Immunoregulation therapy changes the frequency of interleukin (IL)-22⁺CD4⁺ T cells in systemic lupus erythematosus patients

Abstract

T cell and T cell-related cytokine abnormalities are involved in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study showed that the interleukin (IL)-22⁺CD4⁺T cells and IL-22 play an important role in the pathogenesis of SLE. In this study, we aimed to investigate the effects of glucocorticoids (GCs) and immunodepressant agents on IL-22 and IL-22-producing T cell subsets in SLE patients. The frequencies of peripheral blood T helper type 22 (Th22), IL-22⁺Th17, IL-22⁺Th1 and Th17 cells and the concentrations of serum IL-22, IL-17 and interferon (IFN)-γ in SLE patients receiving 4 weeks of treatment with cyclophosphamide (CYC), methylprednisolone and hydroxychloroquine (HCQ) were characterized by flow cytometry analysis and enzyme-linked immunosorbent assay (ELISA). The frequencies of Th22, IL-22⁺ Th17 and Th17 cells and the concentrations of IL-22 and IL-17 were reduced in response to the drugs methylprednisolone, cyclophosphamide and hydroxychloroquine for 4 weeks in the majority of SLE patients. However, the percentage of Th1 cells showed no change. No differences in the levels of IL-22 and IL-22⁺CD4⁺ T cells were found between non-responders and health controls either before or after therapy. IL-22 levels were correlated positively with Th22 cells in SLE patients after treatment. These results suggest that elevated IL-22 is correlated with IL-22⁺CD4⁺T cells, especially Th22 cells, and may have a co-operative or synergetic function in the immunopathogenesis of SLE. GC, CYC and HCQ treatment may regulate the production of IL-22, possibly by correcting the IL-22⁺CD4⁺T cells polarizations in SLE, thus providing new insights into the mechanism of GC, CYC and HCQ in the treatment of SLE.

Introduction

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that causes tissue and organ damage and is characterized by abundant autoantibody production, complement activation and immune complex deposition. Proinflammatory cytokines produced by abnormal T helper (Th) cells have been shown to be involved in the pathogenesis of many immune diseases, including SLE 1. Interleukin (IL)-22 is a member of the IL-10 family, produced by special immune cell populations, including Th22, Th1 and Th17 cells, natural killer (NK) cells, NK T cells and lymphoid tissue inducer cells 2–6. Like all members of the IL-10 family, IL-22 exerts its biological effects via members of the cytokine receptor family class 2. The cell surface IL-22 receptor complex is a heterodimeric receptor consisting of IL-22R1 and IL-10R2 chains 7. Considerable evidence suggests that IL-22 plays an important role in the pathogenesis of autoimmune and inflammatory diseases 8–10. IL-22 is produced mainly by CD4⁺T cells, including Th22, Th17 and Th1 cells. Th1 cells have been associated with the development and progression of SLE 11. Th17 cells are inflammatory CD4⁺ T cells that produce IL-17A, but not interferon (IFN)-γ 12. These cells and their secreted cytokines are found to be elevated in SLE 13. The Th22 subset is a more recently identified CD4⁺ T helper subset that is characterized by the secretion of IL-22, but not IL-17 or IFN-γ 5,14,15. Th22 cells show distinct differences in the profile of altered genes compared to other T cells such as Th1, Th2 and Th17 cells, confirming an individual signature for the Th22 subset 14. Our previous study showed that the percentages of Th22, IL-22-producing Th17 and IL-22-producing Th1 cells and plasma IL-22 levels were increased in patients with SLE relative to healthy controls 16. These observations demonstrated that increased IL-22 and the main IL-22-producing CD4⁺ T cells may be involved in the process of these autoimmune diseases.

The main treatment of autoimmune diseases is immunoregulation. The standard treatments for SLE are based on glucocorticoids (GCs) and immunodepressant agents. GCs remain the cornerstone of the treatment for SLE, despite advances in immunosuppressive drugs, therapeutic protocols and development of new drugs. GCs could correct Th1 polarization by altering the Th1/Th2 cytokine profile 17. Cyclophosphamide (CYC) is a well-established immunodepressant known to influence cell cycle and DNA synthesis. Previous research has indicated that CYC can suppress both humoral and cellular immunity. However, the impact of GCs and CYC on IL-22 production in SLE remains unclear. Hydroxychloroquine (HCQ) has been demonstrated to inhibit proinflammatory cytokine production by macrophages and antigen presentation, although the potential effects on T cells remain elusive 18.

In the present study, we extended our observations and analysis of the dynamic changes in IL-22-producing CD4⁺ T cells in the same patients as our previous study 16 and investigated whether immunoregulation therapy has an effect on the production of IL-22. We compared plasma IL-22 levels and the frequency of IL-22-producing CD4⁺ T cells before and after treatment in SLE patients. This was a prospective study.

Materials and methods

A total of 22 new-onset SLE patients were recruited from the in-patient service of the First Hospital of Jilin University between March and October 2011. Eighteen gender- and age-matched healthy volunteers were recruited from the out-patient service of the same hospital (the same patients and healthy volunteers as described in Zhao et al. 16). Individual patients with SLE were diagnosed according to the diagnostic criteria of the American College of Rheumatology 19. The degrees of disease activity in SLE patients were assessed using the SLE disease activity index (SLEDAI), and a score of ≥6 was defined as active disease 20. Subjects were excluded if she/he had a history of myositis, systemic sclerosis or other autoimmune diseases, a recent infection or had received immunosuppressive or GC therapy within the past 6 months. After blood samples were taken, all enrolled patients with SLE received 4 weeks of intravenous drip treatment with 0·4 g/week of CYC 1·0 mg/kg daily of methylprednisolone and oral treatment with 0·2 g twice daily of HCQ. After treatment, blood samples were taken again. We used the SLEDAI to evaluate the response of the patients to the drugs and divided SLE patients into two groups: the drug-response group (SLEDAI < 6·0) and the drug-non-response group (SLEDAI ≥ 6·0).

The First Hospital Ethical Committee of Jilin University approved the study protocol (no. 2011-008). All subjects gave written informed consent prior to study enrolment. The demographic and clinical characteristics of these enrolled subjects are shown in Table 1.

Table 1.

Treatment with glucocorticoid and immunosuppressive agents modulates the clinical profiles of systemic lupus erythematosus (SLE) patients.

Characteristics	Response group (n = 17)		Non-response group (n = 10)		Healthy controls (n = 18)
	Before treatment	After treatment	Before treatment	After treatment
Male/female	2/15	2/15	1/9	1/9	2/16
Age (years), median (range)	23 (18–29)	23 (18–29)	26 (20–32)	26 (20–32)	25 (17–30)
SLEDAI, median (range)	19 (9–38)	4 (2–5)*	12 (2–24)	9 (6–15)	n.d.
Positive anti-dsDNA	15 (88%)	5 (29%)	9 (90%)	7 (70%)	n.d.
Positive anti-Sm	4 (24%)	2 (12%)	5 (50%)	4 (40%)	n.d.
Positive anti-nuclear antibodies	15 (88%)	7 (41%)	8 (80%)	5 (50%)	n.d.
CRP (mg/l)	11·25 (2·99–117)	10·74 (1·59–20·42)	24·45 (3·73–202)	15·25 (2·19–18)	7·2 (0–15)
ESR (mm/h)	5·68 (3·29–7·91)	2·5 (2·32–5·34)*	6·89 (4·10–9·24)	5·63 (3·79–8·73)	3 (0–5)
C3 (units/ml)	0·47 (0·36–0·95)	1·2 (0·79–1·90)*	0·54 (0·38–0·96)	0·96 (0·48–1·63)*	1·12 (0·9–1·8)
C4 (units/ml)	0·08 (0·02–0·14)	0·15 (0·08–0·4)*	0·06 (0·02–0·13)	0·08 (0·05–0·10)	0·26 (0·1–0·4)
WBC (×109/ml) median (range)	3·98 (3·68-9·60)	4·23 (3·70–18·8)	6·77 (3·19–9·18)	11·74 (7·59–15·9)*	6·31 (4·09–9·13)

^*P < 0·05 versus before treatment. The normal range of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) was 0–15 mg/l and 0–5 mm/h, respectively. The normal range of C3 and C4 was 0·9–1·8 and 0·1–0·4 units/ml, respectively. SLEDAI = systemic lupus erythematosus disease activity index; n.d. = not determined; WBC, white blood cells.

Data collection

The baseline demographic and clinical data were collected from hospital records and reviewed by experienced physicians. The data included age, sex and current medications. Routine laboratory investigation included full blood count, the levels of serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). The levels of serum anti-dsDNA and anti-Sm were determined by indirect immunofluorescence using special kits, according to the manufacturer's instructions (Oumeng, Beijing, China). The levels of serum C3, C4, CRP and ESR were determined by scatter turbidimetry using a Siemens special protein analyser (Siemens Healthcare Diagnostics Products, GmbH, Germany).

Isolation and stimulation of peripheral blood mononuclear cells (PBMCs)

Fasted peripheral venous blood samples were collected from individual participants, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK). PBMCs (10⁶ cells/ml) were stimulated in duplicate with phorbol 12-myristate 13-acetate (PMA, 1 μg/ml) and ionomycin (50 μg/ml; Sigma, St Louis, MO, USA) in 10% human sera (AB type) in RPMI-1640 medium at 37^°C in a humidified incubator with 95% air and 5% carbon dioxide for 4 h and cultured for another 2 h in the presence of brefeldin A (BFA, 0·5 μg/ml; Sigma). Another portion of the cells was used as a negative control and cultured in medium without PMA, BFA and ionomycin.

Flow cytometry analysis

The stimulated PBMCs were harvested and stained with peridinin chlorophyll (PerCP)-anti-CD4 (Becton Dickinson, San Diego, CA, USA), followed by fixing with 4% paraformaldehyde (30 min at room temperature) and then permeabilization with 0·5% saponin in 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) (30 min at room temperature). After washing, the cells were stained with fluorescein isothiocyanate (FITC)-anti-IFN-γ, Alexa-Fluor-anti-IL-17 (Becton Dickinson) and phycoerythrin (PE)-anti-IL-22 (R&D Systems, Minneapolis, MN, USA). The proportion of cytokine⁺ T cells was determined by flow cytometry analysis on a BD fluorescence activated cell sorter (FACS)Calibur (Becton Dickinson) using FlowJo version 7·6.2 software (Ashland, OR, USA).

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of plasma IFN-γ, IL-17 and IL-22 in individual participants were determined by ELISA using specific cytokine kits according to the manufacturer's instructions (R&D Systems).

Statistical analysis

All data are expressed as individual values, median and range of each group of subjects. The difference between SLE patients and healthy controls was analysed using the Mann–Whitney U-test. The difference between before and after treatment in the same group was analysed using a paired t-test. The potential correlation between variables was analysed by Spearman's rank correlation test. All statistical tests were performed using spss 19·0 for Windows (SPSS, Inc., Chicago, IL, USA). A two-sided P-value of <0·05 was considered statistically significant.

Results

Reduced Th22 cells correlated with reduced plasma levels of IL-22 in drug-response SLE patients after treatment

SLE patients received drug treatments for 4 weeks. Syndromes of some patients were significantly lightened; however, some were not. Our previous studies showed that IL-22⁺CD4⁺ T cells correlated positively with SLEDAI values. Therefore, we used the SLEDAI to evaluate the response of the patients to the drugs. We divided SLE patients into two groups: the drug-response group (SLEDAI < 6·0) and the drug-non-response group (SLEDAI ≥ 6·0). In the response group, 4 weeks of treatment led to a significant decrease of Th22 cells over the baseline (pretreatment) level (P < 0·0001), similar to that observed in normal controls (P = 0·3909) (Fig. 1). Moreover, the serum levels of IL-22 were also reduced significantly after treatment compared to before treatment (P = 0·0366) and similar to that observed in normal controls (P = 0·3909) (Fig. 2). Further analysis indicated that the percentages of Th22 cells were correlated positively with the concentrations of plasma IL-22 in the response after treatment SLE patients (P = 0·0061, R = 0·4260) (Fig. 2). However, no differences in the levels of IL-22 were found between non-responders and health controls either before or after therapy (P > 0·05) (data not shown).

Fig. 1.

Flow cytometry analysis of the frequency of interleukin (IL)-22⁺CD4⁺ T cells in drug-response patients with systemic lupus erythematosus (SLE) (before and after treatment) and healthy controls. Peripheral blood mononuclear cells (PBMCs) from drug-response patients with SLE before (0 weeks) and after (4 weeks) treatment with glucocorticoids (GC), cyclophosphamide (CYC) and hydroxychloroquine (HCQ) and healthy controls were stimulated ex vivo with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A (BFA) for 6 h. The cells were stained with peridinin chlorophyll (PerCP)-anti-CD4. After being fixed and permeabilized, the cells were stained with fluorescein isothiocyanate (FITC)-anti-interferon (IFN)-γ, Alexa-Fluor-anti-IL-17 and phycoerythrin (PE)-anti-IL-22 and analysed by flow cytometry. The cells were first gated on lymphocytes (R1) and then gated on IFN-γ^–CD4⁺ T cells (R2) to characterize the frequency of T helper type 22 (Th22) (IL-22⁺IL-17^– IFN-γ^–CD4⁺), IL-22⁺IL-17⁺IFNγ^-CD4⁺ or Th17 (IL-22^–IL-17⁺IFNγ^–CD4⁺) cells or on IL-17^–CD4⁺ T cells (R3) to analyse the frequency of IL-22⁺IFN-γ⁺IL-17^–CD4⁺ T cells. Data are representative charts and expressed as individual values of 17 drug-response patients and 18 controls. (a) Flow cytometry analysis of the frequency of cells gated on lymphocytes. (b) Flow cytometry analysis of the frequency of cells gated on PerCP-anti-CD4⁺ and FITC-anti-IFN-γ^-; (c–e) flow cytometry analysis of the frequency of Th22 (I), IL-22⁺IL-17⁺ IFNγ^–CD4⁺ (II) or Th17 (III) cells in drug-response SLE (C: 0 weeks, D: 4 weeks) and healthy controls (e); (f) flow cytometry analysis of the frequency of cells gated on PerCP-anti-CD4⁺ and Alexa-Fluor-anti-IL-17^-; (g–i) flow cytometry analysis of the frequency of IL-22⁺IFNγ⁺ IL-17^-CD4⁺ T cells (IV) in drug-response SLE (g: 0 weeks, h: 4 weeks) and healthy controls (i); j: quantitative analysis.

Fig. 2.

The levels of plasma interleukin (IL)-17 and IL-22 levels and their relationships with IL-22⁺CD4⁺ T cells. The levels of plasma IL-17 and IL-22 in individual participants were determined by enzyme-linked immunosorbent assay (ELISA) and the potential association between the levels of plasma IL-17 and IL-22 and the frequency of IL-22⁺CD4⁺ T cells in CD4⁺ T cells in drug-response patients after treatment was analysed by Spearman's rank correlation test. Data shown are mean values from individual participants from three separate experiments. (a) The levels of plasma IL-22; (b) the levels of plasma IL-17; (c) the correlation analysis (n = 17). There was no significant association between the levels of plasma IL-22 and the frequency of IL-17⁺CD4⁺ T cells in CD4⁺ T cells and between the levels of plasma IL-17 and the frequency of IL-22⁺CD4⁺ T cells in CD4⁺ T cells in drug-response patients with systemic lupus erythematosus (SLE) after treatment (data not shown).

Reduced percentage of IL-22⁺Th17 and IL-22⁺Th1 cells in drug-response SLE patients after treatment

As Th17 and Th1 cells also can produce IL-22, we further determined the frequency of different kinds of IL-22⁺CD4⁺ T cells. Further analysis indicated that the percentages of IL-22⁺ Th17 cells and IL-22⁺ Th1 cells in drug responders were decreased significantly relative to that observed in responders before therapy (P = 0·03, P = 0·0464) (Fig. 1), but higher than that observed in normal controls (P = 0·0422, P = 0·0111) (Fig. 1). However, there was no significant difference in the percentages of IL-22⁺ Th17 cells and IL-22⁺ Th1 cells before and after treatment with drugs in drug-non-responding patients and healthy controls (P > 0·05) (data not shown).

Reduced percentage of Th17 and levels of serum IL-17 in drug-response SLE patients after treatment

Given that Th1 and Th17 cells have been associated with the development and progression of SLE, we also explored the frequencies of Th1 and Th17 cells and the levels of serum IFN-γ and IL-17. Interestingly, we found that the percentage of Th17 cells was reduced significantly in drug-response patients compared with the baseline values (P = 0·0008), but higher than healthy controls (P = 0·0040) (Fig. 1), accompanied by significantly reduced levels of serum IL-17 in those patients (P = 0·0027) (Fig. 2). However, there was no significant difference in the percentage of Th1 cells and in the level of serum IFN-γ before and after treatment with drugs in those drug-responding patients and healthy controls (P > 0·05) (data not shown). In addition, there was no significant difference in the percentages of Th1 and Th17 cells and in the levels of serum IFN-γ and IL-17 before and after treatment with drugs in the drug-non-responding patients and healthy controls (P > 0·05) (data not shown). Collectively, combined GC, CYC and HCQ treatment dramatically improved clinical symptoms, which was associated with a reduction in the frequency of IL-22⁺ and IL-17⁺ Th cells in the patients.

Discussion

Previous studies suggested that IL-22 can play either a protective or a pathogenetic role in chronic inflammatory disorders and autoimmune diseases. Our previous results demonstrated that IL-22 expression and the number of IL-22-positive CD4⁺ cells were increased in the blood of patients with SLE relative to normal controls 16. The GCs and immunodepressant agents are in routine clinical use to treat SLE. GCs could correct Th1 polarization by altering the Th1/Th2 cytokine profile 17. Treatment with GCs reduces the frequency of peripheral blood IL-22 T cells and the levels of plasma IL-22 in patients with acute bacterial infection 21. High-dose dexamethasone reduced IL-22 production in immune thrombocytopenia 22. CYC is a well-established immunodepressant known to influence cell cycle and DNA synthesis. Previous research has indicated that CYC can suppress both humoral and cellular immunity. Treatment of SLE patients with HCQ has been reported to decrease serum levels of IL-6, IL-18 and TNF-α 23. However, the impact of GCs combined with CYC and HCQ on IL-22 production in SLE remains unclear.

Our approach in this study was to evaluate the levels of serum IL-22 and the frequency of different subsets of peripheral blood CD4⁺ cells in SLE patients with a 4-week treatment. We found that after treatment with GC, CYC and HCQ there were 17 drug-responders and 10 non-responders. This may be because SLE is a heterogeneous and complex autoimmune disease with patients showing different symptoms, which may lead simultaneously to the distinct response to the drug. Importantly, we found that plasma IL-22 levels in responders were decreased significantly relative to those observed in responders before therapy and similar to those observed in normal controls. However, no differences in the levels of IL-22 were found between non-responders and health controls either before or after therapy. These results indicate that IL-22 appears to be related to the pathogenesis of SLE, and treatment with GC, CYC and HCQ can reduce the abnormal increased IL-22. In addition, the two subgroups were divided based on SLEDAI score, which indicated that the changes of IL-22 may be related to SLEDAI. These findings were consistent with our previous results.

The mechanism by which GC, CYC and HCQ reduces IL-22 is speculative at this point. Abnormalities in cellular immunity have been observed widely in SLE. It has been reported that an increased Th1/Th2 ratio in the peripheral blood appears to be a key pathogenic mechanism in SLE 11. In addition, recent studies have indicated that up-regulation of Th17 cells may be an important determinant in the evolution of SLE 13. Our previous data suggested that elevated IL-22 levels play an important role in the pathogenesis of SLE 16. Existing research shows that IL-22 expression is found in activated Th cells, including Th1, Th17 and Th22 cells 24. In the present study, we investigated the percentages of the major IL-22-producing Th cell populations and their cytokines before and after GC, CYC and HCQ treatment. Our results indicated that all IL-22⁺CD4⁺ T cells, including Th22, IL-22⁺Th17 and IL-22⁺ Th1 cells, were reduced in the drug-response SLE patients. A positive correlation between plasma IL-22 levels and the percentage of Th22 cells was found in drug responders. Thus, our findings indicate that the reduced IL-22 levels after treatment in drug responders may be correlated mainly with the decreasing numbers of IL-22⁺CD4⁺ T cells, especially Th22 cells. These results were consistent with our previous data, in which marked up-regulation of IL-22⁺CD4⁺ T cell percentages relative to healthy controls and a positive correlation between plasma IL-22 levels and the percentage of Th22 cells were found in untreated SLE patients. Together, these results showed that IL-22 and IL-22⁺CD4⁺ T cells, especially Th22 cells, may play an important role in the pathogenesis of SLE. Consistent with previous studies, significantly reduced levels of serum IL-17 and percentages of Th17 cells were found in the drug-response patients compared with the baseline values. These results suggest that IL-22⁺CD4⁺ T cells and polarization is a potential indicator of response to GC, CYC and HCQ in patients with SLE. Although IL-22⁺ Th1 cells were reduced in the drug-response SLE patient group, there was no significant difference in the percentages of Th1 cells and in the levels of serum IFN-γ before and after treatment with drugs in those patients and healthy controls. These results indicate that IL-22⁺ Th1 cells, but not Th1 cells, may play an important role in the pathogenesis of SLE. It is also possible that the drug treatment duration was too short to observe changes in Th1 and IFN-γ.

Impaired apoptosis of T cells has been reported previously to be involved in the development of autoimmunity. A study by van Loosdregt et al. [25] indicated that HCQ treatment will preferentially induce apoptosis in these self-reactive cells while only mildly affecting the naive T cell repertoire. In addition, GCs can correct Th polarization by altering the cytokine profile and CYC can suppress both humoral and cellular immunity by influencing cell cycle and DNA synthesis 17. Therefore, we hypothesize that GCs combined with CYC and HCQ may correct the abnormal Th subsets in SLE and mildly affect the naive T cell repertoire. These data provide a novel mechanism by which autoimmunity can be modulated by using GCs combined with CYC and HCQ as a promising therapeutic target.

The study by van Loosdregt et al. indicated that because SLE is a heterogeneous and complex autoimmune disease, several abnormalities may be involved in the cellular mechanisms of immune modulation. The precise mechanisms behind drug therapy merit further study. In addition, the impact of other corticosteroids on IL-22 production, such as dexamethasone, remains to be confirmed further.

Taken together, the present data show that elevated IL-22 is correlated with IL-22⁺CD4⁺ T cells, especially Th22 cells, and may have a co-operative or synergetic function in the immunopathogenesis of SLE. GC, CYC and HCQ treatment may regulate the production of IL-22, possibly by correcting the IL-22⁺CD4⁺ T cell polarizations in SLE, thus providing new insights into the mechanism of GC, CYC and HCQ in the treatment of SLE.

Disclosure

The authors declare that there are no conflicts of interest.