Particulate matter10-induced airway inflammation and fibrosis can be regulated by chitinase-1 suppression

Animal model designs

Female BALB/c mice, between 5 and 6 weeks old (Orient, Daejeon, Korea), were maintained at conventional animal facilities under pathogen-free conditions, and 5 mice were assigned to each group. To establish the PM10-induced murine model (PM10 model), PM10 (ERMCZ-120® certified reference material; Sigma-Aldrich, St Louis, MO, USA; 100 μg suspended in 20 μL normal saline was intranasally administered 6 times over 3 weeks. The dose of PM10 was chosen based on a previous study [8]. OVA is a well-known allergen used to induce asthma in a mouse model.2

Dexamethasone was used as positive control since it has strong anti-inflammatory effect [16]. To establish the chronic OVA-induced asthma murine model, mice were challenged intranasally with 30 µL of OVA (1 mg/mL) (Sigma-Aldrich, St Louis, MO, USA) in saline solution 6 times over 3 weeks. An OVA/PM10-treated model was established with the 6 concurrent treatments mentioned above (PM10 and OVA) over 3 weeks. Chitinase-1 inhibitor (CPX, Sigma-Aldrich, St Louis, MO, USA, 100 mg/kg, intraperitoneally) was administered 6 times over 3 weeks and treated with dexamethasone (Sigma-Aldrich, St Louis, MO, USA, 3 mg/kg, intraperitoneally) 3 times in the final week. Dexamethasone, which has a strong anti-inflammatory effect, was used as positive control. Mice body weights were measured weekly. All mice were euthanized 2 days after their last treatment (Fig. 1A).

Fig. 1figure 1

Study scheme (A), weight change (B), airway hyper-responsiveness (C), and bronchoalveolar lavage fluid analysis (D). CPX chitinase-1 inhibitor, Dex dexamethasone, IP intraperitoneally injection, IN intranasally administration, OVA ovalbumin, BALF bronchoalveolar lavage fluid. Data are presented as mean ± standard deviation. *P < 0.05 between them

We divided mice into 5 groups: control (only treated with saline), PM (PM10-treated model), PM/OVA (PM10 and OVA-treated model to maximize airway and lung inflammation), PM/OVA/CPX (PM10, OVA, and CPX-treated model to reveal effects of CPX on inflammation), and PM/OVA/Dex (PM10, OVA, and Dex-treated model to confirm effects of Dex as a positive control).

All experimental procedures of mice studies were approved by the Institutional Animal Care and Use Committee, Animal Research Ethics Board of Yonsei University (Seoul, Korea) (IACUC approval number, 2020-0087) and were performed in accordance with the Committee’s guidelines and regulations for animal care.

Measurement of airway hyper-responsiveness

Airway hyper-responsiveness (AHR) to inhaled aerosolized methacholine (MCh; Sigma-Aldrich, St Louis, MO, USA) was measured using a forced oscillation technique (FlexiVent; SCIREQ, Montreal, QC, Canada) on the euthanasia day, as described in a previous study [17,18,19]. Aerosolized phosphate-buffered saline (PBS) or methacholine at varying concentrations (3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25.0 mg/mL, or 50.0 mg/mL) was administered to mice for 10 s via a nebulizer connected to a ventilator. Then, AHR was assessed by measurements of airway resistance.

Inflammatory cell counting in bronchoalveolar lavage fluid

To collect bronchoalveolar lavage fluid (BALF), lung lavage was performed, using 1 mL of Hank’s balanced salt solution (HBSS) through a tracheal tube. The recovered BALF was centrifuged and resuspended in 300 µL HBSS. Total cell numbers were determined using a hemocytometer and trypan blue staining. BALF cells were centrifuged by cytocentrifugation (Cytospin 3; ThermoFisher Scientific, Waltham, MA, USA) and were pelleted to cytospin slides. The slides were stained with hematoxylin and eosin (H&E Hemacolor®, Merck, Darmstadt, Germany), and a differential count of inflammatory cells was performed (200 cells per slide).

Histological analysis

The remnant lung, i.e., the lung that was not used for BALF collection, was fixed in 4% formalin and embedded in paraffin. Lung sections were cut into 3–4-µm-thick slices and stained with H&E, periodic acid-Schiff (PAS), and Masson trichrome (M&T) for histological analysis. The slides were observed under a light microscope (× 200 magnification). The area of fibrosis was measured by estimating the color-pixel count over the pre-set threshold color on MT-stained slides at 200 × magnification using MetaMorph program (Molecular Devices, Sunnyvale, CA, USA).

Lung homogenate

After collecting BALF, remaining lung tissue was resected and homogenized using a tissue homogenizer (Biospec Products, Bartlesville, OK, USA) in lysis buffer and protease inhibitor solution (Sigma-Aldrich, St Louis, MO, USA). After incubation and centrifugation, supernatants were harvested and passed through a 0.45-micron filter (Gelman Science, Ann Arbor, MI, USA). The final preparations were stored at − 20℃ for cytokine analysis as described previously [17].

Analysis of cytokines

Concentrations of interleukin (IL)-1β, TNF-α, IL-6, IL-13, and TGF-β in lung homogenates were assessed via enzyme-linked immunosorbent assay (R&D Systems, San Diego, USA) according to the manufacturer’s instructions. All samples were assessed in duplicate.

Immunofluorescence study

Lung tissue was fixed with 10% formalin for 24 h. Lung tissue sections were deparaffinized, permeabilized with 10 mM citrate buffer, and blocked with 5% bovine serum albumin. The slide was incubated with anti-chitinase-1 antibodies (1:80; Santacruz, TX, USA) overnight at 4 °C. Slides were washed five times in phosphate-buffered saline (PBS). After washing, the slides were incubated with m-IgGκ BP-conjugated FITC antibody (1:100; santacruz, CA, USA) and mounting medium with PI (Invitrogen, CA, USA) overnight at 4 °C. Images were acquired with Axio Imager M2 microscope (Carl Zeiss).

Analysis of mRNA expression level

Total RNA was extracted from lung tissues using in TRIzol® reagent (Ambion, Life technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed using reverse transcriptase (Invitrogen, Carlsbad, CA, USA) primed with oligo (dT) primer. The synthesized cDNAs were amplified using the SYBR® green PCR master mix (BioRad, California, USA) and forward and reverse primers (Bioneer, Daejeon, Korea) using a real-time PCR system (StepOnePlus, Applied Biosystems, Foster City, CA, USA). The following primers were used for collagen III and collagen I, respectively: GTG AAA CTG GTG AAC GTG GC (F) and ATA GGA CCT GGA TGC CCA CT (R) for collagen III; GAG AGG TGA ACA AGG TCC CG (F) and AAA CCT CTC TCG CCT CCT GC (R) for collagen I.

Library preparation and mRNA sequencing

Total RNA was extracted from lung tissue using Trizol reagent (Invitrogen). mRNA isolation was performed using the Poly(A) RNA Selection Kit V1.5 (LEXOGEN, Inc., Austria). The total RNA is briefly denatured and the polyadenylated 3' ends of mRNAs are hybridized for isolation. The 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 the Illumina indexes 1–12. The enrichment step was carried out using PCR. Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands) to evaluate the mean fragment size. Quantification was performed using the library quantification kit with an ND 2000 Spectrophotometer (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 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). Then, the trimmed reads were mapped to the reference genome using TopHat [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).

Statistical analysis

All results are expressed as the mean ± standard error. The AHR data were analyzed using repeated-measure analysis of variance (ANOVA), followed by a post-hoc Bonferroni test. One-way ANOVA was performed to assess the significance of differences in BALF cell count, cytokine levels, and quantitative fibrosis among groups. All statistical analyses were performed with IBM SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered statistically significant. All RNA sequencing analyses were performed by R (version 4.0.2; R Foundation for Statistical Computing, Vienna, Austria) software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted by “clusterProfiler” R package. Upset plot was plotted by “DOSE” R package, chord plot was plotted by “GOplot” R package and KEGG pathway was described by "pathview” R package.

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