IL-22 production of effector CD4+ T-cells is altered in SLE patients

Background Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T-cell-dependent B-cell activation and altered T-cell response. Co-stimulatory and co-inhibitory molecules regulate and exert T-cell differentiation, survival and cytokine production. CD134+ and PD-1+ T-cells in SLE patients are increased in SLE. The aim of this study was to characterize CD134+ and PD-1+CD4+ T-cells according to their ability to produce IFN-γ, IL-21 and IL-22 in SLE patients. Methods Peripheral blood of 39 SLE patients and 19 healthy controls (HC) was stimulated with phorbol myristate acetate (PMA) calcium ionophore (Ca-Io). The expression of IFN-γ, IL-21 and IL-22 T-cells within the CD134+ and PD-1+ T-cells was analyzed by flow cytometry. Disease activity was assessed by SLE Disease Activity Index. Results Peripheral unstimulated CD134+ and PD-1+ CD4+ T-cells were significantly increased in patients with lupus nephritis. Upon stimulation both, CD134+ and PD-1+ CD4+ T-cells, produced significantly less IFN-γ in SLE patients as compared to HC. The percentages of IL-22 within the CD134+CD4+ T-cells were also significantly decreased in SLE as compared to HC. Conclusion CD134+ and PD-1+CD4+ T-cells have mainly a Th1 effector T-cell signature. A lower proportion produces also IL-21 and IL-22. The impaired capacity to produce IFN-γ and IL-22 in SLE patients may contribute to the pathogenesis of the disease.


Background
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology. The presence of antibodies against dsDNA is a hallmark of SLE. Although the precise pathogenesis of SLE has not been fully elucidated, T-cell-dependent B-cell hyperactivity contributes to the inflammatory pathology of SLE [1]. Crucial for antibody formation is the interaction between CD4+ T-cells and B-cells particularly in germinal centers (GCs), the site of affinity maturation followed by the subsequent generation of memory B-cells and long-lived plasma cells. Recently, the novel cytokine Interleukin (IL)-21 has been found to play a pivotal role in the differentiation and function of B-cells [2,3].
Beside the activation of B-cells via co-stimulatory molecules, T-cells exert effector functions by the production of proinflammatory cytokines. It is commonly accepted that effector lineages can be divided into Th1, Th2 and Th17 cells according to their characteristic cytokines. Effector functions, including cytokine production, expansion and survival, are enhanced by co-stimulatory molecules such as CD134 (OX40) [4,5]. Previously, we demonstrated an increased expression of the co-stimulatory molecule CD134 (OX40) on T-cells in systemic lupus erythematosus (SLE) patients especially with lupus nephritis [6]. Interestingly, also co-inhibitory receptors such as PD-1 were increased in CD4 + CD25 neg T-cells of SLE patients [7]. Both the co-stimulatory molecule CD134 and co-inhibitory molecule PD-1 are expressed on activated T-cells and have been demonstrated to be crucial for the development of follicular T-helper-cells (T FH ) [2]. Zander et al. demonstrated a crosstalk between PD-1 and CD134 (OX40) regarding T-helper-cell differentiation and anti-plasmodium humoral immunity in humans [8].
The CD134/OX40L axis is pivotal for the CD4 + differentiation toward effector T-cells. On the other hand, T-cell activation can be dampened by PD-1/PD-1L interaction [9,10]. Interestingly, Kasagi et al. found that, in NZB/W F1 mice, a model for lupus nephritis (LN), PD-1 was predominantly expressed on kidney-infiltrating CD4 + T-helper cells. After blocking PD-1 with anti-PD-1 monoclonal antibodies (mAbs), there was a significant reduced mortality in the mice [11].
More recently, follicular T-helper-cells (T FH ) were discovered characterized by IL-21 production. Several murine models indicate a crucial role of IL-21 in the pathogenesis of autoimmune diseases. Murine lupus models (BXSB-Yaa mice) were also investigated [12]. Interestingly, BXSB-Yaa mice which are IL-21R deficient show less lupus-like symptoms as compared to their wild-type BXSB-Yaa mice. Human studies provide further evidence that the IL-21/IL-21R pathway plays a major role in the pathogenesis of autoimmune diseases, in particular in SLE.
CD134 and PD-1 have been described as characteristic phenotypic molecules on IL-21-producing T FH -cells. Since we found that the effector T-cell response is altered as compared to HC, we investigated the cytokine pattern of CD134 + and PD-1 + T-cells in SLE. Therefore, the present study aims to investigate the ability of CD134 + and PD-1 + CD4 + T-cells of human SLE patients to produce effector cytokines IFN-γ, IL-21 and IL-22 ex vivo. To investigate this, whole blood was stimulated ex vivo and the percentages of IFN-γ, IL-21 and IL-22 positive T-cells were analyzed.

Study population
39 SLE patients with a mean age of 40 ± 14 years fulfilling at least four of the American College of Rheumatology revised criteria for SLE and 19 age and sex matched healthy controls (age 36 ± 12 years) were enrolled in this study [13]. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Thirty-two patients had inactive disease (SLEDAI score ≤ 4) and seven patients had active SLE (defined as SLEDAI score > 4). Median (range) disease activity for all patients was 2 (0-28). Thirteen patients had a biopsy-proven lupus nephritis. Followup visits were analyzed in six patients. Time interval between these two assessments was 9 ± 6 months. One patient did not receive any immuno-modulating medication at the time of analysis. Thirty-eight patients received immuno-modulating medication (Table 1).

Sample preparation and in vitro stimulation
Methods were performed as described before [14]. In brief, sodium-heparinized venous blood was obtained from patients and healthy donors. Immediately after sampling, 200 µl blood was mixed with 200 µl RPMI1640 (Gibco Life Technologies, Paisley, UK), supplemented with 50 μg/ml gentamycin (Gibco, Scotland, UK), and aliquoted into 5-ml polypropylene tubes (BD Biosciences) (400 μl per tube). To determine the frequency of T-cell subsets, diluted whole blood was stimulated 4 h with 40 nM phorbol myristate acetate (PMA; Sigma-Aldrich, Steinheim, Germany) and 2 nM calcium ionophore (Ca-Io; Sigma-Aldrich) in the presence of 3 μM Brefeldin A. Brefeldin A was used to block intracellular transport mechanisms, thereby leading to an accumulation of cytokines in the cell. As a negative control, one sample remained without stimulation. Next, culture tubes were incubated at 37 °C, 5% CO 2 .

Flow cytometric analysis
The data were analyzed using Kaluza 1.2 flow analysis software (Beckman Coulter).
As stimulation reduces surface expression of CD4 on T-cells, CD4 + T-cells were identified indirectly by gating on CD3-positive and CD8-negative lymphocytes [15]. The unstimulated samples were used as a guide for setting the linear gates to delineate positive and negative populations for the cytokine staining. For surface staining, isotype controls served as controls.

Statistical analysis
Data are presented as mean ± SD unless stated otherwise. The nonparametric Mann-Whitney U test was used to compare data from SLE patients with that of healthy controls, and differences were considered statistically significant at two-sided p values less than 0.05. Paired samples were tested using the Wilcoxon signed rank test. Correlation analysis was performed using Spearman's rank correlation coefficient.

Influence of immunosuppressive treatment
To analyze the cytokine-secreting capacity of CD134 + and PD-1 + CD4 + T-cell subsets dependent on the immunosuppressive treatment, we stratified the patients in two groups. We compared patients who received MMF to patients with an immunosuppressive regimen without MMF. There was no significant difference between these groups regarding effector cytokine production (IFN-γ, IL-21 and IL-22) within CD134 + and PD-1 + CD4 + T-cells (Fig. 5a). To investigate the role of prednisone, we correlated the daily dose of prednisone in mg/day with these T-cell subsets. There was no significant correlation found in these groups (Fig. 5b).

Discussion
The present study confirms our previous observation that CD134 + CD4 + T-cells are increased in the peripheral blood of patients with systemic lupus erythematosus as compared to HC [6]. The proportion is even more increased in patients with lupus nephritis and has been found in inflamed tissue of lupus nephritis biopsies [16]. The observation by Aten et al. that CD134L is abundantly present in proliferative lupus nephritis predominantly along the epithelial side of the glomerular capillary wall highlights the CD134-CD134L pathway as a potential therapeutic target [17]. In this study, we could also find increased expression of PD-1 on peripheral CD4 + T-cells in lupus nephritis patients. A tendency of increased expression of PD-1 on peripheral CD4 + T-cells was found in our previous study which was significant different most likely due to the lower numbers of LN patients included (n = 7) in contrast to 13 patients in the present study [7]. The significance of CD134 for effector functions has been shown by the observation that treatment with a stimulatory anti-CD134 antibody enhances T-cell expansion and differentiation to effector cells in mice [18,19]. This stimulation, apparently, promotes the secretion of IFN-γ and the upregulation of various interleukin (IL)receptors which might lead subsequently to cytokinemediated kidney cell damage [20]. Interestingly, upon stimulation CD134 + and PD-1 + CD4 + T-cells produce significant lower amounts of IFN-γ in SLE patients as compared to HC. This decrease was not associated with lupus nephritis or active disease. Notwithstanding, immunosuppressive treatment could have impact on the capacity to produce effector cytokines. In our present study there was no correlation between the percentages and the daily dose of glucocorticoids. Patients with more intense immunosuppressive treatment did also not show significantly lower capacities to produce IFN-γ. However, individualized immunosuppressive treatment regimens may hamper the interpretation. Another limitation of the present study is the analysis of stimulated CD3 + CD8 − T-cells. This population consists mainly of CD4 + T-cells but significant proportions of γδ T-cells have been reported in particular after treatment [21]. The percentages of IFN-γ and IL-17 within this subset are relatively low. Thus, the exclusion of this subset would probably not change the current results significantly.
Both CD134 + CD4 + T-cell and PD-1 + CD4 + T-cell subsets were characterized by a Th1 cytokine pattern. IL-21 production was surprisingly not different between SLE patients and HC and relatively low with about 15%. This may indicate that CD134 and PD-1 T-cells express IL-21 + in the germinal center (GC) but in peripheral blood the main cytokine response is IFN-γ.
In contrast to our results, Jacquemin et al. reported that IL-12 signals cooperate with OX40 signals to increase the expression of CXCR5 and IL-21 by memory Th-cells [22].
Th22 cell are described as a novel T-cell lineage producing the signature cytokine IL-22. IL-22has been shown to be increased in the peripheral blood of SLE patients with sole lupus skin disease and to be decreased in lupus nephritis patients [23]. The present study reveals also a decrease of IL-22 within the CD134 + CD4 + T-cells. Moreover, the mRNA levels of IL-22 were decreased in the urine of LN patients with proliferative glomerulonephritis and inversely correlated with the histological activity index [24]. The