Identification of histone acetylation in a murine model of allergic asthma by proteomic analysis

Abstract

The pathogenesis of asthma is closely related to histone acetylation modification, but the specific acetylation sites related to this process remain indistinct. Herein, our study sought to identify differentially modified acetylation sites and their expression distribution in cells involved in asthma in lung tissues. The airway hyper-responsiveness, inflammation, and remodeling were assessed by non-invasive whole-body plethysmography, ELISA, and hematoxylin-eosin staining to confirm the successful establishment of the allergic asthma model. Afterward, the differentially modified acetylation sites in asthmatic lung tissues were identified and validated by using proteomics and western blotting, respectively. The immunohistochemistry analysis was applied to reveal the distribution of identified acetylation sites in asthmatic lung tissues. A total of 15 differentially modified acetylation sites, including 13 upregulated (H3K9ac, H3K14ac, H3K18ac, H3K23ac,H3K27ac, H3K36ac, H2B1KK120ac, H2B2BK20ac, H2BK16ac, H2BK20ac, H2BK108ac, H2BK116ac, and H2BK120ac) and 2 downregulated (H2BK5ac and H2BK11ac) sites were identified and validated. Furthermore, immunohistochemical staining of lung tissues showed that nine of the identified histone acetylation sites (H2BK5, H2BK11, H3K18, H2BK116, H2BK20, H2BK120, H3K9, H3K36, and H3K27) were differentially expressed in airway epithelial cells, and the acetylation of identified H3 histones were observed in both eosinophil and perivascular inflammatory cells. Additionally, differential expression of histone acetylation sites was also observed in nucleus of airway epithelial cells, vascular smooth muscle cells, perivascular inflammatory cells, and airway smooth muscle cells. In conclusion, we identified potential acetylation sites associated with asthma pathogenesis. These findings may contribute greatly in the search for therapeutic approaches for allergic asthma.

Keywords: Bronchial asthma; histone acetylation; proteomics; tandem mass tag.

Figure 1.

Establishment of chronic asthma mouse model. (a) Mice inhaled increasing doses of acetyl-β-methylcholine chloride (3.125–50 mg/ml), and AHR was measured. (b) The total and differentiated cell count in the BALF. (c) Cytokines (IL-4, IL-5, and IFN-γ) and IgE concentration in the BALF. (d) The images of lung sections with H&E staining (scale bar, 50 μm) and (e) the percentage of inflamed lung area are shown. Note: (a) and (b) represented lung sections of the control and the model groups, respectively; (a') and (b') respectively represented magnification views of the box in (a) and (b). Compared to the asthma group, *P < 0.05. (A color version of this figure is available in the online journal.) BAL: bronchoalveolar lavage.

Figure 2.

Quantitative proteomic profiling of histone acetylation in lungs of allergic asthma model. (a) Workflow of quantitative proteomics analysis of histone acetylation in lung tissues of normal and asthmatic mice. (b) Changes and distribution of histone acetylation sites. (A color version of this figure is available in the online journal.) LC-MS/MS: liquid chromatography tandem-mass spectrometry; RPLC: reversed phase liquid chromatography; TMT: tandem mass tag.

Figure 3.

Verification of identified histone acetylation sites. The acetylation sites H3K9ac, H3K14ac, H3K18ac, H3K27ac, and H3K36ac were verified by performing western blot experiments for detection of protein expression. Note: Compared to asthma group, *P < 0.05. (A color version of this figure is available in the online journal.)

Figure 4.

Expression of H2BK5ac and H2BK11ac in the lung tissue. Arrows indicate airway epithelial cell. (A color version of this figure is available in the online journal.)

Figure 5.

Expression and localization of H2BK20ac, H2BK116ac, H2BK120ac, H3K9ac, H3K18ac, H3K27ac, and H3K36ac in the lung tissues. (a) nucleus of airway epithelial cell; (b) airway epithelial cells; (c) vascular smooth muscle cell; (d) macrophage; (e) eosinophil. (A color version of this figure is available in the online journal.)

Figure 6.

Expression and localization of H2BK16ac in the lung tissue. (a) Airway epithelial cells; (b) vascular smooth muscle cells; (c) macrophage; (d) eosinophil; (e) perivascular inflammatory cell; (f) airway smooth muscle cell. (A color version of this figure is available in the online journal.)

Figure 7.

Expression and localization of H3K14ac in the lung tissue. (a) airway epithelial cells; (b) vascular smooth muscle cells; (c) macrophage; (d) eosinophil; (e): perivascular inflammatory cell; (f) airway smooth muscle cell. (A color version of this figure is available in the online journal.)