T. Zhao1, B. Liu1, B. Pan1, X. Cheng1, E. Chen1, Y. Li1, H. B. Alam1 1University Of Michigan Hospital,General Surgery/ Surgery/Medical School,Ann Arbor, MI, USA
Introduction: We have previously demonstrated that Tubastatin A (Tub A), a selective inhibitor of histone deacetylase 6 (HDAC6), improved survival, and increased phagocytosis of splenocytes in a mouse model of lethal cecal ligation and puncture (CLP) induced septic shock. The present study was designed to test the hypothesis that inhibition of HDAC6 with Tub A may affect the spleen cell population.
Methods: C57BL/6J mice were subjected to CLP, and 1 h later given an intraperitoneal injection of either Tub A (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO), or DMSO only (n=7/group). Sham-operated animals were treated in an identical fashion, without CLP. Forty-eight hours later, spleen was harvested and homogenized into single cell suspension. Erythrocytes were lysed, and single-cell suspension was made for analysis. The splenocytes were washed, blocked with anti-mouse CD16/32, stained with anti-mouse 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 was acquired on an LSRII Flow Cytometer (BD Biosciences) and analyzed with FlowJo (FlowJo, LLC). Analysis of variance (ANOVA) with the Bonferroni post-hoc test was used for multiple comparisons among 3 groups.
Results: In spleen at 48 h after CLP, it was found that there were no statistical differences in percentages of neutrophils (CD11b+ Gr-1+) and T lymphocytes (CD3+) among three groups. The percentages of innate immune cells and macrophages were not statistically different between sham and vehicle-treated CLP groups. However, there were statistical differences in percentages of innate immune cells (CD11b+; p=0.0444), macrophages (CD11b+ F4/80+; p=0.0019), and B lymphocytes (B220+; p=0.0125) among three groups. Treatment with Tub A significantly increased percentages of innate immune cells (10.8±0.4 vs.6.2±1.3%, p<0.05) and macrophages (7.0±1.1 vs. 2.9±0.3%, p<0.01; Figure 1) as compared to vehicle-treated CLP group. Animals from the vehicle-treated CLP group showed decreased percentage of B lymphocytes compared to sham group (59.1±2.9 vs. 44.3±2.6%, p<0.05).
Conclusion: Selective inhibition of HDAC6 significantly increased macrophage and innate immune cell proportions in spleen in the lethal septic model. These results may explain the beneficial effects of Tub A treatment in a septic model as previously reported.
