Effect And Mechanism Of HDAC11 On Inducing Tolerance

Objective Histone deacetylase 11 (HDAC11) is closely related to cell division cycle, nervous system development, tumorigenesis and immune regulation. It was recently demonstrated that HDAC11 could negatively regulate the expression of IL-10 in mouse macrophages RAW264.7 and induce immune tolerance. Therefore, the aim of this study was to investigate the effect and mechanism of inhibition of histone deacetylase (HDAC11) on expression of endogenous interleukin 10 (IL-10), the subsequent expression of antigen presenting associated molecules on the surface of Kupffer cells (KCs) and inducing tolerance in lipopolysaccharide-treated (LPS-treated) KCs.Methods BALB/c mice-derived KCs were isolated by density gradient centrifugation. At 24h after culturing in RPMI1640 medium with 20% fetal calf serum (FCS), the adherent cells were randomly divided into three groups: (1) blank control group (blank group, cultured in RPMI1640 medium without any treatment for additional 20 h), negative control group (negative group, transfected with control vector) and HDAC11-shRNA group (transfected with HDAC11-shRNA plasmid), and then treated with LPS. 3 hours later, the mRNA and protein expression of HDAC11 and IL-10 in KCs were determined by real-time PCR and western-blot, respectively. The expressions of MHC-II, B7-1, B7-2 and CD40 on the surface of KCs were evaluated by flow cytometry. T cells derived from mice transgenic for TCR that recognizes OVA323-339 peptide were cocultured with the 3 groups of KCs above, T cell proliferation was measured by [3H]-thymidine incorporation. The density of IL-2 in the supernatant of coculture was assessed by enzyme-linked immunosorbent assay (ELISA).Results (1) Real time-PCR: The mean±standard deviation ( X±S) of 2-ΔCt of HDAC11 and IL-10/GAPDH represented their relative mRNA expression levels. In contrast to the two control groups (blank group 0.145±0.012, negative group 0.141±0.013), HDAC11 mRNA level was the lowest in HDAC-shRNA group (0.076±0.007), which reached statistical significance (P<0.05); while the subsequent IL-10 mRNA level was the highest in the HDAC-shRNA group (0.084±0.006) compared to the two control groups (blank group 0.051±0.004, negative group 0.052±0.004), which reached statistical significance (P<0.05). (2) Western blot: X±S of gray-scale value of HDAC11 or IL-10/GAPDH represented their relative protein expression levels. In contrast to the two control groups (blank group 0.373±0.023, negative group 0.365±0.019), HDAC11 protein level was the lowest in HDAC-shRNA group (0.132±0.012), which reached statistical significance (P<0.05); while the subsequent IL-10 protein level was the highest in the HDAC-shRNA group (0.218±0.018) compared to the two control groups (blank group 0.096±0.009, negative group 0.101±0.011), which reached statistical significance (P<0.05). (3) Flow cytometric analysis: The expression of antigen presenting associated molecules on the surface of KCs such as MHC-II, B7-1, B7-1 and CD40 was the lowest in the HDAC-shRNA group compared to the two control groups. (4) T cell proliferation in coculture: Count per minute (CPM) was the lowest in the HDAC-shRNA group (2163±146) compared to the two control groups (blank group 3654±165, negative group 3509±142), which reached statistical significance (P<0.05). (5) ELISA: The density of IL-2 in the supernate of coculture was the lowest in the HDAC-shRNA group (463.7±38.9 pg/mL) compared to the two control groups (blank group 879.2±65.5 pg/mL, negative group 854.4±67.3), which reached statistical significance (P<0.05). All the differences between blank group and negative group above did not reach statistic significance (P>0.05).Conclusion Our data demonstrates that suppression of histone deacetylase 11 can promote expression of endogenous IL-10 in KCs, and then inhibit the ability of KCs to present antigen and T cell proliferation, finally induce tolerance after LPS treatment. Consequently, HDAC11 may play an important role in the immunologically privileged organ--the liver, and our experiment has provided a favourable in vitro element for the application of HDAC11 in the treatments of endotoxemia in end-stage liver disease and tolerance induction following liver transplantation both in animal experiments and clinical settings.