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. 2014 Oct 16;9(10):e110718.
doi: 10.1371/journal.pone.0110718. eCollection 2014.

HDAC6 deacetylase activity is critical for lipopolysaccharide-induced activation of macrophages

Bing Yan  1 , Songbo Xie  1 , Zhu Liu  2 , Jie Ran  1 , Yuanyuan Li  1 , Jian Wang  1 , Yang Yang  1 , Jun Zhou  1 , Dengwen Li  1 , Min Liu  2
Affiliations

HDAC6 deacetylase activity is critical for lipopolysaccharide-induced activation of macrophages

Bing Yan et al. PLoS One. .

Abstract

Activated macrophages play an important role in both innate and adaptive immune responses, and aberrant activation of macrophages often leads to inflammatory and immune disorders. However, the molecular mechanisms of how macrophages are activated are not fully understood. In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation. Our data show that suppression of HDAC6 activity significantly restrains LPS-induced activation of macrophages and production of pro-inflammatory cytokines. Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation. These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HDAC6 activity is important for LPS-induced macrophage activation.
(A) RAW264.7 cells were treated with tubacin (1 µM), NaB (500 µM) or TSA (5 µM) for 4 hours. Cell lysates were then immunoblotted with antibodies against acetylated α-tubulin, α-tubulin, acetylated histone H4, and histone H4. (B) RAW264.7 cells were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0, 2, 6, 12, and 24 hours. (C) Representative images of resting and activated macrophages. (D) Experiments were performed as in panel B, and the percentage of activated cells was analyzed. (E) Primary BMMs were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0 and 6 hours. (F) Representative images of resting and activated BMMs. (G) Experiments were performed as in panel E, and the percentage of activated BMMs was analyzed. (H) Quantitative RT-PCR analysis of relative HDAC6 mRNA level in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 36 hours. (I) Immunoblotting analysis of α-tubulin and acetylated α-tubulin in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours. (J) RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours were stimulated with LPS (300 ng/ml) for 0 and 24 hours. (K) Experiments were performed as in panel J, and the percentage of activated cells was analyzed. ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant (p≥0.05).
Figure 2
Figure 2. HDAC6 regulates LPS-induced M1 response.
(A) RAW 264.7 cells pretreated with tubacin were stimulated with LPS (300 ng/ml) for 24 hours. The culture medium was then collected, and the concentrations of IL-6, TNF-α, and IL-10 were measured. (B) Primary BMMs pretreated with tubacin were stimulated with LPS (300 ng/ml) for 24 hours. The culture medium was then collected, and the concentrations of IL-6, TNF-α, and IL-10 were measured. ***, p<0.001; **, p<0.01; ns, not significant (p≥0.05).
Figure 3
Figure 3. Effects of HDAC6 on actin polymerization and filopodium formation.
(A) RAW264.7 cells were treated with tubacin (1 µM) for 4 hours and then exposed to LPS (300 ng/ml) for 24 hours. F-actin (green), cortactin (red), and nuclei (blue) were then stained, and images were captured with a laser scanning confocal microscope. (B) Experiments were performed as in panel A, and the Pearson’s correlation coefficient between F-actin and cortactin fluorescence pixels were calculated with ImageJ to measure their co-localization. (C) RAW264.7 cells were treated with tubacin for 4 hours and then with LPS for 6 hours. F-actin (green) and nuclei (blue) were then stained. (D, E) Experiments were performed as in panel C, and the filopodium number (D) and length (E) in each group were calculated. (F) RAW264.7 cells were treated with tubacin for 4 hours and stimulated with LPS for the indicated time. F-actin and G-actin were separated by ultracentrifugation and analyzed by immunoblotting. ns, not significant (p≥0.05).
Figure 4
Figure 4. Suppression of HDAC6 activity compromises macrophage adhesion to the extracellular matrix, and this effect is abolished in LPS-stimulated macrophages.
(A) RAW264.7 cells labeled with calcein-AM were treated with tubacin and seeded in 96-well plates pre-coated with fibronectin or BSA. Cells were washed twice with PBS, and the fluorescence images of cells were captured by a fluorescence microscope before and after wash. (B) Experiments were performed as in panel A, and the fluorescent intensity was measured. The percentage of adherent cells were quantified by dividing the intensity of post-wash with that of pre-wash. (C, D) Experiments and quantifications were performed as in A and B, except that cells were treated with LPS following tubacin treatment. *, p<0.05; ns, not significant (p≥0.05).
Figure 5
Figure 5. Inhibition of HDAC6 enhances LPS-induced microtubule acetylation during macrophage activation.
(A) RAW264.7 cells were treated with tubacin for 4 hours and stimulated with LPS for the indicated time. Acetylated α-tubulin and total α-tubulin were then analyzed by immunoblotting. (B) Experiments were performed as in panel A, and the level of tubulin acetylation was quantified and normalized to the 0-hour LPS stimulation of the control group. (C) RAW264.7 cells were treated with tubacin for 4 hours and stimulated with LPS for the indicated time. F-actin (red), acetylated α-tubulin (green), and nuclei (blue) were then stained, and images were captured with a laser scanning confocal microscope.
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Grants and funding

This work was supported by grants (31171334 to DL and 31170820 to ML) from the National Natural Science Foundation of China (http://www.nsfc.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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