THU0057B TNF-ALPHA INDUCES NECROPTOSIS-LIKE DEATH OF MACROPHAGES AND PROMOTES EXTRACELLULAR RELEASE OF 14–3–3ETA
Authors: Gulzhan Trimova, Kaoru Yamagata, Shigeru Iwata, Tong Zhang, Fumi Uemura, Minoru Satoh, Michelle Zaharik, Norma Biln, Shintaro Hirata, Shingo Nakayamada, Yoshiya Tanaka
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> 14-3-3η is an intracellular protein detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA) [1]. While presence of 14-3-3η is both diagnostic for early and established RA [2] and prognostic for radiographic progression [3], the mechanism of 14-3-3η externalization in RA remains unclear. <h3>Objectives</h3> To clarify the mechanism of externalization of 14-3-3η into the extracellular space using human PBMC-derived macrophages (Mϕ). <h3>Methods</h3> Distribution of 14-3-3η in synovial tissue of patients with RA or osteoarthritis (OA) was examined by immunohistochemistry; cellular morphology was studied by confocal microscopy and electron microscopy (EM). Mϕ were stimulated with TNF-α, Diamide (induces ligand-independent TNFR signaling) or IL-6/sIL-6R. Western blotting was used to detect S-358-phosphorylated MLKL (a mediator of necroptosis) and presence of 14-3-3η in Mϕ culture supernatants. <h3>Results</h3> Dense and widespread staining of 14-3-3η was detected in Mϕ of RA, but not OA, synovial tissues. 14-3-3η and peptidylarginine deiminase 4 (PAD4) co-localized in CD68<sup>+</sup> cells (Mϕ) from RA synovial tissue; 14-3-3η was not detected in CD68<sup>+</sup> cells from RA lung tissue or CD4<sup>+</sup> cells (T cells) from RA synovium. The outer space around the nucleus of healthy control Mϕ treated with TNF-α or Diamide, but not IL-6/sIL-6R, demonstrated abnormal actin distribution by phalloidin staining and presence of cellular and organelle swelling by EM. Further, magnified images showed partial destruction of the cell membrane in TNF-α and Diamide-treated cells. Phosphorylation of MLKL was observed between 20 min and 24 h after stimulation of healthy control Mϕ with TNF-α, with maximum signal at 8 h post-stimulation, but was not observed upon IL-6/sIL-6R stimulation at any time point. After 8 h of stimulation no 14-3-3η was detected in the culture supernatant of healthy control Mϕ endogenously expressing 14-3-3η or Mϕ stimulated with IL-6/sIL-6R, while a high concentration of 14-3-3η was detected in culture supernatants in Mϕ treated with TNF-α or Diamide. <h3>Conclusion</h3> 14-3-3η protein was abundant in RA, but not OA, synovial tissues and co-localized with PAD4 in CD68<sup>+</sup> synovial Mϕ; this close proximity to PAD4 may promote citrullination of 14-3-3η <i>in vivo</i>. Treatment of healthy control Mϕ with TNF-α induced phosphorylation of MLKL and cell swelling and disintegration of the plasma membrane characteristic of necroptosis, correlated with the release of intracellular 14-3-3η protein into cellular supernatants. Our results shed light on mechanism of externalization of 14-3-3η and how it achieves elevated levels in RA synovial fluid. <h3>References</h3> [1] Kilani RT, et al. J Rheumatol 2007; 34:1650–7. [2] Maksymowych W,&nbsp;et al.&nbsp;J Rheumatol&nbsp;2014;&nbsp;41:2104-13. [3] Carrier N, et al. Arthritis Res Ther 2016; 18:50. <h3>Competing Interests</h3> M. Zaharik and N. Biln are employees of Augurex. Y. Tanaka, has received speaking fees and/or honoraria from Daiichi-Sankyo, Astellas, Eli Lilly, Chugai, Sanofi, Abbvie, Pfizer, YL Biologics, Bristol-Myers, Glaxo-Smithkline, UCB, Mitsubishi-Tanabe, Novartis, Eisai, Takeda, Janssen, Asahi-kasei and has received research grants from Mitsubishi-Tanabe, Bristol-Myers, Eisai, Chugai, Takeda, Abbvie, Astellas, Daiichi-Sankyo, Ono, MSD, Taisho-Toyama. All other authors have nothing to disclose. <h3>Disclosure of Interests</h3> Gulzhan Trimova: None declared, Kaoru Yamagata: None declared, Shigeru Iwata: None declared, Tong Zhang: None declared, Fumi Uemura: None declared, Minoru Satoh: None declared, Michelle Zaharik: None declared, Norma Biln Shareholder of: Amgen, Employee of: Pfizer, Amgen and Abbott, Shintaro Hirata Grant/research support from: Eli Lilly, UCB, Consultant for: Bristol-Myers Squibb, Jansen, UCB, Paid instructor for: AbbVie, Eisai, Tanabe-Mitsubishi, Speakers bureau: AbbVie, Astellas, Ayumi, Bristol-Myers Squibb, Chugai, Eisai, Eli Lilly, Jansen, Kissei, Pfizer, Sanofi, Takeda, Tanabe-Mitsubishi, UCB, Shingo Nakayamada Grant/research support from: Mitsubishi-Tanabe, Takeda, Novartis and MSD, Speakers bureau: Bristol-Myers, Sanofi, Abbvie, Eisai, Eli Lilly, Chugai, Asahi-kasei and Pfizer, Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics