Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 18;8(11):2927-2938.
doi: 10.7150/thno.25317. eCollection 2018.

Histone deacetylase 6 modulates macrophage infiltration during inflammation

Bing Yan  1 , Songbo Xie  2 , Yang Liu  1 , Wenxuan Liu  1 , Dengwen Li  1 , Min Liu  2 , Hongbo R Luo  3 , Jun Zhou  1   2
Affiliations

Histone deacetylase 6 modulates macrophage infiltration during inflammation

Bing Yan et al. Theranostics. .

Abstract

Mice with histone deacetylase 6 (HDAC6) deficiency grow and develop normally but exhibit impaired immune response. The molecular mechanisms for this phenotype remain largely elusive. Methods: A mouse acute peritonitis model was used to study the infiltration of neutrophils and monocyte-derived macrophages. In vitro cell motility assays were performed to analyze monocyte/macrophage recruitment. Fluorescence microscopy and flow cytometry were performed to examine the phagocytic ability of macrophages. Immunofluorescence microscopy was used to investigate protein localization, protrusion formation, and microtubule acetylation. Results: HDAC6 deficiency does not affect neutrophil infiltration, but instead attenuates the infiltration of monocyte-derived macrophages into the peritoneal cavity. HDAC6 plays a specific role in monocyte/macrophage recruitment. Loss of HDAC6 suppresses the phagocytic capacity of macrophages challenged with E. coli. Lipopolysaccharide stimulation results in the translocation of HDAC6 and cortactin from the cytosol to the cell periphery, promotes the formation of filopodial protrusions, and enhances microtubule acetylation around the microtubule-organizing center, all of which are abrogated by HDAC6 deficiency. Conclusion: These findings implicate HDAC6 in the innate immune response and suggest that it may serve as a promising target for the treatment of macrophage-associated immune diseases.

Keywords: HDAC6; acetylation; infiltration; inflammation; macrophage; migration.

PubMed Disclaimer

Conflict of interest statement

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

Figures

Figure 1
Figure 1
HDAC6 regulates the infiltration of monocytes/macrophages, but not neutrophils, during inflammation. (A-C) Flow cytometric analysis of neutrophil (CD11b+/Gr-1high) and monocyte (CD11b+/Gr-1intermediate) subpopulations in myeloid cells isolated from the bone marrow of wild-type (WT) and HDAC6 knockout (KO) mice (A). Quantification of neutrophils (B) and monocytes (C) in (A). (D-F) Flow cytometric analysis of recruited neutrophils (Ly6G+/Ly6B+) and monocytes/macrophages (Ly6G-/Ly6B+) present in the peritoneal cavities of mice with E. coli-induced acute peritonitis (D). Quantification of neutrophils (E) and monocytes/macrophages (F) in (D). (G-I) Flow cytometric analysis of recruited neutrophils (CD11b+/Gr-1high) and monocytes/macrophages (CD11b+/Gr-1low) present in the peritoneal cavities of mice with LPS-induced acute peritonitis (G). Quantification of neutrophils (H) and monocytes/macrophages (I) in panel G. *P < 0.05; ns, not significant.
Figure 2
Figure 2
HDAC6 is critical for macrophage migration. (A) Representative images of bone marrow-derived macrophages subjected to transwell assays. Scale bar, 50 μm. (B) Quantification of migrated cells from experiments performed as described in (A). (C) Immunoblot analysis of RAW264.7 macrophages treated with tubacin. (D-E) Representative images of RAW264.7 macrophages subjected to transwell assays (D) and quantification of migrated cells (E). Scale bar, 100 μm. (F-G) Representative images of transwell assays using RAW264.7 macrophages treated with tubacin and LPS (F) and quantification of migrated cells (G). Scale bar, 100 μm. (H-J) Representative images of transwell assays using RAW264.7 macrophages treated with tubacin in the presence of M-CSF or serum (H). Quantification of migrated cells induced with M-CSF (I) or serum (J). Scale bar, 100 μm. **P < 0.01; ***P < 0.001.
Figure 3
Figure 3
HDAC6 does not affect neutrophil migration. (A) Neutrophils from wild-type and HDAC6 knockout mice were labeled with CFSE and Dil, respectively, mixed and injected into acute peritonitis mouse models. Cells infiltrating into the peritoneal cavity were analyzed by flow cytometry. (B) Experiments were performed as described in (A), and the relative migration efficiency was determined. (C) Transwell assays comparing migration of neutrophils from wild-type and HDAC6 knockout mice. Scale bar, 100 μm. (D) Experiments were performed as described in (C), and migrated cells were quantified. (E-G) Neutrophils from wild-type and HDAC6 knockout mice were induced with fMLP, and movement tracks were recorded using EZ-TAXIScan (E). Migration velocity (F) and directionality (G) were quantified. (H-J) Neutrophils from wild-type mice were treated with tubacin, and fMLP-induced movement tracks (H), velocity (I), and directionality (J) were analyzed. (K-M) Neutrophils from wild-type and HDAC6 knockout mice were induced with LBT4 and the movement tracks (K), velocity (L), and directionality (M) were analyzed. *P < 0.05; ***P < 0.001; ns, not significant.
Figure 4
Figure 4
Loss of HDAC6 impairs the phagocytic ability of macrophages. (A) Fluorescence staining showing phagocytosis of E. coli by macrophages from wild-type or HDAC6 knockout mice. Scale bar, 20 μm. (B) Experiments were performed as described in (A), and the numbers of E. coli within macrophages were quantified. (C-E) Flow cytometric analysis of GFP-labeled E. coli phagocytosed by macrophages isolated from wild-type or HDAC6 knockout mice (C). GFP-positive cells (D) and fluorescence intensity (E) were quantified. *P < 0.05.
Figure 5
Figure 5
Effects of HDAC6 on LPS-induced cortactin localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.
Figure 6
Figure 6
Effects of HDAC6 on LPS-induced microtubule acetylation. (A) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against α-tubulin and acetylated α-tubulin. Scale bar, 40 μm. (B-C) Experiments were performed as in (A), and the fluorescent intensity ratios of acetylated α-tubulin to α-tubulin in wild-type derived macrophages (B) and HDAC6 knockout-derived macrophages (C) were determined. **P < 0.01; ns, not significant.
Figure 7
Figure 7
Proposed model for HDAC6-mediated macrophage infiltration and phagocytosis. In wild-type unstimulated macrophages, HDAC6 localizes to the cytosol. In response to inflammatory cues, HDAC6 partially translocates to the cell periphery, where it deacetylates α-tubulin and cortactin, thereby enhancing dynamics of microtubule tips and promoting actin assembly. Changes in cytoskeletal dynamics promote macrophage infiltration and phagocytosis. In the absence of HDAC6, cytoskeletal dynamics are not affected, rendering macrophages insensitive to inflammatory cues.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Swirski FK, Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science. 2013;339:161–6. - PMC - PubMed
    1. Montecucco F, Liberale L, Bonaventura A, Vecchie A, Dallegri F, Carbone F. The role of inflammation in cardiovascular outcome. Curr Atheroscler Rep. 2017;19:11. - PubMed
    1. Foley JF. Focus issue: understanding mechanisms of inflammation. Sci Signal. 2013;6:eg2. - PubMed
    1. Tabas I, Glass CK. Anti-inflammatory therapy in chronic disease: challenges and opportunities. Science. 2013;339:166–72. - PMC - PubMed
    1. Chrobok NL, Sestito C, Wilhelmus MM, Drukarch B, van Dam AM. Is monocyte- and macrophage-derived tissue transglutaminase involved in inflammatory processes? Amino Acids. 2017;49:441–52. - PMC - PubMed

Publication types

-