HDAC8-Selective Inhibition by PCI-34051 Attenuates Inflammation and Airway Remodeling in Asthma via miR-381-3p-TGFβ3 axis

doi:10.21203/rs.3.rs-2573090/v1

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HDAC8-Selective Inhibition by PCI-34051 Attenuates Inflammation and Airway Remodeling in Asthma via miR-381-3p-TGFβ3 axis

https://doi.org/10.21203/rs.3.rs-2573090/v1

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Background: Histone deacetylase (HDAC) families regulate a wide range of physical processes and development of several diseases, and the role of HDACs in asthma development and progression is worth further investigation. This study aimed to evaluate HDAC effects in a mouse model of asthma.

Methods: HDAC8 selective inhibitor PCI-34051 was administered to a mouse model of ovalbumin (OVA)-sensitized and challenged asthma. Airway responsiveness, serum cytokines, histological changes of the airway, and expression levels of α-SMA, b-actin, VEGFR, VEGF, GAPDH, HDAC8, TGF-b3, CD 105, p-ERK 1/2, ERK 1/2, PI3K, p-AKT, AKT, and PDK1 were evaluated. The miR-381-3p level was also measured.

Results: All classic histologic and cellular changes of asthma in inflammation and airway remodeling were altered by HDAC8 inhibitor PCI-34051 via regulating the miR-381-3p level and its downstream gene TGF-b3. Inhibition of TGF-b3 further reduced the activation of ERK, PI3K, AKT and PDK1.

Conclusions: HDAC8 inhibitor PCI-34051 exhibits comprehensive control of asthmatic changes, including inflammation and airway remodeling, in a mouse model.

Asthma

Airway remodeling

Histone deacetylase

Inflammation

microRNA

Asthma is typically characterized by chronic airway inflammation. This common respiratory disease affects an estimated 300 million people worldwide and prevalence is increasing (1). Even with similar clinical presentation, including wheezing, shortness of breath and chest tightness indicating airflow limitation, asthma is a heterogeneous disease mechanically, making it difficult to be defined, diagnosed, assessed for severity and managed (2). Current control of asthma mainly relies on glucocorticoids and long-acting β2 agonists. However, large numbers of patients cannot be well-controlled by the available treatments, probably due to the diverse pathogenic processes. Rather than being viewed as a single disease entity, asthma is now considered to be a clinical condition involving multiple pathological mechanisms. The underlying mechanisms can be roughly divided into two categories: inflammation and airway remodeling. New targeted therapies designed for specific epigenotypes may be worthy of being developed based on gaining more detailed knowledge of better management through individualized treatment of these two key contributors of asthma.

Histone deacetylase (HDAC) families include four classes that are nuclear enzymes able to modify chromatin structure by removing acetyl functional groups from the lysine residues of the target proteins. HDACs regulate a wide range of physical processes, including the cell cycle, differentiation and apoptosis (3). In the pathogenesis of respiratory diseases, increased expression of HDACs has been reported in COPD (4), allergic rhinitis (5–7) and asthma (8, 9). In particular, the role of HDACs in asthma development and progression may be worthy of being investigated. Since HDACs activity is reversible by pan- or specific inhibitors, the therapeutic effects of HDACs inhibitors have been evaluated in several diseases. Moreover, to gain a better understanding of the disease mechanism, HDAC inhibitors also can be used to clarify the function of each molecule involved in pathogenesis, and the associations between the molecules.

In the present study, the HDAC8 selective inhibitor PCI-34051 was administered to mice in the ovalbumin (OVA)-sensitized and challenged asthma model, aiming to discover whether and how HDAC8 contributes to asthma development and the possible pathway. Results may help to identify novel treatment targets, and to benefit the asthma population.

Development of OVA-sensitized and challenged asthma model

This study protocol was reviewed and approved by the institutional animal care use committee (IACUC) of our hospital, approval number AF-SOP-07-1.1-01. The OVA asthma model was utilized in the present study, as previously described (10, 11). Briefly, SPF-grade female BABL/c mice aged 6–8 weeks and weighing 20 ± 2 g were housed for 1 week in a SPF-grade room and provided ad libitum access to water and food. Mice were randomly divided into 4 groups, n = 6 for each group, including the health control (con) group, mice with asthma (ova) group, dexamethasone treatment (dex) group, and PCI-34051 treatment (pci) group. To create the asthma model, mice of the ova, dex, and pci groups were sensitized by intraperitoneal injection of 0.2 mL OVA solution [20 µg OVA and 2 mg aluminum hydroxide (both, Sigma-Aldrich, St. Louis, MO, USA) in 0.2 mL normal saline] at Days 0, 7, and 14. The OVA challenge was conducted on day 21 using 2% OVA saline solution by a nebulizer for 30 minutes daily, 3 times per week for 8 weeks, while mice of the con group were given normal saline instead of OVA. In the dex and pci groups, dexamethasone (2 mg/kg, Sigma-Aldrich) or PCI-34051 (0.5 mg/kg, Sigma-Aldrich) was administrated intraperitoneally before each challenge while the con and ova groups were given normal saline as a control.

Airway Responsiveness Measurement

Airway resistance was estimated by non-invasive whole-body plethysmography (Emka Technologies, Paris, France). Enhanced pause (Penh) was recorded for 3 minutes following administration of acetyl-β-methylcholine chloride (Sigma-Aldrich) at concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 mg/ml, as described previously (12).

Cytokine Measurement

Serum samples were prepared from blood collected by orbital puncture after anesthesia (2.5% isoflurane delivered in O₂, 2 L/minute) and centrifuged at 3500 rpm, 4°C for 15 minutes. Measurement of cytokines, including IL-17/IL-17A, IL-17E/IL-25, IL-6, IL-13 and TNF-α, were conducted by Luminex-based immunoassays.

Lung Tissue Preparation

Mice were sacrificed by cervical dislocation after anesthesia 1 hour after the last OVA challenge. The left lung was fixed with 0.1 mL iced 4% polyoxymethylene intratracheally, then removed and fixed in 4% polyoxymethylene for 24 hours. After re-hydration by running tap water for 6 hours, dehydration and embedding in paraffin were performed, and 4‑µm sections were cut for further analysis.

Histopathological Analysis

Inflammation level was evaluated by HE staining and a 5-point scoring system. Fibrosis level was evaluated by collagen deposition/basement membrane thickness using Masson’s trichrome staining. Mucus metaplasia was evaluated by the percentage of mucus-containing goblet cells determined by PAS staining.

Immunofluorescence Staining

The sections were incubated with blocking solution (Beyotime Biotechnology, Shanghai, China) for 1 hour then incubated with the primary antibodies: α-SMA and CD 31 (both, Abcam, Cambridge, MA, USA) at 4°C overnight and the Alexa Fluor-labelled secondary antibodies (Abcam) at 37°C for 1 hour. The nuclei were stained with DAPI (Abcam). The stained sections were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

Western Blotting

Protein was extracted with RIPA buffer, and the concentrations were measured using BCA protein concentration kit (both, Beyotime Biotechnology). Equal amounts of proteins were subjected to gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking, membranes were incubated with the primary antibodies: α-SMA, β-actin, VEGFR, VEGF, GAPDH, HDAC8, TGF-β3, CD 105, p-ERK 1/2, ERK 1/2, PI3K, p-AKT, AKT, and PDK1, followed by incubating with the corresponding HRP-conjugated secondary antibodies (all, Abcam). The immunoblots were visualized using enhanced chemiluminescence (Bio-Rad, Hercules, California, USA). Densitometry was performed using ImageJ (NIH, Bethesda, Maryland, USA) software.

Immunohistochemistry

The sections were de-paraffinized, hydrated, and antigen retrieval, then incubated with the primary antibodies: VEGF, VEGFR, TGF-β3, CD 105, ERK 1/2, and AKT and the corresponding biotinylated secondary antibodies (all, Abcam). Color development was done by the DAB solution (Vector Laboratories, Burlingame, CA, USA), and counterstained with Mayer’s hematoxylin.

Real-time quantitative RT-PCR (RT-qPCR)

Total RNA was extracted by TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using the reverse transcription kit (Roche, Shanghai, China), RT-qPCR was performed by SYBR Green-based reagent and amplified on a CFX real-time PCR detection system (Bio-Rad). U6 and GAPDH were employed as the internal reference for miR-381-3p and VEGFR and VEGF mRNA, respectively.

Fluorescence in situ hybridization (FISH) analysis

The sections were blocked with 3% BSA in 4×saline-sodium citrate then treated with proteinase K (Roche), and hybridized with FITC-labeled miR-381-3p probe and stained with anti-digoxin rhodamine conjugate (Abcam) at 37°C for 1 hour in the dark. The nuclei were stained with DAPI (Abcam). The stained sections were observed under a fluorescence microscope (Olympus).

The Penh values escalated with the increased concentrations of acetyl-β-methylcholine in all groups, and the value in the ova group was significantly higher than those in the other groups, indicating that the trend of Penh value changed to near normal (con group) when mice received treatment, including dexamethasone (dex group) and HDAC8 inhibitor PCI-34051 (pci group). The airway resistance at the fixed dose acetyl-β-methylcholine chloride 50 mg/mL showed the same effect (Fig. 1a). HDAC8 inhibition reduces the inflammation level, fibrosis level and mucus secretion, which were evaluated by HE, Masson’s trichrome, and PAS staining, respectively (Fig. 1b). HDAC8 expression correlates with the pathogenesis of asthma, since the expression level increased in the ova group, and decreased in the dex and pci groups, as evaluated by western blotting (Fig. 1c).

Levels of the important Th 2 inflammatory cytokines, including IL-17A, IL-17E, IL-6, IL-13 and TNF-α, were significantly increased in the ova group, compared to the con group (p < 0.0001) and significantly decreased in the dex and pci groups compared to the ova group (p < 0.0001 for all of the pci group, p < 0.001 for IL-17/IL-17A of the dex group and p < 0.0001 of the others in the dex group), indicating that HDAC8 inhibition reduces Th2-mediated airway inflammation (Fig. 2).

Figure 3 shows the effects of HDAC8 inhibition on airway remodeling through the expression of α-SMA and CD 31, which represent the structural response of airway smooth muscle cells and the microvasculature in asthma. Immunofluorescence staining and western blot indicated that HDAC8 inhibition reduced the increased numbers of smooth muscle cells and endothelial cells. These results mean that the thickening of vessels wall and neovascularization in asthma may be reduced by HDAC8 inhibition. The percentages of vascular wall area, vascular endothelial cells numbers and neogenetic microvessel density all significantly increased in mice of the ova group compared to those in the con group (p<0.001 of the percentage of vascular wall area, the p<0.0001 for the other two). After receiving treatments, both mice of the dex and pci groups showed significant reduction of all the three indicators, compared to those in the ova group (p<0.05 for both treatments in all three indicators)(Fig. 3a). The protein expression of α-SMA significantly increased in mice of the ova group compared to those in the con group (p<0.0001), and both significantly decreased in mice of the dex and pci groups, compared to those in the ova group (p<0.0001). However, the expression of α-SMA of the dex and pci groups were still significantly higher than that of the con group (p<0.001)(Fig. 3b).

Evaluation of VEGF and the receptor VEGFR involved in Th2-mediated airway inflammation and angiogenesis in asthma demonstrated that protein and mRNA levels of VEGF and VEGFR were both reduced by HDAC8 inhibition (Fig. 4). Immunohistochemistry staining of VEGF showed that the expression significantly increased in mice of the ova group compared to those of the con group (p < 0.0001) and significantly reduced in those of the dex and pci groups, compared to the ova group (p < 0.0001)(Fig. 4a). Western blot indicated that both VEGFR and VEGF expression increased significantly in the ova group, compared to that in the con group (p < 0.05 and p < 0.0001, respectively). VEGF expression decreased significantly in the dex and pci groups, compared to the ova group (p < 0.05 and p < 0.01, respectively), however, the levels still significantly higher than that of the con group (p < 0.0001 and p < 0.001, respectively). VEGFR expression decreased significantly in the dex and pci groups, compared to the ova group (p < 0.01 and p < 0.05, respectively)(Fig. 4b). mRNA expression changes of VEGF and VEGFR showed similar trends of the western blot results (Fig. 4c)

The miR-381-3-p level was significantly lower than that of the con group (p < 0.0001). When mice received HDAC8 inhibitor PCI-34051, miR-381-3-p in the asthmatic airway was significantly higher than that in the healthy control (con group)(p < 0.0001) or asthmatic airway (ova group) (p < 0.0001), indicating that HDAC8 may be associated with miR-381-3-p expression in the lungs, represented by FISH and quantitative RT-PCR data (Fig. 5).

The levels of miR-381-3-p regulated molecules TGF-β3 and CD 105 increased in the ova group and decreased in the dex and pci groups (Fig. 6). Immunohistochemistry staining of TGF-β3 showed that the expression significantly increased in mice of the ova group compared to those of the con group (p < 0.0001) and significantly reduced in those of the dex and pci groups, compared to the ova group (p < 0.0001). Western blot indicated the same results (Fig. 6a). Immunohistochemistry staining of CD 105 showed that the expression significantly increased in mice of the ova group compared to those of the con group (p < 0.0001) and significantly reduced in those of the dex and pci groups, compared to the ova group (p < 0.0001). Western blot indicated the same results (Fig. 6b).

In the ova group, the expression of p-ERK1/2, ERK 1/2, PI3K, p-AKT, AKT and PDK1 all increased compared to those in the con group. The levels of all proteins decreased in the dex and pci groups (Fig. 7) Immunohistochemistry staining of p-ERK 1/2 showed that the expression significantly increased in mice of the ova group compared to those of the con group (p < 0.0001) and significantly reduced in those of the dex and pci groups, compared to the ova group (p < 0.0001). Western blot indicated the same results (Fig. 7a). Immunohistochemistry staining of AKT showed that the expression significantly increased in mice of the ova group compared to those of the con group (p < 0.0001) and significantly reduced in those of the dex and pci groups, compared to the ova group (p < 0.0001)(Fig. 7b). Western blot results showed significant increases of PI3K (p < 0.05), p-AKT (p < 0.05) and PDK1 (p < 0.0001) in mice of the ova group, compared to those in the con group. After receiving dexamethasone treatment, expressions of PI3K (p < 0.01), p-AKT (p < 0.05) and PDK1 (p < 0.0001) were significantly lower than those of the ova group. After receiving PCI-34051 treatment, expressions of PI3K (p < 0.01), p-AKT (p < 0.05) and PDK1 (p < 0.0001) were significantly lower than those of the ova group (Fig. 7b).

Results of the present study have shown that TGF-β3 plays a central role in regulating both the signaling of inflammation and airway remodeling in a mouse model of asthma. All classic histologic and cellular changes of asthma were altered by HDAC8 inhibitor PCI-34051 via regulating the miR-381-3p level and its downstream gene TGF-β3. Inhibition of TGF-β3 further reduced the activation of ERK, PI3K, AKT and PDK1.

Inflammation and airway remodeling play vital roles in the occurrence and progression of asthma, both of which are multicellular processes. Eosinophils, T cells, mast cells, neutrophils and dendritic cells are recruited to the site of asthmatic inflammation, and the structural changes in the airway, including epithelial fragility, goblet cell metaplasia, fibroblast hyperplasia, airway smooth muscle mass increase and basement membrane thickening due to collagen deposition are all significant classic features of asthma. The crosstalk between inflammation and airway remodeling is closed and frequent; inflammation leads to airway remodeling by factors secreted by the inflammatory cells, including cytokines, chemokines and growth factors, and drives the changes in the structural cells of the airway (13); the stimulated structural cells also produce factors to increase the immune cell levels in the airway. For example, airway smooth muscle cells produce IL-6, which is required for IL-17A expression and promotes Th17 polarization (14, 15). Interactions between immune cells and airway structure cells increase the complexity of pathogenesis, the asthma epigenotypes, and also disease control, since the treatment target is difficult to identify (13, 16).

MicroRNAs (miRs) are single-stranded RNA molecules that range from 19 to 24 nucleotides, and have been known to be involved in several important physical functions via regulating the expression of target genes post-transcriptionally. In addition, the roles of miRNAs in the pathogenesis of many diseases have also been investigated (17–19). For asthma, in a 4,4’-methylene diphenyl diisocyanate-induced occupational asthma (OA) mouse model, a 2- to 5-fold downregulation of circulating miR-381-3p was observed in mice of the OA group compared to mice of the normal control group. Pathway analysis results indicated that miR-381-3p may be associated with the regulation of the immune system (20). However, the definitive mechanism has not yet been evaluated in this OA model. Our previous in vitro study found that miR-381-3p overexpression in human bronchial smooth muscle cells decreased the expressions of α-SMA, fibronectin 1 and collagen I, which are all markers of airway remodeling, suggesting that miR-381-3p may be a moderator of airway remodeling in asthma (21).

HDAC8 belongs to the class I HDACs. Since overexpression of HDAC8 has been observed in several malignancies (22–24) and several respiratory diseases, including asthma (4, 9), HDAC8 becomes a novel therapeutic target for these diseases. HDAC8 selective inhibitor PCI-34051 with > 200-fold selectivity over the other class I HDACs has been tested for its therapeutic effects in mouse models of asthma, demonstrating that PCI-34051 reduces the inflammation and airway remodeling in asthma. Treating PCI-34051 ameliorated airway hyperresponsiveness was shown to reduce immune cell counts in bronchoalveolar lavage fluid, significantly decrease inflammatory cell infiltration and cytokines, and reduce M2 macrophage polarization of lung tissue in the mouse model of asthma (4, 9, 25–27). The anti-airway remodeling function of PCI-34051 in asthma was reported to regulate smooth muscle differentiation and contraction, decrease α-SMA expression, reduce fibrosis in EMT and bronchial smooth muscle cells hyperplasia, in addition to collagen deposition (21, 26, 27).

When the effects of PCI-34051-induced bronchial epithelial cell-derived exosomes on human bronchial smooth muscle cells were investigated, miR-381-3p expression was upregulated (21). Further genetic analysis and dual-luciferase reporter assay results indicated that TGF-β3 is the target of miR-381-3p after HDAC8 inhibitor PCI-34051 treatment, which is consistent with results of the present study. Results of the present study showed that PCI-34051 increased the miR-381-3p level and decreased the expression of TGF-β3.

The TGF-β superfamily is the key molecule of the signaling pathway in asthma since it is the major mediator in pro-inflammatory responses and fibrotic tissue remodeling in the lung. Produced by epithelial cells, eosinophils, macrophages and fibroblasts, TGF-β involves epithelial transformation, fibrosis, airway smooth muscle proliferation, and microvascular changes associated with airway wall thickness, mucus production, collagen deposition and cytokine dysregulation in an allergenic asthma model, including OVA sensitization and challenge and house dust mite induction models (28, 29). IL-13 is known to induce TGF-β expression, then increase α-SMA expression, collagen deposition and IL-6 production (30, 31). Furthermore, IL-6 induces VEGF expression and then promotes neovascularization in asthma (14, 32, 33). All of these changes were observed in the present study, and it may be concluded that TGF-β3 plays a central role in regulating both the signaling of inflammation and airway remodeling in asthma. This signal may be enlarged or multiplied because not only TGF-β3 increased, but the receptor also increased, since one of the components of TGF-β3 receptor CD105 level in the ova group was observed to increase.

The TGF-β-centered signaling pathway has been investigated in several studies. It has been reported that TGF-β1 regulates airway smooth muscle hyperplasia in asthma via phosphorylation of PI3K and ERK, which both induce airway smooth muscle cell proliferation (34, 35). PCI-34051 treatment recovered the increases of TGF-β3, p-ERK, ERK, PI3K, p-AKT, AKT and PDK1 in the asthmatic airway to normal levels, indicating the important role of HDAC8 in asthma, and the potential therapeutic effect of HDAC8 inhibitor PCI-34051 by controlling the key modulator TGF-β3.

HDAC8 inhibitor PCI-34051 exhibits comprehensive control of asthmatic changes, including inflammation and airway remodeling, in a mouse model. All classic histologic and cellular changes in the inflammation and airway remodeling of asthma are altered by HDAC8 inhibitor PCI-34051 via regulating the miR-381-3p level and its downstream gene TGF-β3. As a key moderator of asthma occurrence and progression, inhibition of TGF-β3 reduces the activation of ERK, PI3K, AKT and PDK1, which may lead to improving asthma through reduced airway responsiveness, inflammation infiltration, fibrosis and mucus secretion, microvasculature changes and Th2 inflammatory cytokines, including IL-17A, IL-17E, IL-6, IL-13 and TNF-α.

Ethics approval

This study protocol was reviewed and approved by the institutional animal care use committee (IACUC) of our hospital, approval number AF-SOP-07-1.1-01.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

All authors declare no conflict of interests.

Financial support

This work was supported by the National Natural Science Foundation of China [grant number 82170038].

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HDAC8-Selective Inhibition by PCI-34051 Attenuates Inflammation and Airway Remodeling in Asthma via miR-381-3p-TGFβ3 axis

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Abstract

Figures

Introduction

Methods

Development of OVA-sensitized and challenged asthma model

Airway Responsiveness Measurement

Cytokine Measurement

Lung Tissue Preparation

Histopathological Analysis

Immunofluorescence Staining

Western Blotting

Immunohistochemistry

Real-time quantitative RT-PCR (RT-qPCR)

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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