KIF2A decreases IL-33 production and attenuates allergic asthmatic inflammation

Animals

Specific pathogen-free female C57BL/6 J mice aged 6 to 8 weeks were obtained from Nanjing Medical University (Nanjing, China). All mouse experiments and tissue sample collections were carried out in accordance with the guidelines and procedures approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IRB: 1709011).

Cell lines and cell culture and treatment

The human bronchial epithelial cells lines 16-HBE, mouse pulmonary epithelial cell lines MLE-12 and alveolar epithelial type 2 (AT2) were cultured in DMEM/F12 (Gibco, Massachusetts, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, USA), 100 μg/mL penicillin, and 100 μg/mL streptomycin (60162ES76, Yeasen, Shanghai, China). All incubations were carried out in 5% CO2 in air at 37 °C. For the experiments, cells were seeded and grown to 80–90% confluency in 24-well culture plates (3524, Corning, New York, USA) and exposed to whole-body HDM extract from Dermatophagoides pteronyssinus (Greer Laboratories, Lenoir, USA) for the indicated durations. Autophinib (HY-101920, MedChemExpress, New Jersey, USA) was used as an autophagy inhibitor in the study. Usnic acid (HY-N0656A, MedChemExpress, New Jersey, USA) was used to inhibit the phosphorylation of mTOR downstream effectors P70S6K. Cell lysates and cell-free supernatant were collected for further analysis.

AT2 cell isolation and culture

A crude single lung cell suspension was isolated from 6 to  8-week-old B6 mice as previously described [18]. Mice were anaesthetized by intraperitoneal injection of pentobarbital sodium (70 mg/kg), and the lung vasculature was perfused through the right ventricle with PBS. The trachea was punctured, and the lungs were injected with 1 ml digestion buffer (0.25% trypsin/2 mM EDTA (25200056, Gibco, Massachusetts, USA) and 1 mg/ml elastase (Worthington) for 5 min. Immediately following digestion, 0.5 mL of 45 °C 1% low melting agarose in Ca2+ Mg2+-free PBS was instilled into the lungs for 2 min. The lungs were removed from the thorax and digested for 45 min at room temperature on a shaker. The lobes were then separated, minced, and digested with 0.01% DNase (Sigma) for 10 min at room temperature. The resulting cell suspensions were filtered first through a 100 μm filter and then a 40 μm filter (Falcon, Cockeysville, MD).

AT2 cells were isolated using the “panning method” which purifies cells using IgG-coated plates [19]. Briefly, three 100-mm bacteriologic plastic dishes (430,167, Corning, New York, USA) were coated with 3 to 5 ml each of 0.5 mg/ml IgG (Sigma), and IgG was allowed to adhere for 3 h at 37 °C. The cell suspension was added at a density of 20 to 30 × 106 cells/10 ml medium/100-mm dish. Plates were placed in an incubator for 30 min and then removed from the incubator and carefully and gently tipped back and forth 3 times. The unattached cells were removed and centrifuged at 300g for 10 min. The pellet was resuspended in DMEM/F12 with 10% FBS and cultured in 24-well plates.

Western blot

Total cellular protein or tissue was collected following lysis in RIPA lysis buffer (87,788, Thermo Fisher Scientific, Massachusetts, USA) with Protease Inhibitor Cocktail (78,430, Thermo Fisher Scientific, Massachusetts, USA) on ice and centrifugation for 10 min at 12,000 rpm at 4 °C. The supernatant was then transferred to a new tube and denatured in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (P0015, Beyotime Biotech, China) with heating at 100 °C for 10 min. The supernatant was then stored at − 80 °C. The proteins were separated by 12% SDS-PAGE. After electrophoresis, the separated proteins were transferred to 0.45 µm or 0.22 µm polyvinylidene difluoride membranes (Merck Millipore, USA) using a wet transfer method. Nonspecific sites were blocked with 5% nonfat milk in TBS-Tween 20 (TBST; 25 mM Tris [pH 7.5], 150 mM NaCl, and 0.1% Tween 20) for 1 h, and the blots were incubated with primary antibodies, including anti-β-actin (4970L, Cell Signaling Technology, Massachusetts, USA), anti-KIF2A (ab197988, Abcam, Cambridge, England), anti-LC3B (ab192890, Abcam, Cambridge, England), anti-ATG5 (ab108327, Abcam, Cambridge, England), anti-IL-33 (ab187060, Abcam, Cambridge, England), anti-P-4EBP1 (2855, Cell Signaling Technology, Massachusetts, USA), anti-4EBP1 (9644, Cell Signaling Technology, Massachusetts, USA), anti-p-p70 S6 Kinase (Thr389) (9234, Cell Signaling Technology, Massachusetts, USA), anti- p-p70 S6 Kinase (Ser371) (9208, Cell Signaling Technology, Massachusetts, USA), and anti-p70 S6 Kinase (2708, Cell Signaling Technology, Massachusetts, USA), overnight at 4 °C. HRP-linked anti-rabbit IgG (7074, Cell Signaling Technology, Massachusetts, USA) was used to detect antibody binding. After the membranes were treated with Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck Millipore, USA), the binding of specific antibodies was visualized using a Syngene G:BOX Imaging System and was analyzed with ImageJ.

Reverse transcription and qPCR

Total RNA was isolated from frozen tissues or cells using TRIzol (Invitrogen), and cDNA was synthesized using 5X All-In-One RT MasterMix (G490, Abm, Zhenjiangg China) according to the manufacturer’s instructions as described previously [20]. qPCR analysis was performed using a StepOnePlus Real-Time PCR System (ABI, USA) in conjunction with SYBR Advantage qPCR Premix (63976, TaKaRa Bio). The cycling conditions were 95 °C for 30 s, followed by 95 °C for 5 s and 60 °C for 30 s for up to 40 cycles and dissociation at 95 °C for 5 s, 60 °C for 30 s, and a final extension at 95 °C for 15 s. The relative abundance of gene targets was determined by the comparative CT (cycle threshold) number normalized against the tested β-actin comparative CT. The primers used are shown in Table 1.

Table 1 Primers in the studyHDM sensitization/challenge protocol

C57BL/6 J mice were randomly divided into 4 groups: control, HDM, HDM treated with an empty vector (Vehicle) (PS100001, OriGene Tech, Rockville, MD, USA), and HDM treated with pCMV6-KIF2A (MR210157, OriGene Tech). Mice in the latter 3 groups were exposed intratracheally to HDM according to the established 14-day model [21]. The mice anesthetized with isoflurane received 100 μg HDM in 40 μl of normal saline (NS) on Day 0 and 10 μg HDM in 40 μl of normal saline (NS) on Days 7–11 intratracheally to induce allergic lung inflammation. Mice exposed to 40 μl NS according to the HDM protocol served as controls. To explore whether KIF2A was involved in allergic airway inflammation pathogenesis, 50 µg pCMV6-KIF2A-ORF or vehicle was complexed with in vivo-jetPEI™ (201-50 g, Polyplus Transfection, New York, USA) in 200 µl of 5% glucose solution and injected into the tail vein before the HDM challenge on days 6 and 10 [22]. The mice were sacrificed 3 days after the final challenge, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected for analyses.

Measurement and analysis of airway responsiveness

Mice were anesthetized with 70 mg/kg pentobarbital and 1.8 g/kg urethane followed by 0.5 mg/kg pancuronium bromide, and the mice were tracheotomized 72 h after the final challenge [23]. Airway hyperreactivity (AHR) was measured in response to increasing doses of acetylcholine via a flexiVent FX system with an integrated FX1 module (Scireq, Montreal, QC, Canada) under general anesthesia as described previously [24].

Bronchoalveolar lavage fluid and serum analysis

After AHR measurement, whole blood was collected without anticoagulant and incubated for 2 h at room temperature, and serum was isolated by centrifugation at 2000g for 10 min. Bronchoalveolar lavages were conducted by insertion of a cannula into the trachea, which was fixed by a suture. BAL fluids were taken by slow injection and subsequent aspiration of PBS-EDTA solution (1 mM EDTA, PBS, 500 μl × 3; 85 to 90% of the lavage volume was recovered) with a 1 -mL syringe. BALF from each mouse was centrifuged at 500g for 10 min at 4 °C, cell pellets were resuspended in 100 µl phosphate-buffered saline (PBS), and differential cell counts were performed using standard morphological criteria after Wright-Giemsa staining. The BALF was collected, divided into equal portions, and frozen at − 80 °C until further preparation.

Lung histology

Lungs were fixed in 4% paraformaldehyde; the left lung of each mouse was embedded in paraffin according to standard procedures. Sections (5 µm) were mounted on slides for histological or immunohistochemistry (IHC) analysis. Hematoxylin and eosin staining was used to evaluate changes in lung inflammation. Mucus secretion was assessed by PAS staining. Images were visualized with a Zeiss Axio Examiner microscope. The severity of peribronchial inflammation was graded semiquantitatively for the following features: 0, normal; 1, few cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; 4, a ring of inflammatory cells 4 cells deep. The numerical scores for the abundance of PAS-positive mucus-containing cells in each airway were determined as follows: 0, < 0.5% PAS-positive cells; 1, 5–25%; 2, 25–50%; 3, 50–75%; and 4, > 75% [20].

ELISA

The levels of IL-4, IL-5, IFN-γ (431105, 430805, 431205, Biolegend, San Diego, CA), Eotaxin, IL-33 (DY420, DY3626, R&D Systems, Minnesota, USA) and IL-13 (96–900-K207, PeproTech, Offenbach, Germany) in lung homogenates and total IgE (555248, BD Biosciences, San Jose, CA) in sera were measured using commercial ELISA kits according to the instructions provided by the manufacturers.

Immunohistochemistry and immunofluorescence

Formaldehyde-fixed mouse lungs were dehydrated, paraffin embedded, and sectioned (5 μm thickness). Sections were rehydrated, quenched with 3% hydrogen peroxide, incubated in citric buffer for antigen retrieval, and blocked with the avidin/biotin blocking system and then 5% normal goat serum, followed by overnight incubation at 4  C with primary anti-KIF2a antibody. Tissue sections were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The staining was visualized with 3,3′-diaminobenzidine (DAB, Vectastain, Vector Laboratories, USA) and background stained with hematoxylin. All KIF2A-stained sections were scanned with a Zeiss Axio Examiner microscope, and representative photos were chosen and presented for each stimulus group in the study.

For immunofluorescence, slides or cells were then fixed with 4% paraformaldehyde, rinsed twice with PBS, and permeabilized in PBS 0.5% Triton X-100. Samples were incubated with 5% normal goat serum for 1 h at room temperature (RT) and stained in blocking buffer with anti-LC3B antibodies (1:500 in blocking buffer), anti-KIF2A (1:100, sc-272471, Dallas, Texas, USA), or anti-Prosurfactant Protein C (1:50, ab170699, Abcam, Cambridge, England) overnight at 4 °C. After samples were washed with PBS, secondary goat anti-rabbit IgG antibodies conjugated with Alexa Fluor 647 (1:1000) or goat anti-rabbit IgG antibodies conjugated with Alexa Fluor 555 (1:1000) were added to the samples and incubated for 1 h in the dark at RT. The samples were finally mounted with DAPI (36308ES20 Yeasen, China).

Transfection

MLE-12 cells were seeded into 24-well plates. Before transfection, 500 ng vehicle or KIF2A-ORF plasmids was mixed with 2.5 μL of Hieff TransTM Liposomal Transfection Reagent (40802ES02, Yeasen, China) in 100 μL of DMEM for 25 min. After 25 min, the plasmid-transfection reagent mixture was added to the wells (100 μL/well). The transfection medium was discarded after 6 h, and the cells were cultured in medium containing 10% FBS for no less than 24 h before further analysis.

Statistical analysis

Results are expressed as the mean ± SEM. Comparisons among three or more groups were performed by one-way ANOVA followed by the Newman-Keuls post hoc test. Significance between two groups was assessed by unpaired or paired t tests. Statistical analyses were performed using GraphPad Prism 7.0

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