title: Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis.
abstract: BACKGROUND
  Interleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.
  
  
  METHODS
  Intracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells.
  
  
  RESULTS
  Active RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.
  
  
  CONCLUSIONS
  Patients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.
authors.affiliation: Rheumatology, Clinical Immunology and Allergy, Faculty of Medicine, University of Crete, 71003, Iraklion, Greece. choulaki@med.uoc.gr.
authors.forename: Christianna
authors.initials: C
authors.lastname: Choulaki
authors.affiliation: Rheumatology, Clinical Immunology and Allergy, Faculty of Medicine, University of Crete, 71003, Iraklion, Greece. litsapap85@yahoo.com.
authors.forename: Garyfallia
authors.initials: G
authors.lastname: Papadaki
authors.affiliation: Rheumatology, Clinical Immunology and Allergy, Faculty of Medicine, University of Crete, 71003, Iraklion, Greece. arrepa2002@yahoo.gr.
authors.forename: Argyro
authors.initials: A
authors.lastname: Repa
authors.affiliation: Rheumatology, Clinical Immunology and Allergy, Faculty of Medicine, University of Crete, 71003, Iraklion, Greece. elenkabouraki@yahoo.gr.
authors.forename: Eleni
authors.initials: E
authors.lastname: Kampouraki
authors.affiliation: Laboratory of Molecular Hematology, Democritus University of Thrace, 68100, Alexandroupolis, Greece. kkampas@hotmail.com.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Kambas
authors.affiliation: Laboratory of Molecular Hematology, Democritus University of Thrace, 68100, Alexandroupolis, Greece. kritis@med.duth.gr.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Ritis
authors.affiliation: Rheumatology, Clinical Immunology and Allergy, Faculty of Medicine, University of Crete, 71003, Iraklion, Greece. gbert@med.uoc.gr.
authors.forename: George
authors.initials: G
authors.lastname: Bertsias
authors.affiliation: Division of Infections and Immunity, Institute of Molecular Biology and Biotechnology-FORTH, Nikolaou Plastira 100, 70013, Iraklion, Greece. boumpasd@med.uoc.gr.|Biomedical Research Foundation of the Academy of Athens, 4 Soranou Ephessiou, 115 27, Athens, Greece. boumpasd@med.uoc.gr.|4th Department of Medicine, Rheumatology and Clinical Immunology, National and Kapodistrian University, Athens, Greece. boumpasd@med.uoc.gr.
authors.forename: Dimitrios T
authors.initials: DT
authors.lastname: Boumpas
authors.affiliation: Rheumatology, Clinical Immunology and Allergy, Faculty of Medicine, University of Crete, 71003, Iraklion, Greece. sidiropp@med.uoc.gr.
authors.forename: Prodromos
authors.initials: P
authors.lastname: Sidiropoulos
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D058847:Inflammasomes / Q000097:blood / Q000276:immunology*; D007962:Leukocytes / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein
chemical_list: D002352:Carrier Proteins; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human