Romidepsin suppresses monosodium urate crystal-induced cytokine production through upregulation of suppressor of cytokine signaling 1 expression

Abstract

Background: Acute gouty arthritis currently is the most common form of inflammatory arthritis in developed countries. Treatment is still suboptimal. Dosage of urate-lowering therapy is often too low to reach target urate levels, and adherence to therapy is poor. In this study, we therefore explore a new treatment option to limit inflammation in acute gout: specific histone deacetylase (HDAC) inhibition.

Methods: Peripheral blood mononuclear cells (PBMCs) were cultured with a combination of monosodium urate crystals (MSU) and palmitic acid (C16.0) in order to activate the NLRP3 inflammasome and induce IL-1β production. HDAC inhibitors and other compounds were added beforehand with a 1-h pre-incubation period.

Results: The HDAC1/2 inhibitor romidepsin was most potent in lowering C16.0+MSU-induced IL-1β production compared to other specific class I HDAC inhibitors. At 10 nM, romidepsin decreased IL-1β, IL-1Ra, IL-6, and IL-8 production. IL-1β mRNA was significantly decreased at 25 nM. Although romidepsin increased PTEN expression, PBMCs from patients with germline mutations in PTEN still responded well to romidepsin. Romidepsin also increased SOCS1 expression and blocked STAT1 and STAT3 activation. Furthermore, experiments with bortezomib showed that blocking the proteasome reverses the cytokine suppression by romidepsin.

Conclusions: Our results show that romidepsin is a very potent inhibitor of C16.0+MSU-induced cytokines in vitro. Romidepsin upregulated transcription of SOCS1, which was shown to directly target inflammatory signaling molecules for proteasomal degradation. Inhibiting the proteasome therefore reversed the cytokine-suppressive effects of romidepsin. HDAC1/2 dual inhibition could therefore be a highly potent new treatment option for acute gout, although safety has to be determined in vivo.

Keywords: Cytokines; Gout; HDAC; Inflammation.

Fig. 1

Screening of different class I HDAC inhibitors for their suppressive effect on C16.0+MSU-induced IL-1β. Freshly isolated PBMCs from healthy volunteers were pre-incubated for 1 h with different HDAC inhibitors: a romidepsin (Romi, HDAC1/2 inhibitor), b entinostat (Entino, HDAC1/3 inhibitor), c Santacruzamate A (SCZM, HDAC2 inhibitor), d RGFP966 (RGFP, HDAC3 inhibitor), and e ITF3107 (ITF, HDAC6 inhibitor). IL-1β production was induced by adding a combination of 50 μM palmitic acid (C16.0) and 300 μg/mL monosodium urate crystals (MSU) for 24 h. Data are represented as percentage change compared to the IL-1β production with C16.0+MSU alone

Fig. 2

Suppressive effects of romidepsin on C16.0+MSU-induced cytokine production in human PBMCs. Freshly isolated PBMCs were pre-incubated with different concentrations of romidepsin for 1 h, after which cytokine production was induced via addition of a combination of 50 μM palmitic acid (C16.0) and 300 μg/mL monosodium urate crystals (MSU). The supernatant was collected for extracellular cytokines (a–c, f). Cells were lysed with 0.5% Triton X-100, and subsequently, the supernatant of the lysate was collected for measurement intracellular cytokines (d–e)

Fig. 3

Effects of romidepsin on cell viability and transcription of IL-1β and NLRP3 inflammasome components. Freshly isolated PBMCs from healthy donors were pre-incubated with different concentrations romidepsin for 1 h, after which a combination of 50 μM palmitic acid (C16.0) and 300 μg/mL monosodium urate crystals (MSU) was added to induce cytokine production. After 24 h of culture, mRNA was measured for IL1B (a), CASP1 (b), NLRP3 (d), and PYCARD (e). Percentages of Annexin V⁺ and PI⁺ were measured by flow cytometry to determine cell viability (c, f)

Fig. 4

Romidepsin-induced increased expression of PTEN and CPT1A is independent of cytokine suppression. PBMCs from healthy volunteers or Cowden syndrome patients were pre-incubated for 1 h with several concentrations of romidepsin (Romi) or etomoxir. Cytokine production was induced by adding a combination of 50 μM palmitic acid (C16.0) and 300 μg/mL monosodium urate crystals (MSU). After 24 h, PTEN (a) and CPT1A (b) mRNA expression was determined by qPCR. IL-1β production was measured by ELISA after addition of etomoxir (c) or romidepsin in healthy volunteers and Cowden syndrome patients (d)

Fig. 5

Romidepsin induced transcription of SOCS1 and inhibited activation of STAT1 and STAT3. Schematic representations of the SOCS1 and SOCS3 genome and their binding sites for class I HDACs were retrieved from the USCS Genome Browser by means of the track “Transcription Factor ChIP-seq (161 factors) from ENCODE with Factorbook Motifs” (a). PBMCs from healthy volunteers were pre-incubated with romidepsin, after which a combination of 50 μM palmitic acid (C16.0) and 300 μg/mL monosodium urate crystals (MSU) was added. After 24 h, mRNA was isolated and qPCR was performed for SOCS1 and SOCS3 (b, e). Cells were stained extracellularly with CD45-KO and intracellularly with either p-STAT1-PE (c, d) or p-STAT3-PE (f, g) for flow cytometry. Gating on CD45+ lymphocytes and monocytes was performed in the forward versus side scatter graph

Fig. 6

Proteasome inhibitor bortezomib reverses cytokine suppression by romidepsin. PBMCs were isolated from healthy volunteers and were pre-incubated for 1 h with bortezomib. Then romidepsin was added for 1 h pre-incubation, after which a combination of 50 μM palmitic acid (C16.0) and 300 μg/mL monosodium urate crystals (MSU) was added for another 24 h. The supernatant was collected for extracellular IL-1β (a). Cells were lysed with 0.5% Triton X-100, and subsequently, the supernatant of the lysate was collected for intracellular IL-1β measurement (b)