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Inhibition of histone deacetylase 6 restores innate immune cells in the bone marrow in a lethal septic model

Zhao, Ting MD; Li, Yongqing MD, PhD; Liu, Baoling MD; Pan, Baihong MD; Cheng, Xin; Georgoff, Patrick MD; Alam, Hasan B. MD

Journal of Trauma and Acute Care Surgery: January 2016 - Volume 80 - Issue 1 - p 34–41
doi: 10.1097/TA.0000000000000897
AAST Plenary Papers
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BACKGROUND We have previously demonstrated that Tubastatin A, a selective inhibitor of histone deacetylase 6 (HDAC6), improves survival and increases circulating monocyte count and bacterial clearance in a lethal model of cecal ligation and puncture (CLP) in mice. The aim of the present study was to characterize the effects of inhibition of HDAC6 on the bone marrow cell population.

METHODS C57BL/6J mice were subjected to CLP and, 1 hour later, given an intraperitoneal injection of either Tubastatin A (70 mg/kg) dissolved in DMSO or DMSO alone (n = 9 per group). Sham-operated animals were treated in an identical fashion, without CLP. Forty-eight hours later, bone marrow cells were flushed out from the femurs and tibias. Erythrocytes were lysed, and a single-cell suspension was made for analysis. Cells were washed; blocked with antimouse CD16/32; stained with antimouse B220 PE-Cy7, CD3 APC-eFluor 780, CD11b FITC, Gr-1 PerCP-Cy5.5, and F4/80 Antigen APC; and subjected to flow cytometry. Data were acquired on an LSRII Flow Cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Flowjo, LLC, Ashland, OR).

RESULTS In comparison with the sham group, CLP animals showed decreased percentage of innate immune cells (CD11b+, 62.1% ± 3.1% vs. 32.9% ± 4.9%, p = 0.0025) and macrophages (CD11b+F4/80+, 44.6% ± 3.4% vs. 19.8% ± 2.6%, p = 0.0002) as well as increased percentage of T lymphocytes (CD3+, 1.1% ± 0.2% vs. 3.3% ± 0.4%, p = 0.0082) in the bone marrow 48 hours after CLP. Treatment with Tubastatin A restored the innate immune cells (32.9% ± 4.9% vs. 54.0% ± 4.1%, p = 0.0112) and macrophages (19.8% ± 2.6% vs. 47.1% ± 4.6%, p = 0.0001) and increased the percentage of neutrophils (CD11b+Gr-1+, 28.4% ± 3.9% vs. 48.0% ± 4.0%, p = 0.0075). The percentages of B (B220+) and T lymphocytes were not significantly altered by Tubastatin A, compared with the vehicle-treated CLP animals.

CONCLUSION Selective inhibition of HDAC6 in this lethal septic model restored the innate immune cell and macrophage populations and increased the neutrophil composition in the bone marrow. These results may explain the previously reported beneficial effects of Tubastatin A treatment in a septic model.

From the Department of Surgery, University of Michigan Hospital, Ann Arbor, Michigan.

Submitted: July 27, 2015, Revised: September 30, 2015, Accepted: October 5, 2015, Published online: October 21, 2015.

T.Z. and Y.L. contributed equally to this study.

Data were presented at 74th annual meeting of the American Association for the Surgery of Trauma (AAST) and Clinical Congress of Acute Care Surgery, September 9–12, 2015, in Las Vegas, Nevada.

Address for reprints: Hasan B. Alam, MD, General Surgery, 2920 Taubman Center/5331 University of Michigan Hospital, 1500 E Medical Center Dr, Ann Arbor, MI 48109–5331; email: alamh@med.umich.edu.

© 2016 Lippincott Williams & Wilkins, Inc.