A hallmark of RA is chronic inflammation and destruction of joints. Dysfunction at various steps of the complex network comprising humoral and cellular immune responses results in perpetuation of inflammatory activation during development of RA, in which pro-inflammatory autoimmune responses are not adequately regulated by anti-inflammatory mechanisms [3]. Here, we show that CKD-506, a novel specific HDAC6 inhibitor, suppressed inflammatory responses by macrophages, improved Treg function, and inhibited inflammatory responses and the joint-destructive properties of FLS in vitro. In vivo, CKD-506 attenuated arthritis in a murine RA model with efficacy comparable with that of tofacitinib. In addition, CKD-506 had a synergistic effect when combined with methotrexate (both drugs were used at a dose that was ineffective when each was used alone).
Pan-HDAC inhibitors exhibit strong anti-inflammatory and anti-proliferative properties in several immune-mediated and hematologic malignancies; however, they have a narrow therapeutic margin due to side effects such as memory loss, cytopenia, and diarrhea [7, 14, 20–22]. In addition, epigenetic changes might have an unpredictable, long-lasting impact on the immune and non-immune cells even after HDAC inhibition is stopped. HDAC6 is localized predominantly to the cytoplasm where it deacetylates cytoplasmic non-histone proteins such as HSP90 and α-tubulin [15, 23, 24]. Hyperacetylation of α-tubulin has anti-inflammatory effects [23, 24]. CKD-506 is a highly specific inhibitor of HDAC6; as such it has pleiotropic effects on both immune cells and FLS, which constitute the main cellular infiltrates in inflamed RA joints. Here, we found that CKD-506 decreased secretion of pro-inflammatory cytokines TNF-α and IL-6 by macrophages via suppression of the NF-κB and AP-1 pathways (Fig. 1). CKD-506 suppressed immune responses directly by suppressing proliferation of activated T cells and indirectly by augmenting the function of regulatory T cells (Fig. 3). Restoration of defective Treg function is crucial for maintenance of self-tolerance and for preventing aggressive autoimmune cells from destroying tissues in RA joints [25, 26]. The inflammatory environment of RA transforms normal resident synoviocytes into tissue-destructive FLS, which produce large amounts of IL-6, IL-8, and extracellular matrix proteinases MMP-1 and MMP-3 [27, 28]. CKD-506 markedly inhibits joint-destructive FLS [18, 29]. The multicellular effects of HDAC6 inhibition by CKD-506 might contribute to the anti-arthritic effects observed in the murine RA model; these effects were comparable with those of tofacitinib, a JAK/STAT signaling inhibitor with proven clinical efficacy (Fig. 4). Strikingly, the combination of low (sub-therapeutic) dose CKD-506 and low (sub-therapeutic) dose methotrexate had a synergistic therapeutic effect on the arthritis severity in the murine model (Fig. 5). A previous report shows that methotrexate restores Treg function by demethylating the upstream enhancer of FoxP3, which might also potentiate restoration of impaired Treg cell function by CKD-506 [29]. While the mechanism underlying synergism needs further investigation, combination treatment might enable reduction of the dose of CKD-506 and reduce the risk of side effects. Long-term drug safety is of particular interest since patients with RA require life-long treatment [30]. Taken together, inhibiting multiple cell types with the novel HDAC6 inhibitor CKD-506 is promising, and the results are consistent with the observed anti-arthritic effects of other HDAC6 inhibitors [31, 32].
A main limitation of the study is that we did not investigate the exact mechanism by which the HDAC6 inhibitor affects multiple steps in the inflammatory response; indeed, many cytosolic and non-cytosolic proteins are substrates for HDAC6, so examining the mechanism is important.
In conclusion, CKD-506, a novel HDAC6 inhibitor, regulates innate and adaptive immune responses and ameliorates experimental arthritis. CKD-506 has potential as a novel treatment option for treatment of RA.