IntroductionSystemic lupus eritematosus (SLE) is a systemic inflammatory disease that caused by alteration of immune system Lupus eritematosus sistemik (LES) (Albar, 2003) . In recent years it has been In recent years, there has been mentioned a new paradigm that underlies the progression of clinical symptoms in patients with LES is an imbalance between pro-inflammatory responses and anti-inflammatory which is characterized by an increase in the number and activity of Th17 cells as effector cells that play a role in the regulation of inflammatory responses and decrease in the number and activity of Treg cells that act as a suppressor (Yang et al, 2010). Th17 cells can infiltrate the skin, lungs and kidneys of SLE patients. Furthermore, previous study also showed that SLE flares might be linked to the expansion of Th17 cells, as Th17 cells are involved in vascular inflammation. Antagonism of Th17 cells with IL-17-blocking antibodies could relieve the IL-17-mediated vascular inflammation in vitro. Those fact open some research to make the balance of Treg and Th17 cells as a promising new therapeutic target in patients with SLE. The imbalance of Treg and Th17 cells in SLE patients suspected to be affected by several factors, one of which is a cytokine. Cytokines IL-6 and TGF- is a major regulator in the production of Th17 and Treg cells. It is known that there is an increasing number of cytokines IL-6 in patients with SLE so it is associated with an increased proportion of proinflammatory Th17 cells and a decrease in the number of antiinflamatoric Treg cells in patients with SLE (Yang et al, 2011).

Vitamin A plays an important role in the development of a balanced immune system. All-trans-retinoic acid (tRA), a predominant vitamin A metabolite, exerts most of the functions attributed to vitamin A. Based on research by the Elias et al (2013) retinoic acid had been proven to have the ability to reduce cell proliferation and function of T helper (Th) 17 and induces proliferation of regulatory T cells (Treg). The vitamin A metabolite all-trans-retinoic acid (RA) is capable of inhibiting the IL-6-driven induction of Th17 cells and promoting anti-inflammatory Treg-cell differentiation (Mucida et al, 2007). Recent study showed that RA enhances TGF- signalling by increasing the expression and phosphorylation of Smad3. RA also inhibits the expression of IL-6Ra, IRF-4 and IL-23R, thus inhibiting Th17 development in vitro (Xiao et al, 2008). Additional studies confirmed that RA can relieve inflammatory diseases through regulation of the Th17 and Treg cell balance (Elias et al, 2013). Based on these facts, we suspect that the levels of vitamin A in the body can affect the pathogenesis of SLE disease either as a cause of imbalances of Th17 and Treg cells either or as a result of the disease itself. Some studies also mention that there is a relationship between low levels of vitamin A with chronic inflammation (Kida et al, 2011). Therefore, the use of retinoic acid is expected to be a therapeutic agent which can regulate the balance of Treg and Th17 cells and have the potential to treat autoimmune diseases such as SLE.

Study to determine the role of retinoic acid in SLE, especially regarding to its role in regulating Treg and Th17 cells are still rare. Based on that fact this study was conducted to determine serum levels of vitamin A and retinoic acid influence on the balance of Th17 cells and Treg cells in patients with SLE in vitro in Indonesia.

Metode

Subjek Penelitian

Subjects of this study were female 30 SLE patients newly diagnosed by rheumatologist based on American College of Rheumatology (ACR) criteria, experienced flare (had systemic lupus erythematosus disease activity index (SLEDAI) score >3), and did not take vitamin A supplementation. These patients were outpatients and hospitalized patients in the Department of Internal Medicine Saiful Anwar Hospital Malang. The patients were in severe infection or in immunodeficiency condition and also had disease which correlate with vitamin A were excluded from the study. Controls in this study were 30 healthy women matched in age, gender and ethnicity to patients, and did not take vitamin A supplementation. Patients and healthy controls were used in this study to compared the level of serum vitamin A and also seek if there were any difference of retinoic acid effects in both SLE patients and healthy controls. This study met the ethical clearance by Ethics Commission of Faculty of Medicine, Brawijaya University. Informed consents were obtained from all subjects participated in this study.

Serum Collection and Vitamin A Level Measurement

4 ml of fresh blood from each subject collected in serum separator tube was centrifuged in 4C for 20 minutes. The supernatant was collected into fresh tubes and stored in -70C until the next process for enzyme-linked immunosorbent assay (ELISA). Furthermore, serum levels of vitamin A will be measured using ELISA KIT (plant name). ELISA measurement procedures performed by protocol according to the factory.Isolation of Peripheral Blood Mononuclear Cells (PBMCs) 8 ml of heparinized blood from each subject was diluted 1:1 with phosphate buffered saline (PBS) in a canonical tube. The mixture was layered over Ficoll (Amersham Biosciences, cat. 17-1440-03) in the ratio of 2:1 in tube. This tube was centrifuged at 1000xg in 4C for 30 minutes. PBMCs harvested from the interface between Ficoll and plasma (the buffy coat) were collected into fresh tube and then washed twice with PBS. These PBMCs were divided into 2 tubes in the ratio of 1:3 for Treg analysis (direct/fresh process) and Th17 analysis (needs activation first), respectively.

Stimulation for Intracellular Staining of IL-17 as A Marker of Th17

Before we analyzed Th17, we performed stimulation for intracellular staining of IL-17, since this cytokine is a marker of Th17. The goal of this procedure was to make IL-17 to be more easily visible. A 10 g/ml solution of LEAFTM Purified Anti-Human CD3 Antibody Clone UCHT1 (Biolegend, cat. 300413) was prepared in sterile PBS. Cell culture medium (RPMI 1640 with 2mM of ultra-glutamine, 100 U/ml of penicillin, 100 g/ml of streptomycin) and35 l Anti-CD3 were dispensed to each well of the 96-well flat-bottom assay plate. About 500.000 PBMCs were added to each well. Plate was covered and incubated at 37C in 5% CO2 and 100% humidity for 3 days. After 3 days, the cells were harvested and collected into fresh tube.

Treg Immunofluorecent Staining Procedure

All reagents for this procedure were produced by Biolegend. Fresh PBMCs were re-suspended with 0.5 ml of Cell Staining Buffer (cat. 420201). 5 l of PerCP Anti- Human CD4 Antibody (cat. 317432) and 20 l of PE Anti- Human CD25 Antibody (cat. 302606) were added to suspension, incubated at room temperature for 15-20 minutes in the dark and then washed twice with 1.5 ml of Cell Staining Buffer with a centrifuge at 350xg for 5 minutes. Cells were re-suspended in 0.5 ml of Cell Staining Buffer. The suspension was added by 1 ml of 1x FoxP3 Fix/Perm Solution (cat. 421401), mixed and incubated at room temperature in the dark for 20 minutes, then centrifuged at 350xg for 5 minutes and the supernatant was discarded. Cells were washed with 1.5 ml of Cell Staining Buffer, and then washed with 1 ml of 1x FoxP3 Perm Solution (cat. 421402) by centrifuge at 350xg for 5 minutes and the supernatant was discarded. Cells were re-suspended in 1 ml of 1x FoxP3 Perm Solution, incubated at room temperature in the dark for 15 minutes, centrifuged at 350xg for 5 minutes and the supernatant was discarded, and pellets were resuspended again in 100 l of 1x FoxP3 Perm Solution. 20l of FITC Anti-Human FoxP3 Antibody (cat. 320106) was added and incubated at room temperature in the dark for 30 minutes. Cells were washed twice with 1.5 ml of Cell Staining Buffer, and then re-suspended in 0.5 ml of Cell Staining Buffer and analyzed by flow cytometer.

Th17 Immunofluorecent Staining Procedure

All reagents for this procedure were produced by Biolegend. After 3 days of activation with anti-CD3 antibody, PBMCs were suspended in 1 ml of Cell Staining Buffer, centrifuged, and then re-suspended with 0.5 ml of Cell Staining Buffer. 20 l of FITC Anti-Human CD4 Antibody (cat. 317408) and 20 l of PE Anti-Human CD3 Antibody (cat. 300308) were added, incubated at room temperature for 15-20 minutes in the dark, and then washed twice with 1.5 ml of Cell Staining Buffer with centrifuge at 350xg for 5 minutes. Cells were resuspended in 0.5 ml of Cell Staining Buffer. Cells were fixed in 0.5 ml of Fixation Buffer (cat. 420801) in the dark for 20 minutes at room temperature, and then centrifuged at 350xg for 5 minutes, the supernatant was discarded. Cells were re-suspended in 0.6 ml of 1x Permeabilization Wash Buffer (cat. 421002) with centrifuged at 350xg for 5-10 minutes. Cells were re-suspended in 200 l of 1x Permeabilization Wash Buffer and added with 5 l PerCP/Cy5.5 Anti-Human IL-17A Antibody (cat. 512314), incubated for 20 minutes in the dark at room temperature, and then washed twice with 1.5 ml of Permeabilization Wash Buffer with centrifuged at 350xg for 5 minutes.Cells were re-suspended in 0.5 ml of Cell Staining Buffer and analyzed by flowcytometry.

Cell isolationTen milliliters of peripheral venous blood were freshly obtained from subjects which had hipovitaminosis A in early study and collected into heparinized vacutainer. Afterwards, CD4+ T cells were separated using RosetteSep human CD4+ T cell enrichment cocktail (RosetteSep, StemCell Technologies) according to manufacturers instruction. The purity of CD4+ T cell isolates were confirmed using flow cytometry analysis (FACScalibur) and had high expression of surface marker CD4+ (>85%).

Culturing and Stimulation of CD4+ T Cells

CD4+ T cells were cultured in 96 well plates with 5 g/ml plate bound anti-CD3 (Biolegend). Cells were alliquoted into 5x105 cells/well with complete RPMI culture medium (Sigma-Aldrich, St. Louis, MO) containing L-Glutamine and 10% fetal bovine serum (Gibco) supplemented with 100 g/ml streptomycin (Gibco), 100 U/ml penicillin (Gibco) and 5 g/ml antiCD28 (R&D systems). All cultured CD4+ T cells were stimulated into Th17 by adding 10 ng/ml IL-6 (Biolegend), 5 ng/ml TGF-1 (Biolegend), 10 g/ml anti-IFN- (Biolegend), and 10 g/ml anti-IL-4 (Biolegend) in cell cultures. Lastly, retinoic acid (nama pabrik) with different concentrations (0.1, 0.2, and 0.3 g/ml) were added in cell cultures. Cells were incubated at 37oC and 5% CO2 for 72 hours [26].

Measurement of Th17 and Treg Percentages using Flow Cytometry

Harvested CD4+ T cells were counted for Th17 and Treg percentages using flow cytometry (FACScalibur). Before detection of Th17 cells, cells were stimulated with 50 ng/ml PMA (Sigma Aldrich) and 1 g/ml Ionomyc in (Sigma-Aldrich) in the presence of Brefeldin A (BD Pharmingen) at 370C for 4 hours. Cells were stained with FITC anti-human CD4 antibody (Biolegend). Afterwards, cells were fixed, permeabilized, and labeled with and PerCP/Cy5.5 anti-human IL-17A (Biolegend). Th17 were cells which expressed CD4+ IL-17A+. For detection of Treg, cells were labeled with PerCP anti-human CD4 antibody (Biolegend) and PE anti-human CD25 antibody (Biolegend). FITC anti-human FoxP3 antibody (Biolegend) was added later after cells were fixed and permeabilized. Treg were cells which expressed CD4+ CD25+ FoxP3+.

Enzyme-Linked Immunoabsorbent (ELISA) Assay for Cytokines Measurement

Cytokines measurements were done to assess Th17 and Treg function by monitoring their cytokine production. Supernatants from CD4+ T cells culture were collected and stored at -800C for cytokine measurements. Interleukin-17A (IL-17A) (R&D systems) and transforming growth factor-1 (TGF- 1) (eBioscience) secretion were measured by ELISA kits according to the manufactures instructions.

Statistical AnalysisDifferences between groups were determined using ANOVA and paired T-test analysis. Data were described as mean SD. Statistical analysis was performed using SPSS for windows version 16.0. p