CASPorter: A Novel Inducible Human CASP1/NALP3/ASC Inflammasome Biosensor
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Background: Following our 2015 elucidation of the CASP1/NALP3 inflammasome mechanism of glucocorticoid (GC)-resistance in pediatric acute lymphoblastic leukemia (ALL) patients, we engineered a cell-based CASP1/NALP3 reporter system suitable for high-throughput screening (HTS) of small molecule libraries, with the purpose of identifying compounds capable of inhibiting the CASP1/NALP3 inflammasome and synergizing with GC drugs for the treatment of GC-resistant ALL patients and various autoinflammatory diseases. Methods: A Dox-controlled system was utilized to induce the expression of the ASC transgene in HEK293 cells while simultaneously overexpressing NLRP3 and CASP1 . ASC/CASP1/NALP3 inflammasome complex formation was confirmed by co-immunoprecipitation (co-IP) experiments. Next, a LV fluorescence-based biosensor ( CASP orter) was transduced in the HEK293-iASC-NLRP3/CASP1 cell line to monitor the real-time activation of CASP1/NALP3 inflammasome in live cells. The applicability and effectiveness of the CASP orter cell line were tested by co-treatment with Dox and four known CASP1/NLRP3 inhibitors (MCC950, Glyburide, VX-765 and VRT-043198). Inflammasome activation and inhibitions were assessed by Western blotting, fluorescence microscopy and flow cytometry (FC) methods. Results: Dox treatment significantly induced ASC expression and increased levels of cleaved and catalytically active CASP1, co-IPs further demonstrated that CASP1 was pulled-down with NLRP3 in HEK293-iASC-NLRP3/CASP1 cells after induction of ASC by Dox treatment. In HEK293-iASC-NLRP3/CASP1- CASP orter cell system, cleavage of the CASP1 consensus site (YVAD) in the CASP orter protein after Dox treatment causing excitation/emission of green fluorescence and the 71% GFP+ cell population increase quantified by FC (78.1% vs 6.90%). Dox-induced activation of the NLRP3 inflammasome was dose-dependently inhibited by Dox co-treatment with four known CASP1/NLRP3 inhibitors. Conclusion: We have established a cell-based CASP1/NLRP3 inflammasome model, utilizing a fluorescence biosensor as readout for qualitatively observing and quantitatively determining the activation of caspase 1 and NLRP3 inflammasomes in living cells and easily define the inhibitory effect of inhibitors with high efficacy. Keywords: cell-based biosensor, NALP3 inflammasome, CASP1, ASCKeywords:
NALP3
NALP3
Interleukin 18
AIM2
Proinflammatory cytokine
Pyroptosis
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瞄准：为了检验 Nalp3 的激活， inflammasome 和它的下游的目标追随者 lipopolysaccharide (LPS ) 在肝导致了刺激。方法：Six-to-eight-week-old C57BL/6 食物喂的老鼠与 0.5 g/g bodyweight LPS intraperitoneally 被注射并且牺牲了 2， 4， 6， 18 或 24 h 以后。导致 LPS 的肝损坏被生物化学的试金证实检测丙氨酸 aminotransferase (中高音) 层次。决定在肝的 LPS 刺激是否导致了 inflammasome 的激活，即时量的聚合酶链反应被用来计算 Nalp3 inflammasome 的部件的 mRNA 表达式。连接酶的 immunosorbent 试金被用来决定 Nalp3 inflammasome 的几个下游的目标的蛋白质表示层次，包括 caspase-1 和 caspase-1， interleukin (IL )-1 和 IL-18 的二个 cytokine 目标。结果：我们发现那 LPS 注射处于由提高的中高音层次显示了的肝损坏结果。这与 mRNA 和 proinflammatory cytokine 肿瘤坏死因素(TNF ) 的蛋白质层次的重要增加被联系 - 在在浆液的 TNF 的肝，以及增加的层次。我们证明 LPS 刺激为 inflammasome 的所有受体部件在肝导致了 mRNA 层次的 upregulation，包括 Nalp3， Nalp1， pannexin-1 和适配器分子联系 apoptosis 像斑点， caspase 招募域域包含蛋白质。我们也为 caspase-1 发现 mRNA 和蛋白质的层次增加， inflammasome 的一个下游的目标。另外， LPS 挑战为 caspase-1， IL-1 和 IL-18 的二个 cytokine 目标在肝导致了 mRNA 和蛋白质的增加的层次。有趣地， pre-IL-1 和 pre-IL-18 的实质的基线表示在肝被发现。Inflammasome 和 caspase-1 激活被 IL-1 和 IL-18 的活跃形式的重要增加在 LPS 刺激以后显示。结论：我们的结果证明 Nalp3 inflammasome 是 upregulated 并且响应 LPS 刺激在肝激活。
NALP3
Interleukin 18
AIM2
Proinflammatory cytokine
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NALP3
Proinflammatory cytokine
AIM2
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NALP3
Pyroptosis
Pyrin domain
AIM2
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NALP3
AIM2
Pseudogout
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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.
Pyrin domain
AIM2
NALP3
Pyroptosis
Proinflammatory cytokine
TRIF
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This article was originally published online on 23 April 2013 The inflammasome is a novel protein complex that stimulates caspase-1 activation to promote the processing and secretion of IL-1β, a pro-inflammatory cytokine, which is among the most biologically important inflammatory mediators in allergic airway diseases. Among the various types of inflammasomes, Cryopyrin/NALP3 has been suggested to be involved in sensing sterile stress response, extracellular ATP, danger-associated molecular patterns (DAMPs), and crystals. Recently inappropriate NALP3 inflammasome activity has been reported in various allergic airway diseases including asthma. Therefore, inhibitors of the NALP3 inflammasome offer considerable therapeutic promise. Recently Omega-3 fatty acid derivates termed resolvins have shown to alter the effects of pro-inflammatory cytokine storm seen in inflammatory diseases. However the ability of resolvins to modulate the effects of inflammasome activation has not been studied. We investigated whether resolvin treatment inhibits inflammasome activation and regulates the functional effects of inflammasome mediated by IL-1β secretion. In our study the inflammasome was activated by ATP and H2O2 (known nflammasome activators in acute lung injury) in the absence or presence of D series resolvins, resolvin D1 and resolvin D2. Inflammasome activation was assessed by analyzing IL-1β release (end product of inflammasome activation) and caspase-1 cleavage (indicator of inflammasome activation) in THP-1 cells. Further inflammasome was activated in the presence or absence of resolvin in THP-1 cells, and supernatants from these cells were added to A549 cells and human primary small airway epithelial cells (HPSAEC) to study inflammasome mediated functional effects. Our results indicate that resolvin treatment ameliorates inflammasome activation as indicated by decreased caspase-1 activity and IL-1β release. In addition resolvin treatment inhibits inflammasome mediated epithelial cell activation as indicated by suppressed IL-8 release and decreased and ICAM expression. These novel findings suggest that resolvins can be used to modulate the inflammasome activity as well as blunt the effects of the IL-1β mediated cytokine storm stemming from inflammasome activation. These results may offer a therapeutic approach to airway diseases such as asthma, where ungoverned pro-inflammatory cytokine secretion exacerbates the disease pathology.
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AIM2
Interleukin 18
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The NACHT, LRR, and PYD-domains-containing protein 3 (NLRP3) inflammasome is an intracellular multi-protein signaling platform that is activated by cytosolic pattern-recognition receptors such as NLRs against endogenous and exogenous pathogens. Once it is activated by a variety of danger signals, recruitment and assembly of NLRP3, ASC, and pro-caspase-1 trigger the processing and release of pro-inflammatory cytokines including interleukin-1β (IL-1β) and IL-18. Multiple intracellular and extracellular structures and molecular mechanisms are involved in NLRP3 inflammasome activation. Gout is an autoinflammatory disease induced by inflammatory response through production of NLRP3 inflammasome-mediated proinflammatory cytokines such as IL-1β by deposition of monosodium urate (MSU) crystals in the articular joints and periarticular structures. NLRP3 inflammasome is considered a main therapeutic target in MSU crystal-induced inflammation in gout. Novel therapeutic strategies have been proposed to control acute flares of gouty arthritis and prophylaxis for gout flares through modulation of the NLRP3/IL-1 axis pathway. This review discusses the basic mechanism of NLRP3 inflammasome activation and the IL-1-induced inflammatory cascade and explains the NLRP3 inflammasome-induced pathogenic role in the pathogenesis of gout.
NALP3
Proinflammatory cytokine
Pathogenesis
Pyrin domain
Interleukin 18
AIM2
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NALP3
AIM2
Proinflammatory cytokine
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Joint bone
NALP3
Pyrin domain
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