POP1 inhibits MSU-induced inflammasome activation and ameliorates gout

Abstract

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

CARD-only proteins regulate in vivo inflammasome responses and ameliorate gout

“… 23 - 26 Thereby, POPs interfere with the polymerization of inflammasome components and consequently prevent inflammasome assembly and inflammation in vivo . 23 , 27 - 30 A comparable competitive binding mechanism has been proposed for the three human COPs, CARD16/Cop/Pseudo-ICE, CARD17/Inca, and CARD18/Iceberg, but targeting the CARD in caspase-1 or ASC instead of the PYD. 31 - 34 However, most of these studies have been performed by overexpression in epithelial cells before the discovery of the inflammasome, and consequently any relevance of COPs in regulating inflammasome responses and inflammatory disease in vivo remains unknown.…”

“… 20 , 21 , 87 We previously demonstrated that this first step of nucleated ASC polymerization is regulated by POP family members by competitive binding to the PYD of either ASC or upstream sensors, there by ameliorating inflammatory disease. 23 , 27 , 29 , 30 Reminiscent of the three POP family members, the three COPs also evolved in higher primates. 36 , 89 However, their precise role is less well described, largely due to inconclusive and inconsistent studies, including CARD18 inhibiting IL-1β release in one but not another study, 34 , 87 studies of non-immune cells, 31 - 33 , 86 or studies of primed but not inflammasome-activated macrophages before the discovery of the inflammasome.…”

“…injection with PBS or MSU crystals (3 mg) MSU. 27 , 30 After 4h, mice were i.p. injected with XenoLight Rediject Inflammation probe (200 mg kg −1 , #760536, PerkinElmer) or luminol (200 mg kg −1 ) from a 50 mg mL −1 stock solution of luminol sodium salt (#A4685, Sigma), dissolved in sterile PBS and stored at −20°C and in vivo bioluminescence was captured by imaging (IVIS Spectrum, PerkinElmer) 10 min post injection with a 5 min exposure on anesthetized mice.…”

“… 23 , 35 , 36 Nevertheless, transgenic expression of human POP1, POP2, and POP3 in macrophages and mice recapitulates their inflammasome inhibitory function observed in humans and ameliorates inflammation in vivo . 23 , 27 , 29 , 30 Hence, core PYD- and CARD-containing proteins are sufficiently conserved, 37 which allows human proteins to interact with structurally conserved mouse proteins and enables in vivo studies of human inflammasome regulators in mice.…”

Uncoupled pyroptosis and IL-1β secretion downstream of inflammasome signaling

“…Since ASC is the central hub in inflammasome signaling, its function is tightly controlled, including in the sensor level ( 232 ), and also in its interactions with other ASC molecules and the sensor/caspase-1 by alternative splicing ( 233 – 235 ), post-translational modifications ( 236 – 241 ), PYD-only proteins ( 242 – 244 ), and CARD-only proteins ( 245 – 247 ). ASC oligomerization and speck formation can be disrupted by gene mutation ( 248 , 249 ), pathogens ( 250 ), bioactive extracts ( 251 – 254 )/derivatives ( 255 – 257 )/metabolites ( 258 ), cytokines ( 259 ), complements ( 260 ), or chemicals ( 261 – 263 ) or further stabilized by L-plastin ( 264 ), inhibiting or facilitating downstream signaling, respectively.…”