Necroptosis is a programmed cell death process occurring in vertebrate cells in response to infection by pathogens which inhibit the classic programmed cell death process apoptosis. Pathogens block apoptosis by their anti-capsase activity resulting in signalling to the necrosome resulting rapid cell death by necroptosis, thus minimizing the spread of the pathogen. other forms of necrotic cell death such as oncosis by a nonphysiological event resulting in cell swelling as opposed to apoptosis where there is cell shrinkage or necroptosis were there is controlled leakage of the cell contents through the plasma membrane.

 First, extrinsic stimulus through the TNF receptor by TNFα signals the recruitment of the TNF receptor-associated death domain (TRADD) which in turn recruits RIPK1. In the absence of active Caspase 8, RIPK1 and RIPK3 auto- and transphosphorylate each other, leading to the formation of a microfilament-like complex called the necrosome. he necrosome then activates the pro-necroptotic protein MLKL via phosphorylation. MLKL actuates the necrosis phenotype by inserting into the bilipid membranes of organelles and plasma membrane leading to expulsion of cellular contents into the extracellular space, see figure. The inflammatory rupturing of the cell releases Damage Associated Molecular Patterns (DAMPs) into the extracellular space. Many of these DAMPs remain unidentified, however, the “find me” and “eat me” DAMP signals are known to recruit immune cells to the damaged/infected tissue. Necrotic cells are cleared from the immune system by a mechanism called pinocytosis, or cellular drinking, which is mediated by macropinosomes, a subcellular component of macrophages. This process is in contrast to removal of apoptotic cells by the immune system in which cells are removed via phagocytosis, or cellular eating.

Necroptosis can also be induced by numerous drugs such as Shikonin, see figure. Necrostatin is known to inhibit necroptosis with a 1 hour pretreatment before exposure shikonin, see figure.

Flow Cytometric Measurement of Necroptosis

Induction of Jurkat cell necroptosis by 0.5 uM shikonin or 100 ng/ml TNF alpha (24 h) can be measured flow cytometrically by the use of a fixable live-dead stain (Zombie NIR), anti-active-caspasse-3-BV650 (apoptosis) and the up-regulation of RIP3 on Caspase-3 negative cells, see figure. This up-regulation of RIP3 during theh necroptotic process can be inhibited by necrostatin-1, see figure.

Mitochondrial Function During Necroptosis

Necroptosis inducing drug, Shikonin causes the remaining live cells to show mitochondrial dysfunction in a third of these cells with the remainign cells displaying a hyper-polarization on the inner mitochondrial membrane, see figure. While pre-treatment with necroptosis inhibitor, Necrostatin reduces this hyperpolrization of the inner mitochondrial membrane, but not the degree of mitochondrial dysfunction, see figure.

Necroptotic Cell Cycle Arrest

Shikonin was found to cuase cell cycle arrest in G2m after 24h of treatment. Pre-treatment with 60uM Necrostatin for 1h reduced the degree of necroptosis but not the degree of cell cycle arrest, see figure.

Publications

D Bergamaschi, A Vossenkaemper, WYJ Lee, P Wang, E Bochukova, G Warnes. Simultaneous polchromatic flow cytometric detection of multiple forms of regulated cell death. Apoptosis, 2019. (DOI: 10.1007/s10495-019-01528-w)

A Vossenkamper, G Warnes. A flow cytometric immunophenotyping approach to the detection of regulated cell death processes. Journal of Immunological Sciences, 2 (5) 2018.

HL Lee, R Pike, MHA Chong, A Vossenkamper, G Warnes. Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1 dependent apoptosis Methods, 1, 134-135, 56-66, 2018.