
==== Front
World Allergy Organ J
World Allergy Organ J
The World Allergy Organization Journal
1939-4551
World Allergy Organization

S1939-4551(24)00087-5
10.1016/j.waojou.2024.100956
100956
Full Length Article
Anti-aminoacyl-tRNA synthetase-interacting multifunctional protein-1 antibody improves airway inflammation in mice with house dust mite induced asthma☆
Kim Sung-Ryeol MD a
Um Yun Jung MSc b
Chung Sook In PhD c
Jeong Kyoung Yong PhD c
Park Hye Jung MD d
Park Kyung Hee MD ce
Park Jung-Won MD ce
Park Sang Gyu PhD sgpark@ajou.ac.kr
b⁎⁎
Lee Jae-Hyun MD jhleemd@yuhs.ac
ce⁎
a Yongin Severance Hospital, Yonsei University College of Medicine, Division of Pulmonology, Allergy and Critical Care Medicine, Department of Internal Medicine, Kyounggi-do, Republic of Korea
b College of Pharmacy, Ajou University, Suwon, Gyeonggi-do, Republic of Korea
c Institute of Allergy, Yonsei University College of Medicine, Seoul, Republic of Korea
d Gangnam Severance Hospital, Yonsei University College of Medicine, Department of Internal Medicine, Seoul, Republic of Korea
e Yonsei University College of Medicine, Division of Allergy and Immunology, Department of Internal Medicine, Seoul, Republic of Korea
⁎ Corresponding author. Division of Allergy and Immunology, Department of Internal Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, 03722 Seoul, Republic of Korea. jhleemd@yuhs.ac
⁎⁎ Corresponding author. College of Pharmacy, Ajou University, 206 World Cup-ro, Yeongtong-gu, Suwon 16499, Gyeonggi-do, Republic of Korea. sgpark@ajou.ac.kr
22 8 2024
9 2024
22 8 2024
17 9 10095621 2 2024
22 7 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background

Several biologics have been developed and used to treat severe asthma. However, commercialized biologics have limitations in treating T2-low asthma because their main target is the T2 inflammation marker. Therefore, there is an unmet need for treating T2-low severe asthma. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is an auxiliary protein in the mammalian multi-aminoacyl-tRNA synthetase complex. AIMP1 also acts as a cytokine and induces the secretion of proinflammatory cytokines. Since anti-AIMP1 has been shown to reduce interleukin (IL)-6, tumor necrosis factor-α, and IL-17A levels in a mouse model, it could be effective in the treatment of T2-low severe asthma.

Methods

Wild-type BALB/c mice were sensitized and challenged with intranasal inoculation of a crude HDM extract. Atliximab, a chimeric AIMP1 antibody, was administered once (20 μg, 40 μg, 100 μg) on Day 14. We evaluated airway hyperresponsiveness (AHR), performed cellular analyses of the bronchoalveolar lavage fluid (BALF), measured inflammatory cytokine levels, and examined peribronchial histological features.

Results

Atliximab reduced AIMP1 levels in asthmatic mice in a dose-dependent manner. AHR and Inflammatory cells such as neutrophils and eosinophils in the BALF decreased in asthmatic mice treated with atliximab. The levels of IL-6, IL-13, and transforming growth factor-β (TGF-β) in the lung tissue decreased in asthmatic mice treated with a high dose of atliximab (100 μg). Atliximab also reduced goblet cell hyperplasia and peribronchial fibrosis.

Conclusions

Atliximab improved asthmatic airway inflammation including neutrophilic inflammation in HDM-induced asthma mice. These data suggest that anti-AIMP1 plays an important role in the treatment of severe T2-low asthma.

Keywords

Asthma
AIMP1
Interleukin-6
Interleukin-13
==== Body
pmcIntroduction

Asthma is a chronic inflammatory airway disease characterized by clinical symptoms of bronchial hyperresponsiveness and reversible airflow obstruction.1 Asthma is common, and its prevalence is increasing.2 It is estimated that asthma affected 334 million people worldwide in 2010.3 Therefore, asthma is a significant social and economic burden.4

Most asthma cases are well controlled with the use of inhaled corticosteroids and long-acting β2-agonists.5,6 However, 3.6–9.5% of patients with severe asthma have poor asthma control, despite the appropriate use of inhalers.7, 8, 9 Therefore, several biologics have been developed for the treatment of severe asthma. Some cases of severe asthma can be effectively controlled by drugs such as omalizumab (anti-IgE), reslizumab, mepolizumab (anti-interleukin[IL]-5), benralizumab (anti-IL5R), and dupilumab (anti-IL4R).10, 11, 12 However, these biologics act on the biomarkers of T2-high asthma and are not effective for the treatment of T2-low asthma, characterized by neutrophilic or paucigranulocytic asthma. Tezepelumab (anti-thymic stromal lymphopoietin) acts not only on T2 inflammation but also on the interaction between structural cells in the airway and immune cells; therefore, it is effective in some cases of severe non-eosinophilic asthma.13 However, to date, no commercialized biologics acting on the biomarkers of T2-low asthma have been developed.

T2-low asthma is related to neutrophilic inflammation in the airway,14 and neutrophilic inflammation is associated with corticosteroid refractoriness and asthma severity.15 Therefore, developing biologics that target cytokines related to neutrophilic inflammation in the airway is expected to solve a significant portion of the unmet need for the treatment of severe asthma, which exhibits the characteristics of T2-low asthma.

Aminoacyl-tRNA synthetase (ARS)-interacting multifunctional protein 1 (AIMP1) is an auxiliary protein in the mammalian multi-ARS (MARS) complex,16 which plays a critical role in the assembly and stability of MARS.17 AIMP1 acts as a scaffold protein for arginyl-tRNA synthetase and glutaminyl-tRNA synthetase among the 9 aminoacyl tRNA synthetases of MARS to form subcomplex I of MARS,16 and is capable of binding to tRNA to help the tRNA dock with AIMP1-bound ARS.18

AIMP1 functions as a cytokine and stimulates monocytes/macrophages via the activation of p38 mitogen-activated protein kinases (MAPKs), ERK, and NF-kB by inducing the secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-8, and macrophage chemotactic protein-1. AIMP1 also induces the maturation of dendritic cells and enhances the production of IL-6 and IL-12.19

Atliximab, a chimeric AIMP1 antibody, effectively reduced IL-6 and TNF-α levels in macrophage cell study.20 Because IL-6, TNF-α, and IL-17A play important roles in airway neutrophilia,21, 22, 23, 24 anti-AIMP1 is expected to be effective in the treatment of neutrophilic asthma. Therefore, in this study, we aimed to evaluate the effect of atliximab on airway inflammation in a house dust mite (HDM)-induced asthma mouse model, which is a representative T2-low asthma model.25

Materials and methods

Construction of the asthma model

To construct the mouse model with HDM-induced asthma, 5-week-old female BALB/c mice were purchased from Orient Bio Inc. D. farinae allergen extracts were purchased from Prolagen (Seoul, Korea). The protein concentration of these extracts is 1057 μg/mL. The major allergen contents are 20.3 μg/mL for Der f 1 and 28.4 μg/mL for Der f 2, with an endotoxin level of 8985 EU/mL26 Mice were housed under specific pathogen-free conditions (room temperature of 21°C-24°C, relative humidity between 45% and 70%, and a 12-h light/dark cycle). After a 1-week acclimation period, Dermatophagoides farinae was administered intranasally (30 μg/30 μL) twice per week for the first 2 weeks and once per week for the next 2 weeks to induce asthma (Fig. 1). The mice were weighed every week during the experimental period (4 weeks). They were divided into 5 groups: sham (negative control), asthmatic control mice (positive control), asthmatic mice treated with low dose atliximab (20 μg), asthmatic mice treated with medium dose atliximab (40 μg), and asthmatic mice treated with high dose atliximab (100 μg). Each treatment group comprised 5 mice.Fig. 1 Timeline of the model and treatment. HDM, house dust mite; sham, negative control mice

Fig. 1

All experiments were repeated twice, and the procedures were approved by the Institutional Animal Care and Use Committee at Yonsei Medical College of Medicine (Seoul, Korea), which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (approval number: 2021-0154).

Assessment of airway hyperresponsiveness (AHR)

Mice were anesthetized with pentobarbital sodium intraperitoneally, ventilated (flexiVent 5.1H; SCIREQ, Montreal, Canada), and challenged with a saline aerosol, followed by low to high concentrations (0, 3.125, 6.25, 12.5, 25.0, and 50.0 mg/mL) of methacholine (MCh; Sigma-Aldrich). Aerosols were generated using an ultrasonic nebulizer (Omron Healthcare, Kyoto, Japan) and delivered to the inspiratory line of the flexiVent using a biased flow of air. Airway hyperresponsiveness (AHR) was assessed using the forced oscillation technique (FlexiVent 5.1; SCIREQ; Montreal).27

Bronchoalveolar lavage fluid (BALF) analysis

After AHR was measured, bronchoalveolar lavage fluid BALF was collected through the tracheal cannula using Hank's Balanced Salt Solution (HBSS; Gibco). The BALF was centrifuged for 3 min at 1500 rpm and 4°C. Whole cells were resuspended in HBSS and counted using a hemocytometer. Cells were smeared on slides by cytocentrifugation (Cytospin 3; Thermo). The slides were stained with hematoxylin and eosin (H&E) and 200 inflammatory cells (neutrophils, eosinophils, lymphocytes, and macrophages) were counted. To minimize the effects of subjective bias in cell classification, a blind outcome assessment was performed.28

Cytokine analysis

To assay cytokines, the right lung was homogenized in 1.5 mL RIPA buffer (Thermo) containing a protease inhibitor solution (Sigma-Aldrich) using TissueLyser II (Qiagen). The lung homogenates were centrifuged at 14 000×g for 20 min, and the supernatants were collected and stored at −20°C. The concentrations of AIMP-1, IL-1β, IL-6, IL-13, TNF-α, and transforming growth factor-β (TGF-β) were measured using an ELISA kit (R&D Systems; DuoSet), according to the manufacturer's instructions. Optical density at 450 nm was measured using a VersaMax microplate reader (Molecular Devices).

Immunohistochemistry

The left lung was fixed in 10% formalin for 24 h and embedded in paraffin for histological and immunohistochemical analyses. The paraffin-embedded sections were sliced to a thickness of 3–4 μm and stained with H&E and periodic acid-Schiff (PAS) to identify goblet cell hyperplasia. Masson's trichrome staining was used to assess fibrosis. Each stained slide was photographed using an upright microscope (BX53F; Olympus) equipped with a digital camera (U-TV0.63XC; Olympus). Quantification was conducted using Metamorph software (Molecular Devices).

Briefly, the PAS-stained slides were placed under a light microscope at 200× magnification. The goblet cells in the selected bronchi were counted. Finally, goblet cells per micrometer of basement membrane were calculated and statistically analyzed. The area of fibrosis was measured using the color pixel count over the preset threshold color for the entire field, which contained several bronchovascular bundles.

Statistical analysis

Numerical data are expressed as mean ± standard error of the mean. The results were analyzed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp Armonk). One-way ANOVA was used to compare the treatment groups. Body weight and AHR, which are repeated measures, were analyzed using a repeated-measures ANOVA. A P-value of <0.05 was considered statistically significant.

Results

AIMP1 levels were elevated in asthma mice and inhibited by atliximab

The concentration of AIMP1 was higher in asthmatic mice than in sham mice (Fig. 2A). AIMP1 levels tended to gradually decrease dependent on the dose of atliximab. There was no statistical difference in AIMP1 levels between the asthmatic control mice and mice treated with low-dose atliximab. AIMP1 levels of mice treated with medium-to high-dose atliximab were lower than those of the asthmatic control mice and mice treated with low-dose atliximab. There was no statistical difference in AIMP1 levels between the mice treated with medium-to high-dose atliximab and sham mice.Fig. 2 Serum AIMP1 level and AHR in HDM-induced asthma mice. Intraperitoneal atliximab treatment reduced serum AIMP1 (A) and AHR (B) in HDM-induced asthma mice in a dose-dependent manner. Data are presented as mean ± SEM. AIMP-1, aminoacyl-tRNA synthetase-interacting multifunctional protein-1; AHR, airway hyperresponsiveness; HDM, house dust mite; SEM, standard error of the mean ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001

Fig. 2

Atliximab reduced AHR

The AHR in the asthmatic control mice was significantly higher than that in the sham mice (Fig. 2B). All asthmatic mice treated with atliximab showed a decrease in AHR compared to the asthmatic control mice. AHR tended to decrease as the dose of atliximab increased in the mice treated with atliximab. There was no significant difference in AHR between the asthmatic mice treated with medium-to high-dose atliximab and the sham mice.

Atliximab reduced airway inflammatory cells and cytokines

Inflammatory cells in the BALF were significantly increased in the asthmatic control mice compared to sham control mice (Fig. 3). Atliximab significantly reduced a number of inflammatory cells in a dose-dependent manner. The total number of inflammatory cells in BALF was the lowest in asthmatic mice treated with high-dose atliximab, followed by those treated with medium and low doses. These trends were observed for each cell type, including macrophages, lymphocytes, eosinophils, and neutrophils. The number of inflammatory cells did not differ statistically between the mice treated with medium-to high-dose atliximab and sham mice.Fig. 3 Airway inflammation in the bronchoalveolar lavage fluid. Intraperitoneal atliximab treatment reduced Inflammatory cells in the bronchoalveolar fluids of HDM-induced asthma mice in a dose-dependent manner. (A) total cell, (B) macrophage, (C) eosinophil, (D) neutrophil, (E) lymphocyte. Data are shown as mean ± SEM. SEM, standard error of the mean; BAFL, bronchoalveolar lavage fluid; HDM, house dust mite; sham, negative control mice.∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001

Fig. 3

The results of the cytokine analysis are shown in Fig. 4. TGF-β levels were lower in the mice treated with atliximab than in the asthmatic control mice in a dose-dependent manner. The TGF-β level in mice treated with atliximab was not different statistically from that in sham mice (Fig. 4A). The IL-6 level in mice treated with low-to medium-dose atliximab was not different statistically from that in asthmatic control mice. However, the IL-6 level in the mice treated with high-dose atliximab was lower than that in asthmatic control mice. The IL-6 level in the mice treated with high-dose atliximab was not different statistically from that of sham mice (Fig. 4B). There was no significant difference in IL-1β levels between the mice treated with atliximab and asthmatic control mice (Fig. 4C). The IL-13 level in the mice treated with high-dose atliximab was statistically lower than that in asthmatic mice, and the IL-13 level in the mice treated with low-to medium-dose atliximab was not statistically differ from that in asthmatic control mice. The IL-13 level in asthmatic mice treated with atliximab was statistically higher than that in sham mice (Fig. 4D). There was no significant difference in TNF-α levels between the asthmatic mice treated with atliximab and asthmatic control mice (Fig. 4E).Fig. 4 Cytokine levels of TGF-β, IL-6, IL-1β, IL-13, and TNF-α in the lungs. Intraperitoneal atliximab treatment reduced TGF-β (A) in HDM-induced asthma mice in a dose-dependent manner. However, only a high dose of atliximab reduced the levels of (B) IL-6, (C) IL-1β, (D) IL-13, (E) TNF-α. Data are shown as mean ± SEM. SEM, standard error of the mean; TGF-β, transforming growth factor β; IL, interleukin; TNF-α, tumor necrosis factor-α; HDM, house dust mite. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001

Fig. 4

Atliximab improved peribronchial histological features

Histopathological changes in the asthmatic lung tissues are shown in Fig. 5. Inflammatory cell infiltration observed in the peribronchial and perivascular areas markedly decreased, depending on the dose of atliximab (Fig. 5A). Mucous gland hyperplasia was significantly lower in the medium- and high-dose atliximab groups than in asthmatic control group (Fig. 5B and D). Peribronchial fibrosis also decreased in a dose-dependent manner (Fig. 5C and E). Peribronchial fibrosis in the high-dose atliximab group was not statistically different from that in the sham mice group.Fig. 5 Immunohistochemistry features of the peribronchial tissues. Intraperitoneal atliximab treatment ameliorated peribronchial and perivascular inflammatory cell infiltration (H&E), goblet cell hyperplasia (PAS), and peribronchial fibrosis (trichrome) of HDM-induced asthma mice. Data are presented as the mean ± SEM. SEM, standard error of the mean; HDM, house dust mite; sham, negative control mice; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff

Fig. 5

Discussion

The major allergen content of D. farinae allergen extracts used in this study contains more Der f 2 than Der f 1 (Der f 1: 20.3 μg/mL, Der f 2: 28.4 μg/mL), and these allergen extracts have a significant amount of endotoxin (endotoxin level: 8985 EU/mL).26 Der f 2 can activate the TLR4 signaling pathway through binding with LPS.29 The activation of TLR4 signaling induces mixed inflammatory profiles in the airway observed in T2-low asthma.30 Therefore, this study could be conducted using an appropriate T2-low asthma model.

In the results, AIMP1 levels were higher in mice with HDM-induced asthma than in sham mice and were effectively decreased by atliximab treatment. Atliximab reduced peripheral airway resistance and decreased the number of inflammatory cells in the BALF of asthmatic mice, as well as improved goblet cell hyperplasia and peribronchial fibrosis. Atliximab significantly reduced TGF-β, IL-6, and IL-13 levels at a high dose. In particular, TGF-β and IL-6 levels in the mice treated with high-dose atliximab were not statistically different from those in sham mice.

The effect of atliximab in improving airway inflammation is associated with a decrease in IL-6 levels. IL-6 recruits neutrophils to the airway as a proinflammatory mediator,31 promotes smooth muscle cell proliferation in asthma, and contributes to airway remodeling.14 Therefore, IL-6 is known to be an important cytokine in T2-low asthma and is increased in neutrophilic asthma.14 Because AIMP1 induces IL-6 expression by activating dendritic cells,19 the decrease in IL-6 levels in the mice treated with high-dose atliximab appears to be associated with the effect of anti-AIMP. IL-6 levels have been reported to significantly decrease in previous studies on anti-AIMP.20,32 In a previous case study, a humanized IL-6 receptor monoclonal antibody, tocilizumab, was used in 2 patients with severe asthma and effectively controlled severe persistent steroid-resistant asthma in both cases.33 Therefore, atliximab may be applicable as an anti-IL-6 drug in cases of T2-low asthma.

The reduction of TGF-β may have also played an important role in the improvement of airway inflammation by atliximab. Th17 cells can be activated by IL-6 and TFG-β. Decreased IL-6 and TGF-β in this study can induce the reduction of IL-17 and Th17. The reduction of Th17 cells by atliximab has been confirmed in a previous study using lupus-prone mice.32 Th17 cells play an important role in airway neutrophilia.24 IL-17 increases the expression of proneutrophil chemokines, such as granulocyte colony-stimulating factor (G-CSF) and chemokine (C-X-C motif) ligand 1 (CXCL1).24 IL-17A secretes IL-8, a potent chemokine for neutrophils, from the bronchial epithelium.24 IL-8 levels in tracheal aspirates of patients with acute severe asthma are correlated with airway neutrophilia levels.34

Improvement of airway inflammation may also be related to a decrease in NF-κB activation. AIMP1 is known to activate NF-κB which plays a central role in regulating the expression of inflammatory genes in airway cells.35,36 NF-κB is activated in the airway tissue and inflammatory cells after intranasal HDM challenge in mice,37 which activates neutrophil chemokines, including IL-1β, resulting in the characteristics of T2-low asthma.38 In this study, IL-1β tended to decrease in the mice treated with high-dose atliximab compared to the asthmatic control mice. Therefore, it is presumed that the inhibition of NF-κB by anti-AIMP1 leads to the suppression of neutrophil chemokines and helps improve airway inflammation.

Improvement of eosinophilic inflammation by anti-AIMP has not been identified in previous studies, and the mechanism is not clear. It is estimated that the role of AIMP1 in activating MAPKs is related to the improvement of eosinophilic inflammation by anti-AIMP1. AIMP1 activates various immune cells through 3 MAPKs, namely ERK1/2, JNK, and p38.39 MAPKs are critical for immune cell metabolism, migration, production of pro-inflammatory mediators, survival, and differentiation.40 In particular, p38 MAPK is thought to play an important role in airway inflammation. It is an important mediator of signal transduction that responds to a wide range of extracellular stressors, such as UV radiation, osmotic shock, hypoxia, proinflammatory cytokines, and oxidative stress.41 p38 MAPK induces the differentiation and activation of Th2 cells, thereby promoting the release of Th2 cytokines (IL-4, IL-5, and IL-13).42 p38 MAPK-dependent phosphorylation of GATA-3 also stimulates ILC2 to produce IL-5 and IL-13,43 and p38 MAPK exerts many proeosinophilic functions, including inhibition of eosinophil apoptosis and induction of eosinophil differentiation, chemotaxis, and secretory activity.42 p38 MAPK also contributes to T2-low neutrophilic inflammation of the airways.15 Fitzgerald et al. reported that IL-6 production in lung fibroblasts was dependent on the activation of p38 MAPK.44 In addition, p38 MAPK signaling upregulates the expression of intercellular adhesion molecule-1 (ICAM-1) in lung vascular endothelial cells and enhances the release of TNF-α from neutrophils, thus promoting the accumulation of these cells in the airways.45,46 Therefore, p38 MAPK is involved in both eosinophilic and neutrophilic airway inflammation. Jaiswal et al. reported that p38 MAPK contributed to mixed (eosinophilic and neutrophilic) asthma in obese mice sensitized to HDM.47 AIMP1 is known to positively regulate p38 MAPK signaling.48 The use of anti-AIMP1 seems to have contributed to the improvement of mixed inflammation by leading to the inhibition of the p38 MAPK pathway.

There are limitations in this study. Low to medium doses of atliximab did not significantly reduce cytokines. This result might be associated with the relatively lower reduction in AIMP in the mice treated with low-to medium-dose atliximab compared to those treated with a high dose (Fig. 2A). In previous studies, a significant effect of atliximab was also found in the group treated with high-dose atliximab (100 μg).20,32 Another limitation is that our study does not reveal the detailed mechanism of the anti-AIMP1 effect on asthma. Further research is warranted to elucidate these detailed mechanisms.

Conclusion

In this study, atliximab improved asthmatic airway inflammation, including neutrophilic inflammation, in our mouse model of HDM-induced asthma. The effects of atliximab are primarily attributed to the inhibition of IL-6 and TGF-β. These data suggest that anti-AIMP1 plays an important role in the treatment of severe T2-low asthma. Further research is warranted to elucidate the detailed mechanisms of atliximab's effects and to explore its potential for clinical application.

Abbreviations

AIMP1, Aminoacyl-tRNA synthetase-interacting multifunctional protein 1; AHR, Airway hyperresponsiveness; ARS, Aminoacyl-tRNA synthetase; BALF, Bronchoalveolar lavage fluid; CXCL1, C-X-C Motif Chemokine Ligand 1; G-CSF, Granulocyte colony-stimulating factor; H&E, Hematoxylin and eosin; HDM, House dust mite; ICAM-1, Intercellular adhesion molecule-1; IL, Interleukin; MAPKs, Mitogen-activated protein kinases; MARS, Mammalian multi-ARS; PAS, Periodic acid-Schiff; TGF-β, transforming growth factor-β; TNF-α, Tumor necrosis factor-α.

Funding

This study was supported by a faculty research grant from Yonsei University College of Medicine (6-2022-0100 ).

Availability of data and materials

The datasets generated and analyzed during the current study are not publicly available but are available upon reasonable request. Requests for access to these datasets should be directed to [Jae-Hyun LEE, jhleemd@yuhs.ac]. All relevant materials are included in the manuscript.

Author contributions

This paper has co-first authors: Sung-Ryeol KIM & Yun Jung UM.

Both authors have contributed equally to this work and share first authorship.

Conceptualization: Jae-Hyun LEE, Sang Gyu PARK.

Data curations: Kyong-Yong JEONG, Kyung Hee PARK.

Formal analysis: Sung-Ryeol KIM, Yun Jung UM, Hye Jung PARK.

Funding acquisition: Sung-Ryeol KIM, Jae-Hyun LEE.

Investigation: Sook In CHUNG.

Methodology: Sung-Ryeol KIM, Jae-Hyun LEE, Sang Gyu PARK.

Project administration: Jae-Hyun LEE.

Resources: Yun Jung UM, Sang Gyu PARK, Jung-Won PARK.

Validation: Sang Gyu PARK.

Writing-original draft: Sung-Ryeol KIM, Yun Jung UM.

Writing-review & editing: Jae-Hyun LEE, Sang Gyu PARK.

We would like to clarify that no artificial intelligence (AI) tools or systems were used in the research and preparation of this manuscript. All aspects of the research, including the formulation of research questions, data collection and analysis, and the writing of the manuscript, were solely conducted by the authors independently.

Ethics approval

This study was conducted in accordance with the ethical standards and guidelines for animal research. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Yonsei University College of Medicine, Seoul, Korea. This committee is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The approval number for this study is 2021-0154. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution.

Authors’ consent for publication

All authors have reviewed the final version of the manuscript and consent to its publication.

Originality and plagiarism

The work submitted here is original and has been written by the stated authors. We confirm that this work has not been copied from others or plagiarized in any form and is not under consideration for publication elsewhere.

Multiple, redundant, or concurrent publication

We confirm that this work has not been published elsewhere and is not under consideration in any other journal. We understand the ethical implications of redundant or concurrent publication.

Acknowledgment of sources

All sources used in the creation of this manuscript are duly acknowledged. Any citation or quotation has been properly referenced, respecting the intellectual property rights of the original authors.

Authorship of the paper

All individuals who have made a significant contribution to the conception, design, execution, or interpretation of this research work are listed as co-authors. Furthermore, all co-authors have seen and agree with the contents of the manuscript and its submission to this journal.

Declaration of competing interest

The authors declare that there are no conflicts of interest regarding the publication of this paper. The authors have no relevant financial or non-financial interests to disclose.

☆ Full list of author information is available at the end of the article
==== Refs
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