Histone Deacetylase 8 Regulates NF-kB-Related Inflammation in Asthmatic Mice Through H3K9 Acetylation
Bronchial asthma is a chronic respiratory disease characterized by airway inflammation, airway remodeling, and airway hyperresponsiveness. During an asthma attack, numerous inflammatory genes are activated, leading to the increased expression of inflammatory mediators, which ultimately contribute to airway hyperresponsiveness and remodeling. Nuclear factor-kB (NF-kB), a transcription factor composed of dimers from the Rel family, plays a central role in regulating the expression of these inflammatory genes. Histone deacetylases (HDACs) are key enzymes that regulate the deacetylation of histones and non-histone proteins, influencing gene expression and cellular processes. Previous studies have demonstrated that HDAC inhibitors, including HDAC6 inhibitor Tubastatin A HCl, HDAC8 inhibitor PCI-34051, and broad-spectrum HDAC inhibitor Givinostat, can effectively alleviate airway inflammation, remodeling, and hyperresponsiveness in asthma. However, the specific mechanisms by which HDAC8 regulates inflammation in asthma remain unclear. This study aimed to elucidate the role of HDAC8 in asthma pathogenesis, particularly its regulation of NF-kB-related inflammation through histone H3 lysine 9 (H3K9) acetylation.
Experimental Design and Methods
The study utilized female BALB/C mice (specific-pathogen-free grade, 6–8 weeks, 18–22 g) purchased from Liaoning Changsheng Biotech Co., Ltd. All procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Forty-eight mice were randomly divided into four groups: normal control group, asthma group, dexamethasone group, and PCI-34051 group. Mice in the asthma, dexamethasone, and PCI-34051 groups were sensitized with ovalbumin (OVA) and aluminum hydroxide gel via intraperitoneal administration on days 1, 8, and 15. Seven days after the last sensitization, OVA atomization was performed three times per week for eight weeks. Dexamethasone (2.0 mg/kg) and PCI-34051 (0.5 mg/kg) were administered via intraperitoneal injection 30 minutes before stimulation. The normal control group received saline instead of OVA.
Airway responsiveness was measured using non-invasive whole-body plethysmography, and enhanced pause (Penh) values were recorded following stimulation with acetyl-b-methacholine chloride at increasing concentrations. Bronchoalveolar lavage fluid (BALF) was collected, and total cell counts were determined. Differential cell counts were performed using Wright-Giemsa staining. Levels of interleukin-4 (IL-4), interleukin-5 (IL-5), interferon-g (IFN-g) in BALF supernatant, and immunoglobulin E (IgE) in serum were measured using enzyme-linked immunosorbent assay (ELISA) kits. Lung tissues were fixed, embedded in paraffin, and stained with hematoxylin and eosin (H&E), Alcian blue-periodic acid Schiff (AB-PAS), and Masson’s trichrome to evaluate inflammatory infiltration, goblet cell metaplasia, and collagen deposition. Inflammatory infiltration was graded on a scale from 0 to 4 based on the distribution and clustering of inflammatory cells around the bronchus.
Nuclear HDAC8 activity in lung lysates was measured using a fluorometric assay kit. The expression levels of HDAC8 and H3K9 acetylation in lung tissue were assessed using western blotting and immunohistochemistry. The activation level of the NF-kB signaling pathway was evaluated by measuring the expression of NF-kB, phosphorylated NF-kB, inhibitor of kB (IkB), and phosphorylated IkB using western blotting. Statistical analysis was performed using SPSS 23.0 software, and comparisons between groups were conducted using t-tests. P-values < 0.05 were considered statistically significant.
Results
The study revealed that HDAC8 was highly expressed in mouse epithelial cells, alveolar interstitial cells, and inflammatory cells around the airways during asthma pathogenesis. Quantitative analysis showed that the expression level and enzymatic activity of HDAC8 significantly decreased in asthmatic lung tissue under inhibition by dexamethasone or PCI-34051. Treatment with PCI-34051 reduced airway hyperresponsiveness, as evidenced by decreased Penh values at a methacholine concentration of 50 mg/mL. Additionally, PCI-34051 treatment decreased levels of serum IgE, IL-4, and IL-5, and reduced the total number of inflammatory cells and eosinophils in BALF. Histopathological staining demonstrated that PCI-34051 significantly decreased inflammatory cell infiltration, mucus accumulation, and subepithelial collagen deposition in asthmatic lung tissue.
The NF-kB signaling pathway was found to be activated during the inflammatory response in asthma, and NF-kB-related inflammatory gene expression was closely associated with the acetylation level of the H3K9 site. The H3K9 acetylation level was significantly higher in asthmatic lung tissue compared to normal tissue and was predominantly distributed in airway epithelial cells and inflammatory cells. The activation level of the NF-kB signaling pathway was also higher in asthmatic lung tissue, with upregulated expression of NF-kB, phosphorylated NF-kB, IkB, and phosphorylated IkB. Treatment with dexamethasone or PCI-34051 effectively inhibited H3K9 acetylation and NF-kB pathway activation.
Discussion
HDAC8, a member of the Class I HDAC family, plays a significant role in regulating apoptosis, proliferation, and differentiation of T cells, as well as the differentiation and contraction of smooth muscle cells. Previous studies have shown that HDAC8 regulates the acetylation levels of histones at H3K9, H2BK12, and H3K18, influencing downstream gene transcription. In the context of asthma, the activation of the NF-kB signaling pathway in lung tissue directly regulates the expression of numerous inflammatory genes. Acetylation modifications of histone-related sites, particularly H3K9, can alter the spatial conformation between histones and chromatin, exposing promoter regions of target genes to NF-kB binding. This study supports the hypothesis that HDAC8 regulates the acetylation level of H3K9 in lung tissue, thereby affecting the activation of the NF-kB pathway and related inflammation.
The findings suggest that the anti-inflammatory effects of HDAC8 inhibitors are mediated through the regulation of H3K9 acetylation and modulation of NF-kB pathway activation. However, the exact epigenetic mechanisms by which HDAC8 influences the expression of NF-kB-related inflammatory genes remain to be fully elucidated. Further research is needed to explore these mechanisms and to develop targeted therapies for asthma based on HDAC8 inhibition.
Conclusion
This study demonstrates that HDAC8 plays a critical role in regulating NF-kB-related inflammation in asthmatic mice through the modulation of H3K9 acetylation. Inhibition of HDAC8 with PCI-34051 effectively reduces airway hyperresponsiveness, inflammatory cell infiltration, and the expression of inflammatory mediators in asthma. These findings highlight the potential of HDAC8 inhibitors as therapeutic agents for asthma and provide insights into the epigenetic mechanisms underlying airway inflammation.
doi.org/10.1097/CM9.0000000000001963
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