Inhibition of HDAC Enzymes Contributes to Differential Expression of Pro-Inflammatory Proteins in the TLR-4 Signaling Cascade

Abstract

Class I and II histone deacetylases (HDAC) are considered important regulators of immunity and inflammation. Modulation of HDAC expression and activity is associated with altered inflammatory responses but reports are controversial and the specific impact of single HDACs is not clear. We examined class I and II HDACs in TLR-4 signaling pathways in murine macrophages with a focus on IκB kinase epsilon (IKKε) which has not been investigated in this context before. Therefore, we applied the pan-HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) as well as HDAC-specific siRNA. Administration of HDACi reduced HDAC activity and decreased expression of IKKε although its acetylation was increased. Other pro-inflammatory genes (IL-1β, iNOS, TNFα) also decreased while COX-2 expression increased. HDAC 2, 3 and 4, respectively, might be involved in IKKε and iNOS downregulation with potential participation of NF-κB transcription factor inhibition. Suppression of HDAC 1-3, activation of NF-κB and RNA stabilization mechanisms might contribute to increased COX-2 expression. In conclusion, our results indicate that TSA and SAHA exert a number of histone- and HDAC-independent functions. Furthermore, the data show that different HDAC enzymes fulfill different functions in macrophages and might lead to both pro- and anti-inflammatory effects which have to be considered in therapeutic approaches.

Keywords: HDAC; acetylation; inflammation; macrophages.

Figure 1

Effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) on cell proliferation and apoptosis induction. (A): Cytotoxicity and cell proliferation were assessed by the sulforhodamine B (SRB) assay after 6 and 24 h incubation time, respectively, at different concentrations of TSA and SAHA (6 h: n = 4; 24 h: n = 3). The cell number of vehicle-treated cells was set as “1” indicated by the dotted line. (B): Flow cytometric analysis of cell cycle distribution and apoptosis induction 4 and 24 h after treatment with TSA and SAHA (n = 4). (C): TUNEL staining 24 h after TSA and SAHA administration. A preparation without enzyme served as negative control, cells treated with DNase as positive control. The picture shows representative stainings from three independent incubations. Scale Bar: 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001; statistically significant difference as compared to vehicle-treated control.

Figure 2

Histone-deacetylase (HDAC) activity after TSA and SAHA treatment. (A): Western blot analysis of nuclear histone H3 acetylation after 4 h incubation with TSA and SAHA at the indicated concentrations. The pictures show representative blots. (B): Densitometric analysis of all blots (n = 4–5/group). For better comparison, the acetylation levels were normalized to vehicle-treated control cells which have been set as “1” indicated by the dotted line. * p < 0.05, ** p < 0.01 statistically significant difference as compared to vehicle-treated control. AcH3: Acetyl histone H3; PCNA: proliferating-cell-nuclear-antigen.

Figure 3

Acetylation of the IκB kinase epsilon (IKKε) promoter. (A): RAW264.7 cells were incubated for 16 h with TSA and SAHA. ChIP analysis of nuclear extracts was carried out with antibodies against acetylated histone H3 and histone H3 followed by qRT-PCR analysis using primers against the IKKε promoter sequence. (n = 6), (B): IKKε mRNA expression after treatment with TSA and SAHA (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001 statistically significant difference as compared to vehicle control.

Figure 4

Effects of HDACi on lipopolysaccharide (LPS)-induced inflammatory gene expression. (A): mRNA expression analysis of inflammatory genes 4 h after LPS incubation with and without addition of TSA and SAHA at the indicated concentrations. (B): Western blot analysis of inflammatory genes after treatment of cells with LPS with and without SAHA or TSA for 6 h, respectively. The blots show one representative result, the diagrams the densitometric analysis of all blots (n = 5–6), * p < 0.05, ** p < 0.01, *** p < 0.001, statistically significant difference as compared to LPS-treated control.

Figure 5

Effects of HDAC inhibition on LPS-induced NF-κB transcription factor activity. (A): p65 transcription factor analysis as assessed by transcription factor ELISA (TransAM, left panel), nuclear p65 translocation (middle panel) and cytosolic I-κBa degradation (right panel) by western blot analysis (n = 6). The diagrams show the densitometric analysis of all blots. (B): Immunofluorescence staining of RAW264.7 cells after vehicle treatment or incubation with LPS for 30 min with and without addition of TSA and SAHA. The pictures show representative results from three independent incubations. Scale Bar: 20 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, statistically significant difference as compared to LPS-treated control.

Figure 6

Analysis of LPS-induced inflammatory genes and NF-κB transcription factor activity after knock-down of HDACs by siRNA. (A): mRNA expression analysis of inflammatory genes after LPS incubation of RAW264.7 cells with and without knock-down of different HDAC enzymes (n = 5–6), (B): Western blot analysis of inflammatory genes after treatment of control and HDAC knock-down cells with LPS. The blots show one representative result, the diagrams the densitometric analysis of all blots (n = 5–6). (C): Densitometric analysis of western blot analyses for p65 nuclear translocation and cytosolic I-κBa degradation after treatment of RAW264.7 cells with HDAC siRNAs and LPS. Actin served as loading control for cytosolic extracts, PCNA as control for nuclear extracts, (n = 4), * p < 0.05, ** p < 0.01, *** p < 0.001, statistically significant difference as compared to LPS-treated control.

Figure 6

Analysis of LPS-induced inflammatory genes and NF-κB transcription factor activity after knock-down of HDACs by siRNA. (A): mRNA expression analysis of inflammatory genes after LPS incubation of RAW264.7 cells with and without knock-down of different HDAC enzymes (n = 5–6), (B): Western blot analysis of inflammatory genes after treatment of control and HDAC knock-down cells with LPS. The blots show one representative result, the diagrams the densitometric analysis of all blots (n = 5–6). (C): Densitometric analysis of western blot analyses for p65 nuclear translocation and cytosolic I-κBa degradation after treatment of RAW264.7 cells with HDAC siRNAs and LPS. Actin served as loading control for cytosolic extracts, PCNA as control for nuclear extracts, (n = 4), * p < 0.05, ** p < 0.01, *** p < 0.001, statistically significant difference as compared to LPS-treated control.

Figure 7

Effects of TSA and SAHA on mRNA stability of LPS-induced inflammatory genes. mRNA stability was assessed by blocking transcription with actinomycin (AmD) (1 µg/mL). RAW264.7 cells were incubated with LPS for 24 h before TSA and SAHA were added together with actinomycin for 2 and 4 h. The time point of actinomycin addition was set as baseline (“1”). (n = 4). * p < 0.05, ** p < 0.01, statistically significant difference as compared to LPS/actinomycin-treated control.