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. 2023 Jun 19;13(11):3568-3581.
doi: 10.7150/thno.82535. eCollection 2023.

The HDAC10 instructs macrophage M2 program via deacetylation of STAT3 and promotes allergic airway inflammation

Yu Zhong  1 , Tong Huang  1 , Jiewen Huang  1 , Jingyun Quan  1 , Guomei Su  1 , Zhilin Xiong  1 , Yingying Lv  2 , Shihai Li  1 , Xianwen Lai  1 , Yuanyuan Xiang  1 , Qu Wang  3 , Lianxiang Luo  3 , Xiao Gao  1 , Yiming Shao  4 , Jing Tang  5 , Tianwen Lai  1   2
Affiliations

The HDAC10 instructs macrophage M2 program via deacetylation of STAT3 and promotes allergic airway inflammation

Yu Zhong et al. Theranostics. .

Abstract

Background: Perturbation of macrophage homeostasis is one of the key mechanisms of airway inflammation in asthma. However, the exact mechanisms remain poorly understood. Objectives: We sought to examine the role of histone deacetylase (HDAC) 10 as an epigenetic regulator that governs macrophage M2 program and promotes airway inflammation in asthma, and to elucidate the underlying mechanisms. Methods: Peripheral blood and airway biopsies were obtained from healthy individuals and asthmatic patients. Asthma was induced by exposure to allergen in mice with myeloid-specific deletion of Hdac10 (Hdac10fl/fl-LysMCre) mice. HDAC10 inhibitor Salvianolic acid B (SAB), STAT3 selective agonist Colivelin, and the specific PI3K/Akt activator 1,3-Dicaffeoylquinic acid (DA) were also used in asthmatic mice. For cell studies, THP1 cells, primary mouse bone marrow derived macrophage (BMDMs) were used and related signaling pathways was investigated. Results: HDAC10 expression was highly expressed by macrophages and promoted M2 macrophage activation and airway inflammation in asthmatic patients and mice. Hdac10fl/fl-LysMCre mice were protected from airway inflammation in experimental asthma model. Hdac10 deficiency significantly attenuated STAT3 expression and decreased M2 macrophage polarization following allergen exposure. Mechanistically, HDAC10 directly binds STAT3 for deacetylation in macrophages, by which it promotes STAT3 expression and activates the macrophage M2 program. Importantly, we identified SAB as a HDAC10 inhibitor that had protective effects against airway inflammation in mice. Conclusions: Our results revealed that HDAC10-STAT3 interaction governs macrophage polarization to promote airway inflammation in asthma, implicating HDAC10 as a therapeutic target.

Keywords: HDAC10; STAT3; airway inflammation; asthma; macrophage..

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Analysis of HDAC10 expression in asthmatic patients and mice. (A, B) Representative IHC staining of HDAC10 in the airway from asthmatic patients. Images were captured at ×400 magnification and quantification of IHC was done by using Image J software. (C, D) Representative results for coimmunostaining of HDAC10 and CD206 in the lung sections from patients with asthma. Images were captured at ×400 magnification and quantification of IHC was done by using Image J software. (E) Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood samples of study subjects. The HDAC10 expression in PBMC was analyzed by using quantitative RT-PCR (qRT-PCR). (F) Schematic illustrating an established house dust mite (HDM)/lipopolysaccharide (LPS)-induced asthma mouse model (n = 6-8 for each group). (G) qRT-PCR analysis of the expression of Hdac10 in mouse lung tissue. Results were normalized to those of the gene encoding β-actin, and relative expression was calculated by the change-in-threshold method. (H, I) Representative IHC images of HDAC10 in lung tissue of Hdac10fl/fl mice following allergen induction. Images were captured at ×400 magnification and quantification of IHC was done by using Image J software. (J, K) Western blot analysis of HDAC10 and macrophage M2 marker Arg1 expression in lung tissues of Hdac10fl/fl mice following allergen induction and quantification was done by using Image J software. (L, M) Results for coimmunostaining of HDAC10 and macrophage M2 marker CD206 in allergen-induced lung sections. Images were captured at ×400 magnification and quantification was done by using Image J software. Data are shown as means ± SEM. ***P < 0.001 and ****P < 0.0001 versus Control (unless otherwise noted) by two-tailed unpaired Student's t test. Data are representative of three independent experiments with similar results (A, C, H, and L) or are from three independent experiments (G and J). See also Figure S1.
Figure 2
Figure 2
Hdac10 deficiency protected mice against allergic airway inflammation. (A) Schematic illustrating the genetic approach used to generate macrophages-conditional knockout of Hdac10 (Hdac10fl/fl-LysMCre) mice. (B) Hdac10 deficiency was confirmed by assessing genomic DNA. (C-E) Hdac10 deficiency was assessed in BMDMs from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice using qRT-PCR and Western blotting analysis. Quantification was done by using Image J software. (F, G) Representative result for coimmunostaining of F4/80 and HDAC10 in the lung sections from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice. Images were captured at ×400 magnification and quantification was done by using Image J software. (H-M) Total BAL fluid (BALF) cells, differential cell counts, and histologic analysis of the lung sections were performed with hematoxylin and eosin staining to visualize inflammatory cell recruitment from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice treated with allergen. Images were captured at ×200 magnification and quantification was done by using Image J software. Data are shown as means ± SEM (n = 4-6 mice/group). **P < 0.01 and ***P < 0.001 versus Control (unless otherwise noted) by two-tailed unpaired Student's t test or one-way ANOVA, followed by Tukey's multiple comparisons test. Data are representative of three independent experiments with similar results (F and H) or are from three independent experiments (C, D, L and M). See also Figure S2.
Figure 3
Figure 3
Hdac10 deficiency inhibited the macrophage M2 program. (A, B) Flow cytometry analysis (BD Biosciences) of macrophages derived from lung tissues of Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen induction. Quantification of CD206+ cells was analyzed with FlowJo (TreeStar). (C, D) Western blotting analysis of macrophage M2 marker Arg1 expression in the lung homogenates of Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen induction. Quantification was done by using Image J software. (E-G) qRT-PCR analysis of macrophage M2 markers Arg1, Fizz1 and Ym1 mRNA expression in the lung tissue of Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen exposure. (H, I) Flow cytometry analysis (BD Biosciences) of macrophage M2 marker CD206 expression in BMDMs following IL-4 stimulation. Quantification of CD206+ cells was analyzed with FlowJo (TreeStar). (J-L) Western blotting analysis of HDAC10 and Arg1 expression in the BMDMs after IL-4 induction. Quantification was done by using Image J software. (M-P) qRT-PCR analysis was conducted for Hdac10 mRNA and macrophage M2 markers Arg1, Fizz1, and Ym1 mRNA expression in the BMDMs after IL-4 induction. Data are shown as means ± SEM (n = 4-6 mice/group). **P < 0.01 and ***P < 0.001 versus Control (unless otherwise noted) by two-tailed unpaired Student's t test or one-way ANOVA, followed by Tukey's multiple comparisons test. Data are representative of three independent experiments with similar results (C and J) or are from three independent experiments (A, E-H, and M-P). See also Figure S3.
Figure 4
Figure 4
Hdac10 deficiency attenuated allergen-induced PI3K/Akt signaling in macrophages. (A-C) Representative IHC staining of p-Akt and p-P85 in the airway from asthmatic patients. Images were captured at ×400 magnification and quantification of IHC was done by using Image J software. (D-F) Representative IHC staining of p-Akt and p-P85 in the lung tissue from asthmatic mice. Images were captured at ×400 magnification and quantification of IHC was done by using Image J software. (G) Western blotting analysis of p-Akt, p-mTOR and p-P85 expression in the BMDMs from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen exposure. (H, I) Representative photomicrographs of lung inflammation expression are shown. Images were captured at ×200 magnification and quantification was done by using Image J software. (J-L) The expression of inflammatory cytokines in the lung homogenate of mice was analyzed by using qRT-PCR. (M) qRT-PCR analysis was conducted for macrophage M2 markers Arg1 mRNA expression in the lung homogenate of mice. Data are shown as means ± SEM (n = 4-6 mice/group). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus Control (unless otherwise noted) by two-tailed unpaired Student's t test or one-way ANOVA, followed by Tukey's multiple comparisons test. Data are representative of three independent experiments with similar results (A, D, G, and H) or are from three independent experiments (J-M). See also Figure S4.
Figure 5
Figure 5
Hdac10 deficiency suppressed STAT3 to attenuate M2 program. (A, B) Representative IHC staining of STAT3 in the lung tissue from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen induction. Images were captured at ×400 magnification and quantification of IHC was done by using Image J software. (C, E) Western blotting analysis of p-STAT3 and STAT3 expression in the lung homogenates of Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen induction. Quantification was done by using Image J software. (F, G) Representative result for coimmunostaining of F4/80 and STAT3 in the lung sections from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice after allergen induction. Images were captured at ×400 magnification and quantification was done by using Image J software. (H, I) Representative result for coimmunostaining of F4/80 and STAT3 in the BMDMs from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice. Images were captured at ×400 magnification and quantification was done by using Image J software. (J-L) Western blotting analysis was conducted for p-STAT3 and STAT3 expression in the BMDMs after allergen stimulation. Quantification was done by using Image J software. (M) BMDMs from Hdac10fl/fl and Hdac10fl/fl-LysMCre mice were transfected with pSTAT3-TA-luc plasmid for 24 h, and then treated with HDM (100 μg/ml) for another 24 h. Luciferase assay was used to detected STAT3-TA-luc fusion protein to asses STAT3 transactivation activity. Data are shown as means ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus Control (unless otherwise noted) by two-tailed unpaired Student's t test or one-way ANOVA, followed by Tukey's multiple comparisons test. Data are representative of three independent experiments with similar results (A, C, F, H and J) or are from three independent experiments (M).
Figure 6
Figure 6
STAT3 activator Colivelin exacerbated allergic airway inflammation in Hdac10 deficiency mice. (A) Schematic overview of experimental design for STAT3 activator Colivelin for asthmatic mouse. (B) The expression of p-STAT3, STAT3 and Arg1 in the lung homogenate from mice treated with allergen or Colivelin was analyzed by using Western blotting. (C, D) Representative photomicrographs of lung inflammation expression is shown. Images were captured at ×200 magnification and quantification was done by using Image J software. (E-G) The expression of inflammatory cytokines in the lung homogenate of mice was analyzed by using qRT-PCR. (H-J) qRT-PCR analysis was conducted for macrophage M2 markers Arg1, Fizz1, and Ym1 mRNA expression in the lung homogenate of mice. (K-N) BMDMs were transfected with Control or STAT3 plasmid for 24 h and then treated with HDM for another 24 h. Arg1, Fizz1, Cxcl1, and Cxcl2 mRNA expression were assessed by qRT-PCR analysis. Data are shown as means ± SEM (n = 4-6 mice/group). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus Control (unless otherwise noted) by one way ANOVA, followed by Tukey's multiple comparisons test. Data are representative of three independent experiments with similar results (B and C) or are from three independent experiments (E-N). See also Figure S4.
Figure 7
Figure 7
HDAC10 directly interacted with STAT3 and targeted STAT3 for deacetylation. (A) Confocal microscopy of the location of endogenous HDAC10 (Red) and STAT3 (Green) in THP1 cells treated with HDM for 24 h. DAPI, DNA binding dye. Images were captured at ×400 magnification. (B, C) Immunoblot (IB) analysis of endogenous HDAC10 or STAT3 in THP1 cells infected with HDM, assessed before (input) or after IP with IgG (control) or antibody to HDAC10 or STAT3. (D, E) IB analysis of exogenous HDAC10 or STAT3 in THP1 cells transfected with HA-tagged STAT3 alone or together with Flag-tagged HDAC10, assessed before (input) or after IP with antibody to HA or Flag. (F) IB analysis of HDAC10 and STAT3 interaction in a GST pull-down assay. (G) IB analysis of STAT3 acetylation (Ack) in THP1 cells transfected with HA-tagged STAT3 alone or together with histone deacetylase inhibitor Trichostatin A (TSA), assessed before (input) or after IP with antibody to STAT3 and Ack. (H) THP1 cells were transfected with Control siRNA or Hdac10 siRNA for 24 h and then treated with HDM (100 μg/ml) for 24 h, and assessed before (input) or after IP with antibody to STAT3 and Ack. (I) THP1 cells were transfected with pcDNA3.1 or pcDNA-HDAC10 plasmid for 24 h and then treated with HDM (100 μg/ml) for 24 h, and assessed before (input) or after IP with antibody to STAT3 and Ack. Data are shown as means ± SEM. Data are representative of three independent experiments with similar results.
Figure 8
Figure 8
The HDAC10 inhibitor Salvianolic acid B (SAB) prevented allergen-induced airway inflammation. (A) Schematic overview of experimental design for (B-Q) in an asthma mouse model. (B-E) Representative photomicrographs of HE staining, PAS staining, and Masson staining in the lung tissue of mice are shown. Images were captured at ×200 magnification and quantification was done by using Image J software. (F) The expression of HDAC10, STAT3, and Arg1 in the lung homogenate of mice was analyzed by using Western blotting. (G-I) qRT-PCR analysis was conducted for macrophage M2 markers Arg1, Fizz1, and Ym1 mRNA expression in the lung homogenate of mice. (J-L) The expression of inflammatory cytokines in the lung homogenate of mice was analyzed by using qRT-PCR. (M, N) The mRNA expression of Arg1 and Ym1 in BMDMs was conducted by qRT-PCR analysis. (O-Q) The levels of inflammatory cytokines in BMDMs was conducted by qRT-PCR analysis. Data are shown as means ± SEM (n = 4-6 mice/group). **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus Control (unless otherwise noted) by one-way ANOVA, followed by Tukey's multiple comparisons test. Data are representative of three independent experiments with similar results (B and F) or are from three independent experiments (G-Q). See also Figure S4.
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