*Corresponding author: firstname.lastname@example.org
Rheumatoid arthritis (RA) is a chronic
systemic musculoskeletal disease in
which various joints in the body are
inflamed, leading to swelling, pain,
stiness, and a possible loss of function.1,2 RA is
caused by an imbalance in the number or function
of T helper 17 (Th17) cells and regulatory T
(Treg) cells.3,4 Increased oxidative stress also
participates in the pathogenesis and severity of
Oxidative stress in RA can be caused by
deteriorating oxygen supply to the joint tissues
thereby supporting increased reactive oxygen species
(ROS), causing oxidative damage that further
promotes inflammation. Hence, oxidative stress
and inammation are inseparably connected.7,8
Hypoxia-inducible factor-1 alpha (HIF-1α) is a
key transcriptional regulator that enables cellular
metabolic adaptation to low levels of oxygen.9 As
a transcription factor, it inuences and regulates
the expression of dozens of genes involved in
maintaining homeostasis such as changes in oxygen
concentration (oxygen-dependent) and independent
signals.10,11 Superoxide dismutase (SOD) antioxidant
is a protein that functions as important defense
mechanisms for oxidative stress and acts as a rst-
original article O M J , V. 35, N. 1: e90
Eects of Hyperbaric Oxygen on T helper
17/regulatory T Polarization in Antigen
and Collagen-induced Arthritis: Hypoxia-
inducible Factor-1α as a Target
1,2,3*, Joewono Soeroso3, Mohammad Guritno Suryokusumo4
and Tedy Juliandhy5
1Department of Hyperbaric, Drs. Med. R. Rijadi S., Phys. Naval Health Institute, Surabaya, Indonesia
2Department of Physiology, Hang Tuah University, Surabaya, Indonesia
3Department of Biochemistry, Unit of the Experimental Animal, Airlangga University, Surabaya, Indonesia
4Department of Hyperbaric, Pembangunan Nasional University, Jakarta, Indonesia
5Department of Electrical Engineering, Hang Tuah University, Surabaya, Indonesia
Received: 1 January 2019
Accepted: 2 May 2019
Hypoxia-Inducible Factor 1;
T-Lymphocytes, Regulator y;
Rheumatoid Factor; Arthritis,
Objectives: We sought to investigate and prove the eect of hyperbaric oxygen therapy
(HBOT) on T helper 17 (17)/regulatory T (Treg) cell polarization through changes
in the expression of hypoxia-inducible factor-1 alpha (HIF-1α) in rheumatoid arthritis
(RA) animal model. Methods: We used antigen and collagen-induced arthritis (ACIA)
as a RA animal model. Sixteen male BALB/c models of ACIA mice were divided
into two groups, the non-HBOT group as the control group and the HBOT group
as the treatment group. Expression of HIF-1α, 17 anti-cluster dierentiation 196
(CD196), and Treg anti-interleukine 2 receptor β-chain cells (IL-2Rβ) in tissue from
the le knee joint tissue were determined histologically. Oxidative stress and systemic
inammation were assessed by levels of superoxide dismutase (SOD), interleukin 17a
(IL-17a), C-reactive protein (CRP), and rheumatoid factor (RF) using the enzyme-
linked immune-sorbent assay. e degree of arthritis was assessed by clinical scoring of
paw swelling and the diameter of paw swelling. Results: We found a signicant decrease
(p < 0.050) in the expression of HIF-1α, 17 (CD196), IL-17a, RF levels, and the
clinical scores and the diameter of paw swelling when comparing both groups. ere
was no signicant decrease in the level of CRP in the treatment group compared to
the control group. e expression of Treg (IL-2Rβ) increased signicantly (p < 0.050)
and the level of SOD increased but not signicantly (p > 0.050) in the treatment group
compared to the control group. Conclusions: HBOT has eects on the polarization
of 17 to Treg through a decrease in expression of HIF-1α in mice with ACIA.
HBOT is recommended for use as a support therapy for RA in combination with
OMAN MED J, VOL 35, NO 1, JANUARY 2020
*Corresponding author: email@example.com
T H, .
line component of the defense system against free
radicals or reactive species.12
Interleukin 17a (IL-17a) is an inflammatory
biomarker for RA, which has a strong association
with C-reactive protein (CRP).13 CRP is a member
of the pentraxins protein family, which is composed
of ve 23-kDa subunits and its levels can increase
by 1000-fold or more with infection, inammation,
and tissue injury. CRP levels correlate with morning
stiness, pain, fatigue, grip strength, articular index,
Rheumatoid factor (RF) is the most common
laboratory serologic marker for the diagnosis of
RA.15 Although RF can be detected in patients
with other connective tissue diseases, RF isotypes
are helpful in the management of RA patients
from the time of diagnosis until deciding on the
choice of therapeutic strategy. RF testing in RA
patients has a sensitivity of 60% to 90% and a
specicity of 85%.16
RA causes high-intensity pain and severe
suering because it is oen not diagnosed or treated
quickly enough.17 Joint destruction occurs in the rst
six months aer illness, and permanent defects occur
two to three years later if le untreated.18 Extra-
articular manifestations of RA occur in 17.8–40.9%
of RA patients and 1.5–21.5% are usually associated
with increased morbidity and mortality such as
cardiopulmonary and kidney due to developing the
disease itself or the eects of the drugs given.19,20
Years of aggressive treatment are needed to control
symptoms, manage pain, and stop the development
of RA. Standard therapy in the form of drugs and
even surgery has not provided optimal results.21
Hyperbaric oxygen therapy (HBOT) is based
on administering pure oxygen to the patient while
undergoing increased ambient pressure.22 HBOT
can be a new breakthrough for creating appropriate
treatment strategies and reducing the adverse eects
of the drugs used, so HBOT research on RA needs
to be developed again. e Naval Health Institute
of the Indonesian Navy has used HBOT to treat
diving cases of decompression sickness, arterial gas
embolism, and gas poisoning, and even as an adjunct
therapy in some cases of clinical diseases such as
wound healing, diabetes mellitus with gangrene gas,
and osteomyelitis. Several studies have shown that
HBOT has a specic mechanism that can reduce
inflammation, but the mechanism of reducing
arthritis in RA is still unclear.
We conducted a randomized, control study between
January and April 2018. We used BALB/c mice, male,
weighing 20–30 grams, aged 10–14 weeks, healthy
during the adaptation phase with the characteristics
of clear-eyed, shiny fur, agile movements, and
e nature of this experiment was invasive and
fatal to the experimental animals. Random sampling
was used to select 16 mice with collagen-induced
antigen and arthritis (ACIA) from a total of 20. e
selected mice were divided into two groups with
each group consisting of eight mice. e rst group
was the control group (non-HBOT) and the second
group was the treatment group (HBOT).
We used ACIA as an animal model of RA
because ACIA is an animal model that is very similar
to RA in humans. Mice were injected with 100 g
of methylated bovine serum albumin (mBSA) in
50 L phosphate buffer saline (PBS), which was
emulsied with 50 L complete Freund adjuvant
subcutaneously and 200 ng of Bordetella pertussis
toxin (PTx) intraperitoneally.
Seven days later, mice were injected with 50 g
mBSA and 100 g collagen type II (CI I) in 50 L PBS
were emulsied with 50 L of incomplete Freund
adjuvant (IFA) and 200 ng PTx subcutaneously.
On day 14 of the study, mice were injected with 50
g mBSA and 100 g CII in 50 L PBS emulsied
with 50 L IFA subcutaneously and 200 ng PTx
intraperitoneally. On day 28 of the study, mice were
induced with 50 g mBSA dissolved in 20 µL PBS
into the le cavity of the knee joint intra-articular
(ipsilateral) and the right knee cavity (contralateral)
was injected with 20 L PBS intraarticularly.
Twenty-one days later (day 49 of the study), we
got research animals with the characteristics of RA
disease (ACIA animal models).
We used Treatment Table 9 (USN TT9), the
hyperbaric oxygen table dosing protocol developed
by the United States Navy (USN). e treatment
group was given normal air exposure for 10 minutes
at 2.4 atmospheres absolute (ATA) pressure. en
given oxygen exposure of 100% 3–4 L/min for
90 minutes divided by 3 × 30 minutes intervals 2
× 5 minutes breathing with normal air at 2.4 ATA
pressure. After that, the pressure was reduced to
1 ATA while breathing normal air without using
oxygen for 10 minutes. e treatment group received
HBOT for 10 consecutive days.
T H, .
All mice were anesthetized with ketamine (300
mg/kg body weight + xylazine 40 mg/kg body
weight) intraperitoneally aer 30 minutes of exposure
to HBO. Blood and joint tissue samples were taken
aer 10 minutes to ensure no pain response. Blood
was taken using a syringe on the heart ventricle for
enzyme-linked immune-sorbent assay (ELISA)
examination, and synovial tissue was taken by using a
scalpel for immunohistochemical examination. e
mice were killed by neck dislocation.
The expression of HIF-1α was determined
using the anti-HIF-1α mouse monoclonal
immunoglobulin G1 (IgG1) antibody, the anti-
cluster differentiation 196 (CD196) mouse
monoclonal IgG1 antibody for the expression of
17, and anti-interleukine 2 receptor β-chain cells
(IL-2Rβ) mouse monoclonal IgG2b antibody (all
from Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) for expression of Treg. We used the avidin-
biotin complex for staining. Immunoexpression was
calculated from the average number of brown cells
in ve large visual elds at 400 × magnication on a
e level of serum RF was measured using IgG
RF Mouse ELISA kit (LifeSpan BioSciences, Inc.,
USA). e level of serum CRP was measured using
mouse CRP antibody kit (LifeSpan BioSciences,
Inc., USA), and level of IL-17a in plasma was
measured using mouse IL-17a antibody (Bioassay
Technology Laboratory system, Korain Biotech
Co., Ltd, Shanghai, China) with an enzyme-linked
immunosorbent assays (ELISA) reader (Zenix-320
microplate reader). e examination of plasma SOD
levels was measured using a mouse SOD ELISA kit
(LifeSpan BioSciences, Inc., USA) with an ELISA
reader (Spectrophotometer Zhimadzu) analyzers.
e concentrations of RF (IU/mL), CRP (g/mL),
IL-17a (ng/mL), and SOD (L/mL) captured were
Figure 1: Immunohistochemical staining of hypoxia-inducible factor-1α (HIF-1α) in the (G1) non-
hyperbaric oxygen therapy (HBOT) and (G2) HBOT groups. Living cells that expressed the HIF-1α protein
(positive cells) were stained brown and blue cells (hematoxylin stain) showed no expression of the HIF-1α
protein. Magnication (a) = 50 ×, (b) = 400 ×.
OMAN MED J, VOL 35, NO 1, JANUARY 2020
T H, .
determined by measuring absorbance at 450 nm
using a spectrophotometer.
e degree of arthritis was assessed by clinical
scoring of paw swelling and the diameter of the paw
was measured by digital calliper (Mitutoyo, Japan).
e clinical scoring of the paw was graded as follows:
0 = normal and no swelling; 1 = erythema and mild
edema; 2 = erythema and moderate edema; 3 =
erythema and severe edema; 4 = maximal swelling
and deformation leading to incapacitated limb. e
calliper was placed across the paw at widest point
with a level of accuracy up to 0.05 mm (the number
of strips on the slider scale was 20 so that 1 mm: 20
= 0.05 mm). e values were expressed as the mean
of the two paw diameters of mice.
All data were analyzed using SPSS Statistics
(IBM Corp. Released 2013. IBM SPSS Statistics
for Windows, Version 22.0. Armonk, NY: IBM
Corp.). All values were expressed as mean±standard
deviations (SD). A value of p < 0.050 was considered
e normality test, using the Shapiro-Wilk test showed
the expression of HIF-1α, CD196, IL-2Rβ, SOD levels,
IL-17a, CRP, RF, clinical scoring of paw swelling, and
diameter of paw swelling had normal distribution (p >
0.050). e results of Lavene test showed expression
of HIF-1α, SOD levels, IL-17a, RF, and the clinical
scoring of paw swelling and the diameter of paw
swelling had homogeneous variance (p > 0.050), but
the expression of CD196, IL-2Rβ, and the level of CRP
did not have homogeneous variance (p < 0.050).
The independent t-test showed there was a
signicant decrease in the expression of HIF-1α in
HBO group (8.7±2.2, p < 0.001) compared to non-
HBO group (69.9±3.1, p < 0.050) [Figure 1].
Figure 2: Immunohistochemical staining of anti-cluster dierentiation 196 (CD196) in the (G1) non-
hyperbaric oxygen therapy (HBOT) and (G2) HBOT groups. Living cells that expressed the CD196 protein
(positive cells) were stained brown and blue (hematoxylin stain) cells showed no expression of the CD196
protein. Magnication (a) = 50 ×, (b) = 400 ×.
T H, .
The Mann-Whitney test determined that the
expression of CD196 also decreased signicantly in
the HBO group (4.8±1.4, p < 0.001) compared to
non-HBO group (40.0±4.7, p < 0.050) [Figure 2].
e Mann-Whitney test showed the expression
of IL-2Rβ increased signicantly in the HBO group
(5.0±1.3, p < 0.001) compared to the non-HBO
group (48.2±17.9, p < 0.050) [Figure 3].
e dierences in the results of oxidative stress,
systemic inammation, and degree of arthritis in
experimental groups are shown in Table 1. In the
independent t-test showed there was no signicant
Table 1: Dierences of the results of systemic inammation, oxidative stress, and degree of arthritis in the
control and treatment groups.
Variables G1 G2 p-value
SOD, L/mL 0.012 ± 0.001 0.014 ± 0.001 0.093
IL-17, ng/mL 0.2 ± 0.0 0.1 ± 0.0 < 0.001
CRP, g/mL 47.3 ± 18.8 40.4 ± 10.0 0.599
RF, IU/mL 0.012 ± 0.001 41.2 ± 6.8 < 0.001
Clinical scoring of paw swelling 13.6 ± 1.9 8.3 ± 1.9 < 0.001
Diameter of paw swelling, mm 6.9 ± 0.4 6.1 ± 0.4 0.001
SOD: superoxide dismutase; IL-17: interleukin 17; C RP: C-reactive protein; RF: rheumatoid factor.
Data shown as mean±SD; p < 0.050 was considered statistically signicant.
G1: non-HBOT group; G2: HBOT group.
Figure 3: Immunohistochemical staining of anti-interleukine 2 receptor β-chain cells (IL-2Rβ) in the (G1)
non-hyperbaric oxygen therapy (HBOT) and (G2) HBOT groups. Living cells that expressed the IL-2Rβ
protein (positive cells) were stained brown and blue cells (hematoxylin stain) showed no expression of the
IL-2Rβ protein. Magnication (a) = 50 ×, (b) = 400 ×.
OMAN MED J, VOL 35, NO 1, JANUARY 2020
T H, .
increase (p = 0.093) in SOD levels, there was a
signicant decrease (p < 0.001) in the level of IL-
17a, RF, and clinical scoring of paw swelling in the
treatment group compared to control group. e
diameter of paw swelling also decreased signicantly
in the treatment group compared to the control
group [Table 1]. ere was no signicant decrease
in CRP levels between the two groups.
Research on RA continues to develop. Research on
the use of antioxidants or antioxidant-containing
foods combined with drugs, such as methotrexate,
and the addition of natural polyphenol antioxidants,
including silibinin can function to increase the
eect of drugs and reduce oxidative stress.23,24 Our
study limited the use of HBOT alone to determine
the eect and basic mechanism of oxygen use on
the improvement of hypoxic cells and to see the
extent of the role of HBOT in polarization or
changing of phenotype from Th17 cells to Treg
cells. In the future, research on the use of HBOT
in combination with drugs, biological agents, or
other antioxidants needs to be considered and
Several HBOT studies on RA have been carried
out. Clinical improvement due to decreased serum
immune complexes and T lymphocyte function was
found in patients with RA aer 21 sessions of HBOT
under 1.7 ATA for 40 minutes.25 HBOT 70% at 1.5
ATA for three hours daily for two weeks reduced
neuropathic pain in eight Sprague-Dawley male
mice RA models.26 In contrast to previous studies,
this study examined the strategy of autoimmune
anti-rheumatic therapy involving polarization of
17 cells (CD196) into Treg (IL-2Rβ) through
changes in expression of HIF-1α aer HBOT. is
study used higher oxygen levels and pressure than
previous studies but was still based on safe doses. It
has been suggested that if the pressure used to deliver
hyperbaric oxygen did not exceed 3 ATA (equivalent
to 20 meters in seawater) and the duration of
treatment for elective therapy did not exceed two
hours then this HBOT was still considered safe.27
is study used HBOT with dosage according
to USN TT9 breathing 100% oxygen for 3 × 30
minutes intervals 2 × 5 minutes breathing normal
air at 2.4 ATA for 10 consecutive days. Higher doses
were expected to provide more optimal results.
Some theories stated that HBOT was considered a
safe treatment modality but carried a risk because
of hyperoxia and increased pressure. HBOT acted
as a chemical agent that could aect the oxidants
and antioxidants system. e main mechanism for
HBOT was based on the generation of intracellular
ROS. Previously, it has been stated that respiration
with high oxygen concentrations and pressure greater
than 1 ATA would increase ROS production.28,29
We thought that HBOT could lead to increased
ROS and oxidative stress if given at excessive
doses; therefore, this study was limited to 10
Studies of the effects of HBOT on RA on
oxidative stress has also been carried out previously
using oxygen levels and lower pressures compared to
this study.30,31 HBOT increased the SOD activity,
decreased the value of lipoperoxide, and improved
the erythrocyte sedimentation rate and the Lansbury
articular index in patients with RA.30 HBO oxygen
36% at 1.25 ATA for three weeks reduced derivative
reactive oxygen metabolites and CRP in collagen-
induced arthritis type II mice.31
e results of the study on oxidative stress levels
could be seen in SOD levels. The level of SOD
increased but not signicantly in the HBOT group
compared to non-HBOT group. We hypothesized
that HBOT increased ROS production but, if the
level was not excessive, it could be useful because
ROS could also act as a cellular messenger in many
signal transduction pathways and induce other
cytoprotective genes.32,33 is seemingly c ontroversial
eect was strongly inuenced by therapeutic doses,
and the length and interval of exposure. ROS was
related to the amount of oxygen present, but HBOT
paradoxically induced the activity of antioxidant
enzymes such as SOD. e nuclear factor erythroid
2-related factor 2 (Nrf2)/Keap1 pathway was the
main regulator of redox homeostasis. Nrf2 had a
major contribution to the regulation of defense
systems in various antioxidants as a cytoprotective
response to endogenous and exogenous pressures
caused by ROS.34 SOD functioned to suppress or
prevent the formation of ROS in cells by rapidly
neutralizing any molecule with the potential to
develop into free radicals or any free radicals with the
ability to induce other radical production.35 is fact
was consistent with the concept of mitochondrial
hormesis, which states the production of ROS
could induce a positive response that was increased
T H, .
resistance to stress, and actually cause oxidative stress
Understanding HIF expression in RA joints
allows us to better understand the level at which
they are activated based on the severity of the disease,
and how it aects certain cell types that contribute
to perpetuating this disease. RA triggers the
accumulation of HIF-1α and HIF-2α chains, so that
the target of RA therapy was HIF-1α and HIF-2α.37
We chose the HIF-1α variable instead of HIF-2α in
this study because a previous study stated that HIF-
1α was widely expressed and was believed to play an
important role in the hypoxic response compared to
HIF-2α.38 Under normoxia or hyperoxia conditions,
HIF-1α was more easily degraded than HIF-2α,
which was more stable.39,40 Aer HBOT, HIF-1α
was hydroxylated by prolyl hydroxylase domain
proteins, recognized by the ubiquitin E3 ligase, and
directed to the proteasome for degradation.41,42
The results of this study showed significant
decreases in HIF-1α and 17 (CD196) expression,
and a signicant increase in the expression of Treg
(IL-2Rβ) in the treatment group compared to the
control group. e mechanism of HBOT hyperoxia
or normoxia aer exposure to HBOT caused HIF-
1α activity to decrease, which caused polarization or
dierentiation of phenotypes from 17 (CD196)
to Treg (IL-2Rβ). Decreasing the expression of HIF-
1α activated FOXP3 gene expression and finally
degraded RORγt in 17/Treg progenitor when
committing to polarization from 17 to Treg so
that the number of 17 (CD196) decreased.
IL-17a levels decreased signicantly and CRP
levels decreased but not signicantly in the treatment
group compared to the control group. This is in
accordance with previous studies that found a
relationship between serum IL-17a levels and CRP.
IL-17a is inducer CRP from mouse smooth muscle
cells and hepatocytes.43,44 CRP levels decreased, but
not signicantly, because many factors also inuence
CRP levels. ROS is also thought to play a role in
increasing CRP levels.45 We suspect that ROS as a
result of exposure to HBOT could also play a role in
CRP metabolism. Increased blood levels of IL-17a
from mice with RA were of limited use as biomarkers
to show disease activity.46
RFs are a family of autoantibodies directed to
the Fc portion of IgG. ey are locally produced
in RA by B cells present in lymphoid follicles and
germinal center-like structures that develop in
inamed synovium.47 ey are heterogeneous and
usually composed of IgM. Because of this, most
assays detect only IgM. RFs are used as a marker in
individuals with suspected RA or other autoimmune
conditions. Detection of IgM RFs is also helpful as a
prognostic index, and some studies have shown that
immunosuppressive treatment can decrease serum
RF levels. However, the clinical usefulness of RFs in
monitoring disease activity and treatment response is
limited. In this study, the level of IgM RF decreased
signicantly in the treatment group compared to the
control group. Decreasing the number of 17 aer
exposure to HBOT caused a signicant decrease in
IL-17a production in the treatment group compared
to the control group. is resulted in a direct decrease
in autoreactive B cell proliferation and a decrease
in dierentiation and plasma cell activity,16 which
resulted in a decrease in the production of RF.
Hypoxia had been shown to induce an
inflammatory response, in this case, ACIA. The
reduction of arthritis clinically after exposure to
HBOT could be seen from the results of clinical
scoring and diameter of paw swelling, which both
decreased significantly in the treatment group
compared to the control group. e decrease in the
amount and function of Th17 caused a decrease
in the production of pro-inammatory cytokines
IL-17a, IL-17f, IL-21, IL-22, interferon γ, and
so the arthritis decreased.48
We recommend the use of HBOT as a supporting
therapy in RA. Our study provides new insights into
therapeutic interventions in human autoimmune
The authors declared no conflict of interests. The Ethics
Committee of Naval Health Institute, Indonesian Navy stated
that this study was feasibly approved (Animal Ethical Clearance
Certificate No.009/AECC/NHI/IX/2017). This study
received support from the Department of Physiology, Faculty
of Medicine, Hang Tuah University, Surabaya, Indonesia;
Department of Hyperbaric, Drs. Med. R. Rijadi S., Phys. Naval
Health Institute, Indonesian Navy, Surabaya, Indonesia; and
Department of Biochemistry, Unit of the Experimental Animal,
Faculty of Medicine, Airlangga University, Surabaya, Indonesia.
is research was funded by the Faculty of Medicine, Hang Tuah
University, Surabaya, Indonesia.
OMAN MED J, VOL 35, NO 1, JANUARY 2020
T H, .
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