 40 with rheumatoid arthritis (RA) and 40 with systemic lupus erythematosus (SLE), attending the Transfusion Medicine or the Rheumatology Clinic at the Policlinico Tor Vergata, Rome, Italy, were tested for Tα1 content by means of a commercial enzyme‐linked immunosorbent assay (ELISA) kit. Data were analysed in relation to demographic and clinical characteristics of patients and controls. A gender difference was found in the HC group, where females had lower serum Tα1 levels than males (P < 0·0001). Patients had lower serum Tα1 levels than HC (P < 0·0001), the lowest were observed in PsA group (P < 0·0001 versus all the other groups). Among all patients, those who at the time of blood collection were taking disease‐modifying anti‐rheumatic drugs (DMARD) plus steroids had significantly higher Tα1 levels than those taking DMARD alone (P = 0·044) or no treatment (P < 0·0001), but not of those taking steroids alone (P = 0·280). However, whichever type of treatment was taken by the patients, serum Tα1 was still significantly lower than in HC and there was no treatment‐related difference in PsA group. Further prospective studies are necessary to confirm and deepen these observations. They might improve our understanding on the regulatory role of Tα1 in health and disease and increase our knowledge of the pathogenesis of chronic inflammatory autoimmune diseases.     

Altered homeostasis of CD4(+) FoxP3(+) regulatory T-cell subpopulations in systemic lupus erythematosus

The role of naturally occurring regulatory T cells (Treg), known to be phenotypically heterogeneous, in controlling the expression of systemic lupus erythematosus (SLE) is incompletely defined. Therefore, different subpopulations of CD4(+) FoxP3(+) Tregs in patients with active or inactive SLE were investigated and compared with those of healthy subjects and patients with ankylosing spondylitis (AS). Characterization of different subsets of circulating CD4(+) FoxP3(+) Tregs was examined using flow cytometry. CD4(+) CD25(high) T cells were sorted and examined for suppressive activity in vitro. The results showed first that a significant decrease in the frequency of CD4(+) CD25(high) FoxP3(+) T cells was present in patients with active SLE (n = 58), compared with healthy controls (n = 36) and AS patients (n = 23). In contrast, the frequencies of CD25(low) FoxP3(+) and CD25(-) FoxP3(+) CD4(+) T cells were significantly increased in patients with active SLE by comparison with the control subjects. The elevation of these two putative Treg subpopulations was associated with lower plasma levels of complement C3 and C4 in patients with SLE. In addition, the ratios of the three subsets of CD4(+) FoxP3(+) Tregs versus effector T cells (CD4(+) CD25(+) FoxP3(-)) were inversely correlated with the titer of anti-double-stranded DNA IgG in patients with inactive, but not active, SLE. These results suggest that the pathogenesis of SLE may be associated with a defect in the homeostatic control of different Treg subsets.     

核转录因子FOXP3在肺癌组织中的表达及意义

目的： 检测核转录因子FOXP3在不同病变肺组织中的表达，分析CD4+ CD25+ 调节性T细胞在不同病变肺组织中的浸润情况，并进一步探讨不同病理类型的肺癌组织中FOXP3表达的异同。方法: 使用RT-PCR及Western blotting方法，检测21例肺癌、7例支气管扩张、5例炎性假瘤、3例结核球以及15例病灶旁手术切除正常肺组织共153份标本中FOXP3 mRNA及蛋白的表达。结果: 肺癌、肺良性病变及病灶旁正常肺组织FOXP3 mRNA及蛋白的表达阳性分别为41/63、8/45、0/45（P<0.05），肺癌与肺良性病变组织均有FOXP3 mRNA及蛋白表达，分析其平均吸光度A值之间有显著差异（P<0.01）。病灶旁正常肺组织FOXP3 mRNA及蛋白无表达。不同病理类型肺癌组织中均有FOXP3 mRNA及蛋白的表达，分析其平均吸光度A值之间无显著差异（P>0.05）。结论: FOXP3可作为CD4+ CD25+调节性T细胞的一种标志物。肺癌与肺良性病变组织均有FOXP3 mRNA及蛋白表达，FOXP3在肺癌组织中表达强于肺良性病变组织。     

自身免疫性风湿病患者皮肤溃疡的临床特点与治疗策略

目的研究自身免疫性风湿病（ARDs）合并皮肤溃疡患者的临床特点,治疗方法和效果。方法回顾性调查1998年至2008年中山大学附属第一医院ARDs合并皮肤溃疡住院患者的病例资料,分析ARDs患者皮肤溃疡的临床特点,各种治疗方法的效果。结果共有67例ARDs合并皮肤溃疡住院患者,126个溃疡,男女比例为1：3．1（16：51）,平均年龄（50．9±19．2）岁。70％患者溃疡发生时处于疾病活动期。绝大部分患者伴有多种器官系统受累和基础疾病,其中血象改变和低白蛋白血症常见。大部分溃疡在应用糖皮质激素和缓解病情抗风湿药物控制病情活动后保守治疗愈合或好转,或通过手术修复愈合。结论ARDs患者皮肤溃疡的发生及其临床表现与ARDs本身密切相关,治疗上应首先控制ARDs病情活动,再结合全身和局部情况决定手术或非手术治疗,并辅以综合治疗。     

Inverse correlation between CD4+ regulatory T-cell population and autoantibody levels in paediatric patients with systemic lupus erythematosus

CD4(+) CD25(+) regulatory T cells (Tregs) are critical in maintaining self-tolerance and preventing organ-specific autoimmunity. Their role in paediatric systemic lupus erythematosus (SLE), an autoimmune disease characterized by inappropriate regulation of hyperactivated B and T cells, has not been clearly defined. Using flow cytometry to determine cell populations and real-time polymerase chain reaction to assay mRNA expression for FOXP3, CTLA-4, and GITR, we characterized CD4(+) CD25(+) T cells in paediatric SLE patients and healthy subjects. The frequency of CD4(+) CD25(+) Tregs was significantly decreased in patients with active SLE compared with patients with inactive SLE and with controls (7.27% +/- 2.50%, 9.59% +/- 2.80% and 9.78% +/- 2.11%, respectively; P = 0.027 and P < 0.001, respectively), and was inversely correlated with disease activity, as assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 scores (r = -0.59, P = 0.001) and serum anti-double-stranded DNA levels (r = -0.65, P < 0.001). Our preliminary investigations found elevated surface expression of GITR in CD4(+) CD25(+) T cells, elevated mRNA expression of CTLA-4 in CD4(+) T cells and higher amounts of mRNA expression for FOXP3 in CD4(+) cells in patients with active SLE compared with patients with inactive disease and controls. We demonstrated reduced CD4(+) CD25(+) Treg levels were inversely correlated with disease activity, indicating a defective Treg population in paediatric SLE patients. The differences in the expression of FOXP3, CTLA-4 and GITR imply the possible role of CD4(+) Tregs in the pathogenesis of SLE.     

Langerhans cell histiocytosis in Chinese adults: absence of BRAF mutations and increased FOXP3+ regulatory T cells

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by the proliferation of abnormal Langerhans cells. Previous studies mainly focused on children with LCH. However, there is limited information on the clinical and pathological aspects of LCH in adults. Therefore, this study aimed to investigate the clinical and pathological aspects of LCH in Chinese adults. The results showed that the average age of 18 LCH patients was 35.22 ± 16.57 years old. The ratio of male to female was 3.5:1.14 patients (77.8%) had single-system involvement and 4 patients (22.2%) had multi-system diseases. The skin (38.9%) and lungs (44.4%) were the mainly affected organs. No BRAF mutations were detected in the lesions of 18 cases. The number of FOXP3⁺ Tregs was significantly increased in LCH. In conclusion, clinical features of LCH in adults are distinct from those in children. Adult LCH has a relatively good prognosis and presents as a benign disease. Immune regulation plays an important role in the pathogenesis of adult LCH.     

Disease activity assessment of rheumatic diseases during pregnancy: a comprehensive review of indices used in clinical studies

Pregnancy requires a special management in women with inflammatory rheumatic diseases (RDs), with the aim of controlling maternal disease activity and avoiding fetal complications. Despite the heterogeneous course of RDs during pregnancy, their impact on pregnancy largely relates to the extent of active inflammation at the time of conception. Therefore, accurate evaluation of disease activity is crucial for the best management of pregnant patients. Nevertheless, there are limitations in using conventional measures of disease activity in pregnancy, as some items included in these instruments can be biased by symptoms or by physiological changes related to pregnancy and the pregnancy itself may influence laboratory parameters used to assess disease activity. This article aims to summarize the current literature about the available instruments to measure disease activity during pregnancy in RDs. Systemic lupus erythematosus is the only disease with instruments that have been modified to account for several adaptations which might interfere with the attribution of signs or symptoms to disease activity during pregnancy. No modified-pregnancy indices exist for women affected by other RDs, but standard indices have been applied to pregnant patients.The current body of knowledge shows that the physiologic changes that occur during pregnancy need to be either adapted from existing instruments or developed to improve the management of pregnant women with RDs. Standardized instruments to assess disease activity during pregnancy would be helpful not only for clinical practice but also for research purposes.     

CD4~+Foxp3~+T细胞亚群在系统性红斑狼疮中的免疫抑制功能研究

采用五色流式细胞术检测系统性红斑狼疮(systemic lupus erythematosus,SLE)患者和健康对照组的CD4+Foxp3+T细胞及其细胞亚群;以CFSE染色法分析SLE患者和健康对照组CD4+Foxp3+T细胞各亚群的免疫抑制功能。结果发现SLE组CD4+Foxp3+T细胞(占CD4+T细胞的比例)显著高于对照组(12.03%±1.523%vs 6.410%±0.4353%,t=3.790,P<0.01),但其CD4+T细胞的增殖却比对照组活跃(PI:4.052%±0.4004%vs 2.528%±0.2322%,t=3.293,P<0.05)。根据Foxp3和CD45RA表达强度的差异,可将CD4+Foxp3+T细胞清晰地分成3个亚群:CD45RA+Foxp3low(Ⅰ区)细胞、CD45RA-Foxp3high(Ⅱ区)细胞和CD45RA-Foxp3low(Ⅲ区)细胞,SLE组的Ⅰ区和Ⅲ区细胞数量显著增加(分别为t=2.123,P<0.05;t=3.462,P<0.01),而Ⅱ区细胞与对照组之间差异无统计学意义(0.6056%±0.1879%vs0.6571%±0.1764%,t=0.1999,P>0.05)。SLE患者和健康对照者的Ⅰ区和Ⅱ区细胞在体外均具有免疫抑制功能,而Ⅲ区细胞不具有该功能。SLE患者Ⅰ区细胞的免疫抑制能力弱于对照组(t=2.994,P<0.05),而Ⅱ区细胞和Ⅲ区细胞与对照组之间差异均无统计学意义。提示,SLE患者CD4+Foxp3+T细胞比例升高而免疫抑制功能下降,可能是两大原因造成了这种数量和功能的不一致性:①SLE患者CD4+Foxp3+T细胞数量增加主要由于Ⅲ区细胞增多所致,而这区细胞不具有免疫抑制功能;②在SLE患者,具有免疫抑制功能的Ⅰ区细胞的抑制能力明显减弱。     

The role of IL-17 in systemic lupus erythematosus and its potential as a therapeutic target

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies production and immune complex deposition with systemic clinical manifestations. Interleukin (IL)-17-producing cells play a crucial role in disease pathogenesis and represent an attractive therapeutic target.

Areas covered: This review provides an update on the possibility of targeting IL-17 in SLE. The rational for this approach as well as currently available and future targets are discussed.

Expert opinion: Although human expression studies and animal models indicate that IL-17 blocking may be a promising therapeutic strategy for SLE, direct evidence for IL-17 inhibition in SLE patients is unavailable. Biologic therapies and small-molecule drugs that target IL-17 production are required for the achievement of a favorable clinical effect in SLE patients.     

NLRP3: A promising therapeutic target for autoimmune diseases

NLRP3, a member of nucleotide-binding domain-(NOD) like receptor family, can be found in large varieties of immune and non-immune cells. Upon activation, the NLRP3, apoptosis-associated speck-like protein (ASC) and pro-caspase-1 would assemble into a multimeric protein, called the NLRP3 inflammasome. Then the inflammasome promotes inflammation (through specific cleavage and production of bioactive IL-1β and IL-18) and pyroptotic cell death. Previous studies have indicated the importance of NLRP3 in regulating innate immunity. Recently, numerous studies have revealed their significance in autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc) and inflammatory bowel disease (IBD). In this review, we will briefly discuss the biological features of NLRP3 and summarize the recent progression of the involvement of NLRP3 in the development and pathogenesis of autoimmune diseases, as well as its clinical implications and therapeutic potential.     

Phenotypic and functional characterization of a CD4(+) CD25(high) FOXP3(high) regulatory T-cell population in the dog

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4(+) FOXP3(high) T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3(+) cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4(+) and CD8(+) T cells. Removal of the Con A and continued culture disclosed a CD4(+) FOXP3(high) population, distinct from the CD4(+) FOXP3(intermediate) T cells; very few CD8(+) FOXP3(high) T cells were observed, though CD8(+) FOXP3(intermediate) cells were present in equal abundance to CD4(+) FOXP3(intermediate) cells. The CD4(+) FOXP3(high) T cells were thought to represent activated Treg cells, in contrast to the FOXP3(intermediate) cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3(high) T cells were almost exclusively IFN-γ(-) , whereas the FOXP3(intermediate) cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4(+) T cells showing the highest CD25 expression (CD4(+) CD25(high) ) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4(+) T cells with the lowest CD25 expression (CD4(+) CD25(-) ), which proliferated readily. The CD4(+) CD25(high) FOXP3(high) T cells were able to suppress the proliferation of responder CD4(+) T cells in vitro, in contrast to the CD4(+) CD25(-) cells, which showed no regulatory properties.