1,25(OH)2D3 Inhibit Endothelial Damage by Decreasing Histone and Defensin

Externalization during NETosis in Patients with Systemic Lupus

Erythematosus

Kusworini Handono1*, Benny Arie Pradana2, Radhitio Adi Nugroho2, Yona One
Sidarta2, Ivan Andre Hartono2, Dian Hasanah3, Handono Kalim4, Agustina Tri
Endharti 5, Fatchiyah Fatchiyah6

1
Department of Clinical Pathology, Faculty of Medicine Brawijaya University/Saiful Anwar General
Hospital, Malang, Indonesia
2
Biomedical Sciences, Master’s Program, Faculty of Medicine Brawijaya University, Malang,
Indonesia
3
Department of Internal Medicine, Faculty of Medicine Brawijaya University, Saiful Anwar General
Hospital, Malang, Indonesia
4
Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine Brawijaya
University/Saiful Anwar General Hospital, Malang, Indonesia
5
Department of Parasitology, Faculty of Medicine, Brawijaya University, Malang, Indonesia Commented [T1]: Please check for spelling errors.
6
Department of Biology, Faculty of Mathematics and Natural Science, Brawijaya University, Malang,
Indonesia

*Corresponding Author
Kusworini Handono, MD, PhD
Address :Universitas Brawijaya, Jalan Veteran, Malang 65145 East Java
Email :dr.kusworini@gmail.com
Phone : +62341551 611
Fax : +62341564 755

Keywords: SLE, NETs, Histone, HNP, 1,25(OH)2D3, Endothelial cells

Word count : 4340

1,25(OH)2D3 Inhibits Endothelial Damage by Decreasing Histone and
Defensin Externalization during NETosis in Patients with Systemic Lupus
Erythematosus

Kusworini Handono1*, Benny Arie Pradana2, Radhitio Adi Nugroho2, Yona One
Sidarta2, Ivan Andre Hartono2, Dian Hasanah3, Handono Kalim4, Agustina Tri
Endharti 5, Fatchiyah Fatchiyah6

1
Department of Clinical Pathology, Faculty of Medicine Brawijaya University/Saiful Anwar General
Hospital, Malang, Indonesia
2
Department of Internal Medicine, Faculty of Medicine Brawijaya University, Saiful Anwar General Commented [T2]: Spelling error

Hospital, Malang, Indonesia Commented [T3]: I am not sure, but there is inconsistency
4
here. Please check the first page.
Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine Brawijaya
University/Saiful Anwar General Hospital, Malang, Indonesia
5
Department of Parasitology, Faculty of Medicine, Brawijaya University, Malang, Indonesia Commented [T4]: Spelling error
6
Department of Biology, Faculty of Mathematics and Natural Science, Brawijaya University, Malang,
Indonesia

ABSTRACT
Aim: This study aims to investigate the effects of 1,25(OH)2D3 on NETosis and endothelial cell Commented [T5]: Each sentence must have a subject.

apoptosis in Systemic Lupus Erythematosus (SLE) patients with hypovitamin D. Methods:

Neutrophils of five SLE patients with hypovitamin D were treated with 4 different doses of Commented [T6]: Spelling error

1,25(OH)2D3: P0 (0 nM/control), P1 (1 nM), P2 (10 nM), and P3 (100 nM). Phorbol Myristate
Acetate (PMA) was used to stimulate NETs formation. The supernatant was separated and
cocultured with HUVECs. Externalization of histone and HNP during NETosis was measured by
immunofluorescence. Early and late apoptosis of endothelial cells were measured by flowcytometry
using Annexin V and PI antibody. Results: This study shows that neutrophils treated with 10nM of

1,25(OH)2D3 had significantly lower HNP, histone externalization and early endothelial cell
apoptosis compared to the other doses There was also a positive correlation between histone Commented [T7]: The sentence is hard to digest.
Please consider something like this:
externalization and endothelial cells’ early apoptosis. Conclusion: 1,25(OH)2D3 prevents “This study shows that neutrophils treated with 10nM of
1,25(OH)2D3 had significantly lower HNP, histone externalization
endothelial cell damage by decreasing the NETosis process. and early endothelial cell apoptosis compared to the other doses.”
Keywords:SLE, NETs, Histone, HNP,1,25(OH)2D3, Endothelial cells
Abbreviations
SLE, Systemic Lupus Erythematosus; NETs, Neutrophil Extracellular Traps; HNP, Human
Neutrophil Protein; HUVECs, Human Umbilical Vein Endothelial Cells; PMA, Phorbol Myristate
Acetate.
INTRODUCTION Formatted

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease

characterized by the involvement of multi organ damage with a high mortality rate. The five
years survival rate of 108 SLE patients in Cipto Mangunkusumo Hospital in Indonesia is
88% [1]. SLE is characterized by functional changes of neutrophils, plasmacytoid dendritic
cells, B and T lymphocytes [2],. An abnormal neutrophil has been identified in peripheral
blood mononuclear cells (PBMCs) from SLE patients, known as Low Density Granulocytes
(LDGs), and has been reported to have an important role in SLE pathogenesis [3]. These
LDGs have increased capacity to release Neutrophil Extracellular Traps (NETs). NETs are
released during a unique form of cell death called NETosis. NETosis is one mechanism of
the innate immunity that plays a role in the pathogenesis of SLE [4]. NETosis releases
antimicrobial peptides such as Neutrophil Elastase (NE), Myeloperoxidase (MPO), Human
Neutrophil Protein (defensin/ HNP), chromatins, DNA, and histone which forms the NETs
structure. Besides LDGs, neutrophils from SLE patients also have the ability to produce a
huge amount of NETs [5]. NETs are also involved in triggering autoantibody production Commented [T8]: Please cross check this sentence with the
sentence on line number 6.
and complement activation that worsens the clinical manifestation in SLE patients [6].
Autoantibodies and complement activation causes vasculitis that can be life threatening to
SLE patients [7].
Elevated levels of anti microbial peptides, DNA, and histone from NETs are able to
damage endothelium, which may lead to vasculitis. HNPs are small peptides with
antimicrobial properties that play a role in the innate immunity, as well as during NETosis. It
interacts with platelets and exerts a cytotoxic effect, which leads to endothelial cell damage
[9,10]..On the other hand, histone also plays an important role in the induction of epithelial Commented [T9]: Please consider re-structuring these
sentences.
and endothelial cell death. Histone is a structural protein with various modification For example:
“HNPs are small peptides with antimicrobial properties that play a
mechanisms and has the ability to cause a pathological condition in cells. Circulating role in the innate immunity, as well as during NETosis. It interacts
with platelets and exerts a cytotoxic effect, which leads to
histones in vivo can cause endothelial damage, increase microvascular permeability, endothelial cell damage.”

activate the coagulation process, and increase secretion of IL – 6 [11].

A Recent study has reported correlation between vitamin D deficiency with Commented [T10]: Recent studies? Or A recent study?

autoimmune disease activity [12]. Another study showed that a high percentage of SLE patients
tend to be vitamin D deficient [13]. Our previous studies concluded that 1,25(OH)2D3 levels Commented [T11]: I would consider changing this sentence to:
“Another study showed that a high percentage of SLE patients tend
in SLE patients was significantly lower compared to healthy controls. The decreased to be vitamin D deficient [13].”

serum levels of vitamin D in SLE patients impairthe functions of regulatory T cells,

decrease the functions and modulated activities of dendritic cells and B cells, as well as
increase the levels of autoantibodies [14]. In this present study, we assess the effects of
1,25(OH)2D3 on NETosis and endothelial cells apoptosis.

MATERIAL and METHODS

Sample preparation
The neutrophils were collected from 5 newly diagnosed active SLE (Mex-SLEDAI >
5) female patients, aged 17-36 years (mean=26.40±6.77 yo) with 25(OH)D serum levels of
<30 ng/ml, range 16.8 -29.5 ng/ml, mean=21.6±4.7. The characteristics of the subjects can
be seen on Table 1. Ten milliliters of venous blood from each patient was collected in
vacutainers containingEDTA. The serum was isolated and then used to measure the
concentration of 25(OH)D serum by ELISA in accordance with the manufacturer’s
instructions (NovaTein Bio, USA). Umbilical cord blood was obtained from the placenta of
preterm infants (34-36 wk gestation) delivered by cesarean section in Saiful Anwar
Hospital, Malang. Commented [T12]: There are too many double & triple spaces
in this paragraph.
I have deleted most of them.

Isolation of Neutrophils
Neutrophils from subjects were separated from whole blood using gradient
centrifugation with Polymorphprep (Axis-shield PoC AS).Then collected human venous
blood with EDTA was carefully placed over the Polymorphprep (Axis-shield PoC AS) with
equal volume, then centrifuged at 1400 rpm for 33 minutes at room temperature. A layer of
neutrophils were then harvested from the lower band and purified from erythrocytes with
erythrocyte lysis buffer [15]. Neutrophil cells were confirmed by CD10 and CD14 double
positive, purity was >95% [5]. Number of each patient LDGs was measured from PBMC
band which were confirmed by CD10 and CD14 double positive using flowcytometry
[34].(cloke et.al., 2012)

Neutrophil Treatment
Isolated neutrophils were counted with a haemocytometer and 1 to 2 x 105 cells/ml
was seeded in poly-L-lysine coated coverslip with RPMI 1640 media (Sigma-Aldrich, USA)
containing 2 mM glutamin (Gybco, USA), 10 mM HEPES (Gybco, USA), Penicilin-
streptomicin 10.000 U/ml (Gybco, USA), and 10% Fetal Bovine Serum (Gybco, USA) in
24 wells [5]. Isolated neutrophils were incubated for 15 minutes at 37oC, 5% CO2 and
treated with different concentration of 1,25(OH)2D3 (Sigma) (1 nM, 10nM, and 100nM) for
24 hours.
NETs production and isolation
Isolated neutrophils were also seeded in 24 poly-L-lysine-free wells for NETs
production and endothelial cell death assay. Neutrophils were stimulated with 50 nMPMA
for 4 hours. Medium was removed and wells were washed with RPMI. PMA, at this
concentration, does not promote apoptosis.To collect NETs, 2 ml RPMI per well was
added and NETs (the smear on the wells) was collected by vigorous agitation. After
centrifugation at 650 rpm for 5 min, NETs was collected in the supernatant and stored at –
200C [16].

HUVECs Culture
Endothelial cells were obtained from the HUVECs isolated from human umbilical
cord. Then, the umbilical cord was washed from blood and then a collagenase solution
was inserted to the umbilical vein for about 8 minutes. The solution was then centrifuged at
1600 rpm for 8 minutes to obtain cell pellets. Isolated HUVECs were then cultured with
serum free contained Medium 199, gentamicin, phenol red, glutamine, and newborn calf
serum (NCS) in 24 gelatin coated wells until confluenced [15,16]. They were then
incubated at 37°C and 5% CO2. The purity of isolated cells (>99%) was assessed by FACS
following cell labelling with endothelial-specific marker CD146 (Biolegend) [17].

NETs Immunofluorescence assay

Treated neutrophil culture was stimulated using 20 nM PMA (Sigma) to generate
NETs and incubated for 2 hours. It was then washed with ice-cold PBS and fixed with
paraformaldehyde for 15 minutes. Fixed cells were treated with triton 0,5% for 1 minute
followed by blocking buffer FBS 5% for 30 minutes. The fixed cells were then stained with
mouse anti-human histon (1:100), goat anti-human hnp (1:250), and rabbit anti-human
elastase (1:350) for 60 minutes at 37oC followed by incubation with secondary
fluorochrome-conjugated antibody FITC donkey anti rabbit (1:500), Rodhamin donkey
donkey anti mouse (1: 100) or Rodhamin donkey anti goat (1:100) for 60 minutes. Nuclear
DNA was detected using Hoechst 33342 (1:100) incubated for 10 minutes at room
temperature. Coverslips were mounted in Prolong Antifade Reagent (Invitrogen) then
analyzed using Olympus microscope [5]. The percentage of NETs was calculated as the
average of three to four fields (x40) and quantified as the color intensity using image RGB
analysis. Externalization of histon and defensin at NETs (colocalizing of elastase and
DNA) was counted from red intensity per total intensity of the image.
Endothelial Cell Death Assay
HUVECs in 24-well plate were co-cultured with 200 µl supernatant contained NETs
followed by incubation for 16 hours. Apoptosis assay was done using flowcytometry
(FACScalibur) followed by staining with apoptosis kit (AnnexinV–PI). Cells with
AnnexinV+PI- and AnnexinV+ PI+ were regarded as early and late apoptosis HUVECs,
respectively [5,17].

Ethical Clearance
This study was approved by the Ethics Committee of Faculty of Medicine,
Brawijaya University, Malang and informed consent had been obtained from all patients
with SLE.

Statistical Analysis
Data were analyzed using SPSS 16.0 for Windows using one-way analysis of
variance (ANOVA) with Tukey post-tests for multiple comparisons, p<0.05. The correlation
between histone and defensin externalization with endothelial cells apoptosis were
analyzed using Pearson’s correlation test.

RESULTS and DISCUSSION

Results

Table 1. Characteristics of SLE subjects

Characteristics Means ± SD

Age (Years) 26.40 ± 6.77

Duration of Illness (Years) 4.4 ± 2.4
Anti ds-DNA antobodies level ( IU/ml ) 167.42 ± 7.86
Vitamin D serum level (ng/ml) 21.60 ± 4.74
Number of LDGs ( %) 43.39 ± 2.54
Absorbance of NETs (SD) 0.85 ± 0.15

Effect of 1,25(OH)2D3 treatment on the externalization of Histone during NETosis

The 1,25(OH)2D3 treatment in neutrophil culture of SLE patients reduced the
externalization of histone during NETosis as seen in Figure 1. There was a significant
difference in histon externalization (Figure 2) between P2 and P3 compared with P0 (19.38
± 6.16 %; vs. 27.89 ± 4.85 %; p=0.008 and 20,31 ± 0.82 %; vs. 27.89 ± 4.85%; p=0.016).
However, there was no significant difference at histon externalization between P1 and P0
(22.39 ± 1.39 % vs. 27.89 ± 4.85%; p= 0.066). This result indicated that 1,25(OH)2D3 with
doses of 10 nM and 100 nM were able to decrease the externalization of histon during
NETosis from neutrophils of SLE patients, whereas the 1 nM dose of 1,25(OH)2D3 was
not. There was no significant difference in histone externalization between P2 and P3
(19.38 ± 6.16 % vs. 20,31 ± 0.82 %; p=0.751). This showed that 100nM of 1,25(OH)2D3
was not more effective than 1 nM of 1,25(OH)2D3 to decrease histone externalization.

Hoecst NE Histone Merge

P0

P1

P2

P3

Figure 1 NETs with histone components from lupus Neutrophils during NETosis. Representative images of
P0 (0 nM/control), P1 (1 nM), P2 (10 nM), and P3 (100 nM). Cells were stained for detection of
neutrophil elastase (green), DNA (Hoechst 33342, blue), and histone (red).
 *
*

Figure 2 Precentage of NETs with histone components in control group and treatment groups with different
doses of 1,25(OH)2D3. There were reduced mean precentage of NETs with histone among P2 and P3
compared with P0. P2 was the lowest *Significant (p<0.05) compared to control.

Effect of 1,25(OH)2D3 treatment on the externalization of Defensin during NETosis

Neutrophil culture of SLE patients treated with 1,25(OH)2D3 showed a reduction of
defensin externalization as seen in Figure 3. There were significant differences in defensin
externalization (Figure 4) between P1, P2, and P3 compared with P0 (23.035 ± 0.812%;
p=0.011, 24.232 ± 1.902%; p=0.023, 24.480 ± 2.780%; p= 0.027 vs 32.120 ± 0.813%).
This result indicated that treatment of 1 nM, 10 nM, and 100 nM of 1,25(OH)2D3 could
decrease externalization of defensin during NETosis.

* *

Figure 3.Precentage of NETs with defensin components in control group and treatment groups with different
doses of 1,25(OH)2D3. There were reduced mean precentage of NETs with defensin components among P2
and P3 compared with P0. P2 was the lowest *Significant (p<0.05) compared to control.
Hoecst NE Defensin Merge Formatted: Left, Indent: Left: 0", Hanging: 0.59"
Formatted
Figure4.NETs with defensin components from lupus Neutrophils duringNETosis. Representative images of
P0 (0 nM/ control group), P1 (1 nM), P2 (10 nM), and P3 (100 nM). Cells were stained for
detection of neutrophil elastase (green), DNA (Hoechst 33342, blue), and defensin (red).
Formatted

Effect of 1,25(OH)2D3 treatment on early apoptosis of endothelial cells

The 1,25(OH)2D3 treatment incultured neutrophils of SLE patients reduced
endothelial cells early apoptosis due to NETosis as seen in Figure 5. There was a
significant difference in early apoptosis (Figure 6) between P2 and P0 (2.62 ± 0.53 %; vs.
6.17 ± 1.90 %; p=0.001), but there was no significant difference between P1 and P3
compared to P0 (4.61 ± 0.28% vs. 6.17 ± 1.90 %; p= 0.266 and 3.97 ± 0.59 vs. 6.17 ±
1.90 %; p=0.069). This result indicated that only 10nM of 1,25(OH)2D3 was able to
decrease the early apoptosis due to NETosis.
The 1,25(OH)2D3 treatment did not reduce late apoptosis. There was no significant
difference between P0 and treatment groups P1, P2, P3(1.00 ± 0.65 % vs. 1.57 ± 0.15 %,
0.83 ± 0.29 %, and 1.57 ± 0.45%, p=0.144). This indicated that 1,25(OH)2D3 was not able
to decrease the late apoptosis due to NETosis in neutrophils of SLE patients.
Furthermore, there was a moderate positive correlation between early apoptosis
and histon externalization (r=0.759 p=0.004). This result indicated that a reduction in
histone externalization due to 1,25(OH)2D3 treatment can reduce early apoptosis during
NETosis in neutrophils of SLE patients.
 P0 P1 P2 P3
(control) (1nM) (10nM) (100nM)

Figure5.The 1,25(OH)2D3 decreased in early apoptosis. Percentage of early apoptosis showed at lower right
quadrant. Percentage of Late apoptosis showed at upper right quadrant

A B

Figure6.Effect of 1,25(OH)2D3 treatment onearly and late apoptosis of endothelial cells during NETosis (A).
There were reduced mean of early apoptosis among P2 compared with P0. *Significant (p<0.05).Late
apoptosis percentage (B). No significant result among each treatment groups.

Discussion
Various studies revealed that NET formation may be an important process in
modification of autoantigen and exposure to the immune system, as well as in the
induction of tissue damage [18,19]. Increased and aberrant NET formation may have an
important role in the development and progression of autoimmune diseases and another
chronic inflammatory disorders [14,18]. Neutrophils from SLE patients (LDGs) have
greater ability to form NETs with increased expression of many neutrophil proteins and
enzymes that have important role in NET formation and autoimmunity induction. These
NETs also express double stranded DNA (dsDNA), a key autoantigen in pathogenesis of
SLE. NETs itself is composed of DNA, histone, neutrophil elastase (NE), myeloperoxidase
(MPO), human neutrophil protein (HNP) and other granular proteins which will be
externalized during NETosis [14,18].
 Our study showed that histone externalization during NETosis in neutrophil of SLE
patients was decreased by 1,25(OH)2D3 treatment in 10 nM and 100 nM doses. These
results indicated that 1,25(OH)2D3 can inhibit NETs formation in neutrophils of SLE
patients by various possible mechanisms. A study by Hirsch et al, showed that
1,25(OH)2D3 reduced the production of inflammatory mediators and reactive oxygen
intermediates in neutrophils through induction of 5-LOX (5-Lipoxygenase) gene and
suppressed COX-2 (Cyclooxygenase-2) gene expressions [15]. In another study,
1,25(OH)2D3 was able to decrease macrophages activity through inhibition of transcription
listericidal protein coding genes, gp91 Phox (Cybb). gp91Phox is a component of the
phagocytic oxidase (Phox) or also called NADPH oxidase [20]. Phox itself is a key enzyme
in the NETs formation [21]. 1,25(OH)2D3 is also capable to inhibit the PI3K/AKT/mTOR
pathway due to its interaction with VDR and its capability to increase the transcription rate
of PTEN (Phosphatase and Tensin Homolog) and DDIT4 (DNA-damage-inducible
transcript 4) [22]. McInturff et al, 2012, found that inhibition of the mTOR pathway
decreased NETs formation and subsequently bacterial killing ability [23]. Some of the
above mechanisms might explain how 1,25(OH)2D3 was able to reduce NETosis .
Our study showed that defensin externalization during NETosis was decreased by
1,25(OH)2D3 treatment on lupus neutrophils. Defensin is an antimicrobial peptide that may
have a role in the innate and adaptive immunity. Study by Kim et.al,(2009) showed that
treatment with calcipotrion (a vitamin D analogue), decreased expression of defensins.
Vitamin D improved antimicrobial peptide externalization efficiency that regulate the innate
immunity. This study used keratinocyte culture that was induced by UVB, LPS, and TNF-
α. Reduced expressions of defensin was probably caused directly by the effect of
calcipotriol [24]. Another mechanism that was probably related to the reduced defensin
externalization is CYP24, a mitochondrial enzyme that initiates the catabolism of vitamin
D. Abnormal CYP24 regulation was found in several autoimmune disease such as multiple
sclerosis [25].
Our study also showed that 1,25(OH)2D3 reduced early apoptosis of endothelial
cells but not late apoptosis. NETs have been found to induce cell death via several
components, one of which is a histone. In addition to its function as a structural protein
with various modification mechanisms, histone can induce a pathological condition in cells.
Circulating histones in vivo can cause endothelial damage, increase microvascular
permeability, activate the coagulation process, and increase secretion of IL – 6 [26]. In a
previous study by Abrams et al, 2011, C reactive protein was able to prevent endothelial
damage through the inhibition of histone integration on the membrane and thus prevent
the influx of Ca2+. Histones that fail to integrate do not induce Ca2+ influx, therefore unable
to induce cell damage. Saffardzeh et al.(2012) showed the same pathological role of Commented [T13]: Please check the spelling of the Author’s
name. It is inconsistent with the bibliography.
histone proteins, which is one component of chromatin in NETosis. In this study, histones
and MPO were able to cause damage to the alveolar epithelial and endothelial cells. This
is proven by the decrease in epithelial and endothelial cell damage due to NETs when
given anti-histone antibodies in the culture medium [16].
Histone probably caused early apoptosis due to its potential to integrate with
plasma membrane and induce calcium influx. Increased calcium influx into the
mitochondrion or binding of the protein Bid to the mitochondrial membrane, will induce a
permeability transition in the membrane of an adjacent mitochondrion. This willresult in the
release of cytochrome c, followed by the formation of the apoptosome in which caspases
are activated [27]. Early apoptosis itself is indicated by phosphatidylserine (PS)
translocation from the inner membrane to the external membran and recognized by
Annexin V [28].
There are several mechanisms in which defensin may induce apoptosis. Some of
the immune complexes in Lupus serum were formed in association with NETs. NETs
contain autoantibodies to antimicrobial peptides such as LL37, MPO, PR3 and defensin.
That autoantigens can be recognized by ANCA (anti-neutrophil cytoplasmic antibodies).
ANCAs interact with their antigen and cause neutrophil activation. This activation leads to
neutrophil degranulation, cytokine production, and endothelial damage. The presence of
autoantibodies can activate autoreactive T-helper cells and B cells, which can then induce
small vessel vasculitis [8,29].
In this study, we also found a moderate correlation between early apoptosis with
histone externalization. This indicated that an increase in externalizing of histones can
increase the likelihood of early apoptosis. This result has similarities with the research
conducted by Saffardzeh.,et al, 2012, who found that histones and myeloperoxidase are Commented [T14]: Please check the spelling of the Author’s
name. It is inconsistent with the bibliography
predominantly responsible for the NET - mediated cytotoxicity.
In this study there was no significant difference in late apoptosis among each
group. This is probably caused by several mecanisms. PI that bind DNA as a sign of late
apoptosis in this study may not be able to pass through the cell membrane that is still
moderately intact [28]. Based on Wolbers et al, 2004, the apoptotic turnover rate depends
on the substances used to induce apoptosis, while the type of the cell determines the way
of the transition within the apoptotic cascade [30]. Histones as an agent which is
suspected to induce cell death in this study, may not be able to damage the integrity of the
cell at 16 hours of exposure as in previous studies conducted by Villaneuva et al, 2011.
The optimal vitamin D level in serum was >30 ng/ml (>75 nM) [29]. In this study,
100 nM 1,25(OH)2D3 treatment had a lower significant result than using 10nM in
decreasing histone externalization. This condition reveals that after 1,25(OH)2D3 has
reached the optimal dose, any higher dose would give the opposite effects. The dose of 1
nM equals to 0.4 ng/ml [31], therefore 100 nM equals to 40 ng/ml. If this 100 nM dose is
given to treat the culture, it would probably give a toxic or opposite effect [32,33]. Similarly,
in another study conducted using dendritic cells, 100 nM of 1,25(OH)2D3 treatment gave
inverse effect, indicated by an increasing percentage of Th17 cells and levels of IL-17A [2].

CONCLUSION
1,25(OH)2D3, especially the dose of 10 nM, significantly inhibited histone and
defensin externalization during NETosis and endothelial cells early apoptosis. Increasing
doses however, may give inverse results due to opposite effects.

ACKNOWLEDGEMENTS
This study was supported by the Directorate General of Higher Education research
grant of 2013-2014. We thank Dr. dr. Loeki Enggar Fitri, M.Kes, Sp.ParK for the
methodological advice on this study.

DISCLOSURES
The authors declare that there is no conflict of interest regarding the publication of
this paper.

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