Molecular foundations of cellular injury









  • Cell death is an important physiologic homeostatic mechanism critical for host survival.



  • Regulated cell death pathways are evolutionarily conserved processes and include caspase-dependent (apoptosis, pyroptosis) and caspase-independent (necroptosis, mitochondrial permeability transition mediated-regulated necrosis, ferroptosis, and parthanatos) mechanisms.



  • Mechanisms of cell death are important in many critical illnesses, including sepsis, solid organ transplant, ischemia-reperfusion injury, acute kidney injury, acute respiratory distress syndrome, trauma and traumatic brain injury, and multiple-organ dysfunction syndrome.



  • Apoptosis is the least immunogenic regulated cell death pathway.



  • Autophagy is a recycling mechanism for a cell to reuse damaged proteins and organelles.



  • Understanding mechanisms of regulated cell death is important, as these signaling pathways are targets for developing treatments for human critical illness.



Cell death can occur via either regulated or unregulated pathways. Exposure of cells to extreme physical or chemical environmental conditions—such as high temperatures and pressures, shear stress, dangerous pH variations, and steep osmotic gradients—results in accidental or unregulated cell death. In contrast, the host has evolutionarily conserved machinery dedicated to removing unneeded or dangerous cells via multiple complementary mechanisms. Together, these are termed regulated cell death (RCD).


On a chronic basis, the need to balance cellular proliferation with cellular elimination is straightforward, since continued production of new cells without any method to eliminate older or unnecessary cells would end up with an untenable state of an ever-increasing number of cells in the body (especially for rapidly proliferating cell types). In addition, the body needs a method to rapidly respond to an acute insult, such as would be seen in a critically ill patient. For example, tissue injury and inflammation associated with infectious organisms result in the release of pathogen-associated molecular patterns (PAMPs) and dying host cells release danger-associated molecular patterns (DAMPs) to alert surrounding host cells of a threat. Both PAMPs and DAMPs alert the body to the presence of microbes and endogenous danger, respectively. In turn, each can activate signaling pathways that may culminate in RCD.


RCD is directly relevant to critical care since cell death occurs in multiple processes in the intensive care unit, including (but not limited to) sepsis, trauma, ischemia-reperfusion injury, oncology, autoimmune, autoinflammatory diseases, and multiple-organ dysfunction syndrome (MODS). Notably, RCD is neither “good” nor “bad” but needs to be understood within the context of the underlying physiologic state. RCD is clearly adaptive over the course of a host’s life when appropriately regulated, although it can be maladaptive when machinery is either over- or underactive. While RCD can also be beneficial in the acute setting, excessive induction of RCD can be maladaptive, such as when excessive death of lymphocytes in sepsis leads to an immunosuppressed host. As such, in selected clinical scenarios, RCD represents a therapeutic target that could be potentially harnessed to disrupt pathologic inflammation–cell death circuits resulting in shock, organ failure, and, ultimately, patient death.


The understanding of RCD has expanded recently, and guidelines have been formulated to define RCD by molecular mechanisms and machinery involved in each process. RCD pathways are categorized as to whether they are immunogenic and caspase-dependent ( Fig. 83.1 ). Immunogenic cell death is a form of RCD that is sufficient to activate an adaptive immune response in an immunocompetent host. Caspases are a family of cysteine-aspartic proteases that have an essential role in some forms of RCD, specifically apoptosis and pyroptosis. The objective of this chapter is to introduce a framework to understand the multiple forms of RCD, highlight key rodent studies demonstrating either a direct causative linkage between RCD and mortality or important mechanistic insights, and to review data relevant to human critical illness.




• Fig. 83.1


Overview of regulated cell death (RCD) signaling pathways. RCD pathways may be classified as caspase dependent or independent. Apoptosis and pyroptosis are caspase dependent; however, apoptosis is the least immunogenic of RCD pathways due to the presence of an intact plasma membrane. Apoptosis can be instigated by extrinsic signals initiated by cell surface death receptors or by intrinsic mitochondrial stress signals. Pyroptosis is an immunogenic form of RCD that requires two stress signals sensed by a multiprotein inflammasome complex, of which there are several types, resulting in activation of proinflammatory cytokines, IL-1β and IL-18. Necroptosis, mitochondrial permeability transition-medicated regulated necrosis (MPT-RN), ferroptosis, and NETosis are also immunogenic caspase-independent forms of RCD. ADP, Adenosine diphosphate; AIF, apoptosis-inducing factor; DAI, DNA-dependent activator of IFN-regulatory factors; GPX4, glutathione peroxidase 4; IFN, interferon; IL, interleukin; MOMP, mitochondrial outer membrane permeabilization; NET, neutrophil extracellular trap; PAR, poly(ADP-ribose); PARP1, PAR polymerase 1; PKR, protein kinase R; pMLKL, phosphorylated mixed-lineage kinase domain-like protein; RIPK, receptor-interacting serine/threonine-protein kinase; STAT3, signal transducer and activator of transcription 3; TLR, Toll-like receptor; TRIF, TIR domain-containing adaptor protein inducing IFN-β.

(Modified from 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.)


Caspase-dependent forms of regulated cell death


Apoptosis and pyroptosis are caspase-dependent forms of RCD. Apoptosis, the most widely studied method of RCD, is considered less immunogenic than pyroptosis because there is no disruption of the plasma membrane and therefore no release of cytosolic contents into the interstitial space to act as DAMPS, thereby instigating an inflammatory response. Rather, apoptotic cells and their organelles shrink in size; their nuclei condense (pyknosis) and fragment (karyorrhexis); the cell membrane phospholipid, phosphatidylserine, is exposed to the outside environment and acts as an “eat-me” signal; and cell cytoplasm forms blebs without disruption of the plasma membrane, with eventual formation of apoptotic bodies that are engulfed by phagocytes. By contrast, activation of pyroptosis results in proinflammatory cytokine maturation and necrotic cell death characterized by cellular swelling, plasma membrane disruption, and release of cytosolic contents into the interstitial space. , Thus, pyroptosis is a highly immunogenic RCD pathway.


Apoptosis


Apoptosis can be triggered by either an extrinsic pathway involving cell surface receptor-ligand interactions or by an intrinsic pathway involving transient mitochondrial outer membrane permeabilization disruption. The extrinsic pathway is most commonly activated by the interaction of Fas ligand (FasL) with Fas or tumor necrosis factor-α (TNF-α) interaction with TNF receptor-1 (TNFR-1; Fig. 83.2 ). Adaptor proteins interact with death domains on the intracellular portion of FasL, resulting in the recruitment of a multiprotein death-inducing signaling complex (DISC) that recruits and activates the initiator caspase, pro-caspase-8. Activation of caspase-8 leads to activation of the executioner caspase complex containing caspase-3, caspase-6, and caspase-7. The intrinsic pathway is initiated by multiple inciting factors that stimulate the release of mitochondrial cytochrome c into the cytosol due to transient mitochondrial outer membrane permeabilization (MOMP) from cellular stress. Cytochrome c interacts with an adaptor protein, apoptotic protease-activating factor–1 (APAF-1), which recruits the initiator caspase-9 and leads to the formation of a caspase-activating multiprotein complex called the apoptosome (see Fig. 83.2 ). Once formed, the apoptosome activates the executioner caspases. Within the intrinsic apoptosis pathway, the balance between a complex family of pro- and antiapoptotic proteins regulates cytochrome c release from the mitochondria. Proapoptotic family members such as Bax and Bak promote cytochrome c release whereas antiapoptotic proteins, such as Bcl-2 and Bcl-xL, suppress mitochondrial 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.




• 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.


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. B and CD4 + T lymphocytes are disproportionately lost in septic adults. 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. , 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. , Spleen biopsies from adults with sepsis demonstrate decreased numbers of CD4 + and CD8 + T cells as well as immunohistochemical findings of increased inhibitory receptor and ligand expression from the spleen and lung from deceased septic versus nonseptic patients. 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 do not progress to ARDS, and the bronchoalveolar lavage from ARDS patients can induce distal lung epithelial cell death.


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. Both intrinsic and extrinsic pathways play a role in sepsis-induced apoptosis, and pathways differ depending on which model of sepsis is examined. 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. , 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.


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. , Similar increases in survival have been shown in mice in which proapoptotic Bim has been knocked out. Administration of the polycaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD) or the caspase-3 specific inhibitor M-971 results in similar improvements in outcome. ,


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. 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)-γ production is blocked. In contrast, apoptotic cells not only increase mortality, they also prevent IFN-γ 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, 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. Notably, either lymphocyte overexpression of Bcl-2 (prosurvival, antiapoptotic protein) or germline deletion of Bim (proapoptotic protein) in mice with cancer results in elevated mortality, contrary to what is seen in previously healthy septic hosts.


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. , In addition, administering systemic epidermal growth factor (EGF) normalizes intestinal Bid expression and apoptosis and mortality following CLP. , These protective effects appear to be modulated through the intestinal epithelium, as similar results are seen if EGF is selectively overexpressed in intestinal enterocytes. 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 CD4 + T cells in Rag –/– mice restores apoptosis back down to wild-type levels.


Respiratory epithelial cells are resistant to apoptosis when exposed to P. aeruginosa , undergoing cell death in vitro. However, P. aeruginosa pneumonia induces respiratory epithelial apoptosis in mice through activation of the Fas/FasL system, 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. , While lung apoptosis has also been demonstrated following CLP in multiple mouse strains, recently it has been questioned whether CLP causes lung injury. 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. , 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-3-independent (endothelial) manner.


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. Pyroptosis results in rapid plasma-membrane rupture and release of proinflammatory intracellular contents. 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-κB (NF-κB), mitogen-activated protein kinase (MAPK), and IFN-regulatory factor (IRF)-dependent pathways and inflammatory cytokine production, including IFN-α, IFN-β, TNF, interleukin (IL)-12, IL-6, IL-8, and pro-IL-1β. 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. In humans, inflammasome activation and pyroptosis are 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.


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 serine-threonine protein kinases receptor-interaction proteins 1 and 3 (RIPK1 and RIPK3). , RIPK3 phosphorylation of mixed-lineage 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. ,


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. Notably, deletion of RIP3 results in a substantial survival advantage in mice undergoing CLP. 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 wild-type controls. 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-α driven. Interestingly, inflammatory colon lesions in these animals are prevented by deletion of the necroptotic protein RIP3. TNF-α-dependent 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-α as a mediator. , 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.


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. 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. 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. 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. 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. In wild-type mice undergoing CLP, autophagy in the kidney was slowed in the first 24 hours after the induction of sepsis. The administration of rapamycin accelerated autophagy and protected the kidney from tubular epithelial injury during CLP-induced sepsis. Although mechanistic details of autophagy are still being studied, autophagy has been shown to regulate necroptosis, apoptosis, and pyroptosis in a context-specific manner. , , Therapies that activate autophagy during critical illness could accelerate recovery and/or attenuate continued damage of critical illness and speed organ recovery.


May 20, 2021 | Posted by in RHEUMATOLOGY | Comments Off on Molecular foundations of cellular injury
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