Effects of chitinase-1 inhibitor in obesity-induced and -aggravated asthma in a murine model

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Life Sciences 334 (2023) 122163

Available online 25 October 2023

Effects of chitinase-1 inhibitor in obesity-induced and -aggravated asthma in a murine model

Heejae Han

^a

, Yong Jun Choi

^a

, Hyerim Hong

^a

, Chi Young Kim

^a

, Min Kwang Byun

^a

,

Jae Hwa Cho

^a

, Jae-Hyun Lee

^a^,^b

, Jung-Won Park

^a^,^b

, Taylor A. Doherty

^c

, Hye Jung Park

^a^,^c^,^*

aDepartment of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea

bInstitute of Allergy, Yonsei University College of Medicine, Seoul, Republic of Korea

cSection of Allergy and Immunology, Department of Medicine, University of California, San Diego, La Jolla, CA, USA

A R T I C L E I N F O Keywords:

Chitinase Obesity Airway Asthma

A B S T R A C T

Aims: Despite recent investigations on the role of chitinase in asthma, its role in obesity-induced asthma has not been evaluated. Therefore, we investigated the roles of chitin, chitinase-1, and a chitinase-1 inhibitor (compound X, CPX) in a murine model.

Main methods: We assigned C57BL/6 mice to the ovalbumin (OVA) model or obesity model group. In the OVA model, mice received intraperitoneal OVA twice within a 2-week interval and intranasal OVA for 3 consecutive days. Additionally, chitin was intranasally administered for 3 consecutive days, and CPX was intraperitoneally injected three times over 5 days. In the obesity model, a high-fat diet (HFD) was maintained for 13 weeks, and CPX was intraperitoneally injected eight times over 4 weeks.

Key findings: In the OVA model, chitin aggravated OVA-induced airway hyper-responsiveness (AHR), increased bronchoalveolar lavage fluid (BALF) cell proliferation, increased fibrosis, and increased the levels of various inflammatory cytokines (including chitinase-1, TGF-β, TNF-α_,_IL-1β, IL-6, IL-4, and IL-13). CPX treatment significantly ameliorated these effects. In the obesity model, HFD significantly increased AHR, BALF cell proliferation, fibrosis, and the levels of various inflammatory cytokines. Particularly, compared to the control group, the mRNA expression of chitinase, chitinase-like molecules, and other molecules associated with inflammation and the immune system was significantly upregulated in the HFD and HFD/OVA groups. Immunofluorescence analysis also showed increased chitinase-1 expression in these groups. CPX significantly ameliorated all these effects in this model.

Significance: This study showed that CPX can be an effective therapeutic agent in asthma, especially, obesity- induced and -aggravated asthma to protect against the progression to airway remodeling and fibrosis.

1. Introduction

Obesity is an important risk factor for the induction and exacerbation of asthma with features of fibrosis. [1] The underlying mechanisms that explain how obesity induces and aggravates asthma are as follows. 1) Obesity can induce mechanical effects that cause chronic lung compression. [2] 2) Obesity is known to induce neutrophilic inflammation or pauci-inflammation. [3] 3) Metabolic and vitamin D de- ficiencies have additional aggravating effects. [4,5] 4) Obesity can aggravate airway remodeling and fibrosis. [6,7] Recently, the global

prevalence of obesity has increased. In addition, obese patients with asthma typically do not respond to standard asthma medications, including inhalation of corticosteroids, which is a critical treatment option. [1,8] Many researchers have investigated specific drugs for patients with asthma having obesity, and we investigated the effectiveness of a new drug in an obesity-asthma model. [3] However, such a drug has yet to be proven clinically useful.

Chitinase-1 is known to play a critical role in the innate immune response in the respiratory tract, [9] and its expression is upregulated in inflammatory diseases, especially asthma. [10] Furthermore, it is

* Corresponding author at: Department of Internal Medicine, Yonsei University, College of Medicine, Gangnam Severance Hospital, 211, Eonju-ro, Gangnam-gu, 06273 Seoul, Republic of Korea.

E-mail addresses: [email protected] (H. Han), [email protected] (Y.J. Choi), [email protected] (H. Hong), [email protected] (C.Y. Kim), [email protected] (M.K. Byun), [email protected] (J.H. Cho), [email protected] (H.J. Park).

Contents lists available at ScienceDirect

Life Sciences

journal homepage: www.elsevier.com/locate/lifescie

https://doi.org/10.1016/j.lfs.2023.122163

Received 1 July 2023; Received in revised form 4 October 2023; Accepted 6 October 2023

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Life Sciences 334 (2023) 122163

considered to augment TGF-β1 signaling and fibrosis, [11] and its raised levels are considered a biomarker for interstitial lung disease. Therefore, chitinase-1 inhibitors have been investigated recently, with respect to their clinical utility in inflammatory and fibrotic diseases; moreover, prior studies have shown their chemotherapeutic potential as a fungi- cide, pesticide, and anti-asthmatic agent. [12] Recent studies have also explored the therapeutic effects of chitinase-1 inhibitors in asthma and interstitial lung fibrotic diseases. [13] Previously, we reported significant effects of a chitinase-1 inhibitor in particulate matter 10-induced airway inflammation and asthma, in a mouse model. [14] However, the detailed roles of chitin and chitinase-1 in obesity-induced or -aggravated asthma remain unknown.

In this study, we investigated the roles of chitin and chitinase-1 in a murine model of obesity and asthma. We used a chitinase-1 inhibitor (compound X, CPX) to treat obesity-induced and -aggravated asthma models.

2. Materials and methods 2.1. Animal model designs

To establish a classic asthma model and an obesity-associated asthma model, C57BL/6 mice were assigned to an ovalbumin (OVA) and obesity model group, respectively. In the OVA group, significant airway hyper- responsiveness (AHR) and abundant type 2 inflammation were induced in the airways of mice, as described previously [15]. In the OVA model, a mixture of OVA (20 mg per mouse; Sigma-Aldrich, St. Louis, MO, USA) and Imject Alum (100 μL per mouse; Thermo Scientific, Rockford, IL, USA) was intraperitoneally injected twice within a 2-week interval.

After 1 week, OVA (20 mg per mouse) was administered intranasally for 3 consecutive days, as described previously [3]. Chitin from shrimp shells (Sigma, St. Louis, MO, USA) was suspended in distilled water and sonicated five times at 30 % output power for 3 min using a sonicator.

After filtration with a 40-μM sterile cell strainer (BD Biosciences, San Jose, CA, USA), 25 μg chitin was intranasally administered for 3 consecutive days. CPX (100 mg/kg; Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected three times daily for 5 days, as described previously [16]. On the last day of the CPX challenge and 2 days after the last challenge with OVA and/or chitin, the mice were euthanized.

In the obesity model group, the mice were fed a high-fat diet (HFD, D12492; Research Diets, Inc., New Brunswick, NJ; fat accounting for 60

% of the calories) for 13 weeks. Lean mice were fed a normal chow diet

(D12450B; Research Diets, Inc.; fat accounting for 10 %). OVA was then administered as described above. CPX was intraperitoneally injected 8 times over 4 weeks. Two days after the last OVA challenge or on the day of the last challenge with CPX, the mice were euthanized (Fig. 1).

In this study, C57BL/6 male mice were used, and ≤5 animals were raised in one cage. The animal room was maintained at 22 ^◦C and a humidity of 50 % ±10 %, and the light-dark cycle was advanced every 12 h. All experimental procedures in these mouse model studies were approved by the Institutional Animal Care and Use Committee, Animal Research Ethics Board of Yonsei University (Seoul, Korea) (IACUC approval number, 2019–0332) and were performed in accordance with the Committee’s guidelines and regulations for animal care.

2.2. Measurement of airway hyper-responsiveness

AHR to inhaled aerosolized methacholine (MCh; Sigma-Aldrich, A- 2251, St Louis, MO, USA) was measured using a forced oscillation technique (FlexiVent; SCIREQ, Montreal, QC, Canada) on the day of the euthanasia, as described previously. [17–19] Aerosolized PBS or methacholine at varying concentrations of 6, 12, 25, 50, and 100mg/mL was administered to the mice via a nebulizer connected to a ventilator.

2.3. Inflammatory cell count in bronchoalveolar lavage fluid

For bronchoalveolar lavage (BAL) fluid (BALF) analysis, the right lung underwent lavage. The total cell numbers were determined using a hemocytometer and trypan blue staining. BALF cells were centrifuged via cytocentrifugation (Cytospin 3; Thermo Fisher Scientific, Waltham, MA, USA) and pelleted to cytospin slides. The slides were stained with hematoxylin and eosin (H&E) (H&E Hemacolor®; Merck, Darmstadt, Germany), and a differential count of inflammatory cells was performed (200 cells per slide).

2.4. Immunofluorescence staining analysis

The lung tissue was fixed in 10 % formalin for 24 h. Lung tissue sections were deparaffinized, permeabilized with 10 mM citrate buffer, and blocked with 5 % bovine serum albumin. The slides were incubated with anti-chitinase-1 antibodies (1: 80; Santacruz, sc-271,460, TX, USA) overnight at 4^◦C. The slides were washed five times in PBS and then incubated with m-IgGκ BP-conjugated FITC antibody (1:100; Santacruz, sc-516,140, CA, USA) and a mounting medium with PI (Invitrogen, P- 36962, CA, USA) overnight at 4 ^◦C. Images were acquired with an Axio Imager M2 microscope (Carl Zeiss) for immunofluorescence (IF) staining analysis. Quantification of IF positive area was conducted by estimating the color-pixel count over the pre-set threshold color for the entire field containing several bronchial tubes on IF.

2.5. Histological analysis

For pathological analysis, the left lungs were filled with 4 % formalin solution and embedded in paraffin. Lung sections were cut and stained with H&E, periodic acid-Schiff (PAS), and Masson’s trichrome (MT) for histological analysis. Tissue sections were examined using an Olympus BX40 microscope and an Olympus U-TV0.63XC digital camera (Olympus BX53F, Center Valley, PA, USA). Images were acquired using the cellSens Standard 1.6 image software. Quantification analysis was conducted using Metamorph® (Molecular Devices). Proliferative cell infiltration and fibrosis was measured by estimating the color-pixel count over the pre-set threshold color for the entire field containing several bronchial tubes on H&E- or MT-stained slides at 200×magnification. Proliferation of goblet cell was measure by estimating the number of PAS positive cells per 100 μM basement membrane lengths.

At least 10 fields were reviewed per animal, and one representative field per animal was selected and analyzed.

Fig. 1. Study scheme of chitin-treated model and obesity model

CPX, chitinase-1 inhibitor; D, day; HFD, high-fat diet; IP, intraperitoneal injection; IN, intranasal administration; NCD, normal chow diet; OVA, ovalbumin; W, week.

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2.6. Enzyme-linked immunosorbent assay using lung homogenates After collecting BALF, the remaining lung tissue was resected and homogenized using a tissue homogenizer (Biospec Products, Bartles- ville, OK, USA) in lysis buffer and protease inhibitor solution (Sigma- Aldrich, St. Louis, MO, USA). After incubation and centrifugation, su- pernatants were harvested and passed through a filter [17]. The concentrations of chitinase-1 (Fine biotech, EM0939, Wuhan, China), hydroxyproline (Biovision, K555-100, CA, USA), collagen-1 (Fine biotech, EM6446), interleukin (IL)-1β (DY401), TNF-α (DY410), IL-4 (DY404), IL-6 (DY406), IL-13 (DY413), and TGF-β (DY1679) in lung homogenates were assessed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, San Diego, USA) according to the manufacturer’s instructions.

2.7. Fasting glucose level and glucose tolerance test

Mice were starved overnight for fasting glucose level analysis and oral glucose tolerance test (GTT), which were performed at weeks 0 and 13, respectively. Fasting glucose was measured before the start of the GTT, which was performed after oral glucose administration (2 g/kg body weight; Sigma-Aldrich, G7528, St. Louis, MO, USA). Blood from the tail vein was collected at 0, 30, 60, 90, and 120 min after glucose injection to determine glucose levels using an Accu-Check glucometer (Roche, Mannheim, Germany).

2.8. Full mRNA sequencing

Total RNA was extracted from the lung tissue using TRIzol reagent (Invitrogen). Isolated mRNAs were used for cDNA synthesis. Libraries were prepared using the NEBNext Ultra II Directional RNA Seq Kit (NEW ENGLAND BioLabs Inc., UK). Indexing was performed using Illumina indexes 1–12. The enrichment step was performed using polymerase chain reaction (PCR). Subsequently, libraries were checked using an Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands) to evaluate the mean fragment size. Quantification was performed using a library quantification kit with an ND 2000 Spectro- photometer (Thermo Inc., DE, USA) and StepOne Real Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA).

Quality control of the raw sequencing data was performed using FastQC (Simon, 2010). Adapter and low-quality reads (<Q20) were removed using FASTX_Trimmer (Hannon Lab, 2014) and BBMap (Bushnell, 2014). The trimmed reads were then mapped to the reference genome using the TopHat software. [20] Gene expression levels were estimated by calculating fragments per kb per million reads (FPKM) using Cufflinks. [21] The FPKM values were normalized based on a quantile normalization method using EdgeR within R (R development Core Team, 2016).

2.9. Statistical analysis

All results are expressed as mean ±standard error. AHR and weight Fig. 2. Airway hyper-responsiveness (A), BALF cell count (B), level of chitinase-1 in lung homogenates (C), and chitinase-1 expression in immunofluorescence staining (D) in chitin-treated model (n =5 in each group)

Bar, dot, and line graphs present mean ±standard error.

BALF, bronchoalveolar lavage fluid; Cht, chitin; CPX, chitinase-1 inhibitor; OVA, ovalbumin; TC, total cell; MC, macrophages; Lym, lymphocytes; Eos, eosinophils;

Neu, neutrophils.

* P <0.05 between them analyzed by repeat-measures ANOVA (for AHR) and one-way ANOVA (for others) followed by post-hoc Bonferroni test.

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Life Sciences 334 (2023) 122163

data were analyzed using repeated-measures analysis of variance (ANOVA), followed by a post-hoc Bonferroni test. One-way ANOVA was performed to assess the significance of the differences between contin- uous variables. All statistical analyses were performed using IBM SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). P-values <0.05 were considered statistically significant.

3. Results

3.1. CPX ameliorated OVA- and OVA/chitin-induced AHR, inhibited BALF cell proliferation, and increased level of chitinase-1

Compared to the control group, the OVA and OVA/chitin (Cht) groups showed significantly more AHR. CPX completely ameliorated this effect (Fig. 2A). OVA treatment induced significant BALF cell proliferation with eosinophil dominance. Chitin significantly aggravated this effect (Fig. 2B). The level of chitinase-1 in the lung homogenates was significantly increased in the OVA and OVA/Cht groups, while CPX counteracted this effect (Fig. 2C). Chitinase-1 was highly expressed in bronchial epithelial cells, as per IF staining results. CPX also downregulated the expression of chitinase-1 (Fig. 2D & Supplementary Fig. 1A).

3.2. CPX ameliorated OVA- and OVA/chitin-induced fibrosis

Compared to the control group, the OVA and OVA/Cht groups showed increased peribronchial cell infiltration (on H&E staining), number of goblet cells (on PAS staining), and fibrosis (on MT staining) (Fig. 3A, Supplementary Fig. 1B & C). The quantitative fibrosis area, assessed using metamorph, and hydroxyproline and collagen-1 levels increased significantly in the OVA/Cht group compared to the control and OVA groups. CPX completely ameliorated this effect (Fig. 3B-D).

3.3. CPX reduced OVA- and OVA/chitin-induced increased levels of inflammatory cytokines

In the OVA group, the TGF-β and IL-1β levels in lung homogenates were significantly higher than those in the control group. These effects were significantly aggravated by the addition of chitin. The IL-13 levels increased significantly upon addition of chitin to OVA. Compared to the control group, the IL-4 levels were significantly higher in the OVA/Cth group but not in the OVA group. CPX treatment significantly reduced these levels (Fig. 4).

Fig. 3. Pathologic findings (H&E, PAS, and MT; all ×200 magnification) (A), quantitative fibrosis area (B), level of hydroxyproline in lung homogenates (C), and level of collagen-1 in lung homogenates (D)

Bar and line graphs present mean ±standard error.

Cht, chitin; CPX, chitinase-1 inhibitor; OVA, ovalbumin; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; MT, Masson’s trichrome.

* P <0.05 between them analyzed by one-way ANOVA followed by post-hoc Bonferroni test.

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3.4. CPX ameliorated HFD- and HFD/OVA-induced AHR and BALF cell proliferation

The HFD induced a significant increase in weight, gross appearance, volume of internal organs, and lipid accumulation in the liver. More- over, HFD induced a significant increase in fasting glucose and glucose intolerance (Fig. 5D&E). Additionally, in comparison with the control group, HFD and HFD/OVA induced significant AHR. Treatment with CPX significantly ameliorated this effect. HFD/OVA induced significant BALF cell proliferation with eosinophil dominance. CPX ameliorated this effect (Fig. 6A-B).

3.5. CPX reduced HFD- and HFD/OVA-induced increased levels of chitinase-1

Compared with the control group, the HFD group showed a significant increase in chitinase-like 3 mRNA expression. Compared with the control group, the HFD/OVA group showed significantly higher mRNA expression levels of chitinase-1 (chitinase, acidic 1), chitinase-like 1, chitinase-like 3, and chitinase-like 4. Compared with the HFD group, the HFD/OVA group showed higher mRNA expression levels of chitinase-1, chitinase-like 1, chitinase-like 3, and chitinase-like 4. The levels of chitinase-1 in lung homogenates increased significantly in the HFD group, and CPX completely recovered these levels (Fig. 6C–D). Ac- cording to the results of the IF staining, CPX also downregulated

chitinase-1 expression, mainly in the epithelial cells (Fig. 6E & Supple- mentary Fig. 2A).

3.6. CPX ameliorated HFD- and HFD/OVA-induced fibrosis and increase in levels of inflammatory cytokines

Compared with the control group, the HFD/OVA groups showed greater peribronchial cell infiltration and proliferation of goblet cells (Supplementary Fig. 2B & C). Quantitative fibrosis area assessed using metamorph, and levels of hydroxyproline and collagen-1 were significantly higher in the HFD and HFD/OVA groups compared to the control group. CPX ameliorated these effects (Fig. 7). Compared to that in the control group, the mRNA expression of IL-1, IL-17, TNF-α, TGF-β, collagen type IV, and fibronectin was upregulated in the HFD and HFD/

OVA groups (Fig. 6C). The levels of TGF-β, TNF-α, IL-1β, IL-6, IL-4, and IL-13 in lung homogenates increased in the HFD and HFD/OVA groups, while CPX counteracted these effects (Fig. 7).

4. Discussion

This study elucidated CPX to be a promising agent for the treatment of obesity-induced and -aggravated asthma. The level of chitinase-1 increased significantly in both the typical (OVA model) and obese asthma models. Inhalation of chitin induces an increase in chitinase-1 levels, which aggravates airway inflammation and fibrosis. Chitinase-1 Fig. 4. TGF-β (A), IL-1β (B), IL-6 (C), TNF-α (D), IL-13 (E), and IL-4 (F) levels in lung homogenates

Cht, chitin; CPX, chitinase-1 inhibitor; OVA, ovalbumin

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was secreted primarily by bronchial epithelial cells. We also revealed that the inhibition of chitinase-1 using CPX can ameliorate obesity- induced and -aggravated asthma (Fig. 8).

Chitin is widely distributed throughout nature: it is found in the exoskeletons of crabs, shrimp, and insects; the cell walls of fungi; house dust mites (HDM); and the digestive tract linings of many insects.

[22,23] In addition, chitin derivatives are widely used in medical practice (artificial skin, contact lenses, and surgical stitches). [24]

Therefore, humans are frequently exposed to chitin or chitin derivatives in daily life. Previous studies have shown that small chitin particles can stimulate alveolar macrophages and natural killer cells [25], which leads to the modulation of innate and adaptive immune responses. [23]

However, whether such exposure enhances or suppresses Th1/Th2 immune response remains controversial. Strong et al. demonstrated direct intranasal administration of chitin microparticles to downregulate the allergic response to HDM and fungus in a murine model of allergy. [26]

However, Choi et al. demonstrated that HDM-derived chitin enhances airway hypersensitivity to inhaled allergens. [27] This study showed that inhalation of chitin aggravated airway inflammation and peribronchial fibrosis. The inhalation of chitin also enhances the production

of Th1 and Th2 immune-associated inflammatory cytokines. Due to these risks of chitin exposure in asthma, we believe that patients with asthma should be cautious regarding exposure to chitin or chitin derivatives in daily life.

Chitinase can bind and degrade chitin, providing homeostatic defense and clearance mechanisms. An increase in chitinase levels has been extensively documented in a wide range of inflammatory diseases.

[28] There are two active endochitinases in humans: chitotriosidase (chitinase-1) and acidic mammalian chitinase (AMCase). Of these, chitinase-1 is one of the proteins most abundantly secreted by macrophages and epithelial cells. It plays a critical role in host defense against chitin in infectious and inflammatory diseases. However, an increase in chitinase-1 levels in the liver induces hepatic fibrosis and cirrhosis; [29]

is associated with conditions, such as Gaucher’s disease, asthma, and atherosclerosis; [9] and may play a significant role in enhancing Th2- mediated inflammation in asthma. [30] The current study used an OVA model to demonstrate that inhalation of chitin increases levels of chitinase-1 and aggravates asthma with Th1 and Th2 inflammation and fibrosis. We also showed that obesity-associated asthma exhibits increased levels of chitinase-1 and aggravated AHR, inflammation, and Fig. 5.Weight changes (A), outward appearance and internal organs (B), levels of fasting glucose immediately before the first day (on Day 0, baseline) and the final day (on sacrifice day in week 13, final) (C), and glucose intolerance test in obesity model (D). (n =5 in control group; n =7 in HFD group; n =10 in HFD/CPX group;

n =7 in HFD/OVA group; n =10 in HFD/OVA/CPX group) Bar, dot, and line graphs present mean ±standard error.

Cht, chitin; CPX, chitinase-1 inhibitor; HFD, high-fat diet; OVA, ovalbumin

* P <0.05 between them analyzed by repeat-measures ANOVA (for weight) and one-way ANOVA (for others) followed by post-hoc Bonferroni test.

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fibrosis. This study supports previous studies showing a significant role of chitinase-1 in asthma models.

Obesity asthma is a major form of non-type 2 asthma. Patients with asthma and obesity do not respond to typical asthma medications, including inhalable corticosteroids, and generally exhibit severe symptoms and poor quality of life. [1] Despite numerous attempts toward researching specific medications to treat obesity-related asthma, none have been developed. Fukushima et al. revealed that chitinase was highly expressed in diabetic mice compared to non-diabetic mice. [31]

Ahangari et al. showed that chitinase-3 like-1 is induced by a high-fat diet and Th2 inflammation and that this induction simultaneously generates obesity and asthma. [14] Specjalski et al. demonstrated that chitinase-3-like protein 1 correlates with the clinical features of asthma and obesity. [32] There is insufficient scientific evidence to support the role of chitinase-1 in obesity; however, several studies have indicated this possibility. We showed that chitinase-1 can be correlated with the aggravation of obesity-induced asthma. In addition, this study revealed the therapeutic potential of chitinase-1 inhibitors in obesity. We hope that this study will help inform the development and introduction of new treatment options for obesity-associated asthma.

Kasugamycin (CPX), which was used in this study as a chitinase-1 inhibitor, is a natural compound produced by Streptomyces kasugaensis.

It is also a well-known aminoglycoside antibiotic. It achieves chitinase-1 inhibition by interacting with the carboxyl group of a conserved aspartate in glycoside hydrolase family 18 chitinases. [33] A recent study showed that kasugamycin is a novel chitinase-1 inhibitor with strong antifibrotic effects in pulmonary fibrosis. [34] Another study demonstrated that kasugamycin ameliorated particulate matter-induced airway inflammation and fibrosis. [35] Our study further broadens the clinical applicability of kasugamycin.

Chitinase-1 is mainly produced by activated macrophages and

epithelial cells [9]. The absolute macrophage count in the HFD group was higher than that in the HFD/OVA group; however, the chitinase-1 levels were higher in the HFD/OVA group than in the HFD group. We speculated that this could be explained by the following two reasons.

First, the ratio of the macrophage count to the total cell count was notably higher in the HFD group than that in the HFD/OVA group. Thus, macrophages played a crucial role in the HFD group, whereas the HFD/

OVA group had a different set of critical cells, namely, eosinophils. We speculate that the abundance of activated macrophages secreting chitinase-1 might be higher in the HFD group, in comparison with the HFD/OVA group. Second, in this study, IF staining showed that the epithelial cells form a crucial secretory source of chitinase-1, while the contrary may be the case for macrophages. Thus, in our model, epithelial cells could potentially be either redundant or a more dominant source of chitinase-1 compared to macrophages.

This study has some limitations. First, we did not measure the biochemical levels of some obesity-related molecules, such as liver en- zymes, creatinine, and lipid makers, as this study concentrated on asthma and airway inflammation. These parameters might have been affected by OVA administration and obesity, and we cannot ignore the possibility of their involvement in the chitinase-associated pathway.

Second, we measured solely the mRNA levels and not the miRNA levels.

Recent studies revealed that miRNAs play a key role in lung cellular functions and are involved in asthma and obesity. A future investigation to quantify the miRNA levels will be helpful for exploring detailed mechanisms. Third, we could not find other critical mediators involved in the chitinase-1-associated pathway. Finally, we did not employ a mode with a natural aeroallergen such as house dust mite or fungal allergens which can be a focus of future studies. Detailed mechanisms of how chitinase-1 is activated and suppressed should be investigated.

This study suggests a novel mechanism and treatment option for Fig. 6.Airway hyper-responsiveness (A), BALF cell count (B), mRNA expression levels of chitinase-1 (C), level of chitinase-1 in lung homogenates (D), and chitinase- 1 expression in immunofluorescence staining (E)

Bar, dot, and line graphs present mean ±standard error.

BALF, bronchoalveolar lavage fluid; Cht, chitin; CPX, chitinase-1 inhibitor; HFD, high-fat diet; OVA, ovalbumin; TC, total cells; MC, macrophages; Lym, lymphocytes;

Eos, eosinophils; Neu, neutrophils

* P <0.05 between them analyzed by repeat-measures ANOVA (for AHR) and one-way ANOVA (for others) followed by post-hoc Bonferroni test.

# P <0.05 with others.

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Fig. 7. Pathologic findings (H&E, PAS, and MT; all ×200 magnification) (A), quantitative fibrosis area (B), level of hydroxyproline in lung homogenates (C), and level of collagen-1 in lung homogenates (D). TGF-β (E), TNF-α (F), IL-1β (G), IL-6 (H), IL-4 (I), and IL-13 (J) levels in lung homogenates

Cht, chitin; CPX, chitinase-1 inhibitor; HFD, high-fat diet; OVA, ovalbumin; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; MT, Masson’s trichrome

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obesity-associated asthma. Chitin and chitinase-1 play significant roles in OVA- and obesity-induced asthma. We demonstrated an increase in levels of chitinase-1 in a disease model, using various methods, including mRNA sequencing, ELISA, and IF staining. We also demonstrated that a chitinase-1 inhibitor can protect against obesity-induced and -aggravated asthma from progression to airway remodeling and fibrosis. Notably, an in-depth investigation of the mechanism by which this occurs is warranted.

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.lfs.2023.122163.

Prior meeting presentation None.

Animal ethics approval

All experimental procedures of mouse model studies were approved by the Institutional Animal Care and Use Committee, Animal Research Ethics Board of Yonsei University (Seoul, Korea) (IACUC approval number, 2019–0332) and were performed in accordance with the Committee’s guidelines and regulations for animal care.

Consent for publication Not applicable.

Funding

This study was supported by the Research Grant from Gangnam Severance Hospital, Yonsei University College of Medicine. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1I1A1A01055117). In addition, this work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00209884).

CRediT authorship contribution statement

H. Han analyzed and interpreted the data, drafted and revised the article, and approved the final version of the article for publication.

Y.J. Choi, H. Hong, J. Lee, C.Y. Kim, M.K. Byun, and J.H. Choi generated, collected, and analyzed the data, contributed to the draft, revised the article, and approved the final version of the article sub- mitted for publication.

H.J. Park is the corresponding author, who provided critical opin- ions regarding the concept and design of the study, interpreted the data, drafted and revised the article, and approved the final version of the article for publication.

Declaration of competing interest

The authors declare that they have no competing interests.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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%JKVKPCUG KPJKDKVQT

Fig. 8. Summary of study results.

OVA, ovalbumin; HFD, high-fat diet.

(10)

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