Tumour necrosis factor alpha promotes secretion of 14-3-3η by inducing necroptosis in macrophages

Arthritis Res Ther. 2020 Feb 12;22(1):24. doi: 10.1186/s13075-020-2110-9.

Abstract

Background: 14-3-3η is an intracellular protein also detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA). It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3η and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study.

Methods: The source of 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-α. Extracellular 14-3-3η protein levels were examined by western blotting.

Results: Macrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R.

Conclusions: Macrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid.

Keywords: 14-3-3η; Macrophage; Necroptosis; Rheumatoid arthritis; TNF-α.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / immunology
  • 14-3-3 Proteins / metabolism*
  • Arthritis, Rheumatoid* / immunology
  • Arthritis, Rheumatoid* / metabolism
  • Arthritis, Rheumatoid* / pathology
  • Humans
  • Macrophages / immunology
  • Macrophages / metabolism*
  • Macrophages / pathology
  • Necroptosis / immunology*
  • Synovial Fluid / immunology
  • Synovial Fluid / metabolism
  • Synovial Membrane / immunology
  • Synovial Membrane / metabolism
  • Synovial Membrane / pathology
  • Tumor Necrosis Factor-alpha / immunology
  • Tumor Necrosis Factor-alpha / metabolism*

Substances

  • 14-3-3 Proteins
  • Tumor Necrosis Factor-alpha
  • YWHAH protein, human