Inhibition of HDAC Activity by ITF2357 Ameliorates Joint Inflammation and Prevents Cartilage and Bone Destruction in Experimental Arthritis
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Inhibition of histone deacetylases (HDAC) has been shown to modulate gene expression and cytokine production after stimulation with several stimuli. In the present study, the antiinflammatory effect of a potent HDACi, ITF2357, was explored in different experimental models of arthritis. In addition, the bone protective effect of ITF2357 was investigated invitro. Treatment of acute arthritis (Streptococcus pyogenes cell wall (SCW) arthritis) with ITF2357 showed that joint swelling and cell influx into the joint cavity were reduced. Furthermore, the chondrocyte metabolic function was improved by treatment of ITF2357. The production of proinflammatory cytokines by synovial tissue was reduced after ITF2357 treatment. To examine the effect of HDAC inhibition on joint destruction, ITF2357 was applied to both rat adjuvant arthritis and mouse collagen type II arthritis. ITF2357 treatment both ameliorates the severity scores in arthritis models and prevents bone destruction. In an in vitro bone destruction assay, ITF2357 was highly effective at a dose of 100 nmol/L. In conclusion, inhibition of HDAC prevents joint inflammation and cartilage and bone destruction in experimental arthritis.
Although synthetic histone deacetylases inhibitors (HDACi) hyperacetylate histones via the conserved N-terminal lysines present on histones, they also hyperacetylate cytoplasmic proteins, including transcription factors. Therefore, unraveling chromatin and thus permitting transcription factors to bind to DNA is a prominent property of HDACi, but not their only mechanism of action. Thus, the ability of HDACi to acetylate cytoplasmic proteins may affect cellular functions independent of their role on gene expression. In humans, there are 18 distinct HDACs (1) and their specific targeting with synthetic HDACi may have a role in treating diseases, particularly in chronic diseases such as degenerative joint diseases. Also, in order to be used during chronic disease states, such as occurs with inflamed joints, HDACi need to be safe for long-term use.
Inhibitors of HDAC are used widely in medicine. For example, valproic acid is used chronically to treat epilepsy and obsessive disorders (2,3). Whereas several synthetic inhibitors of HDACs have been developed to bring about terminal differentiation of cancer cells and to increase proapoptotic genes (4), to our knowledge the only HDACi used in humans as an antiinflammatory agent is ITF2357 (givinostat) (14). ITF2357, a Class I and II HDACs inhibitor (14), was given to children with systemic onset juvenile idiopathic arthritis (SOJIA) at a dose of 1.5 mg/kg for 12 weeks. The study exhibited no organ toxicity and achieved significant reduction in parameters of systemic disease but particularly the number of painful joints (5).
In general, micromolar concentrations of synthetic HDAC inhibitors are required to increase the expression of several proapoptotic genes in malignant cells (4,6, 7, 8). However, HDACi also exhibit immunosuppressive and anti-inflammatory properties at lower concentrations (9). For instance, nanomolar concentrations of ITF2357 are sufficient to reduce the production and/or activity of proinflammatory cytokines in vitro or to exert a potent effect in animal models of inflammatory and autoimmune diseases (9, 10, 11, 12, 13, 14, 15, 16, 17). Other HDACi, such as SAHA, share with ITF2357 many of the antiinflammatory properties in models of disease in experimental animals (12,14).
In the present study, we have evaluated the effect of ITF2357 in several models of arthritis, ranging from acute to chronic models of arthritis. In addition, the effect of ITF2357 was studied on both TNFα- and IL-1β-induced bone resorption.
HDAC Inhibitors Decrease Cytokine Production by RA Synovial Cells and RA Tissue Explants
Recently, it was demonstrated that HDAC expression and activity in synovial tissue of RA patients correlates positively with the concentration of TNFα (18). In addition, it was shown that HDAC inhibitors suppress cytokine production by RA synovial tissue explants and that both TNFα- and LPS-induced cytokine and chemokine production were decreased by inhibition of HDAC activity (19). However, HDAC inhibition did not influence the spontaneous cytokine production of RA synovial macrophages or intact synovial tissue explants. Interestingly, HDACi induced apoptosis in RA synovial macrophages by suppressing antiapoptotic Bfl-1 protein (19). It has been reported that HDACi activate either the extrinsic or the intrinsic death pathway or both of these pathways (20). In addition, it was demonstrated that HDACi induce growth arrest in RA synovial fibroblasts through induction of p21 and suppression of NF-κB nuclear accumulation (21). HDACi suppress cytokine production without an effect on cell death, which is of high interest. This indicates that it might be possible to uncouple inhibition of cytokine production and induction of apoptosis (14).
Inhibition of HDAC Activity Suppresses Experimental Arthritis
Since it was demonstrated that HDACi suppress the production of proinflammatory cytokines, several studies have been performed to investigate their anti-inflammatory properties in models of arthritis. Anti-type II collagen antibody-induced arthritis (CAIA) is based on the formation of immune complexes in the joints of susceptible mice. The onset of this experimental model of arthritis is highly dependent on cytokines, such as TNFα and IL-1. Both prophylactic and therapeutic treatment of CAIA with HDACi (TSA or FK228) suppressed arthritis severity (22,23) and reduced joint pathology. Of interest, enzymes that promote joint destruction such as matrix metalloproteinases (MMP)-3 and MMP-13 were reduced compared with vehicle. It is known that these MMPs are induced by TNFα but predominantly IL-1β. HDAC inhibition will lead to suppression of these cytokines and the resultant reduction of MMP expression or activity can account for the benefit of HDACi in models of rheumatoid arthritis.
Inhibition of HDAC activity by ITF2357 modulates several in vivo models of inflammation, such as LPS-induced shock, Con A-induced hepatitis, DSS colitis and even traumatic brain injury (24,14,25). In these models, it was demonstrated that IL-1 production is reduced after treatment with ITF2357. IL-1 is the classical proinflammatory cytokine that drives cartilage destruction during chronic joint inflammation. Of interest, in the beginning of the cytokine era, IL-1 was also named by Jeremy Saklatvala “catabolin,” referring to its potent cartilage destructive properties (26). Thereafter, it was demonstrated that IL-1 contributes to joint inflammation and severe cartilage destruction (27). IL-1 exerts potent arthritogenic activity when injected directly into murine knee joints, whereas TNFα induces joint swelling and influx of cells into the joint space (28). Cartilage exposure to IL-1, both in vitro and in vivo, inhibits matrix proteoglycan synthesis and promotes cartilage destruction by upregulation of matrix MMPs. Irreversible cartilage damage elicited by MMPs is one of the hallmarks of joint destruction in inflammatory arthritis. The role of IL-1 was underlined further by elegant studies using IL-1 deficient mice. They revealed that IL-1β is the pivotal mediator that directs chronic joint inflammation, but both IL-1α and IL-1β promote cartilage damage (29,30).
Protective Effect of ITF2357 on Acute Joint Inflammation and Cartilage Catabolism
Protective effect of ITF2357 on acute joint inflammation and cartilage catabolism.
Inhibition (%) of synovial cytokine production after ITF2357 treatment
Induction of SCW arthritis results in a dramatic suppression of chondrocyte metabolic function as measured by the incorporation of radiolabeled 35S-sulphate into the patellar cartilage. As an example, an inhibition of chondrocyte proteoglycan (PG)-synthesis of 44% is seen in Figure 1C. Oral treatment with 1 and 10 mg/kg ITF2357 dose dependently reduced the suppression of chondrocyte PG synthesis compared with the control group (27% and 13%, respectively, versus 44% in the control group). It is known that inhibition of chondrocyte PG synthesis due to joint inflammation, progressively leads to severe cartilage damage. IL-1β appears to be the most potent cytokine studied to date with respect to chondrocyte PG-synthesis. Overexpression of IL-1β results in irreversible cartilage destruction (27). It is likely that the same modulation of IL-1 by HDACi as seen in other models of inflammation accounts for the reduced inhibition of chondrocyte PG-synthesis seen here, and hence in a protective effect against cartilage catabolism during arthritis. This was confirmed by the finding that treatment with 1 mg/kg or 10 mg/kg ITF2357 produced less IL-1β, namely 17% and 62%, respectively, compared with SCW-control animals (Table 1). Interestingly, other proinflammatory cytokines (for example, IL-1α and IFNγ) including arthritogenic ones such as IL-6 and IL-12 or chemokines MIP-1α KC were dose dependently inhibited by ITF2357, suggesting a broad antiinflammatory effect for the drug (Table 1).
In SCW-induced arthritis, influx of predominantly PMNs can be noted in the joint cavity of inflamed joints (Figure 1D). Figure 1E shows the cellular influx at day 2 of SCW-induced arthritis and demonstrates that treatment with ITF2357 (10 mg/kg) reduces the number of inflammatory cells in the joint cavity. In contrast, no protective effect was noted on PG loss from the cartilage layers with either 1mg/kg or 10mg/kg ITF 2357 as measured by Safranin O staining. However, since inhibition of HDAC activity results in protection against inhibition of chondrocyte PG synthesis, the overall effect on cartilage of ITF2357 is beneficial to the cartilage structure.
ITF2357 Inhibits Severe Bone Destruction in Collagen-Induced Arthritis and Adjuvant-Induced Arthritis
Since inhibition of HDAC activity by ITF2357 resulted in amelioration of acute joint inflammation and cartilage damage (improved chondrocyte proteoglycan synthesis) the effect of the drug was studied in arthritis models expressing severe joint destruction (bone and cartilage), namely murine type II collagen-induced arthritis (CIA) and rat adjuvant-induced arthritis (AA). Joint destruction is the hallmark of RA and one of the main targets for targeted therapies in RA.
Histopathology evaluation of the effect of givinostat administration (10 mg/kg) on joint inflammation and tissue damages in the CIA model at day 28.
Bone and cartilage damage
Dilatation of joint cavity
Necrotic and inflammatory deposits in the joint cavity
Since rat AA displays more severe prominent bone destruction compared with murine CIA, rats with AA were treated with 5–50 mg/kg ITF25357. Prophylactic treatment with 50 mg/kg/day showed an optimal antiinflammatory effect when compared with vehicle control (Figure 2C). This suppressive effect was almost similar to leflunomide treatment (positive control) and resulted in strong reduction of bone pathology (data not shown).
Several reports confirmed the above findings that inhibition of HDAC strongly reduced disease severity in models of experimental arthritis (25). In line with our results, prominent protection against bone destruction was seen after treatment with HDACi (33).
Bone Protective Effect of ITF2357
Recently, it was shown that HDACi inhibit osteoclastogenesis and bone resorption by suppressing the induction of c-Fos by RANKL (37). In addition, HDACi induce IFN-β and thereby suppress osteoclastogenesis and bone destruction (38). In addition, HDACi are potent MMP inhibitors which are involved in both cartilage and bone destruction (39, 40, 41). Taken together, these data indicate that HDACi may be a future therapy for chronic joint diseases, in which bone destruction is difficult to treat.
Collectively the data from the literature and the results on ITF2357 presented here demonstrate that HDACi have a strong antiinflammatory and antidestructive effect in several models of arthritis. ITF2357, in particular, suppressed both acute and chronic arthritis. In the SCW model, joint swelling, the inhibition of chondrocyte proteoglycan synthesis and the influx of inflammatory cells to the inflamed joints were suppressed dose dependently by oral treatment with the drug. TNFα and IL-1β in the joint cavity also were reduced by ITF2357. This is consistent with the role played by the two cytokines in this model where joint swelling and cell influx are driven by TNFα, and IL-1β is crucial in the inhibition of chondrocyte PG synthesis. Inhibition of TNFα and IL-1β is not the only mechanism by which ITF2357 exerts its protective effect in the SCW-model, since other proinflammatory cytokines including arthritogenic IL6 and IL12 and chemokines were downmodulated equally by the drug (Table 1).
In addition to the suppressive effect in the acute model of arthritis, ITF2357 and other HDACi are protective in models of chronic arthritis. Remarkably, the efficacy seen upon administration of the drug before arthritis induction is comparable to that obtained when the treatment begins after the onset of the disease (25),33 and Figure 2). Furthermore, as with the SCW model, HDAC inhibition reduces bone destruction in these models of chronic arthritis (33 and Table 2; see Figure 2).
Inhibition of bone resorption also was observed in vitro where ITF2357 suppressed calcium release induced in mouse calvaria by IL-1β or TNFα or other stimuli (Figure 3). Thus, not only is HDAC inhibition able to downregulate the expression of proinflammatory cytokines, it also is repressing their destructive effects on the bone. This is consistent with the results from LPS-induced activation of human PBMC where ITF2357, vorinostat and other HDACi suppress monocyte/macrophage production of TNFα, IL12, IL-1β, etc. and their activity on lymphocytes (9).
One of the critical questions for the treatment of human arthritic conditions with HDACi is whether or not sufficient circulating concentrations can be achieved by safe doses of these drugs. Approximately 500 patients with different pathologies have been treated to date with ITF2357. Safe doses range from 1.4 to 2.1 mg/kg/day. Owing to differences in the metabolic and adsorption processes, the Cmax and AUC values of a dose of 1.0 mg/kg/day ITF2357 in humans (including children) corresponds to 25–30 and 40 mg/kg/day, respectively, in rodents. Therefore the efficacious doses of 10 or 50 mg/kg/day seen here in the various models of experimental arthritis are consistent with a human use. Indeed, in a clinical trial in juvenile idiopathic arthritis (JIA), givinostat (ITF2357) was given to children at 1.5 mg/kg (in two divided doses) for 12 weeks (5). The drug was well tolerated and the overall arthritis score was suppressed in these children.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
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