Abstract
Mature erythrocytes (red blood cells (RBCs)) undergo the programmed cell death (PCD) pathway of necroptosis in response to bacterial pore-forming toxins (PFTs) that target human CD59 (hCD59) but not hCD59-independent PFTs. Here, we investigate the biochemical mechanism of RBC necroptosis with a focus on the mechanism of induction and the minimal requirements for such RBC death. Binding or crosslinking of the hCD59 receptor led to Syk-dependent induction of vesiculated morphology (echinocytes) that was associated with phosphorylation of Band 3 and was required for Fas ligand (FasL) release. FasL-dependent phosphorylation of receptor-interacting protein kinase 1 (RIP1) in combination with plasma membrane pore formation was required for execution of RBC necroptosis. RIP1 phosphorylation led to the phosphorylation of RIP3, which was also critical for RBC necroptosis. Notably, RBC necroptosis was mediated by FasL and not by other candidate inducers, including tumor necrosis factor alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL). Other types of RBC damage, such as eryptotic damage, failed to induce necroptosis when combined with hCD59 crosslinking. This work sheds light on the requirements for this recently discovered PCD in RBCs and provides a clear picture of the biochemical mechanism of induction of RBC necroptosis.
Highlights
Proteins that induce plasma membrane pores are a common biological theme and may be pathogen produced, such as bacterial pore-forming toxins (PFTs), or host produced, such as the membrane attack complex (MAC) of the complement system.[3,4] Pore formation may kill cells through osmotic lysis or may activate target cell signaling pathways including proinflammatory, membrane repair, and programmed cell death (PCD) modules.[4]
We previously demonstrated that RBC necroptosis, induced by the human CD59 (hCD59)-specific PFTs vaginolysin (VLY) and intermedilysin (ILY), is dependent on Fas-FasL signaling, as a monoclonal antibody targeting FasL significantly inhibited RBC death.[1]
The hCD59-independent PFTs, PLY and alpha toxin (A-tox), did not induce phosphorylation of Syk, consistent with the results of Band 3 phosphorylation and echinocyte formation. These results suggest that hCD59 signaling activates Src family kinases, which lead to Syk-dependent vesiculation that is required for FasL-dependent RBC necroptosis
Summary
Proteins that induce plasma membrane pores are a common biological theme and may be pathogen produced, such as bacterial PFTs, or host produced, such as the membrane attack complex (MAC) of the complement system.[3,4] Pore formation may kill cells through osmotic lysis or may activate target cell signaling pathways including proinflammatory, membrane repair, and PCD modules.[4] the specific mechanisms linking bacterial PFTs to RBC necroptosis have not been described. We show that hCD59 signaling, induced by receptor crosslinking, results in Syk-dependent phosphorylation of Band 3 leading to vesicle (echinocyte) formation and release of FasL. The FasLdependent phosphorylation of RIP1/RIP3 produces RBC death by necroptosis only when combined with functional pore formation. We show that RBC necroptosis is mediated only by FasL and not by other known necroptotic mediators/stimuli, including tumor necrosis factor alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL)
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