998 SECTION VII I Pediatric Critical Care Metabolic and Endocrine cytochrome c release Communication between the extrinsic and intrinsic apoptosis pathways occurs through caspase 8 activation of the p[.]
998 S E C T I O N V I I I Pediatric Critical Care: Metabolic and Endocrine Extrinsic FasL apoptosis Fas Anti-apoptotic Bcl-2 pathway Bcl-xL Pro-apoptotic pathway Bax/Bak TNF TNF-R1 Caspase-8 RIP1 Bid P P Caspase-3 RIP1 RIP3 P MLKL P MLKL P MLKL Caspase-9 Intrinsic apoptosis Cytochrome C + Apaf-1 Apoptosis Normal cell Necroptosis P MLKL P MLKL Apoptosome Necroptosis Cellular contents Apoptotic bodies DNA fragmentation Membrane blebbing Release of cellular contents • Fig 83.2 Apoptosis and necroptosis signaling pathways Apoptosis can by initiated by extrinsic receptor- mediated pathways (1) or intrinsic mitochondrial stress signaling (2) mechanisms Extrinsic activation commonly occurs by FasL/Fas interactions at the plasma membrane that leads to the recruitment of death domain containing adaptor proteins (not shown) and the formation of the DISC complex (not shown), leading to the activation of caspase-8 Active caspase-8 cleaves procaspase-3, resulting in the executioner phase of apoptosis Alternatively, cleavage of the proapoptotic Bcl-2 family member Bid amplifies apoptosis signaling through the mitochondrial (intrinsic) pathway The intrinsic pathway is also induced by cytotoxic stress, leading to the release of cytochrome c and formation of the apoptosome, which, in turn, activates caspase-3 When caspase-8 activation is inhibited, death by necroptosis occurs through the serine-threonine kinase that functions as the initiator of the pathway Phosphorylation of RIP1 leads to phosphorylation of RIP3 and MLKL Phosphorylated MLKL translocates to the plasma membrane and alters membrane permeability, resulting in release of cytosolic contents into the extracellular space, inflammation, cell swelling, and eventual cell lysis due to changes in membrane permeability DNA, Deoxyribonucleic acid; FasL, Fas ligand; MLKL, mixed-lineage kinase domain-like protein; RIP, receptor-interaction protein; TNFR-1, tumor necrosis factor receptor-1 cytochrome c release Communication between the extrinsic and intrinsic apoptosis pathways occurs through caspase-8 activation of the protein Bid, leading to cytochrome c release and apoptosome formation Apoptosis is extensively altered in human critical illness Hotchkiss et al demonstrated that septic patients who died in a surgical intensive care unit and underwent immediate autopsy have markedly increased lymphocytic and gut epithelial apoptosis compared with nonseptic critically ill adults.10 B and CD41 T lymphocytes are disproportionately lost in septic adults.11 Similar findings are found in gut epithelial and lymphocyte apoptosis in adults with shock and trauma, with the degree of apoptosis increased with higher severity of injury.12,13 Children and neonates who die from sepsis are also noted to have prolonged lymphopenia and apoptosis-associated T- and B-cell depletion of lymphoid organs compared with children and neonates who die from noninfectious causes.14,15 Spleen biopsies from adults with sepsis demonstrate decreased numbers of CD41 and CD81 T cells as well as CHAPTER 83 Molecular Foundations of Cellular Injury immunohistochemical findings of increased inhibitory receptor and ligand expression from the spleen and lung from deceased septic versus nonseptic patients.16 While airway neutrophils are resistant to apoptosis in patients with acute respiratory distress syndrome (ARDS), perforin/granzyme and Fas/FasL are elevated in adults who die from ARDS compared with those who not progress to ARDS, and the bronchoalveolar lavage from ARDS patients can induce distal lung epithelial cell death.17–20 Animal Studies on Apoptosis While human studies demonstrate an association between apoptosis and mortality, they cannot establish causation In contrast, animal studies can directly prove a link between cell death and mortality Similar to human autopsy studies, apoptosis is primarily localized to lymphocytes, dendritic cells, and the gut epithelium in animal models of sepsis In cecal ligation and puncture (CLP), a murine model of fecal peritonitis, and Pseudomonas aeruginosa pneumonia, maximal lymphocytic and intestinal apoptosis occurs 24 hours after onset of septic insult.21–24 Both intrinsic and extrinsic pathways play a role in sepsis-induced apoptosis, and pathways differ depending on which model of sepsis is examined.25 Animals with gene-specific knockouts of multiple members of the proapoptotic Bcl-2 family members (mitochondrial pathway) or Fas-associated death domain transgenic mice (receptor mediated) demonstrate significant decreases in sepsis-induced splenocyte apoptosis.26,27 While apoptosis, whether initiated by the mitochondrial or the receptor-mediated pathway, converges into a single common pathway mediated by caspase-3, it should be noted that there are likely alternate pathways that are independent of caspase-3 given that caspase-3 knockout mice exhibit a small degree of apoptosis.28 Increased lymphocytic apoptosis appears to be detrimental to survival in sepsis Overexpression of Bcl-2 in transgenic mice in either T lymphocytes or B lymphocytes markedly improves survival in multiple strains of inbred mice subjected to CLP, a mouse model of fecal peritonitis.28,29 Similar increases in survival have been shown in mice in which proapoptotic Bim has been knocked out.27 Administration of the polycaspase inhibitor N-benzyloxycarbonylVal-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD) or the caspase-3 specific inhibitor M-971 results in similar improvements in outcome.29,30 The mechanisms that account for worse outcomes with increasing lymphocytic apoptosis appear to involve immunosuppression Although immunosuppression in sepsis is multifactorial, the ongoing loss of immune effector cells in both the innate and adaptive compartments likely plays a significant role In addition to the loss of cells, there is an upregulation of T regulatory cells and myeloid-derived suppressor cells.30 This upregulation of immunosuppressive cells appears to be driven in part by the production of IL-10, an antiinflammatory immunosuppressive cytokine Notably, adoptive transfer of necrotic cells improves survival in septic animals; however, this benefit is lost if interferon (IFN)-g production is blocked.31 In contrast, apoptotic cells not only increase mortality, they also prevent IFN-g production Interestingly, a preexisting immunosuppressive state may alter the immune apoptotic response Septic mice with preexisting pancreatic adenocarcinoma have a higher mortality when compared with noncancerous septic mice,32 and the presence of malignancy not only impairs tumor-specific immune responses but also impairs pathogen-specific responses, resulting in a state of generalized immunosuppression.33 Notably, either lymphocyte overexpression of Bcl-2 (prosurvival, antiapoptotic protein) or germline deletion of 999 Bim (proapoptotic protein) in mice with cancer results in elevated mortality, contrary to what is seen in previously healthy septic hosts.34 Prevention of gut epithelial apoptosis also improves survival in preclinical models of sepsis, as overexpression of Bcl-2 in gut epithelium decreases mortality in mice subjected to CLP or P aeruginosa pneumonia.22,35 In addition, administering systemic epidermal growth factor (EGF) normalizes intestinal Bid expression and apoptosis and mortality following CLP.36,37 These protective effects appear to be modulated through the intestinal epithelium, as similar results are seen if EGF is selectively overexpressed in intestinal enterocytes.38–40 The immune system directly impacts apoptosis in the intestinal epithelium in critical illness Rag–/– mice, which lack lymphocytes, have a fivefold higher level of gut epithelial apoptosis after CLP compared with wild-type mice, and adoptive transfer of CD41 T cells in Rag–/– mice restores apoptosis back down to wild-type levels.41 Respiratory epithelial cells are resistant to apoptosis when exposed to P aeruginosa, undergoing cell death in vitro.42 However, P aeruginosa pneumonia induces respiratory epithelial apoptosis in mice through activation of the Fas/FasL system,43 and respiratory apoptosis appeared to be essential for survival in this study, with rapid sepsis-induced mortality in Fas or FasL-deficient mice that lack bronchial apoptosis Alveolar and bronchiolar apoptosis is also present in rats with Streptococcus sanguis or Streptococcus pneumoniae type 25 pneumonia.44,45 While lung apoptosis has also been demonstrated following CLP in multiple mouse strains, recently it has been questioned whether CLP causes lung injury.46 Acute lung injury causes increased death in multiple cells within the lung, and intratracheal injection of lipopolysaccharide induces apoptosis in alveolar cells, neutrophils, and macrophages.45,47 This process is associated with upregulation of Fas in alveolar and inflammatory cells, and lung injury can be blocked by administration of an anti-Fas antibody Both epithelial and endothelial apoptosis also occur in the lung in a rat trauma–hemorrhagic shock model in a caspase-3-dependent (epithelial) and caspase-3independent (endothelial) manner.48 Pyroptosis Pyroptosis is an inflammatory or immunogenic form of RCD that depends on the formation of membrane pores by members of the gasdermin protein family often as a consequence of inflammasome (made up of inflammatory caspase) activation Pyroptosis relies on the activation of caspase-1 and caspase-11, which are involved in cytokine maturation and necrotic cell death, respectively.2 Pyroptosis results in rapid plasma-membrane rupture and release of proinflammatory intracellular contents.49 Caspase-1-dependent plasma membrane pores dissipate ionic gradients, producing a net increased osmotic pressure, water influx, cell swelling, and, eventually, osmotic lysis Danger to the host is sensed extracellularly through Toll-like receptors (TLRs) and intracellularly by Nod-like receptors (NLRs) resulting in initiation of signaling cascades that activate nuclear factor-kB (NF-kB), mitogen-activated protein kinase (MAPK), and IFN-regulatory factor (IRF)-dependent pathways and inflammatory cytokine production, including IFN-a, IFN-b, TNF, interleukin (IL)-12, IL-6, IL-8, and pro-IL-1b.49 Low levels of active caspase-1 stimulate cell-survival responses, control intracellular bacterial growth, and mediate inflammatory cytokine production However, when caspase-1 activation passes a threshold, cells undergo pyroptosis and release inflammatory cellular contents.49 In humans, inflammasome activation and pyroptosis are 1000 S E C T I O N V I I I Pediatric Critical Care: Metabolic and Endocrine associated with multiple-organ damage after trauma, including ARDS, acute liver injury, acute kidney injury, myocardial dysfunction, secondary brain and spinal cord injury, and endothelial barrier permeability and coagulopathy.50 Caspase-Independent Forms of Regulated Cell Death Necroptosis Necroptosis is an immunogenic caspase-independent form of RCD that is initiated through a variety of receptors, including death receptors, viral nucleotide sensing receptors, TLRs, and IFNs via intracellular signaling that occurs through the serinethreonine protein kinases receptor-interaction proteins and (RIPK1 and RIPK3).49,51 RIPK3 phosphorylation of mixedlineage kinase domain-like protein (MLKL) results in the trafficking of activated MLKL to the cell membrane, which it then permeabilizes, allowing for cell swelling, calcium influx, and necrotic cell death (see Fig 83.2) Necroptosis is seen in animal models of critical illness, including viral and bacterial infections, severe inflammatory response syndrome, sepsis neuronal disorders, and ischemia-reperfusion injury Necroptosis has been confirmed in human disorders and mechanistic aspects, and clinical relevance has been reviewed.51,52 Animal Studies—Necroptosis Necroptotic pathways of cell death have been implicated in preclinical models in a number of clinical disease processes, including sepsis, viral infections, stroke, myocardial infarction, pancreatitis, acute kidney injury, and inflammatory bowel disease.53–58 Notably, deletion of RIP3 results in a substantial survival advantage in mice undergoing CLP.59 When mice deficient in apoptosis-inhibitors are subjected to influenza infection, they suffer significant lung epithelial cell destruction via RIP3 and display increased mortality compared with wildtype controls.60 Further, mice deficient in the apoptotic adaptor FAS-associated death domain protein (FADD) develop a severe and erosive colitis similar to that seen in Crohn colitis and, like human colitis, the inflammation in FADD-deficient mice is largely TNF-a driven Interestingly, inflammatory colon lesions in these animals are prevented by deletion of the necroptotic protein RIP3.57 TNF-adependent skin conditions can also be cured in animals by deleting necroptotic proteins, suggesting that these pathways may be involved in a host of human diseases that prominently feature TNF-a as a mediator.61,62 Beyond tissue-specific processes, deletion of RIP1 has been shown to cause perinatal lethality associated with severe systemic inflammation at birth with cell necrosis via RIP3-MLKL.63 Autophagy Autophagy is a lysosomal degradation system that is triggered by starvation, exercise, or cellular stress that recycles proteins and organelles (such as mitochondria in a process termed mitophagy) into metabolic precursors to promote cell survival (Fig 83.3) This catabolic process occurs by the formation of intracellular vesicles created from isolated portions of cell membranes that engulf cytoplasmic content and fuse with lysosomes, forming an autophagolysosome that can remove damaged organelles, toxic protein aggregates, and intercellular bacteria.64–67 Autophagy is proposed to be an adaptive response to critical illness that provides a crucial cellular repair process that is essential to reversal of organ dysfunction.64–67 Insufficient autophagy in prolonged critical illness may result in incomplete clearance of cellular damage due to illness and exacerbated by hyperglycemia and could explain lack of recovery from organ failure in prolonged critically ill patients.68 In mice deficient in liver-specific autophagy who underwent CLP, there was more hepatocyte apoptosis, more mitochondrial damage with increased reactive oxygen species (ROS) in hepatocytes, more systemic inflammation, less autophagic vacuoles, and an accelerated time to death compared with wild-type mice who also underwent CLP.69 These findings implicate liver autophagy as a protective mechanism in sepsis-induced organ failure through degradation of damaged mitochondria and in the prevention of apoptosis.69 In wild-type mice undergoing CLP, autophagy in the kidney was slowed in the first 24 hours after the induction of sepsis.70 The administration of rapamycin accelerated autophagy and protected the kidney from tubular epithelial injury during CLP-induced sepsis.70 Although mechanistic details of autophagy are still being studied, autophagy has been shown to regulate necroptosis, apoptosis, and pyroptosis in a context-specific manner.4,5,71 Therapies that activate autophagy during critical illness could accelerate recovery and/or attenuate continued damage of critical illness and speed organ recovery.68 Mitochondrial Permeability Transition–Mediated Regulated Necrosis The mitochondrial permeability transition (MPT) leads to RCD in response to intracellular perturbations such as severe oxidative stress and cytosolic Ca21 overload.72 MPT refers to the abrupt increase in the permeability of the inner mitochondrial membrane to small-molecular-weight solutes, resulting in the rapid dissipation of the mitochondrial membrane potential, osmotic breakdown of both mitochondrial membranes, uncoupling of oxidative phosphorylation, release of intramitochondrial ions and metabolites, mitochondrial matrix swelling, and, ultimately, cell death.73,74 Perturbations in key mitochondrial functions—such as adenosine triphosphate (ATP) production, Ca21 homeostasis, oxygen-derived free radical production, and the MPT—are implicated in mitochondrial dysfunction associated with sepsis-induced MODS as demonstrated in murine CLP.75 Cells are hypothesized to respond in a graded manner to MPT induction For example, when only a few mitochondria initiate MPT, mitophagy is stimulated As more mitochondria undergo MPT, apoptosis or necrosis RCD pathways may be initiated depending on the level of ATP depletion.76 Cells with MPT activation that are able to produce ATP switch their metabolism from oxidative phosphorylation to glycolysis and undergo RCD by apoptosis In contrast, cells that are dependent on oxidative phosphorylation and cannot shift toward glycolysis for ATP production become ATP depleted and undergo necrosis.76 The identity of the MPT pore is elusive.73,77 However, cyclosporine A (CSA), a potent immunosuppressive compound, inhibits the MPT by binding to cyclophilin D.2,75,76 In addition, overexpression of the antiapoptosis protein Bcl-2 inhibits MPT-induced apoptosis.75 Ferroptosis Ferroptosis is a type of RCD dependent on iron and triggered by blockade of the Xc- Cys/Glu antiporter, which allows for the exchange on extracellular L-Cys for intracellular L-Glu across the plasma membrane.49,74 As Cys is a precursor for glutathione (GSH), GSH levels are depleted with lack of import of L-Cys into CHAPTER 83 Molecular Foundations of Cellular Injury Nutrient deprivation Nutrient abundance 1001 Growth factors and insulin PI3K-1 mTORc1 AKT PDK1 AMPK ATG9L ATG14L VPS34 ULK ATG13 Beclin-1 VPS15 FIP200 ATG101 PI3K-III complex ULK complex Phagophore Recycling Autolysosome ATG16L ATG12 Lysosome ATG5 LC3-II LC3-II LC3-II LC3-II ATG3 LC3-I LC3 Elongation ATG4 Autophagosome maturation • Fig 83.3 Autophagy is a normal mechanism to remove damaged organelles, recycle proteins, and eliminate intracellular pathogens Autophagy is initiated by several mechanisms that sense nutrient deprivation, such as adenosine monophosphate–activated protein kinase, which inhibits mTOR, growth factor receptors, and pathogen invasion, such as toll-like receptors (not shown) Proteins involved in autophagy (ATG) complexes lead to initiation and elongation of the autophagosome, which fuses with a lysosome to create an autolysosome Hydrolases within the autolysosome degrade the cellular debris and pathogens into building blocks for the cell to reuse the cytosol Reduced levels of GSH cause a loss-of-function of glutathione peroxidase (GPX4), resulting in ROS from irondependent Fenton-type reactions, which leads to the accumulation of oxidized lipids.74 Ferroptosis has been shown to contribute to neuronal death, and inhibitors of ferroptotic death improve outcome after controlled cortical impact in a mouse model of traumatic brain injury.78,79 Parthanatos Parthanatos, derived from the Greek word for death, is a form of RCD that is triggered by extreme genomic stress resulting in the accumulation of poly(ADP-ribose) (PAR) and the nuclear translocation of apoptosis-inducing factor (AIF) for mitochondria.80 Parthanatos is also known as poly(ADP-ribose) polymerase-1 (PARP-1) dependent cell death PARP-1 is an adenosine diphosphate (ADP)-ribosyl transferase enzyme that transfers ADP ribose groups from NAD1 to their targets in response to deoxyribonucleic acid (DNA) damage.74 Extreme damage of DNA causing breaks and changes in chromatin structure induce the parthanatos pathway Genomic damage is recognized by the PARP-1 enzyme causing an upregulation and accumulation of PAR PAR is a negatively charged linear or branched polymer of variable length that can either covalently or noncovalently bind and alter the function of proteins, including histones.81 PARP-1 modulates DNA repair, DNA transcription, and mitosis through the binding of PAR to its target proteins One target of PAR is AIF, which causes translocation of the mitochondrially located AIF to the nucleus, where it induces chromatin condensation, DNA fragmentation, and cell death.82 The mechanism of how AIF induces DNA fragmentation is an active area of research After PAR is synthesized by PARP-1, it is degraded through a process catalyzed by the enzyme poly(ADP-ribose) glycohydrolase (PARG).83 PARG protects against PAR-mediated cell death and its deletion increases toxicity through the accumulation of PAR.82 Parthanatos is a distinct form of RCD apart from apoptosis and necroptosis Unlike apoptosis, parthanatos causes large-scale, rather than small-scale, DNA fragmentation and does not form apoptotic bodies.84 While parthanatos involves loss of cell membrane integrity, it is not accompanied by cell swelling as is found in necroptosis.85 In a rat renal allograft model, ischemia-reperfusion injury in the renal allograft results in pulmonary injury due to increased expression and activation of PARP-1, RIPK1, and RIPK3 in the lung following renal engraftment.86 1002 S E C T I O N V I I I Pediatric Critical Care: Metabolic and Endocrine NETosis Neutrophil extracellular traps (NETs) undergo an ROS-dependent process triggered by microbial and sterile activators that results in the release of elastase and myeloperoxidase from neutrophil granule to the nucleus that, in turn, promotes histone degradation and chromatin decondensation.87–89 This process, termed NETosis, leads to the extrusion of a meshwork of chromatin fibers dotted with granules that contain antimicrobial molecules that trap and kill extracellular bacteria and fungi.90,91 NETosis, which results in cell death, is accompanied by ruptures in the plasma membrane and neutrophil lysis.90 However, there is also evidence for neutrophils releasing NETs while maintaining an intact plasma membrane wherein neutrophils lacking a nucleus continue vital functions such as chemotaxis, phagocytosis, and bacterial killing.92–94 Cell Death as a Therapeutic Target RCD pathways can theoretically be targeted via mechanism of death (caspase mediated or independent) or their degree of immunogenicity Therapies targeting caspase have not been successfully translated to patients to date due to toxicity and off-target effects, although these remain a potential avenue for future research In addition, because of the link between inflammation and RCD, these pathways represent potential therapeutic targets to prevent the progression of shock and MODS However, cross-talk among RCD pathways is poorly understood, and blocking one pathway of cell death may simply trigger another (which may be even more maladaptive).95–99 For example, mice with a conditional deletion of caspase-8 in the intestinal epithelium spontaneously developed inflammatory lesions in the terminal ileum, had increased susceptibility to developing colitis, and had epithelial cell death in the small intestinal crypts.53 Epithelial cell death was associated with increased expression of RIP3, and death could be inhibited by blockade of necroptosis.53 Midgestational death of mice deficient in FADD or caspase-8 was reversed by elimination of RIP1 or RIP3, indicating an entwined relationship among RCD pathways.53 Therefore, mitigation of organ failures and death require further mechanistic insights and may potentially be more successful by intervening on multiple RCD pathways simultaneously.49,100 If novel drug therapies are to treat critically ill humans in the future, a thorough understanding of the intricate, interconnected pathways of RCD must be established both in animal models and in the context of humans Key References Boomer JS, To K, Chang KC, et al Immunosuppression in patients who die of sepsis and multiple organ failure JAMA 2011;306(23): 2594-2605 Bortolotti P, Faure E, Kipnis E Inflammasomes in tissue damages and immune disorders after trauma Front Immunol 2018;9:1900 Galluzzi L, Vitale I, Aaronson SA, et al Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018 Cell Death Differ 2018;25(3):486-541 Hotchkiss RS, Coopersmith CM, McDunn JE, Ferguson TA The sepsis seesaw: tilting toward immunosuppression Nat Med 2009;15(5): 496-497 Hotchkiss RS, Strasser A, McDunn JE, Swanson PE Cell death N Engl J Med 2009;361(16):1570-1583 Linkermann A, Green DR Necroptosis N Engl J Med 2014;370(5):455465 Linkermann A, Stockwell BR, Krautwald S, et al Regulated cell death and inflammation: an auto-amplification loop causes organ failure Nat Rev Immunol 2014;14(11):759-767 Vanden Berghe T, Kaiser WJ, Bertrand MJ, et al Molecular crosstalk between apoptosis, necroptosis, and survival signaling Mol Cell Oncol 2015;2(4):e975093 Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P Regulated necrosis: the expanding network of non-apoptotic cell death pathways Nat Rev Mol Cell Biol 2014; 15(2):135-147 The full reference list for this chapter is available at ExpertConsult.com ... increased death in multiple cells within the lung, and intratracheal injection of lipopolysaccharide induces apoptosis in alveolar cells, neutrophils, and macrophages.45,47 This process is associated... Hydrolases within the autolysosome degrade the cellular debris and pathogens into building blocks for the cell to reuse the cytosol Reduced levels of GSH cause a loss-of-function of glutathione peroxidase... activation of the Fas/FasL system,43 and respiratory apoptosis appeared to be essential for survival in this study, with rapid sepsis-induced mortality in Fas or FasL-deficient mice that lack bronchial