Cell death is an important biological process for development, homeostasis, and host defense. Many new types of cell death modalities have been defined and characterized over the past decade. Cell death can be classify as Regulated Cell Death (RCD) or accidental cell death (ACD). Regulated cell death involves gene encoded machinery which act in a coordinated sequence of events triggered by extracellular or intracellular signals. These signals can be targeted and modulated by pharmacological interventions. Physiological forms of RCD are broadly referred to as programmed cell death (PCD).

RCD was synonymous with apoptosis but it is now recognized that RCD also many other newly discovered programmed cell death pathways. Several types of regulated cell death have been recently identified, which share common morphological features but employ distinct molecular pathways. Some these includes necroptosis, pyroptosis, and necroptosis. Distinguishing between different forms of non-apoptotic cell death can be challenging. These pathways are activated in response to different triggers and are executed by distinct biochemical pathways.

Necroptosis

Necroptosis is a form of RCD caused by the formation of the necrosome complex and is caspase-independent form of cell death unlike apoptosis. Necroptosis is induced by upstream cell death receptors (DRs) such as the superfamily receptors of tumor necrosis factor TNF Receptor 1 (TNFR1) , CD95, TRAIL-R1, and TRAIL-R2, can transmit a cell death signal upon ligand binding via a conserved cytosolic death domain. Necroptosis is marked by rupture of the plasma membrane and release of pro-inflammatory damage-associated molecular patterns. Targeting components of the necroptotic pathway –either by chemical inhibition or with transgenic models can be used to confirm if cell death is dependent on necroptosis. Necroptosis can be inhibited by Necrostatin-1 (Nec1). Viruses encode several different types of caspase-8 inhibitor, suggesting that prevention of caspase-8-dependent apoptosis provides an advantage to the viruses. In these situations, necroptosis appears to be as a back-up cell death process to limit viral replication.

Pyroptosis

Pyroptosis is a type of regulated necrotic cell death principally described in monocytes and macrophages. This form of RCD is induced by inflammatory caspases, namely caspase-1 as well as Caspase-11 in mouse cells or Caspase-4 and Caspase-5 in human cells. Both caspase-1 and caspase-11 (or caspases-4, -5) induce pyroptosis by proteolytically processing Gasdermin D (GSDMD) yielding an N-terminal fragment that opens pores on the plasma membrane leading to cell death. Among the proteins released are the innate cytokines IL-1β and IL-18, which together with other cellular proteins, lead to tissue inflammation. Pyroptosis often occurs after microbial infections, and serves to initiate the immune response, as well as to kill the infected cell. Cells undergoing pyroptosis demonstrate nuclear condensation associated with DNA damage, cell swelling, and, ultimately, cell lysis associated with release of IL-1β. The mechanisms responsible for this process involve intracellular sensors of bacterial products and formation of the inflammasome. Increased expression of inflammasome genes can be an indication of pro-inflammatory responses in cells. Pyroptosis can be studied by looking at caspase activation, GSDMA cleavage, or by inhibiting or ablating key components of the pyroptotic pathway. Pyroptosis can be evaluated through the quantification of released LDH, the visualization of membrane integrity loss by fluorescence microscopy and detection of IL-1b. Active caspases are cleaved from their inactive pro-caspase forms during pyroptosis. Caspase cleavage can be detected by western blot, using a specific caspase antibody. Demonstrating dependence on caspase 1, 11, 4 or 5 is essential to distinguish pyroptotic cell death from other forms of necroptosis and apoptosis. Caspase 1 activity can be ablated by chemical inhibition with Pan Caspase Inhibitor, Z-VAD-FMK.

Ferroptosis

Ferroptosis is a form of regulated cell death that occurs as a consequence of lethal lipid peroxidation arising from an iron-dependent reactive oxygen species (ROS) accumulation. A number of methods are available to detect the presence of ROS. Ferroptic cells usually exhibit cell membrane rupture and the release of intracellular contents. Ferroptosis can be also triggered by loss or decreased activity of Glutathione peroxidase 4 (GPX4), an enzyme that can reduce hydrogen peroxide, organic hydroperoxides, and lipid peroxide. In the absence of GPX4, uncontrolled lipid peroxidation occurs through the accumulation of lipid radicals, lipid peroxy radicals. Lipid peroxidation alters the chemistry of lipids, reducing their ability to form functional cellular membranes and so can result in loss of membrane integrity and cell death. Fragmentation of lipid alkoxyl radicals also produces reactive aldehydes such as malondialdehyde and 4-hydroxynonenal (4-HNE). Cell death occurring exclusively by ferroptosis can be suppressed by iron chelators, lipophilic antioxidants, inhibitors of lipid peroxidation, and depletion of polyunsaturated fatty acids and this correlates with the accumulation of markers of lipid peroxidation. The presence of ferroptosis can be confirmed by looking at whether cell death is prevented by inhibitors, and by measuring lipid peroxides. Useful assays for studying ferroptosis include measuring Glutathione Peroxidase activity.

Figure 1: Jurkat cells were induced with 100 μM pyocyanin (general ROS inducer, panel A), or 1 μM of tbutyl-hydroperoxide (peroxide inducer, panel B), stained with ROS-ID® Total ROS Detection Kit and analyzed using flow cytometry. Untreated cells were used as a control. Cell debris were ungated. The numbers in the inserts reflect the mean green fluorescence of the control and treated cells.

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