Abstract
IntroductionInterleukin-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.MethodIntracellular 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.ResultsActive 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.ConclusionPatients 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.
Highlights
Interleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation
Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus adenosine triphosphate (ATP) was higher in rheumatoid arthritis (RA) patients (n = 20) versus controls (n = 18) (p < 0.02 for both)
Activation of NLRP3-inflammasome results in higher IL-1β secretion by peripheral blood cells in active RA We have shown that NLRP3-inflammasome-related proteins are overexpressed in freshly, unstimulated peripheral blood cells from RA patients with active disease
Summary
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. Inflammasomes are cytosolic multiprotein complexes that drive the production of inflammatory cytokines, mainly interleukin-1β (IL-1β) and IL-18, in response to pathogens or danger signals [1]. Inflammasomes are composed of a danger sensor, an adaptor protein that is mainly the apoptosis-associated speck-like protein containing a CARD (ASC protein), and caspase-1. There is remarkable diversity in the sensor proteins that form the inflammasomes, leading to the assembly of distinct complexes specialized to sense various signals. NALP3-inflammasome assembly results in ASC polymerization, procaspase-1 self-activation to the active protease, which catalyzes pro-IL1β maturation [3]
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