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Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation

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Abstract

Objective and design

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.

Materials or subjects

Human neutrophils and PBMCs were isolated from peripheral blood of healthy volunteers. PBMCs were differentiated into macrophages in vitro using human M-CSF.

Treatment

Human neutrophils were pretreated with macrophage-conditioned media, neutrophil-conditioned media, recombinant human IL-1β or anakinra prior to stimulation by MSU crystals.

Method

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.

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Abbreviations

NETs:

Neutrophil extracellular traps

AggNET:

Aggregated neutrophil extracellular traps

PMN:

Polymorphonuclear neutrophils

IL-1β:

Interleukin-1 beta

PBMC:

Peripheral blood mononuclear cells

NLRP3:

NACHT, LRR, and PYD domains-containing protein 3

ROS:

Reactive oxygen species

MSU:

Monosodium urate

MPO:

Myeloperoxidase

NE:

Neutrophil elastase

HNE:

Human neutrophil elastase

M-CSF:

Macrophage colony stimulating factor

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Acknowledgements

We thank the personnel at the UGA University Health Center for blood collection and the College of Veterinary Medicine Imaging Core for technical assistance with confocal microscopy and flow cytometry. We are also grateful to Dr. Pramod Giri (UGA) for his help with optimizing the macrophage differentiation protocol. We are also thankful for Dr. Jeremy Sokolove (Stanford University) for providing clinical grade anakinra (Swedish Orphan Biovitrum; purchased via Stanford University Research Pharmacy) used in this study. This work was supported by the start-up fund of Dr. Rada provided by the Office of Vice President for Research, UGA.

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Authors and Affiliations

  1. Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, 501 D.W. Brooks Drive, Athens, GA, 30602, USA

    Payel Sil, Haley Wicklum, Chandler Surell & Balázs Rada

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  1. Payel Sil
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Correspondence to Balázs Rada.

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Sil, P., Wicklum, H., Surell, C. et al. Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation. Inflamm. Res. 66, 227–237 (2017). https://doi.org/10.1007/s00011-016-1008-0

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  • DOI: https://doi.org/10.1007/s00011-016-1008-0

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