The role of HDAC6 in enhancing macrophage autophagy via the autophagolysosomal pathway to alleviate legionella pneumophila-induced pneumonia

Abstract

Legionella pneumophila (L. pneumophila) is a prevalent pathogenic bacterium responsible for significant global health concerns. Nonetheless, the precise pathogenic mechanisms of L. pneumophila have still remained elusive. Autophagy, a direct cellular response to L. pneumophila infection and other pathogens, involves the recognition and degradation of these invaders in lysosomes. Histone deacetylase 6 (HDAC6), a distinctive member of the histone deacetylase family, plays a multifaceted role in autophagy regulation. This study aimed to investigate the role of HDAC6 in macrophage autophagy via the autophagolysosomal pathway, leading to alleviate L. pneumophila-induced pneumonia. The results revealed a substantial upregulation of HDAC6 expression level in murine lung tissues infected by L. pneumophila. Notably, mice lacking HDAC6 exhibited a protective response against L. pneumophila-induced pulmonary tissue inflammation, which was characterized by the reduced bacterial load and diminished release of pro-inflammatory cytokines. Transcriptomic analysis has shed light on the regulatory role of HDAC6 in L. pneumophila infection in mice, particularly through the autophagy pathway of macrophages. Validation using L. pneumophila-induced macrophages from mice with HDAC6 gene knockout demonstrated a decrease in cellular bacterial load, activation of the autophagolysosomal pathway, and enhancement of cellular autophagic flux. In summary, the findings indicated that HDAC6 knockout could lead to the upregulation of p-ULK1 expression level, promoting the autophagy-lysosomal pathway, increasing autophagic flux, and ultimately strengthening the bactericidal capacity of macrophages. This contributes to the alleviation of L. pneumophila-induced pneumonia.

Keywords: Histone deacetylase 6 (HDAC6); Legionella pneumophila; autophagy; autophagyolysosomal pathway; macrophages.

Graphical abstract

Figure 1.

Effects of L. pneumophila infection on HDAC6 in lung tissue of mice. (a) Immunohistochemical analysis (200×) reveals HDAC6 expression in the lung tissue of mice infected with L. pneumophila after 3days. The brownish-yellow colour indicates positive expression of HDAC6, as indicated by the red arrow. (b) The positive HDAC6 staining in each group was qualified using ImageJ. (c) Impact of L. pneumophila infection on HDAC6 mRNA levels in lung tissue. (d) Influence of L. pneumophila infection on HDAC6 protein levels in lung tissue. (e) Statistical comparison of HDAC6 protein levels before and after L. pneumophila infection. (f) PCR-based genotyping outcomes for select mouse strains: Mice tail RNA gel electrophoresis showing wild-type (WT) with a single 521bp band, heterozygote (Het) with two bands at 380bp and 521bp, and homozygous (KO) with a single 380bp band. NS: normal saline group; L. pneumophila: Legionella pneumophila group, n = 4 mice in each group. Data were presented as mean ± SD. *p < 0.05, **p < 0.01.

Figure 2.

Protective effects of HDAC6 knockout on acute lung injury induced by L. pneumophila in a mouse model. (a) Survival rate of mice in the WT group and HDAC6^−/− group following L. pneumophila infection. (b) Changes in body weight during L. pneumophila infection in the WT and HDAC6^−/− groups. (c) Representative image of mouse lung. (d) Lung wet/dry ratio in the WT and HDAC6^−/− groups after infection by L. pneumophila. (e) L. pneumophila CFUs in the lungs of chimeras on D1,D3,D5 post-infection in the WT and HDAC6^−/− groups. (f) Immunofluorescence labeling for L.pneumophila (red) and nucleus (DAPI, blue) in the in the lung tissue of mice showing significantly decreased L. pneumophila expression in HDAC6^−/− group relative to the WT group. (g) Quantitative analysis of L. pneumophila fluorescence in (f). n = 4 mice in each group at each time point. Data were expressed as mean ± SD. *p < 0.05, **p < 0.01.

Figure 3.

Effects of HDAC6 knockout on lung tissue and expression levels of inflammatory factors in mice infected by L. pneumophila. (a) Representative images of haematoxylin-eosin (HE) staining of lung tissue (200×). (b) Immunohistochemical staining was used to detect the expression level of MPO in lung tissue of mice infected by L. pneumophila after HDAC6 knockout (200×). The brownish-yellow color indicates positive expression of MPO. (c) The positive MPO staining in each group was qualified using ImageJ. (d and e) Western blotting was employed to detect the expression level of MPO in lung tissue of mice infected by L. pneumophila after HDAC6 knockout. (f) The expression levels of inflammatory cytokines (IL-1β, IL-6, and IL-10) in lung tissue of mice infected by L. pneumophila after HDAC6 knockout were detected at mRNA level. (g) The secretion levels of inflammatory factors (IL-1β, IL-6, and IL-10) in lung tissue lavage fluid of mice infected by L. pneumophila following HDAC6 knockout. The data were expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Effects of HDAC6 knockout on the proliferation of L. pneumophila in macrophages. (a) Immunofluorescence staining (400×) of L. pneumophila-infected macrophages, DAPI: nuclei (blue); F4/80: macrophage surface marker (green); L.P (Ab): L. pneumophila antibody (red). (b) HDAC6 mRNA levels in macrophages of the WT and HDAC6^−/− groups following L. pneumophila infection. (c and d) HDAC6 protein expression and statistical analysis in macrophages of the WT and HDAC6^−/− groups following L. pneumophila infection. (e) Pathogen load expressed as CFU in macrophages of the WT and HDAC6^−/− groups following L. pneumophila infection. (f) Immunofluorescence labeling for L. pneumophila, nucleus, and BMDMs shows a significant decrease in L. pneumophila expression in the HDAC6^−/− group compared to the WT group. DAPI: nuclei (blue fluorescence); F4/80: macrophage surface marker (green); L.P (Ab): L. pneumophila antibody (red). (g) Quantitative analysis of L. pneumophila fluorescence in (f). Data are expressed as mean ± SD. Intra-group comparison: #P<0.05, ##P<0.01, ###P<0.001; Inter-group comparison: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

Bioinformatics analysis of HDAC6-mediated signaling pathways in L. pneumophila-infected macrophages. (a and b) Volcano plot and heatmap illustrating the expression and clustering of up-regulated and down-regulated genes in HDAC6^−/− and WT group macrophages after bacterial addition. (c and d) KEGG and GO bubble diagrams depicting enriched pathways and gene ontology terms. (e) Confocal microscopic images (600×) of cells transfected with mRFP-EGFP-LC3 adenovirus and co-cultured with L. pneumophila for 6 h, showing autophagosomes and autolysosomes. (f) Transmission electron microscopy images (1200×, 6000×, 12000×) displaying cellular structures, autophagosomes, and autolysosomes at 6h post-L. pneumophila infection. N: nucleus; M: mitochondria; PS: pseudopod; RER: rough endoplasmic reticulum; LD: lipid droplet; Ly: lysosome; ASS: autophagy-lysosome; AP: autophagosomes.

Figure 6.

Effects of HDAC6 knockout on autophagy in lung tissue of mice infected by L.pneumophila. (a) Immunohistochemical analysis of LC3 protein expression in lung tissue of the WT and HDAC6 knockout groups, the brownish-yellow colour represents positive expression (200×). (b) Statistical results of LC3-positive area in lung tissue using ImageJ software. (c and d) Expression of p62 protein in lung tissue of WT and HDAC6^−/− mice infected with L. pneumophila and corresponding statistical analysis, the brownish-yellow color represents positive expression (200×). (e) Protein expression levels of Beclin1, p62, and LC3 in lung tissue at different time points following L. pneumophila infection. (f-h) Statistical analysis of Beclin1, p62, and LC3 protein expression in lung tissue at different time points. (i-l) mRNA expression levels of Beclin1, p62, and LC3 in lung tissue at different time points following L. pneumophila infection. Data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

Effects of HDAC6 knockout on autophagy of macrophages infected by L. pneumophila. (a) Interaction between autophagy-related genes and HDAC6. (b) Protein-protein interaction (PPI) network showing the interaction between autophagy-related genes and HDAC6 at the protein level. (c) mRNA expression level of ULK1 in WT and HDAC6^−/− groups following L. pneumophila infection based on transcriptome sequencing. (d) Protein expression levels of ULK1 and p-ULK1 in WT and HDAC6^−/− groups after L. pneumophila infection. (e and f) Statistical analysis of protein expression of ULK1 and p-ULK1 in cells. (g) Protein expression levels of Beclin1, Atg5, p62, LC3, LAMP2, and Rab7 in cells at different time points after L. pneumophila infection. (h-m) Statistical analysis of protein expression for Beclin1, Atg5, p62, LC3, LAMP2, and Rab7 in cells. Data were presented as mean ± SD. *p < 0.05.