title: CARD8 is a negative regulator for NLRP3 inflammasome, but mutant NLRP3 in cryopyrin-associated periodic syndromes escapes the restriction.
abstract: BACKGROUND
  NLRP3 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).
  
  
  METHODS
  In 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Until 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.
authors: Sayaka
authors: S
authors: Ito
authors: Yukichi
authors: Y
authors: Hara
authors: Tetsuo
authors: T
authors: Kubota
mesh_terms: D053468:CARD Signaling Adaptor Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D056587:Cryopyrin-Associated Periodic Syndromes / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D058847:Inflammasomes / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009154:Mutation; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D009363:Neoplasm Proteins / Q000378:metabolism*; D014162:Transfection
chemical_list: D053468:CARD Signaling Adaptor Proteins; C455623:CARD8 protein, human; D002352:Carrier Proteins; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D009363:Neoplasm Proteins