The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo - PubMed (original) (raw)

. 2005 Jan-Dec;11(1-12):1-15.

doi: 10.2119/2006-00005.Dinarello.

Gianluca Fossati, Eli C Lewis, Jae-Kwon Lee, Giulia Porro, Paolo Pagani, Daniela Modena, Maria Lusia Moras, Pietro Pozzi, Leonid L Reznikov, Britta Siegmund, Giamila Fantuzzi, Charles A Dinarello, Paolo Mascagni

Affiliations

The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo

Flavio Leoni et al. Mol Med. 2005 Jan-Dec.

Abstract

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.

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Figures

Figure 1

Figure 1

Time course of protein hyperacetylation in LPS-stimulated PBMCs. Resting human PBMCs from a healthy donor were incubated with LPS only (10 ng/mL) or LPS in the presence of ITF2357 (100 nM) or SAHA (100 nM). At the indicated times, the cells were lysed and total protein extracts analyzed by Western blotting using an antibody to acetylated lysines. The results are representative of 1 of 3 donors with similar observations.

Figure 2

Figure 2

Effect of ITF2357 on the secretion of TNFα in LPS-stimulated PBMCs. After an overnight rest, PBMCs were preincubated with increasing concentrations of ITF2357 for 1 h at 37°C, followed by LPS (10 ng/mL) for an additional 20 h. TNFα released into the supernatant was measured, and the percent reduction from LPS only (set at 100%) is indicated on the vertical axis. The data are presented as mean percent change (± SEM) of 6 donors. The dashed line indicates inhibition of 50% of TNFα induced by LPS only. *P < 0.05; **P < 0.01 from LPS only.

Figure 3

Figure 3

Effect of ITF2357 on intracellular IL-1α and secreted IL-1β from LPS-stimulated PBMCs. Freshly obtained PBMCs were preincubated for 1 h with increasing nanomolar concentrations of ITF2357 and then stimulated with LPS (10 ng/mL) for 24 hours. (A) Levels of intracellular IL-1α are shown as the percent change from LPS only (set at 100%) as indicated on the vertical axis. (B) Levels of secreted IL-1β are shown as the percent change from LPS only. The data are the mean percent change ± SEM of 6 donors. The dashed line indicates inhibition of 50%. (C) FACS analysis of percent of total cell PI staining expressed at sub-G1 cell cycle. Mean ± SEM of 4 donors are shown. Cells in sub-G1 cycle are from the same cultures shown in B. *P < 0.05; **P < 0.01 from LPS only.

Figure 4

Figure 4

Caspase 3/7 activation and nucleosome formation. Purified monocytes were incubated with and without ITF2357 (as shown under the horizontal axis). After 1 h, LPS (10 ng/mL) was added to some wells. After 24 h, caspase activation and nucleosome formation were assayed. The data are expressed as fold increase (mean ± SEM, n = 3 donors) over monocytes without ITF2357.

Figure 5

Figure 5

Effect ITF2357 on apoptosis and necrosis in PBMCs. (A) PBMCs in RPMI were incubated with increasing concentrations of ITF2357 for 1 h followed by the addition of LPS (10 ng/mL) to appropriate wells. After 24 h at 37° C, the percentage of annexin-V– and PI-positive PBMCs was determined. The data are the mean ± SEM of 5 separate experiments/donors. (B) FACS analysis of PI and annexin V staining in control PBMCs. (C) PI and annexin V staining in PBMCs incubated with 100 nM ITF2357. (D) PI and annexin V staining in PBMCs incubated with 1000 nM ITF2357. The data in B, C, and D are from 1 representative experiment.

Figure 6

Figure 6

Effect of ITF2357 on apoptosis and necrosis in monocytes. Purified monocytes in RPMI were incubated with increasing concentrations of ITF2357 for 1 h followed by the addition of LPS (10 ng/mL) to appropriate wells. After 24 h at 37° C, the percentage of annexin-V– and PI-positive cells was determined. (A) The data are the mean ± SEM of 3 separate experiments/donors. (B) FACS analysis of PI and annexin V staining in control monocytes. (C) PI and annexin V staining in monocytes incubated with 100 nM ITF2357. (D) PI and annexin V staining in monocytes incubated with 1000 nM ITF2357. The data in B, C, and D are from 1 representative experiment.

Figure 7

Figure 7

Effect of ITF2357 on LPS-induced IFNγ production. Freshly obtained PBMCs were preincubated for 1 h with increasing concentrations of ITF2357 (horizontal axis) and stimulated with LPS (10 ng/mL) for 24 h. Levels of secreted IFNγ are shown in ng/mL on the vertical axis. The data are presented as the mean percent change ± SEM of 4 donors and the dashed line indicates inhibition of 50%. *P < 0.05; **P < 0.01 from LPS only.

Figure 8

Figure 8

Suppression of IL-18/IL-12–induced IFNγ by ITF2357. Freshly cultured PBMCs were pretreated with increasing concentrations of ITF2357 (shown under horizontal axis) for 1 h as described in Figure 7. The cells were stimulated with the combination of IL-18 (10 ng/mL) plus IL-12 (1 ng/mL) (left panel). Matching cultures were similarly stimulated in the presence of IL-1Ra (10 μg/mL, middle panel) or TNF-bp (1 μg/mL, right panel). IFNγ was measured in the supernatants after 24 hours. Data are mean ± SEM of IFNγ in ng/mL of 4 donors. *P < 0.05; **P < 0.01 compared with no ITF2357.

Figure 9

Figure 9

Stimulus-dependent suppression of IFNγ by ITF2357. (A) LPS-induced IFNγ from resting PBMCs stimulated with LPS (10 ng/mL). Resting PBMCs were preincubated with ITF2357 as indicated for 1 h at 37° C before the addition of LPS. Mean ± SEM of IFNγ in pg/mL of 4 donors. (B) After preincubation with ITF2357, the cells from the same donors were stimulated with anti-CD3 (500 ng/mL). After 24 h, IFNγ was measured in the supernatants. Mean ± SEM of IFNγ in pg/mL of 3 donors are shown.

Figure 10

Figure 10

Effect of ITF2357 on IL-6 production. Freshly cultured PBMCs were preincubated with ITF2357 for 1 h and then stimulated with the combination of IL-18 (10 ng/mL) and IL-12 (1 ng/mL) as in Figure 8. Open bars indicate PBMCs at a concentration of 2.5 million cells/mL, and solid bars represent PBMCs at 5 million cells/mL. Mean ± SEM of IL-6 measured in the supernatant of triplicate wells after 24 h are shown for 2 donors. *P < 0.05; **P < 0.01 compared with no ITF2357.

Figure 11

Figure 11

Inhibition of IL-6 by ITF2357 is independent of IL-1β and TNFα. Left panel depicts the dose-response inhibition of IL-18/IL-12–induced IL-6 by increasing concentrations of ITF2357. Freshly isolated PBMCs were preincubated with ITF2357 for 1 h. The combination of IL-18/IL-12 was added for an additional 20 h, and IL-6 was measured in the supernatants. Matching cultures were similarly stimulated in the presence of IL-1Ra (10 μg/mL, middle panel) or TNF-bp (1 μg/mL, right panel). Data are mean ± SEM of IL-6 in ng/mL of 4 donors. *P < 0.05; *P < 0.01 compared with no ITF2357.

Figure 12

Figure 12

Steady-state mRNA levels in PBMCs. Northern blot analysis of specific cytokine mRNA extracted from resting PBMCs at times indicated. Lane 1, unstimulated PBMCs; lane 2, ITF2357 only (50 nM); lane 3, LPS (10 ng/mL); lane 4, LPS in the presence of ITF2357 (50 nM). In addition, LPS-induced TNFα, IL-1β, IFNγ, and IL-8 were measured in the supernatant by specific ELISA, and the percent inhibition of LPS-induced cytokines by ITF2357 is indicated under the respective bands and time points are indicated. ND = not determined. Shown are the results of a single donor. Similar results were obtained in a second donor.

Figure 13

Figure 13

Real-time PCR of steady-state TNFα mRNA. Real-time PCR was performed on RNA extracted from resting PBMCs 4 h after stimulation with LPS (10 ng/mL) in the presence of 50 nM ITF2357. The data were calculated as mRNA in picograms per nanogram of 18S RNA. A value of 1.0 was assigned for the unstimulated control of each donor (indicated as A, B, and C), and the fold change was calculated for each donor.

Figure 14

Figure 14

Effect of oral ITF2357 on LPS-induced circulating cytokines. (A) Mice received ITF2357 by gavage 1 h before intraperitoneal LPS (30 mg/kg). Dose-response of oral ITF2357 on serum TNFα (mean ± SEM) 90 min after LPS is shown. Control mice received an oral dose of water followed by LPS; 6 mice in each group. (B) Using another group of mice, an identical study was performed but blood was obtained 6 h after LPS. Mean ± SEM IFNγ is shown for 6 mice per group. (C) Similar to A and B, mice were pretreated with either dexamethasone (DEX at 10 mg/kg) or increasing doses of oral ITF2357 followed by an intravenous injection of anti-mouse CD3 (10 μg/kg). Serum was obtained 90 min after the anti-CD3 antibody and mean ± SEM TNFα is shown (5 mice per group). *P < 0.05; **P < 0.01 compared with water vehicle.

Figure 15

Figure 15

Effect of ITF2357 on ConA-induced hepatitis in BALB/C mice. BALB/C mice (10 per group) received either a single oral dose (100 μL) of 5 mg/kg of ITF2357 or an equivalent volume of water (Controls). After 30 min, both groups received an intravenous injection of ConA (200 μg per mouse). Mean ± SEM serum ALT are shown after 24 h. **P < 0.01 versus ConA with water vehicle.

Figure 16

Figure 16

Effect of ITF2357 on ConA-induced hepatitis in C57Bl/6 mice. C57BL/6 mice (10 per group) received either a single oral dose of 1 or 10 mg/kg ITF2357 (100 μL), whereas control mice received water (Controls). After 30 min, all mice received an intravenous injection of ConA (200 μg per mouse). Mean ± SEM serum ALT are shown after 24 hours. *P < 0.05 versus ConA with water vehicle.

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