Background

Anti-inflammatory prevention and treatment of attacks of gouty arthritis remain challenging, in part because many patients have incomplete responses or contraindications to one or more of the primary oral anti-inflammatory therapies (colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids) [1, 2]. Moreover, gout flares often increase in frequency in the initial phase of urate-lowering therapy (ULT), thereby contributing to poor adherence to ULT and lack of improvement in health-related quality of life [1, 2]. Arhalofenate is a non-agonist ligand of peroxisome proliferator-activated receptor γ (PPARγ) with weak transactivation but robust transrepression activity [3]. It was first developed as an insulin sensitizer for type 2 diabetes mellitus [3]. Subsequently, arhalofenate was demonstrated to have uricosuric activity, as an inhibitor of URAT1, organic anion transporter 4 (OAT4) and OAT10 [4]. In a recent phase II trial in gout patients, which assessed acute gout flare as the primary endpoint, arhalofenate significantly reduced the risk of acute gouty arthritis in comparison with allopurinol alone, whereas there was no significant difference compared with allopurinol in combination with prophylactic colchicine [5]. The risk for urate-lowering therapy-induced gout flares depends on the degree of serum urate lowering [2]. Hence, this study was performed to directly test and characterize the anti-inflammatory effects of arhalofenate pertinent to gout.

Acute gouty arthritis is a characteristically severe phenotypic inflammatory response to deposits of monosodium urate (MSU) crystals which induce expression of NF-κB-dependent proinflammatory cytokines including pro-interleukin (IL)-1β and multiple chemokines [6, 7]. MSU crystals also stimulate activation of the NLRP3 inflammasome, with consequent maturation and release of IL-1β [6, 7]. This is a central driver of the gouty inflammation cascade which involves recruitment and activation of phagocytes [6, 7]. Core factors that modulate activation of the NLRP3 inflammasome, and experimental gout-like inflammation, include mitochondrial function, autophagy, and AMP-activated protein kinase (AMPK) [8, 9].

Mitochondrial reactive oxygen species (ROS) and oxidized mitochondrial DNA (mtDNA) promote inflammation [10,11,12], mediated by activation of NF-κB [10,11,12] and activation of the NLRP3 inflammasome via dysregulated balance between thioredoxins (TRXs) and thioredoxin-interacting protein (TXNIP) [13]. TRX1 and TRX2, mainly located in the cytoplasm and mitochondria, respectively, control cellular ROS by reduction of disulfides to thiol groups [14]. TXNIP directly binds to TRX and inhibits the reducing activity of TRX through disulfide exchange [14]. However, ROS triggers disassociation of TXNIP from TRX1, promoting direct physical interaction between TXNIP and NLRP3 that leads to activation of caspase-1 and release of mature IL-1β [13].

Autophagy mediates cellular homeostasis by degrading damaged proteins and organelles, including mitochondria [15,16,17]. Although MSU crystals promote autophagosome formation, the crystals also induce impairment of proteasomal degradation leading to accumulation of p62 [17]. As a selective autophagy receptor adaptor protein [17], p62 interacts with LC3-II to facilitate autophagic degradation [17], and also is involved in MSU crystal-induced caspase-1 activation and IL-1β release [18]. One of the major factors promoting autophagy is serine/threonine kinase AMPK [19].

AMPK is a nutritional biosensor that maintains cellular energy balance [19, 20], but nutritional excesses and other factors, including stimulation by MSU crystals and IL-1β, decrease AMPK activity [9]. Significantly, AMPK functions as an NF-κB and NLRP3 inflammasome inhibitor and promotes anti-inflammatory macrophage polarization, and markedly decreases the inflammatory response to MSU crystals in cultured macrophages [21, 22]. Moreover, AMPK transduces colchicine anti-inflammatory effects in vitro [22]. Pharmacologic AMPK activation markedly limits experimental gouty inflammation in the mouse in vivo using the subcutaneous air pouch model [22]. Conversely, MSU crystal-induced inflammation is prominently potentiated in AMPKα1 knockout (KO) mice [22].

Thiazolidinedione PPARγ agonists have been shown to cause phosphorylation and activation of AMPK [36]. Levels of p62 usually inversely correlate with autophagic degradation in later stages of autophagy [36]. Although studies have shown that p62 is increased and translocated to damaged mitochondria in NLRP3 inflammasome-activated cells, the detailed molecular mechanism on the link between the NLRP3 inflammasome and autophagy, especially mitophagy, is not yet fully understood. Interestingly, recent studies reported that, on stimulation of macrophages with NLRP3 inflammasome activators, p62, whose expression is induced via NF-κB, LC3-II, and Parkin, were recruited to the damaged mitochondria, initiating organelle clearance via mitophagy [36, 37]. This “NF-κB-p62-mitophagy” signaling axis represents a key macrophage-intrinsic regulatory mechanism that keeps NLRP3 inflammasome activation in check [37, 38]. Further studies on how arhalofenate acid controls MSU crystal-induced NLRP3 inflammation related to mitophagy will be of interest.

Conclusion

Arhalofenate acid, the active acid form of arhalofenate, exerts anti-inflammatory effects in MSU crystal-treated macrophages. These effects were mediated to a large degree by inducing AMPK activation, and at least in part by associated maintenance of mitochondrial function through activation of AMPK and its downstream signaling and preservation of mitochondrial cristae surface area, and by increasing cellular quality control by promoting autophagy. The results of this study identify basic mechanisms by which arhalofenate treatment decreases acute flares in patients with gout [5].