The role of secreted Hsp90α in HDM-induced asthmatic airway epithelial barrier dysfunction
The dysfunction of airway epithelial barrier is closely related to the pathogenesis of asthma. Secreted Hsp90α participates in inflammation and Hsp90 inhibitor protects endothelial dysfunction. In the current study, we aimed to explore the role of secreted Hsp90α in asthmatic airway epithelial barrier function.
Male BALB/c mice were sensitized and challenged with HDM to generate asthma model. The 16HBE and Hsp90α-knockdown cells were cultured and treated according to the experiment requirements. Transepithelial Electric Resistance (TEER) and permeability of epithelial layer in vitro, distribution and expression of junction proteins both in vivo and in vitro were used to evaluate the epithelial barrier function. Western Blot was used to evaluate the expression of junction proteins and phosphorylated AKT in cells and lung tissues while ELISA were used to evaluate the Hsp90α expression and cytokines release in the lung homogenate.
HDM resulted in a dysfunction of airway epithelial barrier both in vivo and in vitro, paralleled with the increased expression and release of Hsp90α. All of which were rescued in Hsp90α-knockdown cells or co-administration of 1G6-D7. Furthermore, either 1G6-D7 or PI3K inhibitor LY294002 suppressed the significant phosphorylation of AKT, which caused by secreted and recombinant Hsp90α, resulting in the restoration of epithelial barrier function.
Secreted Hsp90α medicates HDM-induced asthmatic airway epithelial barrier dysfunction via PI3K/AKT pathway, indicating that anti-secreted Hsp90α therapy might be a potential treatment to asthma in future.
KeywordsAsthma Epithelial barrier Secreted Hsp90α 1G6-D7 HDM
Human bronchial epithelial cell line 16HBE14o-
Airway epithelial cells
House dust mite
Heat shock protein
- One-way ANOVA
One-way analysis of variance
Transepithelial electrical resistance
Asthma is known to be a chronic airway disease, which is characterized by inflammation, shedding of airway epithelial cells (AECs) and airway remodeling . Chronic inflammation exacerbates intensively once the airway is exposed to antigens. As the first defensive barrier between lung and outer environment, AECs and their contacts play important roles in defense, antigen presentation and quick response to different stimulation .
The tight junctions (TJs) are on the surface of AECs, encircling the subapical regions of lateral cell membranes to regulate permeability and restrict lateral movement of the cell membrane. The functions of TJs rely on the interaction of protein complexes . Below the TJs are the adherens junctions (AJs) composing of E-cadherin, β-catenin, p120 and plakoglobulin. Studies had shown that E-cadherin and β-catenin acted as not only important barrier proteins to anchor AECs, but also crucial signaling proteins for immune response [4, 5, 6].
Hsp90 is a member of HSPs family and is defined as molecular chaperones for a long time . Hsp90 expresses constitutively in eukaryotes and its expression is upregulated in various situations such as in stress, inflammation and adverse stimulations. Hsp90α, one of the four subtypes of Hsp90, exists not only in cytoplasm but also on the surface of certain cells [8, 9]. Evidences showed that Hsp90α is actively secreted to intercellular and tissue space for promoting wound healing, inflammatory mediation, invasion and migration. Hsp90α and Hsp90β have different and non-exchangeable functions during wound healing . Asthmatic AECs are always in a damage and self-repair period, yet the role of secreted Hsp90α in asthma is still unknown.
Our previous studies focused on the disruptions of TJs and AJs in asthma, in which we demonstrated that TDI and HDM could cause dysfunctions of TJs and AJs via VEGF pathway or AKT pathway [11, 12, 13, 14]. We found HDM promoted the secretion of Hsp90α in preliminary experiments, and it has not been confirmed whether the secreted Hsp90α plays an important role in asthma. In this study, we evaluated the secretion of Hsp90α and the expression of epithelial barrier proteins. Our data show that secreted Hsp90α is upregulated at the protein levels in response to HDM in mice and 16HBE cells. We also found that secreted Hsp90α contributes to HDM-induced airway epithelial barrier dysfunction and 1G6-D7 prevents this HDM-induced disruption.
Animals and reagents
All the animal experiments were approved by the Committee on the Ethics of Animal Experiments of Southern Medical University in Guangzhou, China and performed under standard guidelines for the Care and Use of Laboratory Animals. SPF BALB/c mice (male, 6-week old, 20–24 g) were purchased from Southern Medical University. The mice were housed in the laboratory with a 12:12-h light/dark cycle at 24°C in an atmosphere of 40–70% humidity. Food and water were sterilized and all experiments involving animals complied with the ARRIVE guidelines. HDM was purchased from ALK-Abello A/S, methacholine was obtained from Sigma-Aldrich and 1G6-D7 (specific anti-secreted Hsp90α monoclonal antibody, mAb) was developed and contributed by Wei Li’s laboratory [15, 16].
HDM-induced asthma and 1G6-D7 administration
The establishment and evaluation of HDM-induced asthmatic animal model
BALB/c mice were randomly distributed to 4 groups(n = 8 per group): (1) control group; (2) 1G6-D7 group; (3) HDM group; (4) 1G6-D7 + HDM group. In this study mice were delivered to intranasal sevoflurane-anesthesia (Maruishi Pharmaceutical Co. Ltd.), then respectively administered with 10 μl of PBS, HDM(400 U/mouse a day), 1G6-D7 (0.1 μg/μl, 10 μl, dissolved in PBS) or 1G6-D7 + HDM daily for 5 consecutive days, during 8 weeks period. All groups were administered via intranasal inhalation. Furthermore, in the 1G6-D7 + HDM group, mice were pretreated with 1G6-D7 30 min before administration of HDM and the concentrations were described above.
RNAi delivery system, cell culture and treatment
16HBE cells were purchased from Fuxiang Biological Technology Co. Ltd., ATCC, USA. We used the RNAi delivery system to knockdown the Hsp90α (Han Bio, Shanghai, China). The selected RNAi sequence against Hsp90α was 5′-GGAAAGAGCTGCATATTAA-3′ , RNAi was cloned into the lentiviral RNAi delivery vector and the gene transduction efficiency of infected 16HBE cells was monitored based on the co-expressed GFP gene marker in the same vector under a fluorescent microscope. When reached 85% confluence, normal 16HBE cells were maintained in serum-free medium for 12 h, then treated with HDM (400 U/ml) with or without 1G6-D7 (25 μg/ml). The Hsp90α-knockdown 16HBE cells were treated with HDM (400 U/ml) or human recombinant Hsp90α (hrHsp90α, 10 μg/ml, Stressmarq Biosciences Inc.). After 24 h, cells were harvested for cell lysates preparation for 12 h. Condition media was collected to investigate the amount of Hsp90α and Hsp90β. In a further experiment, normal 16HBE cells were treated with HDM (400 U/ml) or hrHsp90α (10 μg/ml), with or without LY294002 (10 μM, Cell signaling technology, CST).
The measurement of epithelial barrier function and immunofluorescence
The measurements of transepithelial electrical resistance (TEER), Permeability (FITC-dextran) and immunofluorescence of E-cadherin and β-catenin were performed as described in our previous study . Primary antibodies anti-E-cadherin, anti-β-catenin and FITC (green or red)-linked anti-rabbit IgG were obtained from Santa Cruz Biotechnology, USA. 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Sigma-Aldrich.
Airway resistance to methacholine, mice euthanasia and necropsy
24 h after the last administration, lung resistance (RL) was assessed to evaluate the airway resistance. Mice were placed in a barometric plethysmo-graphic chamber (Buxco Electronics, Troy, NY) and provocated with vehicle (PBS), followed by increasing concentrations of methacholine (6.25 mg/ml, 12.5 mg/ml, 25 mg/ml, and 50 mg/ml) via nebulizer (Buxco Electronics, Inc., Troy, NY) for 3 min. RL and other data were monitored at the same time. The detail protocols of mice sacrifice, anesthesia, sample collection and sample storage were executed as described in our previous study .
Western blot analysis
The supernatants of cell and the completely homogenized right lung tissue samples were collected and boiled with standard SDS sample buffer. The secreted Hsp90α(Calbiochem, Merck.), secreted Hsp90β(Stressmarq Biosciences Inc.) in conditioned media, Hsp90α, Hsp90β, p-AKT (Thr 308, CST), pan-AKT (CST), p-ERK1/2(CST), ERK1/2(CST), p-JNK (CST), JNK (CST), E-cadherin (Santa Cruz.), β-catenin (Santa Cruz.), occluding (Santa Cruz.), claudin1-2(Santa Cruz.) in cell lysate and tissue lysate were analyzed.
As previously described, mice were sacrificed with pentobarbital (100 mg/kg, i.p.) 1 day after the last airway challenge. Blood samples were allowed to rest for 2 h at room temperature, then centrifuged (3000×g, 20 min), and supernatants were harvested and stored at − 80 °C. IgE (BD Bioscience.), Hsp90α(Cloud-Clone Corp.) in serum and Hsp90α, IL-4 (affymetrix, eBioscience.), IL-5 (affymetrix, eBioscience.), IL-13 (affymetrix, eBioscience.), IL-33 (affymetrix, eBioscience.), IFN-γ (affymetrix, eBioscience.) in BALF were measured by ELISA following the manufacturer’s instructions.
Histopathology and immunohistochemistry
Left lungs were gently infused with 10% Paraformaldehyde to fully inflate all lobes (inflation was judged visually) and immersed in Paraformaldehyde for at least 24 h, then fixed, paraffin-embedded, cut in 4-μm sections, and stained with hematoxylin and eosin (H&E) for blinded histopathologic assessment. Immunohistochemistry for Hsp90α, E-cadherin and β-catenin were performed as previously study described .
Statistical analysis was computed using SPSS (version 19.0). The variables were expressed as mean ± standard deviation. One-way ANOVA accompanied by Bonferonni post hoc test for multiple comparisons were utilized to compare differences among groups. P < 0.05 was considered as statistical significance.
Secreted Hsp90α was released in asthmatic mice and 1G6-D7 alleviated AHR
Secreted Hsp90α participated in epithelial barrier dysfunction of asthmatic mice
Secreted Hsp90α promoted the release of Th2 cytokines in asthmatic mice
HDM promoted secretion of Hsp90α both in 16HBE cells and Sh-Hsp90α 16HBE cells
1G6-D7 attenuated HDM-induced bronchial epithelial hyperpermeability
1G6-D7 partly restored the HDM-induced disorder of E-cadherin and β-catenin
1G6-D7 reduced the expression of p-AKT, p-ERK1/2, p-P38 and LRP-1 induced by HDM stimulation
Secreted Hsp90α promoted barrier dysfunction via PI3K/AKT pathway
In this study, we proved that secreted Hsp90α participated in HDM-induced dysfunction of epithelial barrier, airway resistance and airway inflammation, the function was partly mediated by the PI3K/AKT pathway. 1G6-D7 protected AECs from dysfunction and downstream signaling transduction. The study aimed to offer a potential therapeutic strategy to asthma.
Secreted Hsp90α participated in many diseases like tumor, inflammation [8, 10, 17, 18]. Previous research had demonstrated that Hsp90α and Hsp90β mRNA expressions were increased in peripheral blood mononuclear cells of patients with asthma . Our research confirmed the association between asthma and secreted Hsp90α . Based on the results of our study, we did further research in the mechanisms of secreted Hsp90α in asthma.
Asthma is characterized by airway epithelial barrier dysfunction, Th2-mediated airway inflammation, airway remodeling and AHR [5, 21, 22]. The integrity of the airway epithelial barrier is dependent on cellular integrity and strong cell-cell adhesion mediated by particular junctions [23, 24]. Specifically, E-cadherin complexes with β-catenin to form an adhesive junction (AJ) that is involved in signal transduction, providing the structural support required to form these ligation complexes [6, 14, 22, 25, 26, 27, 28, 29, 30]. We found secreted Hsp90α was induced by HDM and participated in the disruptive effect of HDM in mice and AECs. Furthermore, 1G6-D7,a monoclonal antibody developed in Wei Li’s laboratory and specifically combined to secreted Hsp90α [15, 25, 26, 27],showed protective effects on barrier function and proteins in vitro and in vivo. The intracellular signaling pathways, functions of molecular chaperone and intracellular micro-environment were not affected by 1G6-D7 directly since it’s molecular was too big to enter the cells. We found that HDM-induced dislocations of E-cadherin and β-catenin manifesting by ICF and IHC, while 1G6-D7 had protective effects on these changes. Furthermore, after knocking down Hsp90α, exogenous supplement of human recombinant Hsp90α also caused severe dislocation of E-cadherin and β-catenin. All the above indicated that secreted Hsp90α mignt function in promoting cell migration and inflammation, therefore accelerating the loss of the cell-cell junctions. However, it is worth thinking that HDM reduced the expressions of E-cadherin and β-catenin in mice but not in 16HBE cells, suggesting that there might be more mechanisms to be found in vivo.
In our previous research, we found that Hsp90α is involved in HDM-induced asthmatic airway epithelial barrier disruption . Secreted Hsp90α can be induced by stress proteins and cytokines, which play a key role in inflammation and barrier function [31, 32, 33, 34, 35, 36, 37].. We found that the expression of BALF Hsp90α had been promoted by HDM, while 1G6-D7 reduced this response. Furthermore, we found chronic HDM-stimulation provoked a robust Th2 response accompanied by more modest changes in IFN-γ levels in mice, suggesting a shift in the Th1/Th2 balance toward Th2, which was an vital characteristic in asthma. The blockade of secreted Hsp90α with 1G6-D7 reduced the release of HDM-induced Th2 cytokines. IL-4 was important in CD4+ lymphocyte differentiation and the production of IgE,while IL-13 drived airway hyperresponsiveness, mucus production, and subepithelial fibrosis. IL-5 was an obligate cytokine for the survival and maturation of eosinophils. IgE has a positive correlation with eosinophil inflammation while IL-33 could strongly promote the release of Th2 cytokines [38, 39].Co-administration of 1G6-D7 dramatically reduced serum IgE and BALF IL-33 levels as well as HDM-induced Th2 inflammation stimulated by decreasing the levels of IL-4,IL-5,IL-13. The mechanism of secreted Hsp90α in immune-regulation has not been studied in this report,though in previous study, Hsp90 has been proved to be vital in innate immunity and the antigens cross-presentation . In our study, 1G6-D7 only combined to Hsp90α,ameliorated the Th2 inflammation and airway resistance of asthmatic mice. The impact of extracellular Hsp90α in immune-regulation had already been confirmed in activation of monocytes and other pathological processes . More should be done to prove the role of secreted Hsp90α in asthmatic inflammation and immune-regulation.
Our previous studies had revealed that the signaling pathway of VEGF, PI3K/AKT, MAPK/ERK mediated the dysfunction of AECs [11, 12, 13, 42]. The relationship between AKT, ERK and secreted Hsp90α has not been clearly demonstrated. Studies indicated that cells under a variety of stimulations could actively secrete Hsp90α.It has been confirmed that secreted Hsp90α binded to LRP-1, subsequently activated downstream signaling pathway and eventually caused a series of signal molecules change [10, 18, 43]. In this report, the effect of HDM in promoting activation of LRP-1 and phosphorylation of AKT, ERK and P38 were in accordance with our previous studies, while the co-administration of 1G6-D7 reduced the activation of these proteins. But a supplement of hrHsp90α showed severe effect on AKT signaling molecules in Hsp90α-knockdown cells. Although JNK/p-JNK was reported to associate with asthma, but there were no changes in our data. JNK was a client protein of Hsp90α and the 1G6-D7 had no effect on it, even hrHsp90α did not increase the phosphorylation of JNK. Phosphorylation of JNK usually mediates via TGF-β pathway, but it does not involve in secreted Hsp90α . Recently, it has been proved that AKT was crucial to secreted Hsp90α as it works in various cellular process such as promoting cell motility and wound healing . Therefore, we kept on focusing on the relationship between secreted Hsp90α and PI3K/AKT pathway, and the latter had been proved to be important in epithelial dysfunction induced by HDM in our previous research [13, 42]. As we could see, a PI3K inhibitor LY294002 used in this study protected the AECs from hrHsp90α and HDM induced decrease of TEER and increase of FITC dextran permeability, suggesting that secreted Hsp90α played an important role in dysfuction of airway epitheliel barrier via promoting the phosphorylation of AKT, and the PI3K/AKT pathway was exactly the downstream of it. These results were in accordance with Wei Li′ article, which confirmed the signaling pathway of secreted Hsp90α - LRP-1 - p-AKT in HDFs . But it was strange that Wei Li proved the phosphorylation of AKT was Ser473 but not Thr308, whereas in this report and in our previous studies, we found phosphorylation of AKT at Thr308 played an important role in epithelial dysfunction. Moreover, secreted Hsp90α promoted the activation of AKT at Thr308 but the treatment of 1G6-D7 prevented it in vitro. All the above indicated that secreted Hsp90α promoted the development of asthma by inducing epithelial barrier dysfunction via PI3K/AKT pathway.
In summary, we demonstrated that HDM-induced asthmatic mice and 16HBE cells increased the secretion of Hsp90α, which played an important role in asthma since it induced the epithelial barrier dysfunction via PI3K/AKT pathway. Neutralization of Secreted Hsp90α by 1G6-D7 inhibited the phosphorylation of AKT and ameliorated the bronchial epithelial barrier dysfunction induced by HDM. Therefore, the anti-secreted Hsp90α therapy might be a potential treatment in future.
We declare that there was no commercial interest or conflict of interest for this study. We thank Professor Zou Fei for the support of the experimental sites and experimental equipment.
SC, HD, MZ and CH designed the experiment and wrote the manuscript. HD, CY and CH performed the experiments and analyzed the data. HZ helped with data analysis and revised the manuscript. WL, YH, LL, XW and YW helped revise the manuscript. All authors reviewed the manuscript. All authors read and approved the final manuscript.
The design and performance of this study were funded by the National Natural Science Foundation of China (81670026, 81500023,81470228, 81270087, 81270089). The data analysis of this study was funded by the Precision Medicine Research of The National Key Research and Development Plan of China (2016YFC0905800) and the National 973 Program on Key Basic Research Project of China (973 Program, 2012CB518203). And the writing of this study was funded by the Natural Science Foundation of Guangdong Province(2014A030310325).
Ethics approval and consent to participate
The mice used in our study were treated according to an experimental plan approved by the committee of Southern Medical University on the use and care of animals. All procedures compliedwith the guidelines of the Institutional Animal Ethics Committee for the care and use of laboratory animals.
Consent for publication
All the authors declare no competing interests.
The authors declare that they have no competing interests.
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