title: Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation.
abstract: OBJECTIVE
  Arthritic gout is caused by joint inflammation triggered by the damaging effects of monosodium uric acid (MSU) crystal accumulation in the synovial space. Neutrophils play a major role in mediating joint inflammation in gout. Along with neutrophils, other immune cells, such as macrophages, are present in inflamed joints and contribute to gout pathogenesis. Neutrophils form neutrophil extracellular traps (NETs) in response to MSU crystals. In the presence of MSU crystals, macrophages release IL-1β, a cytokine crucial to initiate gout pathogenesis and neutrophil recruitment. Our research investigated interactions between human macrophages and neutrophils in an in vitro model system and asked how macrophages affect NET formation stimulated by MSU crystals.
  
  
  METHODS
  Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF.
  
  
  METHODS
  Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1β or anakinra prior to stimulation by MSU crystals.
  
  
  METHODS
  Interaction of neutrophils with MSU crystals was evaluated by live imaging using confocal microscopy. The presence of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured by ELISA. NET formation was quantitated by Sytox Orange-based extracellular DNA release assay and NE-DNA ELISA. AggNET formation was assessed by macroscopic evaluation.
  
  
  RESULTS
  We found that crystal- and cell-free supernatants of macrophages stimulated with MSU crystals promote MSU crystal-stimulated NET formation in human neutrophils. This observation was confirmed by additional assays measuring the release of MPO, NE, and the enzymatic activity of NE. MSU crystal-induced NET formation remained unchanged when neutrophil supernatants were tested. IL-1β is a crucial cytokine orchestrating the onset of inflammation in gout and is known to be released in large amounts from macrophages following MSU crystal stimulation. We found that recombinant IL-1β strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1β alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1β is mainly responsible for this effect.
  
  
  CONCLUSIONS
  Macrophage-derived IL-1β enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1β affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra.
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, 501 D.W. Brooks Drive, Athens, GA, 30602, USA.
authors.forename: Payel
authors.initials: P
authors.lastname: Sil
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, 501 D.W. Brooks Drive, Athens, GA, 30602, USA.
authors.forename: Haley
authors.initials: H
authors.lastname: Wicklum
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, 501 D.W. Brooks Drive, Athens, GA, 30602, USA.
authors.forename: Chandler
authors.initials: C
authors.lastname: Surell
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, 501 D.W. Brooks Drive, Athens, GA, 30602, USA. radab@uga.edu.
authors.forename: Balázs
authors.initials: B
authors.lastname: Rada
mesh_terms: D065206:Extracellular Traps; D006073:Gout / Q000276:immunology; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000494:pharmacology; D053583:Interleukin-1beta / Q000276:immunology*; D016209:Interleukin-8 / Q000276:immunology; D019272:Leukocyte Elastase / Q000276:immunology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology*; D009195:Peroxidase / Q000276:immunology; D014527:Uric Acid / Q000494:pharmacology*
chemical_list: C508606:CXCL8 protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D053583:Interleukin-1beta; D016209:Interleukin-8; D014527:Uric Acid; D009195:Peroxidase; D019272:Leukocyte Elastase
keywords: Gout; Interleukin-1 beta; Macrophage; Neutrophil extracellular traps (NETs)