Inhibition of histone deacetylase 6 restores innate immune cells in the bone marrow in a lethal septic model.
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 (CD11bF4/80, 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 (CD11bGr-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.


CONCLUSIONS
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.
Ting
T
Zhao
Yongqing
Y
Li
Baoling
B
Liu
Baihong
B
Pan
Xin
X
Cheng
Patrick
P
Georgoff
Hasan B
HB
Alam
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects*; D004195:Disease Models, Animal; D005434:Flow Cytometry; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006877:Hydroxamic Acids / Q000494:pharmacology*; D007113:Immunity, Innate / Q000187:drug effects*; D007211:Indoles / Q000494:pharmacology*; D008026:Ligation; D008264:Macrophages / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000187:drug effects; D010538:Peritonitis / Q000276:immunology / Q000401:mortality; D011677:Punctures; D011897:Random Allocation; D018805:Sepsis / Q000276:immunology* / Q000401:mortality
D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D007211:Indoles; C553587:tubastatin A