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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

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

References

  1. 1.

    Busso N, So A. Microcrystals as DAMPs and their role in joint inflammation. Rheumatology (Oxford). 2012;51:1154–60.

    CAS  Article  Google Scholar 

  2. 2.

    Neogi T. Clinical practice. Gout. N Engl J Med. 2011;364:443–52.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Mitroulis I, Kambas K, Ritis K. Neutrophils, IL-1beta, and gout: is there a link? Semin Immunopathol. 2013;35:501–12.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Pope RM, Tschopp J. The role of interleukin-1 and the inflammasome in gout: implications for therapy. Arthritis Rheum. 2007;56:3183–8.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Schauer C, Janko C, Munoz LE, Zhao Y, Kienhofer D, Frey B, et al. Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat Med. 2014;20:511–7.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Schorn C, Janko C, Krenn V, Zhao Y, Munoz LE, Schett G, et al. Bonding the foe—NETting neutrophils immobilize the pro-inflammatory monosodium urate crystals. Front Immunol. 2012;3:376.

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;440:237–41.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Oliveira SH, Canetti C, Ribeiro RA, Cunha FQ. Neutrophil migration induced by IL-1beta depends upon LTB4 released by macrophages and upon TNF-alpha and IL-1beta released by mast cells. Inflammation. 2008;31:36–46.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Schett G, Dayer JM, Manger B. Interleukin-1 function and role in rheumatic disease. Nat Rev Rheumatol. 2016;12:14–24.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Schlesinger N. Canakinumab in gout. Expert Opin Biol Ther. 2012;12:1265–75.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Schlesinger N. Treatment of chronic gouty arthritis: it is not just about urate-lowering therapy. Semin Arthritis Rheum. 2012;42:155–65.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Amaral FA, Costa VV, Tavares LD, Sachs D, Coelho FM, Fagundes CT, et al. NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B(4) in a murine model of gout. Arthritis Rheum. 2012;64:474–84.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Rada B, Park JJ, Sil P, Geiszt M, Leto TL. NLRP3 inflammasome activation and interleukin-1beta release in macrophages require calcium but are independent of calcium-activated NADPH oxidases. Inflamm Res. 2014;63:821–30.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Meissner F, Seger RA, Moshous D, Fischer A, Reichenbach J, Zychlinsky A. Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease. Blood. 2010;116:1570–3.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    van Bruggen R, Koker MY, Jansen M, van Houdt M, Roos D, Kuijpers TW, et al. Human NLRP3 inflammasome activation is No 1–4 independent. Blood. 2010;115:5398–400.

    Article  PubMed  Google Scholar 

  16. 16.

    Busso N, So A. Mechanisms of inflammation in gout. Arthritis Res Ther. 2010;12:206.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Cronstein BN, Terkeltaub R. The inflammatory process of gout and its treatment. Arthritis Res Ther. 2006;8(Suppl 1):S3.

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Mitroulis I, Kambas K, Chrysanthopoulou A, Skendros P, Apostolidou E, Kourtzelis I, et al. Neutrophil extracellular trap formation is associated with IL-1beta and autophagy-related signaling in gout. PLoS One. 2011;6:e29318.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Naccache PH, Grimard M, Roberge CJ, Gilbert C, Lussier A, de Medicis R, et al. Crystal-induced neutrophil activation. I. Initiation and modulation of calcium mobilization and superoxide production by microcrystals. Arthritis Rheum. 1991;34:333–42.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Popa-Nita O, Naccache PH. Crystal-induced neutrophil activation. Immunol Cell Biol. 2010;88:32–40.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Pang L, Hayes CP, Buac K, Yoo DG, Rada B. Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps. J Immunol. 2013;190:6488–500.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–5.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Desai J, Kumar SV, Mulay SR, Konrad L, Romoli S, Schauer C, et al. PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling. Eur J Immunol. 2016;46:223–9.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Chhana A, Dalbeth N. The gouty tophus: a review. Curr Rheumatol Rep. 2015;17:19.

    Article  PubMed  Google Scholar 

  25. 25.

    Yoo DG, Floyd M, Winn M, Moskowitz SM, Rada B. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes. Immunol Lett. 2014;160:186–94.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Yoo DG, Winn M, Pang L, Moskowitz SM, Malech HL, Leto TL, et al. Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation. Journal of immunology. 2014;192:4728–38.

    CAS  Article  Google Scholar 

  27. 27.

    Sil P, Yoo DG, Floyd M, Gingerich A, Rada B. High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro. J Vis Exp. 2016;(112). doi:10.3791/52779.

  28. 28.

    Schorn C, Janko C, Latzko M, Chaurio R, Schett G, Herrmann M. Monosodium urate crystals induce extracellular DNA traps in neutrophils, eosinophils, and basophils but not in mononuclear cells. Front Immunol. 2012;3:277.

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Busso N, Ea HK. The mechanisms of inflammation in gout and pseudogout (CPP-induced arthritis). Reumatismo. 2011;63:230–7.

    Google Scholar 

  30. 30.

    Farrera C, Fadeel B. Macrophage clearance of neutrophil extracellular traps is a silent process. J Immunol. 2013;191:2647–56.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Nakazawa D, Shida H, Kusunoki Y, Miyoshi A, Nishio S, Tomaru U, et al. The responses of macrophages in interaction with neutrophils that undergo NETosis. J Autoimmun. 2016;67:19–28.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Warnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis. Science. 2015;349:316–20.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, Wooten MW. Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol Cell Biol. 2004;24:8055–68.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Chenevier-Gobeaux C, Lemarechal H, Bonnefont-Rousselot D, Poiraudeau S, Ekindjian OG, Borderie D. Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1beta and tumour necrosis factor-alpha. Inflamm Res. 2006;55:483–90.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Yan B, Han P, Pan L, Lu W, Xiong J, Zhang M, et al. IL-1beta and reactive oxygen species differentially regulate neutrophil directional migration and Basal random motility in a zebrafish injury-induced inflammation model. J Immunol. 2014;192:5998–6008.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Singh A, Zarember KA, Kuhns DB, Gallin JI. Impaired priming and activation of the neutrophil NADPH oxidase in patients with IRAK4 or NEMO deficiency. J Immunol. 2009;182:6410–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Perretti M, Appleton I, Parente L, Flower RJ. Pharmacology of interleukin-1-induced neutrophil migration. Agents Actions 1993;38 Spec No:C64–5.

  38. 38.

    Brinkmann V, Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol. 2007;5:577–82.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Kaplan MJ, Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol. 2012;189:2689–95.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Mayadas TN, Cullere X, Lowell CA. The multifaceted functions of neutrophils. Annu Rev Pathol. 2014;9:181–218.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Semerad CL, Liu F, Gregory AD, Stumpf K, Link DC. G-CSF is an essential regulator of neutrophil trafficking from the bone marrow to the blood. Immunity. 2002;17:413–23.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Torres R, Macdonald L, Croll SD, Reinhardt J, Dore A, Stevens S, et al. Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis. Ann Rheum Dis. 2009;68:1602–8.

    CAS  Article  PubMed  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Balázs Rada.

Ethics declarations

Conflict of interest

The authors have no financial conflicts of interest to report.

Additional information

Responsible Editor: John Di Battista.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Keywords

  • Neutrophil extracellular traps (NETs)
  • Interleukin-1 beta
  • Macrophage
  • Gout