Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity

Cytokine 72 (2015) 146–153

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Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity Roba M. Talaat a, Sara F. Mohamed a, Iman H. Bassyouni b,⇑, Ahmed A. Raouf c a

Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City University, Egypt Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, El-Kasr El-Aini Hospital, Cairo 12613, Egypt c Biochemistry Department, National Liver Institute (NLI), Menofia University, Egypt b

a r t i c l e

i n f o

Article history: Received 25 September 2014 Received in revised form 6 December 2014 Accepted 31 December 2014

Keywords: Cytokines Rheumatoid arthritis Systemic lupus erythematosus TH17 T-regulatory cells

a b s t r a c t Aim: Imbalance of T-helper-cell (TH) subsets (TH1/TH2/TH17) and regulatory T-cells (Tregs) is suggested to contribute to the pathogenesis of Systemic lupus erythematosus (SLE). Therefore, we evaluated their cytokine secretion profile in SLE patients and their possible association with disease activity. Methods: Sixty SLE patients, 24 rheumatoid arthritis (RA) patients and 24 healthy volunteers were included in this study. Demographic, clinical, disease activity and serological data were prospectively assessed. Plasma cytokines levels of TH1 (IL-12, IFN-c), TH2 (IL-4, IL-6, IL-10), TH17 (IL-17, IL-23) and Treg (IL-10 and TGF-b) were measured by enzyme linked immunosorbent assays (ELISA). Results: SLE patients were found to have significantly higher levels of IL-17 (p < 0.001), IL-6 (p < 0.01), IL12 (p < 0.001) and IL-10 (p < 0.05) but comparable levels of IL-23 and IL-4 and slight reduction (but statistically insignificant) of TGF-b levels compared to controls. IL-6, IL-10 and IL-17 were significantly increased (p < 0.05) with disease activity. The RA group exhibited significantly higher levels of plasma IL-4 (p < 0.01), IL-6 (p < 0.05), IL-17 (p < 0.001), IL-23 (p < 0.01) and TGF-b (p < 0.5) and lower IFN-c (p < 0.001) and IL-10 (p < 0.01) than those of healthy subjects. Conclusion: Our study showed a distinct profile of cytokine imbalance in SLE patients. Reduction in IFN-c (TH1) and TGF-b1 (Treg) with the elevation in IL-6 and IL-17 (TH17) could imply skewing of T-cells toward TH17 cells. Breaking TH17/Treg balance in peripheral blood may play an important role in the development of SLE and could be responsible for an increased pro-inflammatory response especially in the active form of the disease. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Autoimmune rheumatic diseases (ARDs) encompass a wide variety of diseases in which both innate and adaptive immune responses result in autoimmune-mediated tissue destruction. In total, ARDs affect approximately 5% of the population and result in substantial morbidity, and increased mortality. Among the ARDs, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) for which the natural history in humans is best understood [1,2]. SLE is a heterogeneous inflammatory chronic autoimmune disorder characterized by the deposition of immune complexes in different organs which may lead to multisystem involvement [3]. It has a progressive as well as relapsing/remitting nature in many patients and present as a mixture of mild skin or musculoskeletal ⇑ Corresponding author. Tel.: +20 123476767; fax: +20 2 37489467. E-mail addresses: [email protected], [email protected]. eg (I.H. Bassyouni). http://dx.doi.org/10.1016/j.cyto.2014.12.027 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.

and hematological signs and symptoms. It may also involve more serious central nervous system complications and organ damage [4]. Even though the etiology of SLE is unknown, many predisposing factors have been found, including genetic, environmental, infections, and hormonal factors [3]. The immune malfunction that leads to overt SLE is complex, but the pathological hallmarks of SLE alter immune responses to autoantigens with autoantibody production. It produces an endless source of antibodies with various specificities like [3,5,6]. This action is mediated by alterations in the production of some cytokines; complement activation; unrestricted B lymphocyte hyperactivity accompanied by immunoglobulin repertoire changes leading to overproduction of auto-antibodies and impaired cell mediated immunity, which results from both T lymphocyte and antigen presenting cell (APC) abnormalities [3,7]. CD4+ TH lymphocytes can be divided into TH1 (interferon (IFNc), IL-2, and tumor necrosis factor-b (TNF-b) and type 2 (IL-4, IL-5, IL-6, IL-10 and IL-13) subsets on the basis of their cytokine secretion profile [8]. However, human TH1 and TH2 subsets are usually

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defined according to IFN-c/IL-4 production because the synthesis of IL-2, IL-6, and IL-10 is not stringently restricted to a single subset [9]. In addition to well characterized TH1 and TH2 lymphocytes, naive CD4+ T-cells can be differentiated into TH17, a distinct subset of TH cells characterized by expression of the transcription factor (related orphan receptor gamma; RORct) [10]. TH17 cells secrete a profile of potent proinflammatory cytokines, including IL-17, IL21, IL-22 and potent TNF-a and IL-6 upon certain stimulation [11]. Recent studies indicated that IL-17 aids in coordinating tissue inflammation [10–12]. It has been hypothesized that TH17-cells, in particular, play a pivotal role in the initiation and development of autoimmunity. Several studies suggested a pathogenic role of TH17-cells in the progression of SLE [8,13,14]. Another subpopulation of CD4+ lymphocytes is T-regulatory (Tregs) cells. It is thought to suppress autoreactive effector T cells. At present, human Treg cells can be characterized by a CD4+CD25+ and transcription factor forkhead box P3 (FoxP3+) phenotype (CD4 + CD25 + FoxP3+). A decreased frequency and/or an impaired function of these cells might be involved in the development of autoimmune diseases [10,14]. Though the concept of the preventive role of Treg cells in autoimmunity is widely accepted, data regarding SLE are inconsistent [10,15]. Cytokine secretion of Treg that has been reported includes IL-10 and TGF-b [16]. Controversy still exists regarding the contribution of specific TH cell subsets and related cytokines in the pathogenesis of SLE. We hypothesized that measuring circulating cytokines may shed some light on the proposed imbalance between TH cell subsets in SLE. In this context, we conducted this case-control study in order to investigate plasma levels of TH1 (IL-12, IFN-c), TH2 (IL-4, IL-6, IL-10), TH17 (IL-17, IL-23) and Treg (IL-10 and TGF-b) cytokines. The correlations of these cytokines with the disease activity were also demonstrated.

2. Patients and methods 2.1. Study population Sixty consecutive SLE patients (56 women) who fulfilled the American College of Rheumatology (ACR) criteria for the diagnosis of SLE were included in the present study [17]. Their mean age was 28.58 ± 7.3 years (range 17–49 years); the mean duration of SLE was 4.97 ± 3.4 years (range 0.33–15 years). On the day of blood sampling, disease activity was assessed for all the patients using the SLE disease activity index (SLEDAI) [18]. Based on the disease activity [19,20], SLE patients were divided into 28 inactive (SLEDAI < 6) and 32 active (SLEDAI P 6) cases. Treatment of our SLE patients consisted of glucocorticoids in 58 patients (doses > 10 mg in 10 cases); antimalarial in 45 (75%); azathioprine in 38 (60%), cyclophosphamide in 23 (38%), and 4 patients (6.6%) received cyclosporine. The study also included a group of 24 consecutive rheumatoid arthritis (RA) patients as disease control (21 females with mean age of 46.3 ± 9.0 years) fulfilling the EULAR/ACR criteria for diagnosis of RA [21]. RA disease activity was assessed using the Disease Activity Score 28 (DAS28) based on evaluation of 28 joints (tenderness and swelling, joint count) and the erythrocyte sedimentation rate (ESR). All SLE and RA patients attended the Inpatient clinic of the Rheumatology Department at El-Kasr El-Ainy Hospitals, Cairo University over a period of 2 years. Patients with concomitant malignant diseases, infections, diabetes, as well as patients with other autoimmune diseases were excluded. The study also included 24 healthy volunteers as a normal control group (mean age 29.70 ± 6.96 years; 22 female). All investigations were done in accordance with the Menufia University, Health and Human Ethical Clearance Committee

guidelines for Clinical Researches. Local ethics committee approved the study protocol and informed consent was obtained from all participants. 2.2. Sample collection Venous blood was withdrawn in Ethylene Diamine Tetra acetic acid (EDTA) sterile tubes from patients and healthy subjects. Whole

Table 1 The ACR criteria of SLE, clinical manifestations, and serological characteristics. Parameter

SLE patients (N = 60)

Demographic data Age (years) Disease duration (years) Female/male SLEDAI

Mean ± SD 28.58 ± 7.30 4.97 ± 3.41 4/56 8.46 ± 8.4

ACR criteria of SLE Malar rash Discoid rash Photosensitivity Oral Ulcers Arthritis Serositis Renal disorders Neuropsychiatric disorders Haematological disease Anti-nuclear Ab Anti-dsDNA Ab

No (%) 30 (50.0) 8 (9.3) 47 (54.6) 27 (31.4) 47 (78.3) 30 (50.0) 34 (56.7) 22 (36.7) 17 (28.3) 60 (100) 35 (58.3)

Other clinical manifestations Cardiopulmonary disorders Cutaneous vasculitis

No (%) 15 (25) 13 (15.1)

Other serological findings ESR (mm/h) WBC (103/ll) HB (g/dl) Platelets (103/ll) ALT (U/L) AST (U/L) Albumin (g/dl) Creatinine (mg/dl) C3 (mg/dl) C4 (mg/dl)

46.40 ± 30.8 6.47 ± 2.60 11.33 ± 1.90 244.4 ± 76.60 17.40 ± 9.10 21.98 ± 9.80 3.71 ± 0.80 0.77 ± 0.70 60.85 ± 51.50 11.91 ± 10.54

Anti-dsDNA, anti-double stranded DNA; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; ESR, erythrocytes sedimentation rate; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; WBC, white blood count. Table 2 Demographic and laboratory characteristics of RA patients. Parameter

RA (No = 24)

Demographic data Male/female Age (years) Disease duration (years) DAS-28 score

Mean ± SD 4/20 46.3 ± 9.0 7.8 ± 5.5 5.4 ± 1.5

Laboratory investigations ESR (mm/h) WBC (103/ll) HB (g/dl) Platelets (103/ll) ALT (U/L) AST (U/L)

Mean ± SD 48.9 ± 17.0 8.8 ± 4.50 11.01 ± 1.31 382.5 ± 126.7 24.4 ± 19.5 24.3 ± 16.1

Clinical involvement no (%) Erosions ExtraArticular SC. nodules RF Active patients (DAS28 P 4.0)

No (%) 13 (54.1) 9 (37.5) 5 (20.8) 18 (75.0) 16 (66.7)

ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; DAS28, Disease activity score 28; ESR, erythrocytes sedimentation rate; HB, haemoglobin; RF, rheumatoid factor; SC, subcutaneous; WBC, white blood count.

R.M. Talaat et al. / Cytokine 72 (2015) 146–153

a*** a***

RA

IL-12

1400 1200

a** b***

800 600 400

Mean + SEM (pg/ml)

RA

IL-4

a**

25000 20000

b*

15000 10000 5000 Normal

RA

SLE

20

a*** b**

15 10 5 Normal

RA

350

IL-23

300

a**

SLE

250 200 150

b**

100

a* b***

10000 8000 6000 4000 a***

2000 Normal

RA

SLE

RA

SLE

IL6

100 90 80 70 60 50 40 30 20 10 0

a*

a**

RA

SLE

_____________________ Mean + SEM (pg/ml)

IL-10

12000

Normal

Normal

_______________________ Mean + SEM (pg/ml)

a***

0

SLE

Mean + SEM (pg/ml)

Normal

30000

0

25

50

200

0

IL-17

SLE

1000

0

30

0 Normal

Mean + SEM (pg/ml)

Mean + SEM (pg/ml)

IFN

2000 1800 1600 1400 1200 1000 800 600 400 200 0

Mean + SEM (pg/ml)

Mean + SEM (pg/ml)

148

50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0

TGF a*

b***

Normal

RA

SLE

Fig. 1. Plasma levels of IL-6, IL-17, IL-23, IL-4, IL-10, IFN-c, TGF-b, and IL-12 in SLE and RA patients. Results are expressed as mean ± standard error. TGF-b: transforming growth factor and IFN-c: Interferon gamma. a: groups statistically significantly different from controls; b: groups statistically significantly different from RA patients; ⁄ p < 0.05, ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001.

blood was allowed to stand for 30 min and centrifuged at 1500 rpm for 10 min. Plasma samples were separated and divided into aliquots then stored at 80 °C for subsequent cytokine analysis. 2.3. Laboratory and immunological investigations At time of blood sample collection, all patients and healthy controls were subjected to full history taken laying stress on disease duration, drug intake, preceding viral infection and bleeding manifestations. Routine biochemistry tests were collected from

patients’ records. Complete blood count was done using a Coulter counter (T660) and ESR was done by the Westergren method. Rheumatoid factor (RF) was determined by the latex fixation method. Anti-double stranded DNA (anti-dsDNA) antibody using a modified Farr assay. 2.4. Cytokine assay The levels of circulating cytokines IL-4 (0.195–100 ng/ml), IL-10 (0.195–100 ng/ml), IL-12 (0.195–100 ng/ml), IFN-c (0.195–100 ng/

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149

Fig. 2. Correlation between various circulating cytokines in SLE patients.

ml) in plasma of patients and controls were determined by ELISA as previously described [22,23] with slight modifications. A commercial DuoSet ELISA kits were used for detection of total concentrations of IL-6 (9.38–600 pg/ml), and TGF-b (31.2–100,000 pg/ml) (R&D System, Inc., Minneapolis, MN), IL-17 (less than 10 pg/ml) (RayBiotech, Inc. Norcross, GA) and IL-23 (up to 520.8 pg/ml)

(eBioscience, Inc.) according to the manufacturer’s instructions. Briefly, samples or standards were added to a coated microtiter plate and incubated for 2 h at room temperature then overnight at 4 °C. Free sites were blocked with 5% fetal bovine plasma (FBS). Biotinylated polyclonal antibody was added for 2 h at 37 °C. At the end of the incubation period, streptavidine conjugated

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Table 3 Cytokine levels according to Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Cytokine (pg/ml)

Inactive (SLEDAI < 6) n = 28

Active (SLEDAI P 6) n = 32

p Value

IL-6 IL-17 IL-23 IL-4 IL-10 IFN-c IL-17 TGF-b IL-12

43.5 ± 15.4 11.4 ± 1.5 128.1 ± 19.8 14028.6 ± 3013.2 2378.2 ± 559 396.1 ± 190.2 11.4 ± 1.5 25129 ± 2980 1115.4 ± 74.7

91.4 ± 24.5 17.7 ± 2.3 110.2 ± 15.7 19251.2 ± 3328.6 5633.1 ± 1418 444.1 ± 207.7 17.7 ± 2.3 28530.2 ± 2393.6 1097 ± 72

p < 0.05 p < 0.05 NS NS p < 0.05 NS p < 0.05 NS NS

IFN-c, Interferon gamma; TGF b1, transforming growth factor b1.

to horseradish peroxidase was added to the wells. After additional 1 h incubation, hydrogen peroxide (H2O2) and tetramethylbenzidine (TMB) substrate solution (1:1) were added. The reaction was stopped by 1 M HCl stopping buffer. The absorbance of each well was measured at 450 nm using a microplate reader (SunriseTM, Tecan Group Ltd. Männedorf/Switzerland). Each plasma sample was analyzed in duplicates. The ELISA reader-controlling software (Softmax) readily processes the digital data of raw absorbency value into a standard curve from which cytokine concentrations of unknown samples can be derived directly. 2.5. Statistical analysis The Statistical Package for Social Sciences (SPSS) version 19 (LEAD Technology Inc., Charlotte, NC, USA) was used to analyze the data. Continuous variables were summarized through the mean ± standard deviation (SD) and categorical variables using absolute values and percentages. The difference in the averages between the study groups was examined by Kruskal–Wallis nonparametric ANOVA test followed by Mann–Whitney test. Spearman’s rank correlation was used to examine the relationship between two continuous variables. 3. Results 3.1. Patients’ characteristics Demographic and clinical characteristics of SLE and RA patients were summarized in Tables 1 and 2. 3.2. Cytokines secretion profile in relation to SLE and RA diseases As shown in Fig. 1, SLE patients were found to have significantly higher levels of IL-17 (p < 0.001), IL-6 (p < 0.01), IL-12 (p < 0.001) and IL-10 (p < 0.05) but comparable IL-23 and IL-4 and slight statistically insignificant reduction of TGF-b levels compared to controls. A significant decrease in IFN-c (p < 0.001) was reported in SLE patients in relation to normal subjects. Regarding the correlation of cytokines among SLE patients (Fig. 2), IL-6 and IL-23 were positively correlated (r = 0.237, p < 0.05). IL-17 was positively correlated with IL-12 (r = 0.296, p < 0.01) while it was negatively correlated with IFN-c (r = 0.468, p < 0.001). A negative correlation was found between IL-10 with IL-12 (r = 0.434, p < 0.001), IFN-c (r = 0.397, p < 0.001) and TGF-b (r = 0.469, p < 0.001). To evaluate which cytokine was associated with disease activity, SLE patients were subdivided into active and inactive groups according to SLEDAI. As seen in Table 3, IL-6, IL-10 and IL-17 were significantly increased (p < 0.05) in SLE patients with active versus those with inactive disease.

No statistical significant association was found between the variable cytokines in the present study and the medications used in SLE patients (p > 0.05; data not shown). Among the RA patient group, we found significantly higher levels of plasma IL-4 (p < 0.01), IL-6 (p < 0.05), IL-17 (p < 0.001), IL-23 (p < 0.01) and TGF-b (p < 0.5) than those of healthy controls. On the contrary, IFN-c (p < 0.001) and IL-10 (p < 0.01) were significantly decreased. Although it was found slightly increased, we did not observe any difference in the plasma levels of IL-12 between patients and controls. As shown in Fig. 3, IL-17 correlated directly with IL-23 (r = 0.283, p < 0.05), TGF-b (r = 0.357, p < 0.05) and IL-4 (r = 0.435, p < 0.01) while it correlated inversely with IL-10 (r = 0.583, p < 0.001) and IFN-c (r = 0.786, p < 0.001). IL-10 was directly correlated with IFN-c (r = 0.664, p < 0.001) and indirectly correlated with IL-4 (r = 0.413, p < 0.05).

4. Discussion The role of cytokines as effectors or predisposing elements in autoimmune rheumatic diseases has received prominent attention. However, results were frequently contradictory and, more importantly, difficult to incorporate into a conceptual. Thus, our study is concerned with the TH1/TH2/TH17/Treg cytokine secretion profile in SLE patients. In the present study, a significant down regulation of IFN-c in SLE patients could be attributed to the previously observed reduction in frequency and number of TH1 cells in SLE patients [10]. In accordance with our data, production of IFN-c was demonstrated to be decreased in SLE patients [24]. Dolff et al. [14] and Hagiwara et al. [25] have reported a reduction frequency in IFN-c produced by peripheral blood mononuclear cells (PBMCs) in SLE patients. In contrast, other studies have found elevated levels of IFN-c in their SLE patients [3,5,19,26]. Although we had reported a reduction in IFN-c in our patients, active SLE patients had slightly more IFN-c than those with inactive disease. The production of IFN-c by PBMCs from SLE patients is significantly correlated with global disease activity [8,19,24]. Herein, the level of IL-4 was compared with the controls as reported in previous studies [20,27]. Also, it has been found that the percentage of IL-4+ cells within the CD4+T-cells from SLE patients is not statistically different in comparison to healthy controls [14]. In agreement with our data, SLE patients were found to have significantly higher plasma levels of IL-10 [5,6,19,20]. Plasma levels of IL-10 were consistently reported to be increased in SLE [8,19,27,28]. It has been found to be over produced by the B cells and monocytes [29]. Therefore, IL-10 has recently been suggested as a marker of SLE [19]. Liorente et al. [30] has proved that plasma IL-10 levels are higher in SLE patients, and both spontaneous and mitogen-induced IL-10 production has been found to be increased in SLE’s PBMCs [19,30]. It plays a central role in the pathogenesis of SLE, including regulation of growth and differentiation of B cells and auto-antibody production. IL-10 might contribute to a number of the earlier peripheral B-cell abnormalities observed in SLE, including plasma cell expansion [7]. Some have proposed that the immunologic imbalance in SLE may be related to an abnormally high production of IL-10. B lymphocyte hyperactivity may result from autocrine and paracrine effects of IL-10 signaling [3,31]. IL-10 may trigger apoptosis of CD4+ and CD8+ T cells, which might partly explain its pathogenic role in SLE [32]. Plasma IL-10 levels were significantly increased with disease activity which has been shown to be correlated with overall disease activity in several studies [3,19]. The abnormally generated IL-10 might result from elevated toll-like receptor(TLR) -9 expression on B cells, as

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Fig. 3. Correlation between various circulating cytokines in RA patients.

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TLR-9 level of peripheral blood B lymphocytes from SLE patients has been found to be significantly correlated with disease activity and anti-dsDNA antibody [32]. In the present study, we found increased soluble levels of IL-6 and IL-17 with comparable level of IL-23 in plasma of lupus patients compared with those in healthy controls. In line with our results, plasma levels of IL-6, a cytokine that promotes the development of TH17 cells, are higher in SLE patients than in healthy subjects. These findings suggest that the balance of TH17 and TH1 responses as well as IL-6 production is dysregulated in SLE, leading to increased IL-17 production from CD4+ T cells, an increase that may contribute to disease pathogenesis [5]. Several reports suggested that IL-6 plays a critical role in the B-cell hyperactivity, auto-antibodies production and immunopathology of SLE [19,33]. Concerning IL-17, and in accordance with its documented elevation in SLE patients, the frequency of IL-17-producing T cells have been increased in the peripheral blood of SLE patients [5,7,34]. The ratio of TH17 to TH1 cells was higher in SLE patients than healthy controls [10]. These observations indicated that patients with SLE have an aberrant CD4+ T-cell response, resulting in a propensity toward an increased frequency of TH17 cells [7,14]. Consistent with our findings, the plasma IL-23 level was not found to be different between lupus patients and healthy subjects. Plasma levels of other TH17-polarizing cytokines, including IL-23 were similar between the patients and controls [20]. IL-6 and IL23 are involved in developing and/or expanding TH17 cells [7]. The uneven contributions of the p35 subunit (unique to IL-12) and the p40 subunit (shared between IL-12 and IL-23) to the pathogenesis of various experimental autoimmune disorders was confirmed by ensuing studies that implicated IL-23 in the development of organ-specific autoimmune disease by promoting TH17 which invades the target organ causing progression of inflammation [13,35]. Our SLE patients were found to have significantly higher levels of IL-12. This data is in accordance with previous reports [26,36]. This might be indicated to the participation of Dendritic cells (DCs) in SLE as it represents the main IL-12 producer cells [37]. The IL-12 family of cytokines (IL-12, IL-23 and 27) are key players in the regulation of T cell responses. These responses are orchestrated by monocytes, macrophages, and dendritic cells, which normally produce the members of the IL-12 family of cytokines in response to infection [38]. Regarding SLE disease activity, IL-6 and IL-17 were significantly increased with active disease. A functional role of TH17 cells in SLE was proposed based on the demonstration that the level of IL-17 was elevated in active SLE [14]. A strong correlation between the frequency of CD4+IL-17+ T cells and disease activity in SLE patients has been observed [5]. Both the percentage of circulating TH17 cells and the ability to produce IL-17 are increased in active patients [14]. Our data showed a slight reduction of TGF-b1 levels in SLE compared to controls. As previous reports demonstrated, concentrations of plasma TGF-b1 has been decreased in SLE patients in parallel with reduced peripheral Treg cells [32]. Both TH17 and inducible Treg cells require the same cytokine, TGF-b1, during the early stage of differentiation, in the presence of pro-inflammatory cytokines such as IL-6 [5]. CD4 + murine lymphocytes in the presence of low concentrations of TGF-b1 and the pro-inflammatory cytokine IL-6 results in acquisition of the TH17 cell phenotype [39–40]. Becker-Merok et al. [41] has found that patients with SLE had lower levels of TGF-b1 which were found to correlate with disease activity and CD4+, CD8+, and natural killer cell counts [32,41] which may explain why the reduction of TGF-b in lymphoid tissues might lead to immune dysregulation and auto-antibody production [32]. TGF-b alone induced the Treg transcription factor Foxp3

and is essential for the development of Tregs in the periphery. However, the presence of pro-inflammatory cytokines like IL-6 inhibited the induction of Foxp3+ Tregs and simultaneously promoted TH17 cell differentiation [40–42]. Although we had elevated IL-10 plasma levels, the reduction of TGF-b1 consistent with the elevation of IL-6 and IL-17 may reflect a TH17 cell activity rather than Treg. Autoimmunity may result when CD4+ T cell differentiation is biased away from Treg cells toward the TH17 cell phenotype [39]. SLE patients in the active phase of the disease are characterized by a deficiency in Treg cells and decreased Treg/TH17 ratio [10]. Supporting to our data in which a reduction in IFN-c (TH1) and TGF-b1 (Treg) is associated with elevation in IL-6 and IL-17 (TH17), Kleczynska et al. [10] proved that patients with active SLE are characterized by a relative deficiency in TH1 and Treg cells compared to the TH17 subset. Such imbalance is not limited to SLE flares, but is the hallmark of the disease, since also patients with quiescent disease display a TH17/ Treg ratio favoring TH17 cells [10,14,43]. As reported in previous studies [5,20,44] the immunosuppressive therapies (both prednisolone and DMARD) did not correlate with the variable cytokines in SLE patients. This lack of correlation might be explained by the huge variability in SLEDAI and diversity in the type of involvement by SLE. RA is the most common chronic autoimmune disease, with a broken TH17/Treg balance in peripheral blood [45]. Take a look into our RA patients’ samples, we find a significant elevation in most of cytokines that we have measured such as IL-6, IL-17, IL23, IL-4 and TGF-b1. Our data are consistent with previously reported data [46–48] that have stressed on the potential predominant role of TH17 cells in the inflammatory process and chronic progression in RA which coincides with a reduction in frequencies of Treg cells in peripheral blood of RA patients. Sivalingam et al. [47] reported that the pro-inflammatory cytokines (such as IL-6) were significantly elevated in RA patients. Plasma concentrations of IL-6 were significantly elevated in patients with RA compared to those of healthy controls [49]. RA has been observed to be associated with high levels of IL-6 in the synovial membrane and plasma in data of several studies [49,50]. Cascão et al. [46] found a high plasma concentration of IL-17A in RA patients. It has been reported that IL-6 and IL-17 are elevated in the plasma and this correlates with a higher disease activity [46,51].

5. Conclusion Although our study was limited by small sample size, multiple statistical comparisons and cross-sectional design, it supported a role of imbalance in TH1/TH17, TH1/TH2 as well as Treg/TH17 cells (based on their major cytokines secretion profile) in SLE patients. Larger studies with longitudinal measurement of these cytokines may provide information in regard to their relationship with disease activity and clinical response to treatment. We also believe that further functional studies are warranted to determine the mechanism for TH1/TH17/Treg disturbed balance in peripheral blood especially in active form of the disease which in turn will favor the development of therapeutic strategies. References [1] López de Padilla CM, Reed AM. Involvement of dendritic cells in autoimmune diseases in children. Pediatr Rheumatol Online J 2007;11:5–16. [2] National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) report. Understanding Autoimmune Diseases. National Institutes of Health (NIH). 2012; 1–3 [NIH Publication No. 11-7582]. [3] El-Sayed M, Nofal E, al Mokadem S, al Makhzangy I, Gaballah H, Akl H. Correlative study of plasma TH1/TH2 cytokines levels in patients with systemic lupus erythematosus with SLEDAI. Egypt Dermatol Online J 2008;4:3–19.

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