Opposing regulation and roles for PHD3 in lung dendritic cells and alveolar macrophages.

The prolyl hydroxylase domain-containing enzymes (PHDs) are important metabolic sensors of the cell and its environment, which might be employed to alert cells of the immune system. These enzymes regulate the expression of the hypoxia inducible factor (HIF) isoforms and NF-κB, crucial transcription factors controlling cellular metabolism and inflammation. PHD/HIF signaling is activated in the allergic lung and is proposed as a potential druggable pathway. Here, we investigated the regulation and role of the PHD isoforms in CD11c-expressing dendritic cells (DCs) and macrophages (Mϕ), sensors of the environment and crucial antigen-presenting cells in the pathogenesis of asthma. Although PHD2 and PHD3 were expressed in baseline, stimulation with house dust mite (HDM) allergen, hypoxia, and TLR4 ligands induced the expression of PHD3 in DCs. Conditional deletion or overexpression of PHD3 in CD11chi cells had minor effects on DCs and alveolar Mϕ biology in steady state. However, when put into competition with wild-type counterparts in mixed chimeric mice, alveolar Mϕ uniquely required PHD3 for optimal reconstitution of the alveolar space. Using genetic and chemical approaches, we were unable to find a clear role for PHD3 or the other PHD isoforms in DCs in asthma development. These data show cell-specific competitive advantage of PHD3 expression in antigen-presenting cells, but question whether therapeutic manipulation of PHDs in DCs would offer therapeutic benefit in asthma.

Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival.

The IRE1-XBP1 signalling pathway is part of a cellular programme that protects against endoplasmic reticulum (ER) stress, but also controls development and survival of immune cells. Loss of XBP1 in splenic type 1 conventional dendritic cells (cDC1s) results in functional alterations without affecting cell survival. However, in mucosal cDC1s, loss of XBP1 impaired survival in a tissue-specific manner-while lung cDC1s die, intestinal cDC1s survive. This was not caused by differential activation of ER stress cell-death regulators CHOP or JNK. Rather, survival of intestinal cDC1s was associated with their ability to shut down protein synthesis through a protective integrated stress response and their marked increase in regulated IRE1-dependent messenger RNA decay. Furthermore, loss of IRE1 endonuclease on top of XBP1 led to cDC1 loss in the intestine. Thus, mucosal DCs differentially mount ATF4- and IRE1-dependent adaptive mechanisms to survive in the face of ER stress.

Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor α-induced necroptosis.

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

Programmed necrosis from molecules to health and disease.

During the past decade, cell death researchers have witnessed a gradual but deep conceptual revolution: it has been unequivocally shown that necrosis, which for long had been considered as a purely accidental cell death mode, can also be induced by finely regulated signal transduction pathways. In particular, when caspases are inhibited by pharmacological or genetic means, the ligation of death receptors such as the tumor necrosis factor receptor 1 (TNFR1) can lead to the assembly of a supramolecular complex containing the receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) that delivers a pronecrotic signal. Such complex has recently been dubbed necrosome and mediates the execution of a specific instance of regulated necrosis, necroptosis. Soon, it turned out that programmed necrosis occurs in nonmammalian model organisms and that it is implicated in human diseases including ischemia and viral infection. In this review, we first describe the historical evolution of the concept of programmed necrosis and the molecular mechanisms that underlie necroptosis initiation and execution. We then provide evidence suggesting that necroptosis represents an ancient and evolutionarily conserved cell death modality that may be targeted for drug development.

Molecular mechanisms and pathophysiology of necrotic cell death.

Necrotic cell death has long been considered an accidental and uncontrolled mode of cell death. But recently it has become clear that necrosis is a molecularly regulated event that is associated with pathologies such as ischemia-reperfusion (IR) injury, neurodegeneration and pathogen infection. The serine/threonine kinase receptor-interacting protein 1 (RIP1) plays a crucial role during the initiation of necrosis induced by ligand-receptor interactions. On the other hand, ATP depletion is an initiating factor in ischemia-induced necrotic cell death. Common players in necrotic cell death irrespective of the stimulus are calcium and reactive oxygen species (ROS). During necrosis, elevated cytosolic calcium levels typically lead to mitochondrial calcium overload, bioenergetics effects, and activation of proteases and phospholipases. ROS initiates damage to lipids, proteins and DNA and consequently results in mitochondrial dysfunction, ion balance deregulation and loss of membrane integrity. Membrane destabilization during necrosis is also mediated by other factors, such as acid-sphingomyelinase (ASM), phospholipase A(2) (PLA(2)) and calpains. Furthermore, necrotic cells release immunomodulatory factors that lead to recognition and engulfment by phagocytes and the subsequent immunological response. The knowledge of the molecular mechanisms involved in necrosis has contributed to our under-standing of necrosis-associated pathologies. In this review we will focus on the intracellular and intercellular signaling events in necrosis induced by different stimuli, such as oxidative stress, cytokines and pathogen-associated molecular patterns (PAMPs), which can be linked to several pathologies such as stroke, cardiac failure, neurodegenerative diseases, and infections.