Reduction of forkhead box P3 levels in CD4⁺CD25^high T cells in patients with new-onset systemic lupus erythematosus

Abstract

The aim of this study was to quantify and evaluate the forkhead box P3 (FoxP3) expression regulatory T cells in new-onset systemic lupus erythematosus (SLE) patients before and after treatment. Forty-four newly diagnosed and untreated SLE patients, including 24 with active disease (SLEDAI ≥ 10) and 20 with inactive disease (SLEDAI < 5), were enrolled in this study. Twenty-one age- and sex-matched healthy volunteers were also included as controls. Peripheral blood samples were collected and mononuclear cells isolated. The expression of CD25 and FoxP3 in CD4⁺ T cells were analysed with flow cytometry. CD4⁺CD25⁺ (3·95–13·04%) and CD4⁺CD25^high (0·04–1·34%) T cells in peripheral blood in untreated patients with new-onset active lupus were significantly lower than that in the patients with inactive lupus (7·27–24·48%, P < 0·05 and 0·14–3·07% P < 0·01 respectively) and that in healthy controls (5·84–14·84%, P < 0·05). Interestingly, the decrease in CD4⁺CD25^high T cells was restored significantly in patients with active lupus after corticosteroid treatment. There was, however, a significantly higher percentage of CD4⁺FoxP3⁺ T cells in patients with active (5·30–23·00%) and inactive (7·46–17·38%) new-onset lupus patients compared with healthy control subjects (2·51–12·94%) (P < 0·01). Intriguingly, CD25 expression in CD4⁺FoxP3⁺ T cells in patients with active lupus (25·24–62·47%) was significantly lower than that in those patients with inactive lupus (30·35–75·25%, P < 0·05) and healthy controls (54·83–86·38%, P < 0·01). Most strikingly, the levels of FoxP3 expression determined by mean fluorescence intensity in CD4⁺CD25^high cells in patients with active SLE were significantly down-regulated compared with healthy subjects (130 ± 22 versus 162 ± 21, P = 0·012). CD4⁺CD25^high T cells are low in new-onset patients with active SLE and restored after treatment. Despite that the percentage of CD4⁺FoxP3⁺ T cells appear high, the levels of FoxP3 expression in CD4⁺CD25^high T cells are down-regulated in untreated lupus patients. There is a disproportional expression between CD25^high and FoxP3⁺ in new-onset patients with active SLE.

Introduction

CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺ regulatory T cells (T_regs) are instrumental in induction and maintenance of peripheral immune tolerance in rodents and humans [1–4]. In contrast to mice, where the majority of CD4⁺CD25⁺ T cells expressing FoxP3 and exhibiting suppressive activity, it seems that most FoxP3⁺ T_regs exist in the CD4⁺CD25^high subpopulation in humans [5]. Deficiencies in the cell numbers and/or changes of function in T_regs have been implicated in a variety of human diseases, including autoimmune diseases. In this regard, it has been reported that patients with multiple sclerosis exhibit a decreased function of CD4⁺CD25⁺ T_regs[4]. Patients with diabetes and rheumatoid arthritis also show a certain degree of deregulation of peripheral CD4⁺CD25⁺ T_regs[6,7]. Systemic lupus erythematosus (SLE) is a typical autoimmune disease affecting nearly all vital organs and tissues, characterized by a deregulated increase in T cell responses and T cell-dependent autoantibodies in the patients. Although recent advances have begun to suspect that deficiency of frequency or function CD4⁺CD25⁺ T_regs might be involved in development of the disease, the reported data are inconsistent. In addition, most of the patients in previous studies were treated with varieties of medications including corticosteroids, which might interfere with the immune system and complicate the interpretation of the results. In order to understand the role of CD4⁺CD25⁺FoxP3⁺ T_regs in SLE patients, we selected 44 patients with new-onset SLE newly diagnosed without prior treatment with glucocorticosteroid and immunosuppressives, and investigated their CD4⁺CD25⁺FoxP3⁺ T_regs. Unexpectedly, we found that patients with active SLE show a significant decrease in CD4⁺CD25⁺ and CD4⁺CD25^high subpopulation, but a dramatic increase in frequency of CD4⁺FoxP3⁺ T cells, suggesting a discrepancy between CD25 and FoxP3 expression in CD4⁺ T cells in the SLE.

Materials and methods

Patients and healthy control subjects

Forty-four patients with new-onset SLE in the absence of previous treatment with glucocorticoid and immunosuppressants were recruited into the study, including 42 females and two males, with ages ranging between 15 and 53 years (mean age 28·7 ± 9·9 years). All patients fulfilled the SLE criteria of the American College of Rheumatology [8,9]. Lupus disease activity was evaluated using an SLE disease activity index (SLEDAI). The new-onset SLE patients were divided into active and inactive groups according to their SLEDAI. The active group included 24 patients (SLEDAI ≥ 10) and the inactive group included 20 patients (SLEDAI < 5). Twenty-one healthy volunteers, age- and sex-matched, were also enrolled as controls. All patients in the active group were treated with prednisolone ≥ 1 mg/kg/day plus intravenous cyclophosphamide 0·6–1·0 g every 2–4 weeks depending on patients' tolerance and disease activity. The study was approved by the ethical committee of Peking Union Medical College Hospital and informed consent was obtained from every subject.

Reagents and antibodies

The monoclonal antibodies and reagents were obtained from eBioscience (San Diego, CA, USA) unless indicated otherwise: fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (RPA-T4), phycoerythrin (PE)-conjugated anti-human CD25 (BC96), allophycocyanin conjugated anti-human FoxP3 (PCH101) and their respective isotype control antibodies.

Preparation of peripheral blood mononuclear cells and flow cytometry analysis

Peripheral blood of SLE patients before and 1 month after treatment were collected and peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. PBMCs were washed in phosphate-buffered saline containing 2% fetal calf serum and 1 × 10⁶ cells were incubated with FITC-CD4 (20 μl) and PE-CD25 (20 μl) in the dark at 4°C for 30 min. Subsequently, intracellular FoxP3 staining was performed according to the manufacturer's instructions. Stained cells were then analysed by a FACS^sort (BD Biosciences, San Jose, CA, USA). CD4⁺ T cells were gated electronically and the expression of CD25 and FoxP3 was analysed. The CD4⁺CD25^high T cells were defined as reported by Baecher-Allan [5].

Measurement of serum interleukin-6

Serum concentrations of interleukin (IL)-6 were determined by enzyme-linked immunosorbent assay using a human IL-6 reagent kit obtained from eBioscience. The assay was performed according to the manufacturer's instructions.

Statistical analysis

All data were analysed using spss version 13·0 software. One-way analyses of variance were used to compare data with normal distribution and homogeneity of variance. Independent-sample t-tests were used to compare mean between two groups and paired-sample t-tests were used to compare mean before and after treatment. The Mann–Whitney U-test was used to compare data of non-normal distribution; values of P < 0·05 were considered significant, and all tests were two-tailed.

Results

Frequency of CD4⁺CD25⁺, CD4⁺CD25^high and CD4⁺FoxP3⁺ T cells in peripheral blood of new-onset SLE patients and healthy controls

We first compared the frequency of CD4⁺CD25⁺, CD4⁺CD25^high and CD4⁺FoxP3⁺ T cells in the new-onset SLE patients and healthy controls. It was found there was no statistically significant difference in the percentages of CD4⁺CD25⁺ and CD4⁺CD25^high T cells within the CD4⁺ population between the two groups (P = 0·769 for CD4⁺CD25⁺, P = 0·533 for CD4⁺CD25^high) (Fig. 1a and b). However, there was a significant increase of CD4⁺FoxP3⁺ T cells in new-onset SLE patients compared with healthy controls (P = 0·0001) (Fig. 1c).

Fig. 1.

Percentage of CD4⁺CD25⁺ and CD4⁺CD25^high and CD4⁺forkhead box P3 (FoxP3)⁺ T cells between patients with new-onset systemic lupus erythematosus (SLE) (n = 44) and healthy controls (n = 21). (a, b) There was no significant difference between patients with new-onset SLE and healthy controls in the percentage of CD4⁺CD25⁺ or CD4⁺CD25^high T cells within CD4⁺ T cells; (c) increase in CD4⁺FoxP3⁺ T cells in new-onset SLE patients (n = 21) compared with healthy controls (n = 11). The horizontal lines represent mean values.

Decreased frequency of CD4⁺CD25⁺, CD4⁺CD25^high T cells and increased frequency of CD4⁺FoxP3⁺ T cells in new-onset patients with active SLE

However, when the patients were divided into active and inactive SLE, the percentage of CD4⁺CD25⁺ T cells in the new-onset patients with active SLE was decreased significantly compared with healthy controls (P = 0·034) and inactive SLE patients (P = 0·007) (Fig. 2a). The percentage of the CD4⁺CD25^high subpopulation within CD4⁺ T cells was reduced in patients with active SLE compared with those with inactive SLE (P = 0·001), although the decrease was statistically insignificant when compared with healthy controls (P = 0·088) (Fig. 2b). A significant elevation of CD4⁺FoxP3⁺ T cells in both active and inactive new-onset SLE patients compared with healthy controls (P < 0·01 respectively) was also observed (Fig. 2c).

Fig. 2.

The patients with active systemic lupus erythematosus (SLE) exhibit reduced the percentage of CD4⁺CD25⁺ and CD4⁺CD25^high T cells and increased the percentage of CD4⁺forkhead box P3 (FoxP3)⁺ T cells. (a) The percentage of CD4⁺CD25⁺ T cells from patients with active SLE were significantly lower than that from healthy controls or patients with inactive SLE; (b) The percentage of CD4⁺CD25^high T cells from patients with active SLE were significantly lower than from patients with inactive disease. (c) Both active and inactive SLE patients had more CD4⁺FoxP3⁺ T cells than healthy controls. The horizontal lines represent mean values.

Discrepant expression between CD25 and FoxP3 in CD4⁺ T cells in patients with SLE

The aforementioned decrease in CD25^high subsets and increase in FoxP3⁺ subpopulations in CD4⁺ T cells of SLE patients promoted us to analyse further the pattern of CD25 versus FoxP3. When CD4⁺FoxP3⁺ T cells were gated, it was found that cells expressing CD25 were decreased dramatically in patients with active and inactive SLE (range 25·24–62·47% and range 30·35–75·25%) compared with those in healthy controls (range 54·83–86·38%) (P = 0·0009 and P = 0·001). A representative healthy control and an active patient are displayed in Fig. 3a. Of the patients, those with active SLE exhibited significantly lower levels of CD25 expression than was found in inactive patients (Table 1, P = 0·04). Strikingly, when the CD4⁺CD25^high subset was analysed, it was observed that FoxP3 expression was significantly down-regulated in active SLE patients. The reduction of FoxP3 in active SLE patients was demonstrated by the mean fluorescence intensity of FoxP3⁺ T cells compared with healthy control subjects (130 ± 22, n = 10 versus 162 ± 21, n = 11, P = 0·012) (Fig. 3c).

Fig. 3.

Reduction of CD25⁺ in CD4⁺forkhead box P3 (FoxP3)⁺ cells and down-regulation of FoxP3 expression in CD4⁺CD25^high subset in patients with active systemic lupus erythematosus (SLE). (a) CD4⁺FoxP3⁺ T cells were gated and the expression of CD25 is displayed in a representative healthy control and an active SLE patient. (b) CD4⁺CD25^high cells were gated and the expression of FoxP3 is shown in a representative healthy control and an active patient from indicated groups. (c) Mean fluorescence intensity of FoxP3 within CD4⁺CD25^high cells was significantly lower in active SLE patients than in healthy controls (100·52–160·92 versus 122·58–198·10, P = 0·012). FITC, fluorescein isothiocyanate; APC, antigen-presenting cell; PE, phycoerythrin; MFI, mean fluorescence intensity.

Table 1.

Expression of CD25 in CD4⁺forkhead box P3 (FoxP3)⁺ T cells from healthy controls, active and inactive systemic lupus erythematosus (SLE) patients.

Subjects	% CD25 of CD4+FoxP3+ T cells
Healthy controls (n = 11)	72·47 ± 9·42%*
Active SLE (n = 10)	42·49 ± 10·26%†
Inactive SLE (n = 11)	55·00 ± 13·27%

^*Compared with active SLE and inactive SLE P = 0·0009, and P = 0·001;

^†Compared with inactive SLE P = 0·04.

Serum levels of IL-6 increased in patients with active SLE

The serum concentration of IL-6 in patients with active SLE (10·52 ± 5·17 pg/ml) was significantly higher than that in healthy controls (3·82 ± 2·47 pg/ml, P = 0·003) and inactive SLE patients (6·06 ± 3·45 pg/ml, P = 0·046), while there was no statistically significant difference between inactive SLE patients and healthy controls (P = 0·324) (Fig. 4a). In this study, we found that active SLE patients exhibited increased serum levels of IL-6 and decreased FoxP3 expression of CD4⁺CD25^high T cells (Fig. 4b), although linear regression analysis did not show a significant correlation.

Fig. 4.

The active systemic lupus erythematosus (SLE) patients had increased serum concentration of interleukin (IL)-6. (a) The concentration of IL-6 in patients with active SLE was significantly higher than that in healthy controls and patients with inactive SLE. (b) The relative tendency of serum concentration of IL-6 and the expression of forkhead box P3 in CD4⁺CD25^high T cells in active SLE patients.

Effect of glucocorticosteroid and cyclophosphamide on CD4⁺FoxP3⁺, CD4⁺CD25⁺ and CD4⁺CD25^high T cells in active SLE patients

We then examined whether treatment of patients with SLE had any effect on the expression of CD25 and FoxP3 in CD4⁺ T cells in blood. Patients with active SLE were selected because these patients showed the most significant changes in both CD25 reduction and FoxP3 increase. Although it did not reach statistical significance, the percentage of CD4⁺CD25⁺ T cells increased in the majority of active patients (Fig. 5a). Treatment of patients with glucocorticosteroid and cyclophosphamide increases dramatically the percentages of CD25^high cells (Table 2, Fig. 5b). The CD4⁺FoxP3⁺ T cells in the same patients, however, were changed insignificantly (Table 2).

Fig. 5.

CD4⁺CD25⁺ T cells (a) and CD4⁺CD25^high T cells (b) increased after treatment in the majority of active lupus patients.

Table 2.

Increase in CD4⁺CD25^high T cells in patients with active systemic lupus erythematosus after glucocorticosteroid and cyclophosphamide treatment.

	Changes (%)
Cells	Before	After
CD4+CD25+	8·95 ± 2·89%	10·80 ± 3·00%*
CD4+CD25high	0·49 ± 0·33%	0·64 ± 0·33%†
CD4+FoxP3+	12·69 ± 4·87%	11·39 ± 5·81%‡

^*Compared with before treatment, P = 0·061;

^†compared with before treatment, P = 0·024;

^‡compared with before treatment, P = 0·51. FoxP3, forkhead box P3.

Discussion

The immune system has evolved a number of mechanisms to maintain peripheral immune tolerance and to protect against autoimmunity. There is clear evidence that CD4⁺CD25⁺ T_regs is an important component of immune system in controlling autoimmunity. These naturally occurring T cells can control actively and dominantly the activation and function of autoreactive T cells that have escaped from the thymus and can prevent development of the autoimmune disease [1,10]. Recent evidence indicates that CD4⁺CD25⁺ T_regs may even cure certain autoimmune disorders, such as inflammatory bowel disease in animal models [11]. A number of reports have shown that there are relationships between multiple autoimmune diseases and the deficiency of the number or function of T_reg cells. It is generally believed that FoxP3 is a special marker for T_reg cells; it has been identified as a master gene for the cell-lineage commitment, development and function of T_reg cells [12]. The current dogma still maintains that CD4⁺CD25⁺ T cells have a regulative function only when expressing FoxP3.

Systemic lupus erythematosus is a complicated autoimmune disease that has characteristic immunopathogenesis. Although there have been some reports concerning T_reg cells in SLE, the results were inconsistent [13–18]. Liu [13] and Crispin [14] showed that the number of CD4⁺CD25⁺ T_reg decreased, while Alvarado-Sanchez [16] and Suarez [17] found no alteration in the number in SLE patients. Alvarado-Sanchez [16] showed that suppressive function of T_regs in one-third of patients was impaired, whereas Miyara [15] demonstrated that the number instead of the function of CD4⁺CD25⁺ T_reg in active SLE patients was impaired, and Valencia [18] reported that CD4⁺CD25⁺ T_regs from active SLE patients had a reversible defect in suppressive function. One of the reasons for the inconsistency might be related to the treatment received by SLE patients before they were enrolled into the study. To exclude the interference of various treatments, in this study we have examined a possible role of CD4⁺CD25⁺FoxP3⁺ T cells in the pathogenesis and development of SLE by studying patients with new-onset SLE in the absence of treatment. Several novel conclusions can be drawn from the current study. First, patients with active, but not inactive, SLE exhibit a dramatic decrease in the CD4⁺CD25^high subset, although total CD4⁺CD25⁺ T cells are also reduced, as reported previously [14,15]. As CD25 expression also increases in activated/effector T cells after antigen stimulation, not all CD4⁺CD25^high T cells have a regulatory function. We attempted to determine the level of T_reg cells in SLE patients by examining the expression of FoxP3. Our results, however, indicate surprisingly that CD4⁺FoxP3⁺ T cells increase in the patients with SLE, irrespective of disease status. Analysis of those CD4⁺FoxP3⁺ T cells in the patients reveals that the majority of FoxP3⁺ T cells fail to express CD25. Because it has been demonstrated in vitro that FoxP3 can be up-regulated in human CD4⁺CD25^– T cells upon stimulation [19], the increased FoxP3⁺ T cell frequency in SLE patients is due possibly to the induced FoxP3 expression by autoimmunity in vivo. Another possibility is that CD4⁺CD25⁺ T_reg cells could lose CD25 without affecting FoxP3 expression, which has been confirmed in mouse T_reg cells [20].The functional significance of these CD25^–FoxP3⁺ T cells is unknown, however, and is under intensive investigation.

The most significant observation is the reduction of the levels of FoxP3 expression in the CD4⁺CD25^high T subset in untreated patients with SLE. The finding suggests that levels of FoxP3 expression in CD4⁺ T cells in humans might be associated with the regulatory activity of CD4⁺CD25⁺ T cells. In this regard, recent evidence indicates that reduction of levels of FoxP3 in natural CD4⁺CD25⁺ T_regs abrogates their suppressive activity [21,22]. The down-regulation of FoxP3 expression in the CD4⁺CD25^high subset in patients with SLE may reflect the situation in human disease which might be associated with the deficiency of CD4⁺CD25⁺ T_reg function in maintaining peripheral immune tolerance, and consequently pathogenesis, of SLE. Although the underlying mechanisms that account for the down-regulation of FoxP3 expression in CD4⁺CD25⁺ T_regs in lupus is unknown, the fact of high levels of IL-6 in the lupus patients [23,24] and the recent evidence of IL-6 down-regulation of FoxP3 in CD4⁺ T cells may provide a molecular clue to help disclose the molecular events [25]. Lastly, treatment with glucocorticosteroid enhances significantly levels of the CD4⁺CD25^high subpopulation in SLE patients. The relationship between the restoration of CD4⁺CD25^high percentage and the regulatory activity of T_regs remains to be determined.