The crystal-induced activation of NLRP3 inflammasomes in atherosclerosis

Abstract Atherosclerosis is an inflammatory disease, which is accompanied by the deposition of cholesterol-rich lipids and the infiltration of macrophages. Other well-known features of atherosclerotic lesions include the deposition of cholesterol crystals and calcium phosphate crystals; however, their pathophysiological role remains unclear. Recent studies suggest that cholesterol crystals play a pivotal role in activation of NLRP3 inflammasomes, which regulate caspase-1 activation and the subsequent processing of IL-1β, in atherosclerotic lesions. NLRP3 inflammasomes are essential for the initiation of vascular inflammation during the progression of atherosclerosis. Therefore, the regulatory mechanisms of NLRP3 inflammasomes are regarded as potential targets for atherosclerosis treatment. Here, we review the current knowledge regarding the role of NLRP3 inflammasomes in the progression of atherosclerosis and the prospects for therapeutic approaches targeting NLRP3 inflammasomes

Oligomerized CARD16 promotes caspase-1 assembly and IL-1β processing

AbstractIncreasing evidence indicates that caspase recruitment domain (CARD)-mediated caspase-1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)-1β release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo-oligomer. Oligomerized CARD16 formed a filament-like structure with CASP1CARD and a speck with apoptosis-associated speck-like protein containing a CARD. A filament-like structure formed by CARD16 promoted CASP1 filament assembly and IL-1β release. In contrast, CARD17 did not form a homo-oligomer or filaments and inhibited CASP1-dependent IL-1β release. Mutated CARD16D27G, mimicking the CARD17 amino acid sequence, formed a homo-oligomer but failed to form a filament-like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL-1β release. These results suggest that oligomerized CARD16 promotes CARD-mediated molecular assembly and CASP1 activation

Interferon-tau attenuates uptake of nanoparticles and secretion of interleukin-1β in macrophages.

BACKGROUND: Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages. METHODS AND RESULTS: IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody. CONCLUSIONS: Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB

The cardiac glycoside ouabain activates NLRP3 inflammasomes and promotes cardiac inflammation and dysfunction

<div><p>Cardiac glycosides such as digoxin are Na⁺/K⁺-ATPase inhibitors that are widely used for the treatment of chronic heart failure and cardiac arrhythmias; however, recent epidemiological studies have suggested a relationship between digoxin treatment and increased mortality. We previously showed that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, which regulate caspase-1-dependent interleukin (IL)-1β release, mediate the sterile cardiovascular inflammation. Because the Na⁺/K⁺–ATPase is involved in inflammatory responses, we investigated the role of NLRP3 inflammasomes in the pathophysiology of cardiac glycoside-induced cardiac inflammation and dysfunction. The cardiac glycoside ouabain induced cardiac dysfunction and injury in wild-type mice primed with a low dose of lipopolysaccharide (LPS), although no cardiac dysfunction was observed in mice treated with either ouabain or LPS alone. Ouabain also induced cardiac inflammatory responses, such as macrophage infiltration and IL-1β release, when mice were primed with LPS. These cardiac manifestations were all significantly attenuated in mice deficient in IL-1β. Furthermore, deficiency of NLRP3 inflammasome components, NLRP3 and caspase-1, also attenuated ouabain-induced cardiac dysfunction and inflammation. <i>In vitro</i> experiments revealed that ouabain induced NLRP3 inflammasome activation as well as subsequent IL-1β release from macrophages, and this activation was mediated by K⁺ efflux. Our findings demonstrate that cardiac glycosides promote cardiac inflammation and dysfunction through NLRP3 inflammasomes and provide new insights into the mechanisms underlying the adverse effects of cardiac glycosides.</p></div

IL-1β deficiency decreases ouabain-induced macrophage infiltration.

<p>WT and IL-1β^−/− mice were treated with ouabain (2 mg/kg) 12 h after LPS (3 mg/kg) administration and then sacrificed 12 h after ouabain treatment. (A and C) Heart sections were immunohistochemically stained for CD45 and CD68. (B and D) The number of CD45⁺ (leukocytes) and CD68⁺ (macrophages) cells was quantified (n = 4 for each). Data are expressed as the mean ± SEM. *<i>P</i> < 0.05 and **<i>P</i> < 0.01. ^#<i>p</i> < 0.05 vs. vehicle (WT).</p

Ouabain induces NLRP3 inflammasome activation and IL-1β release in macrophages.

<p>(A–C) After priming with or without LPS (100 ng/mL) for 6 h, J774 macrophages were treated with ouabain (A, 50–100 μM; B and C, 100 μM) for 3 h. (A) IL-1β levels in the supernatants were assessed (n = 6 for each). (B) Heart <i>Il1b</i> and <i>Nlrp3</i> mRNA levels were assessed by real-time RT-PCR analysis (n = 4 for each). (C) The processing of pro-IL-1β in the lysates and supernatants was assessed by western blot analysis. ATP was used as a positive control. (D) LPS-primed J774 macrophages were treated with ouabain (100 μM) for 3 h in the presence or absence of YVAD-FMK (20 μM). IL-1β levels in the supernatants were assessed (n = 4 for each). (E and F) After priming with or without LPS (100 ng/mL) for 6 h, primary murine macrophages from WT and NLRP3^−/− mice were treated with ouabain (E, 100 μM; F, 50–100 μM) for 3 h. (E) IL-1β levels in the supernatants were assessed (n = 4 for each). (F) Cell death was assessed by LDH activity in the supernatants (n = 4 for each). Data are expressed as the mean ± SEM. *<i>P</i> < 0.05 and **<i>p</i> < 0.01. ^#<i>P</i> < 0.05 vs. vehicle. ^$<i>P</i> < 0.05 vs. LPS alone and ^§<i>P</i> < 0.05 vs. Ouabain with LPS.</p