cell-fractionation-u937-cells-absence-high-speed

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

cell-fractionation-u937-cells-isopycnic-density-gradient

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

generation-rip1-knockout-u937-cell-line-using-crispr-cas9

Generation of a <em>RIP1</em> Knockout U937 Cell Line Using the CRISPR-Cas9 System

This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated ...

Albany College of Pharmacy and Health Sciences

The cholesterol dependent cytolysins (CDCs) are a family of pore-forming toxins produced by a wide range of bacteria. Some CDCs are important virulence factors for their cognate organisms, but their activity must be tightly regulated to ensure they operate at appropriate times and within the appropriate subcellular compartments. pH-dependent activity has been described for several CDCs, but the mechanism of such regulation has been studied in depth only for listeriolysin O (LLO), which senses environmental pH through a triad of acidic residues that mediate protein unfolding. Here we present data supporting a distinct mechanism for pH-dependence for inerolysin (INY), the CDC produced by Lactobacillus iners. Inerolysin (INY) has an acidic pH optimum with loss of activity at neutral pH. INY pH-dependence is characterized by reversible loss of pore formation with preservation of membrane binding. Fluorescent membrane probe assays indicated that INY insertion into host cell membranes, but not oligomerization, was defective at neutral pH. These data support the existence of a newly appreciated form of CDC pH-dependence functioning at a late stage of pore formation.

Environmental pH modulates inerolysin activity via post-binding blockade.

Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR-Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs . This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.

Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.

Inhalation of ricin toxin is associated with the onset of acute respiratory distress syndrome (ARDS), characterized by hemorrhage, inflammatory exudates, and tissue edema, as well as the nearly complete destruction of the lung epithelium. Here we report that the Calu-3 human airway epithelial cell line is relatively impervious to the effects of ricin, with little evidence of cell death even upon exposure to microgram amounts of toxin. However, the addition of exogenous soluble umor ecrosis actor (TNF)-elated poptosis-nducing igand (TRAIL; CD253) dramatically sensitized Calu-3 cells to ricin-induced apoptosis. Calu-3 cell killing in response to ricin and TRAIL exposure was partially inhibited by caspase-8 and caspase-3/7 inhibitors, consistent with involvement of extrinsic apoptotic pathways in cell death. We employed nCounter Technology to define the transcriptional response of Calu-3 cells to ricin, TRAIL, and the combination of ricin plus TRAIL. An array of genes associated with inflammation and cell death were significantly upregulated upon treatment with ricin toxin and were further amplified upon addition of TRAIL. Of particular note was interleukin-6 (IL-6), whose expression in Calu-3 cells increased 300-fold upon ricin treatment and more than 750-fold upon ricin and TRAIL treatment. IL-6 secretion by Calu-3 cells was confirmed by cytometric bead array analysis. On the basis of these finding, we speculate that the severe airway epithelial cell damage observed in animal models following ricin exposure is a result of a positive-feedback loop driven by proinflammatory cytokines such as TRAIL and IL-6. Ricin toxin is a biothreat agent that is particularly damaging to lung tissue following inhalation. A hallmark of ricin exposure is widespread inflammation and concomitant destruction of the airway epithelium. In this study, we investigated the possible interaction between ricin and known proinflammatory cytokines associated with lung tissue. Using an established human airway epithelial cell line, we demonstrate that epithelial cell killing by ricin is significantly enhanced in the presence of the proinflammatory cytokine known as TRAIL (CD253). Moreover, epithelial cells that are simultaneously exposed to ricin and TRAIL produced large amounts of secondary proinflammatory signals, including IL-6, which in the context of the lung would be expected to exacerbate toxin-induced tissue damage. Our results suggest that therapies designed to neutralize proinflammatory cytokines such as TRAIL and IL-6 may limit the bystander damage associated with ricin exposure.

TRAIL (CD253) Sensitizes Human Airway Epithelial Cells to Toxin-Induced Cell Death.

Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway that is distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which depend on glycolysis. This suggests that increased cellular glucose may prime necroptosis. Here we show that exposure to hyperglycemic levels of glucose enhances necroptosis in primary red blood cells (RBCs), Jurkat T cells, and U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Total levels of RIP1, RIP3, and mixed lineage kinase domain-like (MLKL) proteins were increased following treatment with high levels of glucose in Jurkat and U937 cells and was not due to transcriptional regulation. The observed increase in RIP1, RIP3, and MLKL protein levels suggests a potential positive feedback mechanism in nucleated cell types. Enhanced PCD due to hyperglycemia was specific to necroptosis as extrinsic apoptosis was inhibited by exposure to high levels of glucose. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by treatment with nec-1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD.

Hyperglycemic Conditions Prime Cells for RIP1-dependent Necroptosis.

Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and is classified as a biothreat agent by the Centers for Disease Control and Prevention (CDC). Inhalation, the most potent route of toxicity, triggers an acute respiratory distress-like syndrome that coincides with near complete destruction of the lung epithelium. We previously demonstrated that the TNF-related apoptosis-inducing ligand (TRAIL; CD253) sensitizes human lung epithelial cells to ricin-induced death. Here, we report that ricin/TRAIL-mediated cell death occurs via apoptosis and involves caspases -3, -7, -8, and -9, but not caspase-6. In addition, we show that two other TNF family members, TNF-α and Fas ligand (FasL), also sensitize human lung epithelial cells to ricin-induced death. While ricin/TNF-α- and ricin/FasL-mediated killing of A549 cells was inhibited by the pan-caspase inhibitor, zVAD-fmk, evidence suggests that these pathways were not caspase-dependent apoptosis. We also ruled out necroptosis and pyroptosis. Rather, the combination of ricin plus TNF-α or FasL induced cathepsin-dependent cell death, as evidenced by the use of several pharmacologic inhibitors. We postulate that the effects of zVAD-fmk were due to the molecule's known off-target effects on cathepsin activity. This work demonstrates that ricin-induced lung epithelial cell killing occurs by distinct cell death pathways dependent on the presence of different sensitizing cytokines, TRAIL, TNF-α, or FasL.

TNF Family Cytokines Induce Distinct Cell Death Modalities in the A549 Human Lung Epithelial Cell Line when Administered in Combination with Ricin Toxin.

Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study.

Storage Primes Erythrocytes for Necroptosis and Clearance.

The TNF superfamily of proinflammatory and proapoptotic cytokines influence tissue-wide responses to molecular insults such as small molecules, toxins, and viral infections that perturb cellular homeostasis at the level of DNA replication, transcription, and translation. In the context of acute lung injury, for example, TNF superfamily members like TNF-α and TRAIL can severely exacerbate disease pathophysiology. This chapter describes a systematic approach to optimization of mammalian cell viability assays and transcriptional profiling through nCounter Technology to permit a detailed examination of how TNF-α and TRAIL modulate programmed cell death pathways in concert with ricin toxin, a ribosome-inactivating protein (RIP) and a potent inducer of acute respiratory distress. We compare two widely used luciferase- and colorimetric-based cell viability assays and provide optimization protocols for adherent and non-adherent cell lines. We provide a computational workflow to facilitate downstream analysis of datasets generated from nCounter gene expression panels. While combined treatment with ricin toxin and TRAIL serves as the exemplar, the methodologies are applicable to any TNF superfamily member in combination with any biological agent of interest.

Sensitization of Airway Epithelial Cells to Toxin-Induced Death by TNF Superfamily Cytokines.

We have previously identified a shift from TNF-α-induced apoptosis to necroptosis that occurs under hyperglycemic conditions. This shift involves the downregulation or silencing of caspases and concurrent upregulation of necroptotic proteins leading to activation of the necrosome. In addition, under hyperglycemic conditions in vivo, this shift in cell death mechanisms exacerbates neonatal hypoxia-ischemia (HI) brain injury. Here, we identify two major factors that drive the hyperglycemic shift to necroptosis: (1) reactive oxygen species (ROS) and (2) receptor-interacting protein kinase 1 (RIP1). ROS, including mitochondrial superoxide, led to the oxidation of RIP1, as well as formation and activation of the necrosome. Concurrently, ROS mediate a decrease in the levels and activation of executioner caspases-3, -6, and -7. Importantly, hyperglycemia and mitochondrial ROS result in the oxidation of RIP1 and loss of executioner caspases prior to death receptor engagement by TNF-α. Moreover, RIP1 partially controlled levels of mitochondrial ROS in the context of hyperglycemia. As a result of its regulation of ROS, RIP1 also regulated necrosome activation and caspase loss. Mitochondrial ROS exacerbated neonatal HI-brain injury in hyperglycemic mice, as a result of the shift from apoptosis to necroptosis.

Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis.

The ribosome-inactivating proteins include the biothreat agent, ricin toxin (RT). When inhaled, RT causes near complete destruction of the lung epithelium coincident with a proinflammatory response that includes TNF family cytokines, which are death-inducing ligands. We previously demonstrated that the combination of RT and TNF-related apoptosis inducing ligand (TRAIL) induces caspase-dependent apoptosis, while RT and TNF-α or RT and Fas ligand (FasL) induces cathepsin-dependent cell death in lung epithelial cells. We hypothesize that airway macrophages constitute a major source of cytokines that drive lung epithelial cell death.

Necroptosis of Lung Epithelial Cells Triggered by Ricin Toxin and Bystander Inflammation.

Apoptosis and necroptosis overlap in their initial signaling but diverge to produce non-inflammatory and pro-inflammatory outcomes, respectively. High glucose pushes signaling in favor of necroptosis producing a hyperglycemic shift from apoptosis to necroptosis. This shift depends on receptor-interacting protein 1 (RIP1) and mitochondrial reactive oxygen species (ROS). Here, we show that RIP1, mixed lineage kinase domain-like (MLKL) protein, Bcl-2 agonist/killer (Bak), Bcl-2 associated x (Bax) protein, and dynamin-related protein 1 (Drp1) traffic to the mitochondria in high glucose. RIP1 and MLKL appear in the mitochondria in their activated, phosphorylated states while Drp1 appears in its activated, dephosphorylated state in high glucose. Mitochondrial trafficking is prevented in  KO cells and upon treatment with N-acetylcysteine. Induction of ROS replicated the mitochondrial trafficking seen in high glucose. MLKL forms high MW oligomers in the outer and inner mitochondrial membranes while Bak and Bax form high MW oligomers in the outer mitochondrial membrane in high glucose, suggesting pore formation. MLKL, Bax, and Drp1 promoted cytochrome  release from the mitochondria as well as a decrease in mitochondrial membrane potential in high glucose. These results indicate that mitochondrial trafficking of RIP1, MLKL, Bak, Bax, and Drp1 are key events in the hyperglycemic shift from apoptosis to necroptosis. This is also the first report to show oligomerization of MLKL in the inner and outer mitochondrial membranes and dependence of mitochondrial permeability on MLKL.

Mitochondrial Trafficking of MLKL, Bak/Bax, and Drp1 Is Mediated by RIP1 and ROS which Leads to Decreased Mitochondrial Membrane Integrity during the Hyperglycemic Shift to Necroptosis.

ArticleTotal : 3	Year
Publication title Cited by 4	2019
Publication title Cited by 1	2021
Publication title	2025

Article	Year
Environmental pH modulates inerolysin activity via post-binding blockade. Scientific reports\| PubMed ID: 29367601	2018
Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis. Cell death discovery\| PubMed ID: 29760953	2018
TRAIL (CD253) Sensitizes Human Airway Epithelial Cells to Toxin-Induced Cell Death. mSphere\| PubMed ID: 30258037	2018
Hyperglycemic Conditions Prime Cells for RIP1-dependent Necroptosis. The Journal of biological chemistry\| PubMed ID: 27129772	2016
TNF Family Cytokines Induce Distinct Cell Death Modalities in the A549 Human Lung Epithelial Cell Line when Administered in Combination with Ricin Toxin. Toxins\| PubMed ID: 31374990	2019
Storage Primes Erythrocytes for Necroptosis and Clearance. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 31486324	2019
Sensitization of Airway Epithelial Cells to Toxin-Induced Death by TNF Superfamily Cytokines. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33185865	2021
Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis. Cell death discovery\| PubMed ID: 33298902	2020
Necroptosis of Lung Epithelial Cells Triggered by Ricin Toxin and Bystander Inflammation. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 36695077	2023
Mitochondrial Trafficking of MLKL, Bak/Bax, and Drp1 Is Mediated by RIP1 and ROS which Leads to Decreased Mitochondrial Membrane Integrity during the Hyperglycemic Shift to Necroptosis. International journal of molecular sciences\| PubMed ID: 37239951	2023

Timothy J. LaRocca

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