The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet β cells in vivo and in vitro

In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25-2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC(50) of 25-50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.     

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

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.     

Inhibition of histone deacetylases in inflammatory bowel diseases

This review, comprised of our own data and that of others, provides a summary overview of histone deacetylase (HDAC) inhibition on intestinal inflammation as well as inflammation-mediated carcinogenesis. Experimental colitis in mice represents an excellent in vivo model to define the specific cell populations and target tissues modulated by inhibitors of HDAC. Oral administration of either suberyolanilide hydroxamic acid (SAHA) or ITF2357 results in an amelioration in these models, as indicated by a significantly reduced colitis disease score and histological score. This effect was paralleled by suppression of proinflammatory cytokines at the site of inflammation as well as specific changes in the composition of cells within the lamina propria. In addition, tumor number and size was significantly reduced in two models of inflammation-driven tumorigenesis, namely interleukin (IL)-10-deficient mice and the azoxymethane-dextran sulfate sodium (DSS) model, respectively. The mechanisms affected by HDAC inhibition, contributing to this antiinflammatory and antiproliferative potency will be discussed in detail. Furthermore, with regard to the relevance in human inflammatory bowel disease, the doses of ITF2357 considered safe in humans and the corresponding serum concentrations are consistent with the efficacious dosing used in our in vivo as well as in vitro experiments. Thus, the data strongly suggest that HDAC inhibitors could serve as a therapeutic option in inflammatory bowel disease.     

Specific Inhibition of Histone Deacetylase 8 Reduces Gene Expression and Production of Proinflammatory Cytokines in Vitro and in Vivo

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25–100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC₅₀ of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100–1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.     

Histone deacetylase inhibitors as therapeutic agents for acute central nervous system injuries

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of potentially therapeutic agents for treating acute injuries of the central nervous system (CNS). In this review, we summarize data regarding the effects of HDAC inhibitor administration in models of acute CNS injury and discuss issues warranting clinical trials. We have previously shown that the pan-HDAC inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h after injury. Using a well-characterized, clinically relevant mouse model of closed head injury, we demonstrated that a single dose of ITF2357 administered 24 h after injury improves neurobehavioral recovery and reduces tissue damage. ITF2357-induced functional improvement was found to be sustained up to 14 d after trauma and was associated with augmented histone acetylation. Single postinjury administration of ITF2357 also attenuated injury-induced inflammatory responses, as indicated by reduced glial accumulation and activation as well as enhanced caspase-3 expression within microglia/macrophages after treatment. Because no specific therapeutic intervention is currently available for treating brain trauma patients, the ability to affect functional outcome by postinjury administration of HDAC inhibitors within a clinically feasible timeframe may be of great importance. Furthermore, a growing body of evidence indicates that HDAC inhibitors are beneficial for treating various forms of acute CNS injury including ischemic and hemorrhagic stroke. Because HDAC inhibitors are currently approved for other use, they represent a promising new avenue of treatment, and their use in the setting of CNS injury warrants clinical evaluation.     

Sulfuretin, a major flavonoid isolated from Rhus verniciflua, ameliorates experimental arthritis in mice

AimSulfuretin, a major flavonoid isolated from Rhus verniciflua, is known to have anti-inflammatory effects. However, the mechanisms underlying the anti-inflammatory effect of sulfuretin on rheumatoid arthritis have not been elucidated. In this study we investigated whether sulfuretin treatment modulates the severity of arthritis in an experimental model.Main methodsWe evaluated the effects of sulfuretin on tumor necrosis factor-α (TNF-α)-treated human rheumatoid fibroblast-like synoviocytes (FLS) in vitro and on collagen-induced arthritis (CIA) mice in vivo.Key findingsIn vitro experiments demonstrated that sulfuretin suppressed the chemokine production, matrix metalloproteinase secretion, and cell proliferation induced by tumor necrosis factor-α in rheumatoid FLS. In addition, sulfuretin inhibited the osteoclast differentiation induced by macrophage colony-stimulating factor and receptor activator of NF-κB ligand in bone marrow macrophages. In mice with CIA, early intervention with sulfuretin prevented joint destruction, as evidenced by a lower cumulative disease incidence and an absence of diverse disease features based on hind paw thickness, radiologic and histopathologic findings, and inflammatory cytokine levels. In mice with established arthritis, sulfuretin treatment significantly reduced synovial inflammation and joint destruction. The in vitro and in vivo protective effects of sulfuretin were mediated by inhibition of the NF-κB signaling pathway.SignificanceThese results suggest that using sulfuretin to block the NF-κB pathway in rheumatoid joints reduces both inflammatory responses and joint destruction. Therefore, sulfuretin may have therapeutic value in preventing or delaying the progression of rheumatoid arthritis.     

Effects of the histone deacetylase inhibitor ITF2357 in autoinflammatory syndromes

We explored the effects of the oral histone deacetylase (HDAC) inhibitor ITF2357 in patients with autoinflammatory syndrome. In this prospective open-label pilot study, eight patients were enrolled; one patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS), three patients with hyper-IgD and periodic fever syndrome (HIDS) and four patients with Schnitzler syndrome were closely followed during 90 d of ITF2357 treatment. Three patients with Schnitzler syndrome and one TRAPS patient experienced a partial remission. In four patients, there was no effect. In HIDS patients, there was a tendency toward a higher attack frequency and increasing attack severity. In two patients (one TRAPS and one HIDS), we observed a decrease of acute-phase response without signs of clinical improvement. One patient with Schnitzler syndrome showed a partial response despite an ongoing acute-phase response. In conclusion, ITF2357 monotherapy was able to induce partial response only in patients with Schnitzler syndrome and no response in patients with HIDS.     

Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease

NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with N:(G)-monomethyl-L-arginine (L-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N:-iminoethyl-L-lysine (L-NIL), would have more directed anti-arthritic properties. Whereas both L-NMMA and L-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with L-NMMA ablated synovitis, surprisingly L-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of L-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with L-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific L-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.     

IL-17 derived from juxta-articular bone and synovium contributes to joint degradation in rheumatoid arthritis

The origin and role of IL-17, a T-cell derived cytokine, in cartilage and bone destruction during rheumatoid arthritis (RA) remain to be clarified. In human ex vivo models, addition of IL-17 enhanced IL-6 production and collagen destruction, and inhibited collagen synthesis by RA synovium explants. On mouse cartilage, IL-17 enhanced cartilage proteoglycan loss and inhibited its synthesis. On human RA bone explants, IL-17 also increased bone resorption and decreased formation. Addition of IL-1 in these conditions increased the effect of IL-17. Blocking of bone-derived endogenous IL-17 with specific inhibitors resulted in a protective inhibition of bone destruction. Conversely, intra-articular administration of IL-17 into a normal mouse joint induced cartilage degradation. In conclusion, the contribution of IL-17 derived from synovium and bone marrow T cells to joint destruction suggests the control of IL-17 for the treatment of RA.     

Histone Deacetylase Inhibitors Modulate Interleukin 6-dependent CD4+ T Cell Polarization in Vitro and in Vivo

Histone deacetylase (HDAC) inhibitors have been associated primarily with an anti-proliferative effect in vitro and in vivo. Recent data provide evidence for an anti-inflammatory potency of HDAC inhibitors in models of experimental colitis. Because the balance of T cell subpopulations is critical for the balance of the mucosal immune system, this study explores the regulatory potency of HDAC inhibitors on T cell polarization as a mechanistic explanation for the observed anti-inflammatory effects. Although HDAC inhibition suppressed the polarization toward the pro-inflammatory T helper 17 (Th17) cells, it enhanced forkhead box P3 (FoxP3)⁺ regulatory T cell polarization in vitro and in vivo at the site of inflammation in the lamina propria. This was paralleled by a down-regulation of the interleukin 6 receptor (IL-6R) on naïve CD4⁺ T cells on the mRNA as well as on the protein level and changes in the chromatin acetylation at the IL6R gene and its promoter. Downstream of the IL-6R, HDAC inhibition was followed by a decrease in STAT3 phosphorylation as well as retinoic acid receptor-related orphan receptor γT (RORγT) expression, thus identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. These results directly translated to experimental colitis, where IL-6R expression was suppressed in naïve T cells, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals, resulting in the amelioration of disease. This study indicates that, in experimental colitis, inhibition of HDAC exerts an anti-inflammatory potency by directing T helper cell polarization via targeting the IL-6 pathway.     

Therapeutic effect of a poly(ADP-ribose) polymerase-1 inhibitor on experimental arthritis by downregulating inflammation and Th1 response

Poly(ADP-ribose) polymerase-1 (PARP-1) synthesizes and transfers ADP ribose polymers to target proteins, and regulates DNA repair and genomic integrity maintenance. PARP-1 also plays a crucial role in the progression of the inflammatory response, and its inhibition confers protection in several models of inflammatory disorders. Here, we investigate the impact of a selective PARP-1 inhibitor in experimental arthritis. PARP-1 inhibition with 5-aminoisoquinolinone (AIQ) significantly reduces incidence and severity of established collagen-induced arthritis, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of AIQ is associated with a striking reduction of the two deleterious components of the disease, i.e. the Th1-driven autoimmune and inflammatory responses. AIQ downregulates the production of various inflammatory cytokines and chemokines, decreases the antigen-specific Th1-cell expansion, and induces the production of the anti-inflammatory cytokine IL-10. Our results provide evidence of the contribution of PARP-1 to the progression of arthritis and identify this protein as a potential therapeutic target for the treatment of rheumatoid arthritis.     

Vasoactive intestinal peptide prevents experimental arthritis by downregulating both autoimmune and inflammatory components of the disease

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease of unknown etiology, characterized by chronic inflammation in the joints and subsequent destruction of the cartilage and bone. We describe here a new strategy for the treatment of arthritis: administration of the neuropeptide vasoactive intestinal peptide (VIP). Treatment with VIP significantly reduced incidence and severity of arthritis in an experimental model, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of VIP was associated with downregulation of both inflammatory and autoimmune components of the disease. Our data indicate VIP as a viable candidate for the development of treatments for RA.     

HDAC inhibition in rheumatoid arthritis and juvenile idiopathic arthritis

Rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) are heterogeneous autoimmune diseases characterized by chronic joint inflammation. Methotrexate is used as the gold standard to treat rheumatoid arthritis, yet there are many patients in whom the disease cannot be controlled or who experience unacceptable intolerance. Most of the biologics currently used are effective, but mostly if combined with methotrexate. Long-term possible side effects, such as impaired host defense mechanisms against infection and lymphoma, are distinct disadvantages and a major concern of anticytokine therapies. Parenteral administration is a problem, particularly in children. Thus, there is a need to explore new treatment options. Here we review the properties of histone deacetylase inhibitors (HDACi) as they apply to rheumatoid arthritis by looking at effects on cytokine production, T-cell differentiation and the function of macrophages, dendritic cells, osteoblasts, osteoclasts and synovial fibroblasts. We also review the safety and efficacy of givinostat (ITF 2357) in the treatment of systemic onset juvenile idiopathic arthritis (SOJIA) and its influence on the cytokine networks in SOJIA. Givinostat is an orally active HDACi which was given to children with SOJIA. After 12 wk of treatment, there were significant benefits, particularly in reducing the pain and arthritic component of the disease and decreasing the neutrophilia. CD40L, IL-1α and IFNγ in whole blood lysates decreased at wks 2 and 4 compared with baseline levels. The clinical data are consistent with those from animal models of rheumatoid arthritis and suggest that trials with HDACi are promising as a safe oral alternative to anticytokines and methotrexate.     

Local Delivery of Mesenchymal Stem Cells with Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold Suppress Arthritis in Rats

Mesenchymal stem cells (MSC) have been used recently for the treatment of autoimmune diseases in murine animal models due to the immunoregulatory capacity. Current utilization of MSC requires cells in certain quantity with multiple courses of administration, leading to limitation in clinical usage. Here we efficiently treated collagen-induced arthritis rats with a single local implantation with reduced number of MSC (2∼20% of previous studies) with nano-fiber poly-lactic-co-glycolic acid (nano-fiber) scaffold. MSC seeded on nano-fiber scaffold suppressed arthritis and bone destruction due to inhibition of systemic inflammatory reaction and immune response by suppressing T cell proliferation and reducing anti- type II collagen antibody production. In vivo tracing of MSC demonstrated that these cells remained within the scaffold without migrating to other organs. Meanwhile, in vitro culture of MSC with nano-fiber scaffold significantly increased TGF-β1 production. These results indicate an efficient utilization of MSC with the scaffold for destructive joints in rheumatoid arthritis by a single and local inoculation. Thus, our data may serve as a new strategy for MSC-based therapy in inflammatory diseases and an alternative delivery method for bone destruction treatment.     

Development of proteoglycan-induced arthritis is independent of IL-17

IL-17 is the hallmark cytokine for the newly identified subset of Th cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-gamma are secreted in CIA and EAE, disease is exacerbated in IFN-gamma- or IFN-gamma receptor-deficient mice due to the ability of IFN-gamma to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-gamma. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17-/-) mice and found the onset and severity of arthritis were equivalent in wild-type (WT) and IL-17-/- mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17-/- mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however, histological analysis of the arthritic joints from WT and IL-17-/- mice revealed a similar extent of joint cellularity, cartilage destruction, and bone erosion despite significantly reduced RANKL (receptor activator of NK-kappaB ligand) expression. There were only subtle differences between WT and IL-17-/- mice in proinflammatory cytokine expression, T cell proliferation, and autoantibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and that the production of other proinflammatory mediators is sufficient to compensate for the loss of IL-17 in PGIA.     

Inhibition of monocyte chemoattractant protein-1 ameliorates rat adjuvant-induced arthritis

Chemokines, including RANTES/CCL5 and MCP-1/CCL2, are highly expressed in the joints of patients with rheumatoid arthritis, and they promote leukocyte migration into the synovial tissue. This study was conducted to determine whether the inhibition of RANTES and MCP-1 therapeutically was capable of ameliorating rat of adjuvant-induced arthritis (AIA). Postonset treatment of AIA using a novel inhibitor for endogenous MCP-1 (P8A-MCP-1) improved clinical signs of arthritis and histological scores measuring joint destruction, synovial lining, macrophage infiltration, and bone erosion. Using immunohistochemistry, ELISA, real-time RT-PCR, and Western blot analysis, we defined joint inflammation, bony erosion, monocyte migration, proinflammatory cytokines, and bone markers, and p-p38 levels were reduced in rat AIA treated with P8A-MCP-1. In contrast, neither the dominant-negative inhibitor for endogenous RANTES (44AANA47-RANTES) nor the CCR1/CCR5 receptor antagonist, methionylated-RANTES, had an effect on clinical signs of arthritis when administered after disease onset. Additionally, therapy with the combination of 44AANA47-RANTES plus P8A-MCP-1 did not ameliorate AIA beyond the effect observed using P8A-MCP-1 alone. Treatment with P8A-MCP-1 reduced joint TNF-alpha, IL-1beta, and vascular endothelial growth factor levels. P8A-MCP-1 also decreased p38 MAPK activation in the joint. Our results indicate that inhibition of MCP-1 with P8A-MCP-1 after the onset of clinically detectable disease ameliorates AIA and decreases macrophage accumulation, cytokine expression, and p38 MAPK activation within the joint.