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Phenotypical and Functional Characterization of Neutrophils in Two Pyrin-Associated Auto-inflammatory Diseases

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Abstract

Purpose

Familial Mediterranean Fever (FMF) and Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) are clinically distinct autoinflammatory disorders caused by mutations in the pyrin-encoding gene MEFV. We investigated the transcriptional, phenotypical, and functional characteristics of patient neutrophils to explore their potential role in FMF and PAAND pathophysiology.

Methods

RNA sequencing was performed to discover transcriptional aberrancies. The phenotypical features, degranulation properties, and phagocytic capacity of neutrophils were assessed by flow cytometry. Production of reactive oxygen species (ROS), myeloperoxidase (MPO) release, and chemotactic responses were investigated via chemiluminescence, ELISA, and Boyden chamber assays, respectively.

Results

Neutrophils from PAAND and FMF patients showed a partially overlapping, activated gene expression profile with increased expression of S100A8, S100A9, S100A12, IL-4R, CD48, F5, MMP9, and NFKB. Increased MMP9 and S100A8/A9 expression levels were accompanied by high plasma concentrations of the encoded proteins. Phenotypical analysis revealed that neutrophils from FMF patients exhibited an immature character with downregulation of chemoattractant receptors CXCR2, C5aR, and BLTR1 and increased expression of Toll-like receptor 4 (TLR4) and TLR9. PAAND neutrophils displayed an increased random, but reduced CXCL8-induced migration. A tendency for enhanced random migration was observed for FMF neutrophils. PAAND neutrophils showed a moderately but significantly enhanced phagocytic activity as opposed to neutrophils from FMF patients. Neutrophils from both patient groups showed increased MPO release and ROS production.

Conclusions

Neutrophils from patients with FMF and PAAND, carrying different mutations in the MEFV gene, share a pro-inflammatory phenotype yet demonstrate diverse features, underscoring the distinction between both diseases.

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Data Availability

All data are described in the manuscript and supplementary data.

Code Availability

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References

  1. Masters SL, Simon A, Aksentijevich I, Kastner DL. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu Rev Immunol. 2009;27:621–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. de Jesus AA, Canna SW, Liu Y, Goldbach-Mansky R. Molecular mechanisms in genetically defined autoinflammatory diseases: disorders of amplified danger signaling. Annu Rev Immunol. 2015;33:823–74.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Harapas CR, Steiner A, Davidson S, Masters SL. An update on autoinflammatory diseases: inflammasomopathies. Curr Rheumatol Rep. 2018;20:40.

    Article  PubMed  Google Scholar 

  4. Mathur A, Hayward JA, Man SM. Molecular mechanisms of inflammasome signaling. J Leukoc Biol. 2018;103:233–57.

    CAS  PubMed  Google Scholar 

  5. Hayward JA, Mathur A, Ngo C, Man SM. Cytosolic recognition of microbes and pathogens: inflammasomes in action. Microbiol Mol Biol Rev. 2018;82.

  6. Liston A, Masters SL. Homeostasis-altering molecular processes as mechanisms of inflammasome activation. Nat Rev Immunol. 2017;17:208–14.

    Article  CAS  PubMed  Google Scholar 

  7. Centola M, Wood G, Frucht DM, Galon J, Aringer M, Farrell C, et al. The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyt development and is regulated in response to inflammatory mediators. Blood. 2000;95:3223–31.

    Article  CAS  PubMed  Google Scholar 

  8. Heilig R, Broz P. Function and mechanism of the pyrin inflammasome. Eur J Immunol. 2018;48:230–8.

    Article  CAS  PubMed  Google Scholar 

  9. de Torre-Minguela C, Mesa Del Castillo P, Pelegrin P. The NLRP3 and pyrin inflammasomes: implications in the pathophysiology of autoinflammatory diseases. Front Immunol. 2017;8:43.

    PubMed  PubMed Central  Google Scholar 

  10. Park YH, Wood G, Kastner DL, Chae JJ. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat Immunol. 2016;17:914–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Aubert DF, Xu H, Yang J, Shi X, Gao W, Li L, et al. A burkholderia type VI effector deamidates Rho GTPases to activate the pyrin inflammasome and trigger inflammation. Cell Host Microbe. 2016;19:664–74.

    Article  CAS  PubMed  Google Scholar 

  12. Xu H, Yang J, Gao W, Li L, Li P, Zhang L, et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature. 2014;513:237–41.

    Article  CAS  PubMed  Google Scholar 

  13. Gavrilin MA, Abdelaziz DHA, Mostafa M, Abdulrahman BA, Grandhi J, Akhter A, et al. Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia. J Immunol. 2012;188:3469–77.

    Article  CAS  PubMed  Google Scholar 

  14. Dumas A, Amiable N, de Rivero Vaccari JP, Chae JJ, Keane RW, Lacroix S, et al. The inflammasome pyrin contributes to pertussis toxin-induced IL-1beta synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis. PLoS Pathog. 2014;10:e1004150.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Manukyan G, Aminov R. Update on Pyrin functions and mechanisms of familial mediterranean fever. Front Microbiol. 2016;7:456.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Federici S, Sormani MP, Ozen S, Lachmann HJ, Amaryan G, Woo P, et al. Evidence-based provisional clinical classification criteria for autoinflammatory periodic fevers. Ann Rheum Dis. 2015;74:799–805.

    Article  PubMed  Google Scholar 

  17. Moghaddas F, Llamas R, De Nardo D, Martinez-Banaclocha H, Martinez-Garcia JJ, Mesa-Del-Castillo P, et al. A novel pyrin-associated autoinflammation with neutrophilic dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to familial mediterranean fever. Ann Rheum Dis. 2017;76:2085–94.

    Article  CAS  PubMed  Google Scholar 

  18. Masters SL, Lagou V, Jeru I, Baker PJ, Van Eyck L, Parry DA, et al. Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation. Sci Transl Med. 2016;8:332ra45.

    Article  PubMed  Google Scholar 

  19. Ozen S, Demirkaya E, Erer B, Livneh A, Ben-Chetrit E, Giancane G, et al. EULAR recommendations for the management of familial Mediterranean fever. Ann Rheum Dis. 2016;75:644–51.

    Article  CAS  PubMed  Google Scholar 

  20. Liew PX, Kubes P. The neutrophil’s role during health and disease. Physiol Rev. 2019;99:1223–48.

    Article  CAS  PubMed  Google Scholar 

  21. Nauseef WM, Borregaard N. Neutrophils at work. Nat Immunol. 2014;15:602–11.

    Article  CAS  PubMed  Google Scholar 

  22. Manukyan G, Petrek M, Kriegova E, Ghazaryan K, Fillerova R, Boyajyan A. Activated phenotype of circulating neutrophils in familial Mediterranean fever. Immunobiology. 2013;218:892–8.

    Article  CAS  PubMed  Google Scholar 

  23. Mitroulis I, Kourtzelis I, Kambas K, Chrysanthopoulou A, Ritis K. Evidence for the involvement of mTOR inhibition and basal autophagy in familial Mediterranean fever phenotype. Hum Immunol. 2011;72:135–8.

    Article  CAS  PubMed  Google Scholar 

  24. Apostolidou E, Skendros P, Kambas K, Mitroulis I, Konstantinidis T, Chrysanthopoulou A, et al. Neutrophil extracellular traps regulate IL-1beta-mediated inflammation in familial Mediterranean fever. Ann Rheum Dis. 2016;75:269–77.

    Article  CAS  PubMed  Google Scholar 

  25. Stoler I, Freytag J, Orak B, Unterwalder N, Henning S, Heim K, et al. Gene-dose effect of MEFV gain-of-function mutations determines ex vivo neutrophil activation in familial mediterranean fever. Front Immunol. 2020;11:716.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G. Zymography methods for visualizing hydrolytic enzymes. Nat Methods. 2013;10:211–20.

    Article  CAS  PubMed  Google Scholar 

  27. Metzemaekers M, Vandendriessche S, Berghmans N, Gouwy M, Proost P. Truncation of CXCL8 to CXCL8(9-77) enhances actin polymerization and in vivo migration of neutrophils. J Leukoc Biol. 2020;107:1167–73.

    Article  CAS  PubMed  Google Scholar 

  28. De Buck M, Berghmans N, Portner N, Vanbrabant L, Cockx M, Struyf S, et al. Serum amyloid A1alpha induces paracrine IL-8/CXCL8 via TLR2 and directly synergizes with this chemokine via CXCR2 and formyl peptide receptor 2 to recruit neutrophils. J Leukoc Biol. 2015;98:1049–60.

    Article  PubMed  Google Scholar 

  29. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Marini O, Costa S, Bevilacqua D, Calzetti F, Tamassia N, Spina C, et al. Mature CD10 + and immature CD10 − neutrophils present in G-CSF-treated donors display opposite effects on T cells. Blood. 2017;129:3271.

    Article  Google Scholar 

  31. Sengelov H, Follin P, Kjeldsen L, Lollike K, Dahlgren C, Borregaard N. Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J Immunol. 1995;154:4157–65.

    Article  CAS  PubMed  Google Scholar 

  32. Guma M, Ronacher L, Liu-Bryan R, Takai S, Karin M, Corr M. Caspase 1-independent activation of interleukin-1beta in neutrophil-predominant inflammation. Arthritis Rheum. 2009;60:3642–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mankan AK, Dau T, Jenne D, Hornung V. The NLRP3/ASC/Caspase-1 axis regulates IL-1beta processing in neutrophils. Eur J Immunol. 2012;42:710–5.

    Article  CAS  PubMed  Google Scholar 

  34. Karmakar M, Sun Y, Hise AG, Rietsch A, Pearlman E. Cutting edge: IL-1beta processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and caspase-1. J Immunol. 2012;189:4231–5.

    Article  CAS  PubMed  Google Scholar 

  35. Chen KW, Gross CJ, Sotomayor FV, Stacey KJ, Tschopp J, Sweet MJ, et al. The neutrophil NLRC4 inflammasome selectively promotes IL-1beta maturation without pyroptosis during acute Salmonella challenge. Cell Rep. 2014;8:570–82.

    Article  CAS  PubMed  Google Scholar 

  36. Karmakar M, Katsnelson M, Malak HA, Greene NG, Howell SJ, Hise AG, et al. Neutrophil IL-1beta processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on K+ efflux. J Immunol. 2015;194:1763–75.

    Article  CAS  PubMed  Google Scholar 

  37. Perez-Figueroa E, Torres J, Sanchez-Zauco N, Contreras-Ramos A, Alvarez-Arellano L, Maldonado-Bernal C. Activation of NLRP3 inflammasome in human neutrophils by Helicobacter pylori infection. Innate Immun. 2016;22:103–12.

    Article  CAS  PubMed  Google Scholar 

  38. Mohammadi N, Midiri A, Mancuso G, Patane F, Venza M, Venza I, et al. Neutrophils directly recognize group B streptococci and contribute to interleukin-1beta production during infection. PLoS One. 2016;11:e0160249.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Balci-Peynircioglu B, Akkaya-Ulum YZ, Avci E, Batu ED, Purali N, Ozen S, et al. Potential role of pyrin, the protein mutated in familial Mediterranean fever, during inflammatory cell migration. Clin Exp Rheumatol. 2018;36:116–24.

    PubMed  Google Scholar 

  40. Ramos MV, Ruggieri M, Panek AC, Mejias MP, Fernandez-Brando RJ, Abrey-Recalde MJ, et al. Association of haemolytic uraemic syndrome with dysregulation of chemokine receptor expression in circulating monocytes. Clin Sci. 2015;129:235–44.

    Article  CAS  Google Scholar 

  41. Ravi AK, Plumb J, Gaskell R, Mason S, Broome CS, Booth G, et al. COPD monocytes demonstrate impaired migratory ability. Respir Res. 2017;18:90.

    Article  PubMed  PubMed Central  Google Scholar 

  42. D’Amico G, Frascaroli G, Bianchi G, Transidico P, Doni A, Vecchi A, et al. Uncoupling of inflammatory chemokine receptors by IL-10: generation of functional decoys. Nat Immunol. 2000;1:387–91.

    Article  PubMed  Google Scholar 

  43. Metzemaekers M, Gouwy M, Proost P. Neutrophil chemoattractant receptors in health and disease: double-edged swords. Cell Mol Immunol. 2020;17:433–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Lidar M, Scherrmann J-M, Shinar Y, Chetrit A, Niel E, Gershoni-Baruch R, et al. Colchicine nonresponsiveness in familial Mediterranean fever: clinical, genetic, pharmacokinetic, and socioeconomic characterization. Semin Arthritis Rheum. 2004;33:273–82.

    Article  CAS  PubMed  Google Scholar 

  45. Bonfoco E, Ceccatelli S, Manzo L, Nicotera P. Colchicine induces apoptosis in cerebellar granule cells. Exp Cell Res. 1995;218:189–200.

    Article  CAS  PubMed  Google Scholar 

  46. Dalbeth N, Lauterio TJ, Wolfe HR. Mechanism of action of colchicine in the treatment of gout. Clin Ther. 2014;36:1465–79.

    Article  CAS  PubMed  Google Scholar 

  47. Coelho FM, Pinho V, Amaral FA, Sachs D, Costa VV, Rodrigues DH, et al. The chemokine receptors CXCR1/CXCR2 modulate antigen-induced arthritis by regulating adhesion of neutrophils to the synovial microvasculature. Arthritis Rheum. 2008;58:2329–37.

    Article  PubMed  Google Scholar 

  48. Angelidis C, Kotsialou Z, Kossyvakis C, Vrettou A-R, Zacharoulis A, Kolokathis F, et al. Colchicine pharmacokinetics and mechanism of action. Curr Pharm Des. 2018;24:659–63.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank all patients and healthy volunteers.

Funding

This work was supported by the Research Foundation-Flanders (FWO-Vlaanderen) (G.0808.18 N), a “C1” grant (C16/17/010) from KU Leuven, the Rega Foundation, and received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 779295. M.M. obtained a PhD fellowship supported by the L’Oréal–UNESCO for Women in Science initiative and the FWO-Vlaanderen. E.V.N. is an FWO SB fellow.

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Author notes

  1. Bert Malengier-Devlies and Mieke Metzemaekers contributed equally. Paul Proost, Patrick Matthys, and Carine Wouters contributed equally.

Authors and Affiliations

  1. Laboratory of Immunobiology, Rega Institute, KU Leuven, Leuven, Belgium

    Bert Malengier-Devlies, Lien De Somer, Patrick Matthys & Carine Wouters

  2. Laboratory of Molecular Immunology, Rega Institute, KU Leuven, Leuven, Belgium

    Mieke Metzemaekers, Mieke Gouwy, Maaike Cockx, Lotte Vanbrabant, Noëmie Pörtner, Sofie Struyf & Paul Proost

  3. Division of Pediatric Rheumatology, Department Pediatrics, University Hospitals Leuven, Leuven, Belgium

    Erika Van Nieuwenhove, Lien De Somer & Carine Wouters

  4. Laboratory of Clinical Infectious and Inflammatory Disorders, KU Leuven, Leuven, Belgium

    Albrecht Betrains & Steven Vanderschueren

  5. European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases (RITA), University Hospital Leuven, Leuven, Belgium

    Albrecht Betrains, Lien De Somer, Steven Vanderschueren, Ellen De Langhe & Carine Wouters

  6. Laboratory of Clinical en Epidemiological Virology, KU Leuven, Leuven, Belgium

    Jurgen Vercauteren

  7. Division of General Internal Medicine, University Hospitals Leuven, Leuven, Belgium

    Steven Vanderschueren

  8. Division of Rheumatology, University Hospitals Leuven, Leuven, Belgium

    Ellen De Langhe

  9. Laboratory of Tissue Homeostasis and Disease, KU Leuven, Leuven, Belgium

    Ellen De Langhe

Authors
  1. Bert Malengier-Devlies
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  2. Mieke Metzemaekers
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  3. Mieke Gouwy
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  4. Erika Van Nieuwenhove
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Contributions

All authors contributed to the study conception and design. Erika Van Nieuwenhove, Albrecht Betrains, Lien De Somer, Steven Vanderschueren, Ellen De Langhe, and Carine Wouters were responsible for diagnosis and recruitment of patients and/or collection of clinical data. Bert Malengier-Devlies, Mieke Metzemaekers, Mieke Gouwy, Maaike Cockx, Lotte Vanbrabant, and Noëmie Pörtner performed experiments and analyzed data. Bert Malengier-Devlies and Mieke Metzemaekers performed statistical analysis under supervision of Jurgen Vercauteren. Paul Proost, Patrick Matthys, and Carine Wouters jointly supervised the study. The first draft of the manuscript was written by Bert Malengier-Devlies and Mieke Metzemaekers and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Paul Proost or Carine Wouters.

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Ethical approval related to patient samples is described in the “Patients and Methods” section.

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Reference to informed consent of patients is described in the “Patients and Methods” section.

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Conflict of Interest

Carine Wouters obtained unrestricted grants to KU Leuven from Novartis, Roche, GSK immuno-inflammation and Pfizer.

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Malengier-Devlies, B., Metzemaekers, M., Gouwy, M. et al. Phenotypical and Functional Characterization of Neutrophils in Two Pyrin-Associated Auto-inflammatory Diseases. J Clin Immunol 41, 1072–1084 (2021). https://doi.org/10.1007/s10875-021-01008-4

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