Introduction

Systemic 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. Alltrans-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 resuspended 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 AntiHuman 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 resuspended 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 antiCD3 antibody, PBMCs were suspended in 1 ml of Cell Staining Buffer, centrifuged, and then resuspended 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 isolation
Ten 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 (SigmaAldrich, 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 antihuman 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 Analysis
Differences 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<0.05 was considered significant.

Result (4 kelompok gambar untuk masing2 hasil)

Subject Characteristics and Vitamin A Levels
The mean of SLE patients age was 23.303.1 years old. Most of the clinical manifestations were
malar rash, photosensitivity and arthritis (Table 2). Vitamin A levels in SLE patients were
significantly lower than those in healthy controls (41.24 40.63 ng/mL versus 67.08 5.7, P=
0.02)

Hipovitaminosis A (levels <20 ng/ml) were observed in 11 SLE patients (36%) and 5 healthy
controls (16%), whereas normal levels of vitamin A (levels 20 ng/ml) were observed in 19 SLE
patients (63%) and 25 healthy controls (83%) (Table 1).

Table 1. Vitamin A Levels of SLE Patients and Healthy Controls.

Characteristic
SLE patients
Healty
P value
(N=30)
Control
(N=30)
Age (years)
23.30 3.1
22.97 2.5
Normal level of Vitamin
63%
83%
A
Hypovitaminosis A
36%
16%
Mean of vitamin A levels 41.24 40.63
67.08 5.7
P= 0.02
(ng/ml)

Table 2. Clinical Manifestations of SLE Patients

Clinical Manifestations
Malar Rash
Oral Ulceration
Photosensitivity
Arthritis
Nephritis
Hematologic manifestations
Anti-nuclear antibody (ANA) positive
Anti-dsDNA positive

%
80%
23.3%
36.7%
76.7%
30%
20%
93.3%
93.3%

Effect of In Vitro Treatment of retinoic acid on Th17 and Treg Percentages on SLE Patients
This study showed that retinoic acid treatment could reduce Th17 differentiation from CD4 + T
cell cultures of SLE patients which stimulated by Th17 differentiation cytokines (figure 3A).
There were significant reductions of Th17 percentages from cultures with addition of 0.2, and 0,3
g/ml retinoic acid but not with 0,1 g/ml retinoic acid compared with control (37.881.44% vs.

29.911.44%; p=0.04, 37.881.44% vs. 27.522.41%; p=0.01, 37.881.44% vs. 34.223.55%;

p=0.464, respectively). The lowest result were shown in group with 0,3 g/ml of retinoic acid
administration (figure 3C).
On the other hand retinoic acid also could increase Treg differentiation on CD4 + T cell cultures
from SLE patients which encouraged by Th17 differentiation cytokines (figure 3B). There were
only one group which could increased the Treg percentages, addition of retinoic acid with 0,3
g/ml doses could increased Treg percentages significantly but not for the other two group with
0,1 g/ml and 0,2 g/ml dose of retinoic acid compared with control (13.814.45% vs
34.331.80%; p=0.01, 13.814.45% vs 23.636.08%; p=0.068, 13.814.45% vs 23.482.24%;
p=0.073, respectively). (figure 3C).
This study also revealed that retinoic acid could shift CD4+ T cells differentiation more toward
Treg in environment favored to become Th17. It were shown that there were significant
reduction of Th17/Treg ratios in cultures added 0.1, 0,2, and 0,3 g/ml retinoic acid compared to
control (2.930.92 vs.1.230.22; p=0.011, 2.930.92 vs.1.250.10; p=0.011, 2.930.92 vs
0.800.11; p=0.003, respectively) (figure 3C).
Discussion
Systemic lupus erythematosus (SLE) is an autoimmune disease with persistent inflammation that
damages multiple organs. SLE is initiated by a breach of immunotolerance to self which recently
found to be mediated by imbalance of Treg and Th17 cells. Both Th17 and Treg cells are
involved in the pathogenesis of SLE as the dynamic balance between these cells is damaged in
SLE patients (Yang et al, 2011). Vitamin A has both positive and negative regulatory functions in
the immune system. Recent insights into the role of vitamin A in the promotion and regulation of
multiple immunological pathways draw new attention to the sweeping influence of vitamin A in
immunity. Based on these functions, we suspected a role of vitamin A in several autoimmune
diseases, but research on the role of vitamin A in SLE is still rarely performed. We found that the
levels of vitamin A in SLE patients was significantly lower when compared with healthy
controls. This findings shows that there is a relationship between blood levels of vitamin A with
SLE disease progression. Conditions of vitamin A deficiency is also associated with the
imunophatologic processes during Listeria monocytogenes infection and autoimmune
encephalitis (Mucida et al, 2007; Xiao et al, 2008) where it is in line with our findings.
The exact mechanism of how vitamin A becomes lower in SLE disease still can not be explained.
However, some studies suggest that vitamin A deficiency can lead to Increased inflammatory
responses and tissue damage (Wiedermann et al, 2000). Another study conducted by Kida et al.
(2011) proved that chronic inflammatory conditions was able to reduce the expression of the
enzyme lecithin: retinol acyltransferase (LRAT) which resulted in the disruption of retinoid
storage process in the liver. Another study also mentions that the interference with the LRAT
gene associated with the likelihood of vitamin A deficiency in experimental animals (Diana,
2011). This can explain the possible causes of decreasing levels of vitamin A in patients with
SLE, but there are several other possibility factors that influence level of vitamin in SLE patients
such as diet low in vitamin A, comorbid disease, etc. so we need further research to determine
the exact mechanism role of vitamin A in patients with SLE
Results of our study showed that the percentage of Th17 cells in SLE patients was higher than
healthy controls as well as on Treg cells. As mentioned above, the pathogenesis of SLE is

affected by the imbalance between the two subsets of CD4+ cells that is Th17 and Treg cells. It
was found that imbalance between these two components in SLE is correlated with disease
progression and tissue damages in SLE [6-8]. The same results were mentioned by Dian (2012)
in which the Treg cells in SLE patients is higher than the healthy control. While some others
found increased numbers of Treg cells, but with impaired suppressor function or the resistance of
effector T cells to suppressive action of Tregs (Venigalla et al, 2008). Research conducted by
Magdalena et al (2014) showed just the opposite outcome, in those study revealed a systemic
Th17/Treg imbalance which occurred in SLE patients with low disease activity and in remission.
In that study showed that there is an increase in the percentage of Th17 cells and also decrease of
CD4 +CD25high FoxP3+ Treg cells compared to healthy controls. An increase in the percentage
of Th17 cells and Treg cell depletion is indicated in patients with lupus nephritis (Xing et al,
2012).Regulatory T cells in SLE patients have been previously investigated in many studies, and
the results are still confusing. The inconsistencies may be due to differences in both the
phenotype assigned to Tregs and the degree of SLE activity in those studies.
This study also has demonstrated the there is a negative correlation between levels of vitamin A
and the percentage of Th17 cells but not for Treg cells in cultured CD4+ cells of SLE patients.
The data shows the percentage of Th17 cells were higher in SLE patients with hipovitaminosis A
compared with SLE patients with normal levels of vitamin A, while the percentage of Treg cells
did not differ between the two groups. The correlation between the levels of vitamin A and the
percentage of Th17 cells is thought to occur through the role of vitamin A in regulating Th17 and
Treg cells. Recently all-trans retinoid acid (ATRA), an active metabolite of vitamin A has been
proven to skew Regulatory T cell-T helper 17 cell (Treg-Th17) balance toward Treg in vitro (Xin
et al, 2015). The study conducted by Kevin et al (2008) showed that ATRA and other agonists of
the retinoic acid receptor alpha (RAR) could inhibit the formation of Th17 cells and promote
FoxP3 Treg expression. Other study revealed that RA both in vitro and in vivo inhibits the
development of Th17 cells by interfering with signaling events in the TGF- and IL-6/IL-21/IL23 pathways that are involved in the development and function of these cells. They also
demonstrated that RA in vivo does not increase the frequency of Treg cells significantly in an
autoimmune disease setting but mainly inhibits the generation of effector T cells. It also tell us
that beneficial effect of RA in autoimmunity is mainly due to its inhibition of pathogenic Th17
responses (Xiao et al, 2014).
On the other hand our study showed that administration of RA at dose 0.2 M and 0,3 M in
hipovitaminosis condition were able to reduce Th17 and IL-17A production but only RA with 0,3
M dose could enhance Treg differentiation on CD4+ T cells cultures of SLE patients. RA also
could reduce Treg/Th17 ratio in all of three doses which tells us that RA could regulate the
balance of CD4+ Treg and Th17 cells in SLE patients in vitro. RA also only affect Treg
differentiation with high dose but could easily regulate Th17 production with lower dose. The
mechanism how retinoic acid can regulate the differentiation of Th17 cells and Treg cells in SLE
patients still can not be explained with certainty. Previous research proves that retinoic acid is
able to inhibit the formation of Th17 cells through its role in blocking the action of TGF- to
express IL-6 receptor or IL-6 (Elias et al., 2008). IL-6 itself is a cytokine that needed to induce
the phosphorylation of STAT3 in Th17 cells. STAT3 will produce the cytokines IL-21 and the
induction of IL-23 receptor expression through the ROR-t. Both IL-21 and IL-23 acts to induce
the clonal expansion of Th17 cells (Xiao et al, 2008).

While the mechanism of action of retinoic acid in the modulation of cell differentiation into CD4
+ Treg cells is through histone acetylation at the promoter FoxP3 after binding to receptors RAR
(McGrane, 2007; Kang et al, 2007). Moreover the binding of retinoic acid with RAR along with
TGF-1 may also increase the activation and phosphorylation of smad3 so that will activate
transcription fakor FoxP3 (Xiao et al, 2008).
We suspect that mechanism of retinoic acid in inhibiting the differentiation of Th17 on the other
hand increase the differentiation of Treg in patients with SLE is obtained through its role in
inhibits the expression of IL-6 cytokine receptor. As a result of these obstacles, the IL-6 can not
bind to its receptors to initiate the differentiation of Th17 cells. Inhibition on IL-6 action also will
reinforce the process of differentiation of Treg cells by retinoic acid and TGF- on CD4 + cells.
In addition, based on study conducted by Linglu et al, 2010 administration of retinoic acid along
with TGF-1 in CD4 + cells will trigger the differentiation of Treg cells that have the potent
immunosuppressive ability and resistant to the effects of proinflammatory cytokines such as IL-6
and IL-1.
Higher dose of retinoic acid was needed to enhance the differentiation of Treg cells in cultures of
CD4 + SLE. This is probably due to the administered dose is still not optimal to trigger an
increase in the percentage of Treg cells significantly. In addition it has been found a transcription
factor Pbx-1 and increased expression of Pbx-1 in patients SLE associated with reduced
production of CD4+CD25+FoxP3+ Treg cells followed with an increase the production of T cell
memory that does not have the suppressive ability (Eric et al, 2011). We suspected that Pbx-1
was affecting the ability of retinoic acid in inducing the differentiation of Treg cells in patients
with SLE. But there is still needs another research to determine the exact mechanisms role of
Pbx-1 in modulation of CD4 + T cells in a molecular level of SLE patients.
This research has shown the possibility of retinoic acid to be used as a novel therapeutic agent
for SLE by alleviating the immune response in SLE specifically through modulating balance
between Th17 and Treg. Although retinoic acid has proven capable to modulate Th17/Treg
balance in many other studies, study that tested retinoic acid on the Th17/Treg balance in SLE
has still yet never been done. In this study, we are still unable to determine the exact mechanism
of retinoic acid pathway in modulating Th17 and Treg cells in patients with SLE. We suspect that
retinoic acid works mainly in modulating the expression of IL-6 receptor. Inhibition on IL-6
alone is apparently quite able to inhibit Th17 cell differentiation in cultured CD4 + SLE.
Therefore, this does not rule out the possibility of opening further research in the future to cover
the limitations in this study.