Asthma is a common chronic respiratory disease affecting over 300 million populations worldwide and has become a major global health challenge . As a complex airway disorder, asthma is characterized by various inflammatory cells-mediated immunity, which promotes excessive inflammation, mucus overproduction, airway hyperresponsiveness, and airway remodeling . Therein, the airway epithelium represents the first-line host barrier against foreign substances, which is closely involved in the initial pathogenesis of asthma [3, 4].
Aeroallergens from house dust mites (HDMs) such as Dermatophagoides pteronyssinus are the most prevalent sources of a range of allergens which are highly associated with allergic asthma . Inhaled HDMs trigger airway epithelial cells to immediately express pattern recognition receptors (PRRs) especially Toll-like receptor 4 (TLR4) [6, 7]. Upon allergen recognition, activated and damaged airway epithelia release a variety of proinflammatory cytokines and chemokines, eventually leading to asthma pathogenesis . In particular, HDM allergen challenge contributes to epithelial cells apoptosis, increased epithelium permeability and histological changes, which finally orchestrates airway injury.
High mobility group box 1 (HMGB1) is an important inflammatory mediator released from injured and death cells and believed to be endogenous danger signal for DNA repair, recombinant, cell death and apoptosis, which is responsible for multiple cancers and immune diseases[9, 10]. It has been reported that HMGB1 critically participates in inflammatory development of asthma acting as a ligand of TLR4. Besides, emerging studies revealed that HMGB1 was elevated in induced sputum and plasma in asthmatic patients, and that measures to inhibit HMGB1 were helpful to alleviate airway inflammation in ovalbumin(OVA)-induced asthma model[12, 13]. It is likely that HMGB1 plays an important role in allergic pathophysiological process. But the effect of HMGB1 on HDM-induced airway damage and inflammation is not well elucidated.
Clara cell secretory protein(CC16), also known as CC10, uteroglobin, secretoglobin-1A1, or club cell secretory protein(CCSP), is a 16-kDa homodimeric protein belonging to the secretoglobin superfamily and is mainly secreted by mucosal nonciliated airway epithelial (Clara) cells localized in bronchi and bronchioles. CC16 possesses anti-inflammatory and immunoregulatory properties, and is regarded as an endogenous protective protein against several pulmonary diseases. Previous studies have shown that recombinant CC16 can help to decrease airway inflammatory response in chronic obstructive pulmonary disease (COPD) and acute respiratory disease syndrome (ARDS) [15, 16]. Given that airway damage and inflammation participate in HDM-induced asthma as well, we proposed that CC16 might serve as a helpful regimen to abrogate injured airway epithelium. Moreover, some studies demonstrated that CC16 could inhibit the transcription factor nuclear factor-κB (NF-κB) signaling pathway in airway inflammatory diseases [15, 17]. It is well known that NF-κB pathway is downstream signaling of HMGB1-TLR4 axis and is able to modulate inflammatory cytokine genes expression in asthma . Nevertheless, whether CC16 would protect against proinflammatory effect of HMGB1 remains elusive. In this study, using cultured cells and a HDM-induced murine asthma model, we investigated the participation of HMGB1 together with potential signaling molecules in progression to airway inflammation of asthma. We also explored protective effect of CC16 on airway damage and epithelial cell apoptosis exposed to HDM allergen and the underlying mechanism. This study may shed light on a novel remedial option for HDM-induced asthma.
Materials and Methods
HDM sensitized and challenged model and recombinant CC16 treatment
Healthy female Balb/c wild-type (WT) mice (6-8 weeks old,20-25g) were maintained in pathogen-free animal facility in a 12h light-dark cycle with regular food and water. In order to explore the preventive effect of CC16 on HDM-induced airway inflammation and injury, the WT mice were randomly divided into four groups and were subjected to the following regimens: (1) Control group; (2) HDM group; (3) HDM+CC16-5 group (treated with CC16 at a dose of 5ug/g/body); (4) HDM+CC16-10 group (treated with CC16 at a dose of 10ug/g/body). To establish an animal model of asthma, mice were sensitized with intraperitoneal injection of HDM (Dermatophagoides pteronyssinus) extract (Greer Labs,Lenoir, NC,USA) 100µg in 200µl phosphate buffered saline(PBS) on Days 0,7,and 14 respectively. On Day 21 to 28, mice were challenged by intranasal administration of 100μg HDM (solved in 50µl PBS) daily under chloral hydrate anesthesia. Intranasal treatment of CC16 (5μg/g or 10μg/g) (PeproTech, Rocky Hill, NJ, USA) (dissolved in 20 µl sterile saline) or the same dose of saline were given 30 minutes before each HDM challenge. Mice received PBS instead of HDM in the sensitization and challenge phase as negative control. All mice were sacrificed for endpoint analysis on day 35. The bronchoalveolar lavage fluid (BALF) was collected by injecting and retracting 1 ml of 0.9% saline solution and was then centrifuged for cytokines analysis and differential cell counts. The lung tissues were harvested appropriately for subsequent histopathological examination. All murine experimental procedures were approved by the Animal Research Ethical Committee of Shanghai East hospital.
Adenovirus gene delivery
To determine the in vivo effect of HMGB1 on allergic airway injury, recombinant adenovirus expressing the mouse HMGB1 gene (Ad-HMGB1) or mouse HMGB1 shRNA(Ad-sh-HMGB1) was constructed by OBiO Technology Corp. Ltd. (Shanghai, China). Adenoviral vectors containing no transgene were used as negative control (Ad-GFP). One week before the establishment of asthmatic model, a dose (1x109pfu) of Ad-PGRN, Ad-sh-HMGB1 or Ad-GFP were intratraceally delivered into the mice. The efficacy of interfere was evaluated with qPCR.
The human airway epithelial BEAS-2B cells were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China) and cultured in DMEM/F medium (Hyclone, Camarillo, CA,USA) containing 10% fetal bovine serum(Clark Bioscience, Claymont, DE,USA), 100 U/ml penicillin and 100μg/ml streptomycin(Gibco, Grand Island, NY, USA) at 37℃ with 5% CO2 in humidified air. After reaching 80-90% confluence, the cells were seeded to proper culture slides and acclimated with free serum free-DMEM for subsequent experimental purpose.
In order to knockdown the expression of HMGB1, small-interfering RNAs (siRNAs) targeting human HMGB1 gene (si-HMGB1) and negative control siRNA (si-NC) were synthesized and purchased from GenePharma (Shanghai, China).For overexpressing HMGB1, recombinant pcDNA-HMGB1 plasmid was constructed by cloning the full-length HMGB1 into pcDNA3.1 vectors. Empty vector was used as the negative control. For transfection, BEAS-2B cells were transfected with si-HMGB1, si-NC, pcDNA-HMGB1 or vector using the lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen, Camarillo CA, USA).
Lung histopathology and TUNEL staining
The lung samples were fixed in 10% formalin overnight and embedded in paraffin. Lung sections of 4μm thickness were stained with haematoxylin and eosin(H&E), as well as periodic acid Schiff (PAS) for histopathology analysis. TUNEL staining was performed using the InSitu Cell Death Detection kit (Roche,Switzerland, no.11684817910) to detect the apoptotic cells.
The tissue samples were dewaxed in xylene and rehydrated in graded ethanol solutions. The sections were blocked with normal goat serum and incubated for 20 min at room temperature. Then the sections were immunostained with the primary antibodies against HMGB1(Cat#6893, Cell Signaling Technology, Danvers, MA,USA) at 4℃ overnight. Followed by washing three times with PBS, the sections were incubated with HRP-labeled secondary antibody at 37℃ for 30 min. The stained sections were observed under a light microscopy.
The treated BEAS-2B cells were fixed with 4% paraformaldehyde and permeated with 0.1% Triton X-100 for 10 min. After incubation with anti-HMGB1 at 4℃ overnight, the cells were stained with secondary Alexa Fluor®488-conjugated antibody (Beyotime Biotechnology, Shanghai, China) for 1 hr at 37℃ in the dark. The nuclei were counterstained with DAPI. Images were visualized by using a fluorescence microscope.
Cell Viability Assay
BEAS-2B cells were seeded into a 96-well plate at a density of 3000 cells/well and treated as described above. Then cell viability was assessed with the Cell Counting Kit-8 assay (CCK8; Dojindo Laboratories, Tokyo, Japan) at 0,12,24,48hr, respectively. The optical density(OD) values of the absorbance at 450nm were measured using Riorad microplate reader.
Measurements of IL-4, IL-5, IL-13 (Abcam, Cambridge, UK), HDM specific-IgE (Chondrex, Redmond, USA) and HMGB1 (Arigobio, Taiwan, China) in the BALFs as well as serum CC16 (Biovendor Systems, Candler, USA) were performed with enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s recommendations.
Cell apoptosis was determined using double staining with Annexin V-FITC/propidium iodide (PI). Briefly, the treated cells from different groups were resuspended in a 500 μl binding buffer volume and 5ul of Annexin V-FITC and 5 μl of PI (Beyotime) were added. Subsequently, the cells were incubated for 15 minutes at room temperature protected from light. The phycoerythrin (PE)-conjugated anti-cleaved caspase-3(Cat#9661, Cell Signaling Technology) antigen was used to detect activated caspase-3 of cells according to the manufacturer’s protocol. All data were quantified on FACS Calibur flow cytometer (BD Biosciences, CA, USA).
Western Blot Analysis
Lung tissues or cells were lysed with RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride. The supernatants were collected by centrifuged and total protein concentration was qualified using a BCA Protein Kit (Beyotime Biotechnology, Shanghai, China). Then equal quantities of protein samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes(Meckmillipore,Germany) that were consequently blocked with 5% fat-free milk for 1hr. After that, the membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies were used as follows: anti-HMGB1 (1:1000; Cat#6893),anti-TLR4 (1:1000; Cat#14358), anti-NF-κB (1:1000; Cat#8242), anti-p-NF-κB (1:1000; Cat#3033), cleaved caspase-3 (1:1000; Cat#9661) (Cell Signaling Technology); anti-Bcl-2 (1:1000; Cat#ab182858),anti-Bax (1:1000; Cat#ab32503), and anti-β-actin (1:1000; Cat#ab8227) (Abcam).Following extensively rinsing in TBST, the membranes were incubated with HRP-conjugated secondary antibodies and further detected using ECL chemiluminescent method (Millipore, Billerica, MA, USA). The blots were quantified with ImageJ.
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from lung tissues or cells with TRIzol reagent (Invitrogen) and diluted in nuclease-free DEPC-treated water. After the preparation with DNase treatment (Qiagen, Hilden, Germany), total RNA was reverse transcribed into cDNA which was applied as a template for qRT-PCR reaction. Then relative mRNA levels were quantified by ABI PowerUpTMSYBRTM Green Master Mix (Termo Fisher Scientific, Waltham, MA, USA). PCR conditions consisted of 40 cycles at 95℃ for 15 sec and 60℃ for 1 min, followed by a melting curve analysis. All reactions were performed using an ABI7500 Fast Real-Time PCR System (Applied Biosystems, USA). The primer sequences were as followed: interleukins (IL)-25 forward: 5′-CGTCCCACTTTACCACAACC-3′ and reverse: 5′-ACACACACACAAGCCAAGGA-3′; IL-33 forward: 5´-GTACTTTATGCAACTGCGTTCTGG-3´ and reverse: 5´- CAGACATTGCTTTCTGCACTTTTC-3´; thymic stromal lymphopoietin (TSLP) forward: 5´-TTCACTCCCCGACAAAACATTT-3´ and reverse: 5′-TGGAGATTGCATGAAGGAATACC-3′; HMGB1 forward: 5′-GGGTACTGCCTTGCTTGACA-3′ and reverse: 3′- ATCAGACCCTTTCAGGAGGC-5′. The expression of target gene was calculated by the 2-△△CT method and β-actin was used as a housekeeping gene.
Caspase-3 activity assays
The caspase-3 activity in the lung tissues was evaluated with an assay kit (Beyotime Biotechnology) according to manufacturer’s protocols.
Statistical analysis was performed using the SPSS19.0 software. All data were expressed as mean ±standard error of the mean. The student’s t-test (comparisons between two groups) or one-way ANOVA with Bonferroni post-hoc test were used for analyses. P<0.05 was considered statistically significant.