CARD8 is a negative regulator for NLRP3 inflammasome, but mutant NLRP3 in cryopyrin-associated periodic syndromes escapes the restriction

Sayaka Ito,Tetsuo Kubota,Yukichi Hara

doi:10.1186/ar4483

Sayaka Ito, Tetsuo Kubota + Show 1 more

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https://doi.org/10.1186/ar4483

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Abstract

IntroductionNLRP3 plays a role in sensing various pathogen components or stresses in the innate immune system. Once activated, NLRP3 associates with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 to form a large protein complex termed inflammasome. Although some investigators have proposed a model of NLRP3-inflammasome containing an adaptor protein caspase recruitment domain-containing protein 8 (CARD8), the role of this molecule remains obscure. This study aimed to clarify the interaction between CARD8 and wild-type NLRP3 as well as mutant forms of NLRP3 linked with cryopyrin-associated periodic syndromes (CAPS).MethodsIn here HEK293 expression system, cells were transfected with the cDNAs for inflammasome components. Also used were peripheral blood mononuclear cells (PBMCs) and human monocyte-derived macrophages (HMDMs) from healthy volunteers. The interaction of CARD8 and NLRP3 was studied by immunoprecipitation. The effect of CARD8 expression on IL-1β secretion was assessed by ELISA. CARD8 knockdown experiments were carried out by transfection of the specific siRNA into HMDMs.ResultsIn HEK293 cells, CARD8 interacted with wild-type NLRP3, but not with CAPS-associated mutant NLRP3. CARD8 significantly reduced IL-1β secretion from cells transfected with wild-type NLRP3, but not if they were transfected with mutant NLRP3. In addition, association of endogenously expressed CARD8 with NLRP3 was confirmed in resting PBMCs, and CARD8 knockdown resulted in higher amount of IL-1β secretion from HMDMs.ConclusionsUntil specific stimuli activate NLRP3, CARD8 holds NLRP3, and is supposed to prevent activation by subtle stimuli. However, CAPS-associated mutant NLRP3 is unable to bind with CARD8, which might be relevant to the pathogenesis of CAPS.

Highlights

NLRP3 plays a role in sensing various pathogen components or stresses in the innate immune system
Caspase recruitment domain-containing protein 8 (CARD8) significantly reduced IL-1β secretion from cells transfected with wild-type NLRP3, but not if they were transfected with mutant NLRP3
Association of endogenously expressed CARD8 with NLRP3 was confirmed in resting peripheral blood mononuclear cells (PBMCs), and CARD8 knockdown resulted in higher amount of IL-1β secretion from human monocyte-derived macrophages (HMDMs)

Summary

Introduction

NLRP3 plays a role in sensing various pathogen components or stresses in the innate immune system. NLRP3 associates with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 to form a large protein complex termed inflammasome. Some investigators have proposed a model of NLRP3-inflammasome containing an adaptor protein caspase recruitment domain-containing protein 8 (CARD8), the role of this molecule remains obscure. The NLR (nucleotide binding oligomerization domain-like receptor) protein family plays a role as an intracellular sensor for pathogens and cell injury by detecting conserved structures, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [1,2]. Mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene encoding NLRP3 result in cryopyrin-associated periodic syndromes (CAPS) characterized by recurrent episodes of systemic inflammatory attacks in the absence of infection or autoimmune diseases [10]. As for the mechanism by which those mutations activate NLRP3 leading to autoinflammation, Lee et al have recently noticed the role of the calcium-sensing receptor that regulates NLRP3 inflammasome through Ca2+ and cAMP [12], but other mechanisms might be contributed as well

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