Applications of reconstituted inflammasomes in a cell-free system to drug discovery and elucidation of the pathogenesis of autoinflammatory diseases

Kaneko, Naoe; Iwasaki, Tomoyuki; Ito, Yuki; Takeda, Hiroyuki; Sawasaki, Tatsuya; Morikawa, Shinnosuke; Nakano, Naoko; Kurata, Mie; Masumoto, Junya

doi:10.1186/s41232-017-0040-y

Cited by 9 publications

(12 citation statements)

References 64 publications

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“…To answer this, we performed recently developed cell-free experiments. 17 Cell-free experiments, with no ROS, revealed that IAPP/amylin directly induces the interaction between NLRP3 and ASC confirmed by Alpha and immunoprecipitation, which could be an initial event of NLRP3 inflammasome activation. Consistent with the known function of NLRP3, IAPP/amylin directly interacted with LRR domain (NLRP3-Exon (4-9)) of NLRP3 ( Figures 4 and 5 ).…”

Section: Discussionmentioning

confidence: 82%

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IAPP/amylin deposition, which is correlated with expressions of ASC and IL-1β in β-cells of Langerhans’ islets, directly initiates NLRP3 inflammasome activation

Morikawa

Kaneko

Okumura

et al. 2018

Int J Immunopathol Pharmacol

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Recent findings revealed that type 2 diabetes mellitus (T2D) is a chronic inflammatory disease and an islet amyloid polypeptide (IAPP)/amylin, is deposited within pancreatic islets. IAPP/amylin has been reported to activate NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been shown to recognize pathogens and/or metabolites and complexes with the adopter protein apoptosis-associated speck-like protein containing a caspase-recruitment domain ASC to form a huge complex, called an inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species (ROS) were reported to be involved in activation of NLRP3 inflammasome, we were hypothesized that IAPP could directly activate NLRP3 inflammasome, leading to islets β-cell death. We analyzed expression of the inflammasome components ASC, NLRP3, caspase-1, IL-1β, IAPP/amylin, and insulin immunohistochemically in Langerhans’ islets of autopsy cases. The initial event of NLRP3 inflammasome activation was assessed using a cell-free system consisting of NLRP3 and ASC with the amplified luminescent proximity homogeneous assay. IAPP/amylin deposition in Langerhans’ islets was detected and significantly correlated with expressions of IL-1β and ASC. IAPP/amylin directly interacted with NLRP3 and initiated an interaction between NLRP3 and ASC in a cell-free system. The deposition of IAPP/amylin in β-cells of Langerhans’ islets may act together with the expression level of an inflammasome component, ASC, to regulate IL-1β processing, and directly lead to the dysfunction of β-cells. The interaction between IAPP/amylin and NLRP3 could be an attractive drug target to avoid both inflammation and β-cell death for T2D therapy.

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Section: Discussionmentioning

confidence: 82%

“…Using wheat germ cell-free system–specific expression plasmids, NLRP3-FL-Btn and FLAG-ASC-FL proteins were also synthesized as described previously. 16 , 17 …”

Section: Resultsmentioning

confidence: 99%

IAPP/amylin deposition, which is correlated with expressions of ASC and IL-1β in β-cells of Langerhans’ islets, directly initiates NLRP3 inflammasome activation

Morikawa

Kaneko

Okumura

et al. 2018

Int J Immunopathol Pharmacol

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“…While we may avoid those suppressors and focus on potentially more direct inhibitors of NLRP3 inflammasome, these compounds may offer clues as to how physiological pathways upstream of NLRP3 inflammasome are wired together. In addition, screens designed to identify direct inhibitors of the NLRP3 inflammasome 13,46 may also help minimize discovery of hit compounds that alter cell physiological processes 47 .…”

Section: Discussionmentioning

confidence: 99%

Niclosamide activates the NLRP3 inflammasome by intracellular acidification and mitochondrial inhibition

Tran

Kitami

2019

Commun Biol

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The NLRP3 inflammasome is unique among pattern recognition receptors in using changes in cellular physiology as a mechanism for sensing host danger. To dissect the physiological network controlling inflammasome activation, we screened for small-molecule activators and suppressors of IL-1β release in macrophages. Here we identified niclosamide, a mitochondrial uncoupler, as an activator of NLRP3 inflammasome. We find that niclosamide inhibits mitochondria and induces intracellular acidification, both of which are necessary for inflammasome activation. Intracellular acidification, by inhibiting glycolysis, works together with mitochondrial inhibition to induce intracellular ATP loss, which compromises intracellular potassium maintenance, a key event to NLRP3 inflammasome activation. A modest decline in intracellular ATP or pH within an optimal range induces maximum IL-1β release while their excessive decline suppresses IL-1β release. Our work illustrates how energy metabolism converges upon intracellular potassium to activate NLRP3 inflammasome and highlights a biphasic relationship between cellular physiology and IL-1β release.

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“…In order to identify directly stimulating endogenous ligands, we previously developed a reconstituted NLRP3 inflammasome in a cell-free system [ 5 , 13 ]. In this study, we successfully prepared Aβ oligomers and Aβ fibrils (Fig.…”

Section: Discussionmentioning

confidence: 99%

Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system

et al. 2018

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BackgroundAlzheimer’s disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.MethodsWe prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells.ResultsMonomers, oligomers, and fibrils of Aβ were successfully prepared. Aβ oligomers and fibrils interacted with NLRP3. Aβ oligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβ monomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1β was not secreted according to the cell-based assay.ConclusionOligomerized Aβ originating from non-toxic Aβ monomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer’s disease.Electronic supplementary materialThe online version of this article (10.1186/s41232-018-0085-6) contains supplementary material, which is available to authorized users.

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Applications of reconstituted inflammasomes in a cell-free system to drug discovery and elucidation of the pathogenesis of autoinflammatory diseases

Cited by 9 publications

References 64 publications

IAPP/amylin deposition, which is correlated with expressions of ASC and IL-1β in β-cells of Langerhans’ islets, directly initiates NLRP3 inflammasome activation

IAPP/amylin deposition, which is correlated with expressions of ASC and IL-1β in β-cells of Langerhans’ islets, directly initiates NLRP3 inflammasome activation

Niclosamide activates the NLRP3 inflammasome by intracellular acidification and mitochondrial inhibition

Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system

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