International Medical Journal Vol. 26, No. 6, pp. 479 - 482 , December 2019

IMMUNOLOGY

Hyperbaric Oxygen Effect on Switching Phenotype M1-M2 Macrophage Expression in Antigen and Collagen-Induced

Arthritis

T. Harnanik1,2,3), J. Soeroso3), M. Guritno Suryokusumo4), T Juliandhy5)

ABSTRACTObjective: To evaluate the effect of hyperbaric oxygen (HBO) therapy on switching phenotypes from M1 macrophage cluster

differentiation (CD) 80 to M2 (CD163) through changes in expression of hypoxia-inducible factor-1α (HIF-1α) in antigen and collagen-induced arthritis (ACIA).

Methods: We used male Balb / C mice as models of ACIA. The severity of arthritis was assessed by a clinical arthritis score and with the diameter of the left knee joint measured using a calliper. Interleukine-10 (IL-10) level in plasma was measured by ELISA technique. Expression of HIF-1α, CD80 dan CD163 were measured by immunochemistry technique. We used IBM SPSS statistics version 22.0. Statistical significance test was done by independent t-test.

Results: The level of expression of HIF-1α decreased significantly p = 0.000 (p < 0.05) in the HBO group (33.20 ± 10.12) com-pared to non-HBO group (106.40 ± 21.20). The level of expression of CD80 decreased significantly p = 0.000 (p < 0.05) in the HBO group (69.80 ± 6.71) compared to non-HBO group ((308.10 ± 25.01). The level of expression of CD163 increased signifi-cantly p = 0.000 (p < 0.05) in the HBO group (153.00 ± 8.78) compared to non-HBO group (44.90 ± 2.89). The clinical arthritis score and diameter of the left knee joint of mice decreased significantly (p < 0.05) and IL-10 levels increased significantly (p < 0.05) in the HBO group compared to the non-HBO group.

Conclusion: HBO has an effect on switching the M1 phenotypes to M2 through a decrease in the expression of HIF-1α while increasing IL-10 production thereby reducing arthritis in ACIA. Therefore, HBO has therapeutic potential for the treatment of rheumatoid arthritis.

KEY WORDSHBO, RA, HIF-1α, CD80, CD163

Received on January 16, 2019 and accepted on July 11, 20191) Department of Hyperbaric, Drs. Med. R. Rijadi S., Phys. Naval Health Institute, Indonesian Navy Surabaya, Indonesia2) Department of Physiology, Faculty of Medicine, Hang Tuah University Surabaya, Indonesia3) Department of Biochemistry, Unit of the Experimental Animal, Faculty of Medicine, Airlangga University Surabaya, Indonesia4) Department of Hyperbaric, Faculty of Medicine, Pembangunan National University Jakarta, Indonesia5) Department of Electrical Engineering, Faculty of Engineering and Marine Sciences, Hang Tuah University, Surabaya, Indonesia.Correspondence to: T. Harnanik(e-mail: titut.harnanik1972@gmail.com)

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INTRODUCTION

Rheumatoid arthritis (RA) is a chronic autoimmune disease charac-terized by symmetrical polyarthritis, synovitis, cartilage destruction and bone1,2). Hypoxia or low partial pressure of oxygen (pO2) is an important characteristic of the synovial tissue of RA3). Hypoxia-inducible 1-alpha (HIF-1α) is the main regulator of cellular response to hypoxia4).

The imbalance of proinflammatory cytokine production by macro-phages M1 (CD80) and anti-inflammatory by M2 (CD163) plays a role in the pathogenesis of RA5,6). M1 expresses inflammatory genes, includ-ing tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. M2 produces a variety of immunosuppressive cytokines and growth fac-tors such as IL-10, IL-1ra (IL-1 receptor antagonist) and transforming growth factor-β(TGF-β) which are closely related to their ability to

weaken inflammation and promote the improvement of extracellular tis-sue7).

Hyperbaric oxygen (HBO) therapy is breathing with oxygen-en-riched air at a pressure of more than 1 ATA in a high-pressure air cham-ber8). Several studies have shown that HBO plays an important role in reducing arthritis because it has an anti-inflammatory effect, but the mechanism is still unclear.

We used antigen and collagen-induced arthritis (ACIA) as an RA model because it was an experimental model of experimental animals that more closely described the characteristics of chronic RA humans. ACIA was a combination of two experimental models of antigen-in-duced arthritis (AIA) and collagen-induced arthritis (CIA).

The aim of this study was to evaluate the effect of HBO therapy on switching phenotypes of CD80 to CD163 through change expression of HIF-1α in decreasing arthritis in ACIA animal models. We also evaluat-ed the reduction of arthritis with clinical arthritis score, the diameter of

C 2019 Japan Health Sciences University & Japan International Cultural Exchange Foundation

Harnanik T. et al.480

the knee joint of mice and assessment levels of anti-inflammatory cyto-kines IL-10.

METHODS

Research design This type of study was true experimental because subjects were

chosen randomly, there were control groups and confounding variables could be controlled. The study design was "randomized control group the post-test design". The sampling technique used simple random sam-pling method.

ACIA Model MiceWe used Balb/C mice, healthy during the adaptation phase with the

characteristics of clear-eyed, shiny fur, agile movements and good fae-ces or not soft, weight 20-30 grams, female sex, age 10-14 weeks. Adaptation phase for 14 days at the Laboratory of Biochemistry, Unit of the Experimental Animal, Faculty of Medicine, Airlangga University, Surabaya, Indonesia. 20 Balb/C mice were divided into 2 groups, each

group consisting of 10 mice, 1 group of ACIA without HBO therapy (non-HBO group) and 1 group of ACIA with HBO therapy (HBO group).

We used Baddack's method for making ACIA models. On the first day, mice were induced with ACIA. ACIA mice were injected with 100 μg of methylated bovine serum albumin (mBSA) in 50 μl of phosphate buffer saline (PBS) emulsified with 50 μl of complete in subcutaneous (s.c) and 200 ng toxin Bordetella pertussis (PTx) in complete Freund's adjuvant (CFA) intraperitoneally (i.p.). One week later, mice were injected with 50 μg mBSA and 100 μg bovine type II collagen (CII) in 50 μl PBS emulsified with 50 μl of incomplete Freund’s adjuvant (IFA) in s.c. and 200 ng PTx. One week later, mice were injected with 50 μg mBSA and 100 μg CII in 50 μl PBS emulsified with 50 μl IFA in s.c. and 200 ng PTx i.p. Two weeks later, induced arthritis was achieved by 50 μg mBSA intra-articular (i.a.) injection which was dissolved in 20 μl PBS into the cavity of the left (ipsilateral) knee joint i.a. and the cavity of the right knee (contralateral) injected with 20 μl PBS i.a. RA model animals were obtained after three weeks9).

Arthritis evaluationAnimal models were obtained after being induced ACIA and

showed signs of arthritis in the knee joint of mice. The severity of arthritis was assessed by the clinical arthritis score and the diameter of the knee joint of mice. The clinical arthritis score used a scale of 0-4 for each limb (0 = no swelling; 1 = slight swelling and erythema; 2 = mod-erate swelling and erythema; 3 = severe smelling and erythema; 4 = maximal inflammation with joint rigidity).

The diameter of the knee joint was measured by a calliper (Mitutoyo digital calliper, Japan) with a level of accuracy up to 0.05 mm (the number of strips on the slider scale was 20 strips so that 1 mm: 20 = 0.05 mm). The caliper was placed across the knee joint at widest point. The values were expressed as the mean of the two knee joint diameters of mice.

HBO Therapy ProcedureHBO therapy was done at the Naval Health Institute, Indonesian

Navy, Surabaya Indonesia. The treatment group was given HBO therapy in a high-pressure air chamber which was often called an animal cham-ber. The HBO group was given normal air exposure for 10 minutes to 1.7 ATA pressure. Then given O2 100% 3-4 L/minute exposure for 90 minutes divided by 3 each of 30 minutes intervals 5 minutes breathing normal air at 1.7 ATA pressure. After that, the pressure was reduced to 1 ATA while breathing normal air without using oxygen for 10 minutes. The HBO group received HBO for 10 consecutive days. The treatment group were given HBO therapy in the morning at 07.00 western Indonesia time.

Sampling procedure All experimental animals were euthanized under the Institutional

Animal Care and Use Committee, Euthanasia Guidelines, Document 006 Version 02 February 28, 2017. Euthanasia was performed after 30 minutes of exposure to HBO. The animal was anesthetized with 4 x ket-amine (75 mg / kgBW + xylazine 10 mg / kgBW) intraperitoneal. After 10 minutes there wasn't pain response, surgery and blood were taken using a 3 cc syringe needle on the heart ventricle then animal model were euthanized.

Table 1. Differences clinical arthritis score and diameter of the mice knee joint in the experimental groups.

Group Clinical arthritis score Diameter (mm)

Mean SD p Mean SD p

Non-HBO 13.50 2.17 0.000 6.58 0.75 0.003HBO 7.30 2.31 5.53 0.57

p < 0.05 was considered statistically significant; SD = standard deviations

Figure 1. Differences level of IL-10 in the experimental groups.

Figure 2. The expression of HIF-1α, CD80 and CD163 with an immunochemistry technique using a 200x magnifica-tion light microscope. Some red arrows were examples of brown living cells that express HIF-1α proteins, CD80 and CD163 (positive cells). Blue cells were living cells that did not express HIF-1α, CD80 and CD163 proteins.

Hyperbaric Oxygen Effect on Switching Phenotype M1-M2 Macrophage Expression 481

IL-10 levels in the plasma of ACIA were measured using mouse IL-10 antibody Bioassay Technology Laboratory system (Korain Biotech Co., Ltd, Shanghai, China) with an enzyme-linked immunosor-bent assays (ELISA) reader (Zenix-320 microplate reader). The level of IL-10 captured was determined by measuring absorbance in a 450 nm wavelengths using a spectrophotometer.

The expression of HIF-1α, CD80 and CD163 levels in left knee joint tissue of ACIA animal models used anti-HIF-1α mouse monoclo-nal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-CD80 mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-CD163 mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with immunohistochemistry technique. The method of colouring used the avidin-biotin complex.

RESULTS

We used the IBM SPSS statistics version 22.0 to analyze all data. All data on the descriptive, expressed as mean values and standard devi-ations (SD). A value of p < 0.05 was considered statistically significant.

The normality test, using the Shapiro-Wilk test showed the clinical arthritis score, the diameter of the mice knee joint, IL-10 levels, expres-sion of HIF-1α, CD80, CD163 were normal distribution (p > 0.05). The results of Lavene test showed the clinical arthritis score, the diameter of knee joint, IL-10 levels, expression of HIF-1α, CD80, CD163 had a homogeneous variance (p > 0.05) in the non HBO and HBO groups. The independent t- test showed there was significantly decrease p = 0.000 (p < 0.05) in the result of clinical arthritis score in HBO group (7.30 ± 2.31) compared to non-HBO group (13.50 ± 2.17). The diameter of the knee joint also decreased significantly p = 0.003 (p < 0.05) in the HBO group (5.53 ± 0.57) compared to non-HBO group (6.58 ± 0.75) (Table 1).

The independent t-test showed there was significantly decrease p = 0.020 (p < 0.05) in the level of IL-10 in HBO group (59.75 ± 15.24) compared to non-HBO group (45.07 ± 9.99). The differences level of IL-10 in the experimental groups are shown in Figure 1.

The independent t-test showed there was significantly decrease p = 0.000 (p < 0.05) in the expression of HIF-1α in HBO group (33.20 ± 10.12) compared to non-HBO group (106.40 ± 21.20). The expression of CD80 also decreased significantly p = 0.000 (p < 0.05) in HBO group (69.80 ± 6.71) compared to non HBO group (308.10 ± 25.01) and the expression of CD163 increased significantly p = 0.000 (p < 0.05) in HBO group (153.00 ± 8.78) compared to non HBO group (44.90 ± 2.89) (Figure 2).

DISCUSSION

The concept of dosage of HBO therapy is derived from the defini-tion of HBO as a drug. The dosage of HBO includes O2 levels, depth of pressurization, duration, surface intervals, frequency, air breaks and a number of treatments. This study used 100% oxygen for 3x30 minutes intervals 2x5 minutes breathing normal air for 10 consecutive days. The aim of this study was to evaluate and the effects of HBOT at high pres-sure 1.7 ATA in ACIA models. The pressure used did not exceed 3 ATA (equivalent to a depth of 20 meters in seawater) and the duration of treatment for elective therapy generally did not exceed 2 hours in one session was still considered safe10).

The results of the study could be seen in clinical arthritis score, the diameter of mice knee joint levels, expression of HIF-1α, CD80 and CD163 proteins. There were significant decreases (p < 0.05) in arthritis scores and diameter of mice knee joint levels in HBO group compared to non-HBO group, this condition showed a decrease in clinical joint inflammation in the group treated with HBO therapy.

There was a significant decrease (p < 0.05) in the expression of M1(CD80) macrophage as proinflammatory cells in the HBO group compared to non HBO groups and there was a significant increase (p < 0.05) in the expression of M2 (CD163) macrophage as anti-inflammato-ry cells on HBO group compared to non HBO groups. There was also a significant decrease (p < 0.05) in the expression of HIF-1α in the HBO group, indicating that there was an improvement in oxygen circulation in the ACIA joint that had hypoxia. The results of this study showed a decrease in joint inflammation in groups that received exposure to HBO therapy which could be explained by biomolecular theory.

The therapeutic base of HBO is to increase the amount of oxygen on the entire tissue so as to improve the part of the body including joints

that are experiencing ischemia11). HIF is a transcription factor, a key reg-ulator of oxygen homeostasis in cells. As a transcription factor, its influ-ences and regulate the expression of genes involved in maintaining homeostasis such as changes in oxygen concentration (oxygen-depen-dent) and independent signals12,13). Hypoxia-inducible-1α coordinate transcriptional programs, vascular, metabolic and functional adaptation to O2 deficiency. The mechanism in controlling HIF activity was through inhibiting HIF (FIH) factors, working to prevent interactions between HIFα and p300 co-activator proteins by using oxygen to hydroxylate asparagine residues, so that HIFα transcription did not occur. In hypoxic conditions, there was no hydroxylation of asparagine residues so that HIFα transcription could occur14).

Inflammatory and hypoxic factors were known to increase the expression of HIF-1α. The effects of hypoxia and HIF regulated the expression of inflammatory factors very complex because more than one pathway could be involved in inducing expression thereby regulat-ing a number of important pathophysiological characteristics in RA, including synovial inflammation, angiogenesis and cartilage damage. Differential HIF expression in synovial joints and functional behaviour in different cell types regulated RA progression15). Understanding HIF expression in RA joints allowed us to better understand the level at which they were activated based on the severity of the disease, and how it affected certain cell types that contributed to perpetuating this disease.

The M1 phenotype produced high-level oxidative metabolites (nitric oxide and superoxidation) and pro-inflammatory cytokines that were important for host defence and tumour cell killers but it could cause col-lateral damage to healthy cells or tissues. In contrast, M2 increased angiogenesis and matrix remodelling during the suppression of destruc-tive immunity16). Macrophage could be polarized into the M1 or M2 phenotype depending on the signal in the micro level environment. This divergence was called macrophage polarization17). Phenotypic polariza-tion of macrophage was regulated through inducible nitric oxide syn-thase (iNOS) and arginase expression depending on HIF activation. M1 macrophage expressed the iNOS enzyme and M2 macrophage expressed arginase enzyme.

HIF-1α played a role in switching phenotypes or changes in macro-phage phenotypes. During inflammation, occurred tissue hypoxia where hypoxia affected the function of macrophage. HIF-1α activated iNOS in the M1 macrophage to form Nitric Oxide (NO) and citrulline when hypoxia occurred. This product was needed as resistance to pathogens and inhibited cell proliferation. However, excessive M1 activity could produce collateral damage to healthy cells or tissues18). The condition of normoxia or hyperoxia after HBO decreased expression of HIF-1α caused iNOS activation to decrease so that arginase became active in the M2 macrophage. Both enzymatic pathways (iNOS and arginase) were mutually antagonistic and inhibited other activated enzymes19).

HIF-1α played a role as the main regulator of inflammation in RA20). The conditional absence of HIF-1α in the animal RA model showed improvement in clinical and histological experimental arthritis. Inhibition of the HIF-1 signal also reduced the effect of induced fibro-blast-like synoviocytes (FLS) by synovial hypoxia in RA patients21). To maintain homeostasis, the immune system developed a number of defence mechanisms, such as the production of anti-inflammatory cyto-kines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) by M2 macrophage and T regulatory cells22).

CONCLUSION

HBO O2 100% 3-4 L / min for 3x30 minutes intervals 2x5 minutes of breathing with normal air at a pressure of 1.7 ATA for 10 consecutive days has an effect on switching phenotype M1 (CD80) to M2 (CD163) through a decreased expression of HIF-1α. Improvement of inflamma-tion in the ACIA group receiving HBO therapy both clinically and bio-molecularly can be explained in theory so that it can be used for the development of further research and HBO is expected to be an adjuvant therapy for RA.

ACKNOWLEDGEMENTS

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,

Harnanik T. et al.482

Airlangga University, Surabaya, Indonesia. This research was funded by the Faculty of Medicine, Hang Tuah University, Surabaya, Indonesia.

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