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Differential ubiquitination in NETs regulates macrophage responses in systemic lupus erythematosus
  1. Ana Barrera-Vargas1,
  2. Diana Gómez-Martín2,
  3. Carmelo Carmona-Rivera3,
  4. Javier Merayo-Chalico1,
  5. Jiram Torres-Ruiz1,
  6. Zerai Manna4,
  7. Sarfaraz Hasni4,
  8. Jorge Alcocer-Varela1,
  9. Mariana J Kaplan3
  1. 1Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición, Salvador Zubirán, Mexico City, Mexico
  2. 2Department of Immunology and Rheumatology and Red de Apoyo a la Investigación, CIC-UNAM, Instituto Nacional de Ciencias Médicas y Nutrición, Salvador Zubirán, Mexico City, Mexico
  3. 3Systemic Autoimmunity Branch, Intramural Research Program, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
  4. 4Lupus Clinical Research Unit, Intramural Research Program, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
  1. Correspondence to Dr Mariana J Kaplan, Systemic Autoimmunity Branch/NIAMS, National Institutes of Health, Bethesda, MD 20892, USA; mariana.kaplan{at}nih.gov

Abstract

Objectives To assess if ubiquitinated proteins potentially present in neutrophil extracellular traps (NETs) can modify cellular responses and induce inflammatory mechanisms in patients with systemic lupus erythematosus (SLE) and healthy subjects.

Materials and methods We studied 74 subjects with SLE and 77 healthy controls. Neutrophils and low-density granulocytes were isolated, and NETs were induced. Ubiquitin content was quantified in NETs by western blot analysis, ELISA and immunofluorescence microscopy, while ubiquitination of NET proteins was assessed by immunoprecipitation. Monocyte-derived macrophages from SLE and controls were isolated and stimulated with NETs or ubiquitin. Calcium flux and cytokine synthesis were measured following these stimuli.

Results NETs contain ubiquitinated proteins, with a lower expression of polyubiquitinated proteins in subjects with SLE than in controls. Myeloperoxidase (MPO) is present in ubiquitinated form in NETs. Patients with SLE develop antiubiquitinated MPO antibodies, and titres positively correlate with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (P<0.01), and negatively correlate with complement components (P<0.01). Stimulation of monocyte-derived macrophages with NETs or with ubiquitin led to enhanced calcium flux. In addition, stimulation with NETs led to enhanced cytokine (tumour necrosis factor-α and interleukin-10) production in macrophages from patients with SLE when compared with controls, which was hampered by inhibition of NET internalisation by macrophages.

Conclusion This is the first study to find ubiquitinated proteins in NETs, and evidence for adaptive immune responses directed towards ubiquitinated NET proteins in SLE. The distinct differences in ubiquitin species profile in NETs compared with healthy controls may contribute to dampened anti-inflammatory responses observed in SLE. These results also support a role for extracellular ubiquitin in inflammation in SLE.

  • systemic lupus erythematosus
  • inflammation
  • cytokines

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Footnotes

  • AB-V and DG-M contributed equally.

  • Handling editor Josef S Smolen

  • Contributors AB-V: study design, data acquisition (experiments), data analysis, manuscript preparation. DG-M: study design, data acquisition (experiments), data analysis, manuscript preparation. CC-R: study design, data acquisition (experiments), data analysis. JM-C: data acquisition (experiments), data analysis. JJT-R: data acquisition (experiments), data analysis. ZGM: study design and data acquisition (clinical data). SH: study design and data acquisition (clinical data). JA-V: study design, manuscript revision. MJK: study design, manuscript preparation and revision.

  • Funding This study was supported by the Intramural Research Program at NIAMS/NIH (ZIA AR041199) and a grant from the Consejo Nacional de Ciencia y Tecnología (CONACYT, 255941, SEP-Ciencia Básica 2015).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval The protocol was approved by the local ethics committee at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (Ref. 1243) and at the NIAMS/NIDDK (94-AR-0066).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement No additional data is available.

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