Selective Inhibition of SIRT2 Improves Outcomes in a Lethal Septic Model

Background: Seven isoforms of histone deacetylase Class III have been reported - Sirtuin (SIRT) 1-7. We recently demonstrated that EX-527, an inhibitor of SIRT1, reduces mortality in a mouse model of lethal-cecal-ligation-and-puncture (CLP)-induced septic shock. Our present study was aimed at determining whether selective inhibition of SIRT2, with AGK2, would decrease animal death and attenuate the inflammatory response in a septic model. Methods: Experiment I: C57BL/6J mice were intraperitoneally given either AGK2 (82 mg/kg) in dimethyl sulfoxide (DMSO) or DMSO alone, and 2 h later subjected to CLP. Survival was monitored for 240 hours. Experiment II: mice treated the same way as Experiment I, were grouped into (i) DMSO vehicle, and (ii) AGK2, with sham mice (operating but without any treatment) serving as controls. Peritoneal fluid and peripheral blood were examined at 24 and 48 hours for cytokine production. Samples of blood at 48 h were also allocated to assess coagulability using Thrombelastography (TEG). Morphological changes of bone marrow were evaluated from long bones (femurs and tibias) with hematoxylin and eosin (H&E) staining. Bone marrow atrophy was quantified by a blinded pathologist. Experiment III: cytokines in supernatant of the cultured normal primary splenocytes were measured after the cells were stimulated by lipopolysaccharide and treated with or without AGK2 (10 mu M) for 6 hours. Results: AGK2 significantly reduced mortality and decreased levels of cytokines in blood (TNF-alpha: 298.3 +/- 24.6 vs 26.8 +/- 2.8 pg/ml, p=0.0034; IL-6: 633.4 +/- 82.8 vs 232.6 +/- 133.0 pg/ml, p=0.0344) and peritoneal fluid (IL-6: 704.8 +/- 67.7 vs 391.4 +/- 98.5 pg/ml, p=0.033) compared to vehicle control. Also, AGK2 suppressed the TNF-alpha and IL-6 production in the cultured splenocytes (TNF-alpha: 68.1 +/- 6.4 vs 23.9 +/- 2.8 pg/ml, p=0.0009; IL-6: 73.1 +/- 4.2 vs 49.6 +/- 3.0 pg/ml; p=0.0051). The TEG data showed that the mice subjected to CLP displayed prolonged fibrin formation and fibrin cross-linkage time, slower clot formation, decreased platelet function, and clot rigidity. AGK2 treatment was associated with dramatic improvements in fibrin cross-linkage and clot formation times, without a significant impact on the clot initiation parameters or platelet function. Additionally, AGK2 significantly attenuated the bone marrow atrophy (58.3 +/- 6.5 vs 30.0 +/- 8.2%, p=0.0262). Conclusion: Selective inhibition of SIRT2 significantly improves survival, and attenuates sepsis-associated cytokine storm, coagulopathy, and bone marrow atrophy in a mouse model of lethal septic shock.