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Front. Immunol. | doi: 10.3389/fimmu.2018.00306

RIPK1, RIPK3 and MLKL are activated by sublytic complement and participate in complement-dependent cytotoxicity

Michal Lusthaus1, Niv Mazkereth1, Natalie Donin1 and  Zvi Fishelson1*
  • 1Cell and Developmental Biology, Tel Aviv University, Israel

The complement system participates in the pathogenesis of many diseases. Complement activation produces several active protein complexes and peptides, including the terminal C5b-9 complexes. It was reported that C5b-9 complexes insert into the plasma membrane and cause membrane perturbation, intracellular calcium surge, metabolic depletion, and osmotic lysis. Previously, we showed that complement-dependent cytotoxicity (CDC) is regulated by JNK and Bid. Here, we demonstrate that three mediators in TNFα-induced necroptosis (regulated necrosis), the receptor-interacting protein kinases, RIPK1 and RIPK3, and mixed-lineage kinase domain-like protein (MLKL), are activated by complement and contribute to CDC. Cell treatment with necrostatin-1, a RIPK1 inhibitor, GSK'872, a RIPK3 inhibitor, or necrosulfonamide and GW806742X, MLKL inhibitors, restrain CDC. These findings were confirmed by using specific siRNAs targeting the synthesis of these proteins. Mouse fibroblasts lacking RIPK3 or MLKL were found to be less sensitive to C5b-9 than were wild-type fibroblasts. Enhanced CDC was achieved by RIPK1 or RIPK3 overexpression but not by the overexpression of a RHIM-RIPK1 mutant nor by a kinase-dead RIPK3 mutant. Necrostatin-1 reduces the CDC of wild-type but not of RIPK3-knockout fibroblasts. Cells treated with a sublytic dose of complement exhibit co-localization of RIPK3 with RIPK1 in the cytoplasm and co-localization of RIPK3 and MLKL with C5b-9 at the plasma membrane. Data supporting cooperation among the RIP kinases, MLKL, JNK, and Bid in CDC are presented. These results provide a deeper insight into the cell death process activated by complement and identify potential points of cross-talk between complement and other inducers of inflammation and regulated necrosis.

Keywords: complement, C5b-9, ripk1, ripk3, mlkl, Regulated necrosis

Received: 20 Dec 2017; Accepted: 05 Feb 2018.

Edited by:

Francesco Tedesco, Istituto Auxologico Italiano (IRCCS), Italy

Reviewed by:

Lubka T. Roumenina, INSERM UMRS 1138, Cordeliers Research Center, Complement and diseases team, France
Horea Rus, School of Medicine, University of Maryland, United States  

Copyright: © 2018 Lusthaus, Mazkereth, Donin and Fishelson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Zvi Fishelson, Tel Aviv University, Cell and Developmental Biology, Sackler School of Medicine, P.O.Box 39040, Tel Aviv, 69978, Israel,