Collateral damage: necroptosis in the development of lung injury

Abstract

Cell death is increasingly recognized as a driving factor in the development of acute lung injury. Necroptosis, an immunogenic regulated cell death program important in innate immunity, has been implicated in the development of lung injury in a diverse range of conditions. Characterized by lytic cell death and consequent extracellular release of endogenous inflammatory mediators, necroptosis can be both beneficial and deleterious to the host, depending on the context. Here, we review recent investigations linking necroptosis and the development of experimental lung injury. We assess the consequences of necroptosis during bacterial pneumonia, viral infection, sepsis, and sterile injury, highlighting increasing evidence from in vitro studies, animal models, and clinical studies that implicates necroptosis in the pathogenesis of ARDS. Lastly, we highlight current challenges in translating laboratory findings to the bedside.

INTRODUCTION

Cell death is central in the pathophysiology of acute respiratory distress syndrome (ARDS). While apoptosis has been well described in acute lung injury, now newly described forms of nonapoptotic programmed cell death have been identified as important potential mediators of ARDS (2, 4, 7, 26, 37, 44, 81, 82). Recent investigations of acute lung injury have focused on nonapoptotic programmed cell death pathways such as necroptosis, ferroptosis, parthanatos, and pyroptosis due to their inflammatory and immunogenic downstream effects. Necroptosis, a cell death program involving the receptor interacting kinases 1 and 3 (RIPK1/3) and mixed lineage kinase like (MLKL), leads to lytic cell death with subsequent release of damage-associated molecular patterns (DAMPs) that can activate the innate immune system. Diverse stimuli, both infectious and sterile, can induce necroptosis, and depending on the context and duration of this response, the resulting effects may be either protective or pathological for the host.

Necroptosis has been implicated in the inflammatory response and tissue injury in preclinical models of systemic illness as well as in human disease. Experimental disease models, including cerulein-induced pancreatitis, acetaminophen-induced liver injury, traumatic spinal cord injury, and inflammatory bowel disease, have demonstrated evidence of necroptosis as measured by phosphorylated RIPK3 and MLKL (39, 41, 42, 49). These necroptosis intermediates have also been detected in tissue samples from human diseases, including inflammatory bowel disease, malignancies such as melanoma and lung adenocarcinoma, and ischemia-reperfusion injury during renal and cardiac transplant (21, 38, 41, 42, 54, 55, 65). Mounting in vitro and in vivo evidence shows that diverse pulmonary cell types, including epithelial cells, endothelial cells, and alveolar macrophages, employ this cell death program (16–18, 57). Thus, a closer look at the role of necroptosis in lung injury is warranted.

OVERVIEW OF NECROPTOSIS

Necroptosis is a form of programmed cell death driven by the necrosome, a multiprotein scaffold consisting of receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) that activates the pseudokinase mixed lineage kinase like (MLKL). During RIPK1-dependent necroptosis, RIPK1 undergoes autophosphorylation following activation and interacts with RIPK3 via RIP homotypic interactive motif (RHIM)-RHIM interactions. This RIPK1-RIPK3 complex, or necrosome, directly phosphorylates MLKL. Phosphorylated MLKL oligomerizes and translocates to the cell membrane, forming pores that lead to loss of cellular integrity. Intracellular contents that function as damage-associated molecular patterns (DAMPs), such as HMGB1, mitochondrial DNA, uric acid, histones, and ATP, are released to the extracellular environment, resulting in profound inflammation, recruitment of leukocytes, and a sustained immune response involving stimulation of the innate immune system via pattern recognition receptors (28, 78). While necroptotic cell death forms an important component of host defense by restraining proliferation of intracellular pathogens, this same process results in unrestrained inflammation and tissue destruction (8, 24).

Diverse conditions, including tumor necrosis factor (TNF) receptor signaling, Toll-like receptor (TLR)3 and TLR4 activation, and direct DNA sensing, may induce necrosome formation (19). Interferon-γ and stimuli leading to TNF signaling, including TLR2, TLR3, TLR4, TLR5, and TLR9 activation, initiate RIPK1-dependent necrosome formation (25, 47). Yet another example of highlighting the complexity of investigating the role of necroptosis in disease is that multiple PRR (TLRs) and nucleic acid sensors participate in necroptosis through direct interaction with RIPK3, independent of RIPK1 (5, 25, 47, 66, 69). Adaptor proteins containing RHIM domains such as TIR domain-containing adaptor-inducing interferon-β (TRIF) and Z-DNA-binding protein (ZBP1)/DNA-dependent activator of interferon regulatory factors (DAI) promote RIPK1-independent necroptosis through their direct interaction with RIPK3. The pattern recognition receptors TLR3 and TLR4 can directly interact with TRIF triggering RIPK1-independent necroptosis, and the cytosolic nucleic acid sensor DAI can directly interact with RIPK3 to promote necroptosis (29). Because pattern recognition receptor ligation and nucleic acid sensor engagement in response to various stimuli result in inflammatory signaling and nonnecroptotic cell death, efforts to determine the specific role of necroptosis in disease pathogenesis have been challenging.

Other cell death modalities, including apoptosis, pyroptosis, and parthanatos, employ RIPK1 and RIPK3 signaling in their program, making it particularly challenging to distinguish which cell death pathway contributes most to pathological phenotypes (82). RIPK1 and RIPK3 are critical components of cellular responses to stress or insults that result in either proinflammatory signaling or apoptosis. Apoptosis is a form of immunologically quiescent regulated cell death in which extrinsic or intrinsic signals lead to activation of initiator caspases, which in turn activate executioner caspases to affect nuclear fragmentation, generation of apoptotic bodies, blebbing, cell shrinkage, and clearance by efferocytosis. RIPK1 participates in several intermediate complexes in the apoptosis pathway and can promote cell survival or cell death, depending on the enzymatic activity of ubiquitinases, deubiquinases, and caspase 8 (Fig. 1). For example, following TNF receptor 1 ligation, polyubiquitination of RIPK1 leads to NF-κβ activation, cell survival, and inflammatory signaling. Alternatively, deubiquitination of RIPK1 promotes apoptosis in the presence of caspase 8, whereas the absence of caspase 8 activity predisposes the cell toward necroptosis via RIPK1 autophosphorylation (Fig. 1). Posttranslational modifications of RIPK3 can also promote either apoptosis or necroptosis. Knock-in of kinase-inactivated RIPK3 results in overwhelming apoptotic cell death, suggesting that the noncatalytic functions of RIPK3 promote apoptosis, while RIPK3 homodimerization promotes necroptosis even in the absence of RIPK1 (49). Kinase-inactivated RIPK3 can also promote pyroptosis by promoting the processing and secretion of pro-IL-1β to IL-1β after LPS stimulation, thus activating formation of the inflammasome (31, 32, 46). Depletion or active inhibition of caspases, whether employed by microbes that suppress caspase 8 as a virulence mechanism as seen in viral infection or by pharmacological use in experimental conditions with the pan-caspase inhibitor Z-VAD-FMK, inhibit apoptosis and can precipitate RIPK3-mediated necroptosis spontaneously (20, 50). The intersection of necroptosis and apoptosis in various lung diseases was recently reviewed by Sauler et al. (59) and will not be contrasted in depth in this review. However, the distinction between the inflammatory response generated by lytic cell death programs such as necroptosis and pyroptosis and the immunologically quiescent apoptosis have critical implications for tissue and organism survival following an infectious or sterile insult. Cells may deploy this bidirectional capability in response to differential stimuli; in vitro evidence suggests that apoptosis is employed as a defense against less virulent injuries, while necroptosis and other inflammatory cell death modes may be used during overwhelming injury to alarm surrounding cells and alert the immune system to danger (18). Clearly, the fate of a cell to undergo apoptosis, necroptosis, or other cell death programs reflects a complex balance of factors, including local cellular conditions and specific initiating insults.

Fig. 1.

Necroptosis pathways. Common triggers of necroptosis and their overlap with proinflammatory and apoptosis signaling pathways are illustrated. Tumor necrosis factor-α (TNF-α) ligates TNF receptor, resulting in formation of a complex consisting of TRADD and polyubiquitinated receptor-interacting kinase 1 (RIPK1). This complex (complex I) promotes inflammation and cell survival. Deubiquitnation results in RIPK1 dissociation from complex I. Two complexes can form (depending on caspase 8 activation). Complex IIa consists of activated caspase 8 and leads to apoptosis, while in the presence of caspase 8 inhibition, complex IIb (the necrosome) forms. In complex IIb (the canonical necrosome), RIPK1 engages RIPK3 via interaction of rip homotypic interaction motif (RHIM) domains, resulting in phosphorylation of RIPK3, RIPK3 oligomerization, and mixed lineage kinase like (MLKL) phosphorylation. Phosphorylated MLKL oligomerizes and translocates to the cell membrane, and lytic cell death occurs. Necroptosis can also be initiated through noncanonical necrosomes independent of RIPK1. The Toll-like receptor (TLR) adaptor TRIF (TIR domain-containing, adapter-inducing interferon-β) can be activated following TLR ligation (TLR4 or TLR3). TRIF interacts with RIPK3 via its RHIM domain. DNA-dependent activator of interferon-regulatory factors (DAI) is a cytosolic DNA sensor that can also directly interact with RIPK3 through its RHIM domain. In the presence of caspase 8, DAI may also promote apoptosis (not shown). RIPK3 can also promote inflammasome activation and inflammation (leftmost panel).

Animal models of necroptosis have employed strategies including pharmacological and genetic ablation of RIPK1, RIPK3, MLKL, and DAI (Table 1). As knowledge accrues about the multiple functions of each of these proteins in necroptosis and other vital cell mechanisms, it has become clear that none constitutes a perfect model to test the potential therapeutic potential of necroptosis inhibition in clinically heterogenous syndromes. Determining the predominant cell type or cell death pathway triggered during sterile and infectious models has been elusive in animal studies employing global knockout or pharmacological inhibition of necroptotic pathway proteins. Furthermore, even within a given organism, sterile insults or infections likely result in more than one form of cell death. Indeed, animal models of drug-induced liver toxicity showed that use of Nec-1, a small molecule inhibitor of RIPK1, results in inhibition of necroptosis but an increase in apoptosis and thus exacerbated injury (9). Common small-molecule inhibitors used to inhibit necroptosis include Nec-1 and Nec-1s (RIPK1), GSK’872, dabrafenib (RIPK3), and necrosulfonamide/NSA (MLKL) (14, 39, 68, 76). Because inhibition of RIPK1 and RIPK3 may also inhibit apoptosis, the effects of those experiments cannot definitively prove which cell death pathway is responsible. This complexity is further illustrated by the different phenotypes generated by global Ripk1-, Ripk3-, or Mlkl knockout animal models (1). RIPK1 and RIPK3 may also have necroptosis-independent proinflammatory activity, which is interrupted by MLKL activation and completion of necroptosis (33, 79). Examples of necroptosis-independent activity include the ability to initiate pyroptosis and more recently T cell proliferation (1). Elegant studies utilizing caspase 8 knockout/Ripk3 knockout and caspase 8 knockout/Mlkl knockout mice demonstrated that the nonnecroptotic functions of RIPK3 contribute substantially to inflammation, as deletion of RIPK3 protected mice from increased T cell proliferation, cytokine secretion, and autoimmunity observed in the presence of caspase8/MLKL mice that had intact RIPK3 signaling. These well-designed studies highlight the necessity for more complex model systems to distinguish the necroptotic and nonnecroptotic functions of the RIPK1, RIPK3, and MLKL. Additionally, in vivo studies using tissue or cell-specific knockouts will be required before developing specific therapeutic targets for clinically heterogenous syndromes such as ARDS.

Table 1.

Necroptosis in animal models of lung injury

Injury Model	Cell Type Affected	Mechanism of Necroptosis	Outcomes of Necroptosis Inhibition	Comments
Bacterial pneumonia  S. aureus (17, 18, 34, 35, 73, 83)  K. pneumoniae (27)  S. marascens (16)  S. pneumoniae (16)	Alveolar macrophage Neutrophils Alveolar epithelial cells Bronchial epithelial cells	Pore-forming toxins	Increased alveolar macrophage survival Decreased neutrophil aggregation Decreased bacterial titers RIPK3−/− mice protected in SA and Serratia MLKL−/− mice protected in Serratia	Mouse and non-human primate models
Viral infection  Influenza A (12, 37, 50, 69, 77)	Fibroblasts Alveolar epithelial cells Bronchial epithelial cells	Viral proteins activate DAI ssRNA activates DAI NS1 protein activates MLKL	DAI−/− mice protected from IAV mortality in 1 study, but had higher mortality in another DAI−/− had impaired viral clearance in vivo At low MOI, RIPK3−/− mice protected from H7N9 IAV In higher MOI, RIPK3−/− mice had increased mortality and viremia in H1N1 IAV MLKL−/− mice had normal survival in IAV, but MLKL/FADD−/− mice had increased mortality	Blockade of necroptosis increased apoptosis and vice versa
Sepsis  LPS (33, 39, 57, 72)  CLP (23, 61, 75)  Systemic infection (35)  TNF-α (49)	Alveolar epithelial cells Alveolar macrophages Lung endothelial cells	Increases in phospho-RIPK3 and phospho-MLKL	RIPK3−/− mice protected in CLP one LPS models, but not protected in separate LPS model RIPK3 and MLKL−/− mice had increased mortality in systemic S. aureus model MLKL−/− mice not protected in CLP model RIPK3/MLKL/Caspase 8−/− mice protected in TNF-α model
Ischemia-reperfusion  Kidney transplant (10, 80, 82)	Type II alveolar epithelial cells Alveolar epithelial cells	Increased RIPK1 and RIPK3 expression	Nec-1 treatment-diminished cell death in rat renal IRI model	Ferroptosis also observed
Ventilator-induced lung injury  Hyperoxia (22, 67)  Barotrauma (63)	Alveolar epithelial cells		RIPK3 knockout, GW440139B, and GW′39B decreased RIPK3 and MLKL activation, lung inflammatory cell infiltrate, and alveolar damage in hyperoxia model RIPK3−/− but not MLKL−/− mice protected in VILI mouse model
Toxin  Oleic acid (52, 53)	Lung endothelial cells	Increases in RIPK1, RIPK3, and MLKL expression and necrosome assembly	Nec-1 prevented declines in P/F ratio and lung wet/dry ratio, BAL protein and leukocyte count, and histology

BAL, bronchoalveolar lavage; CLP, cecal ligation and puncture; DAI, DNA-dependent activator of interferon regulatory factors; FADD, Fas-associated death domain; IAV, influenza A virus; IRI, ischemia-reperfusion injury; LPS, lipopolysaccharide; MLKL, mixed lineage kinase like; MOI, multiplicity of infection; RIPK3, receptor-interacting kinase 3; SA, Staphylococcus aureus; ssRNA, single-stranded ribonucleic acid; TNF-α, tumor necrosis factor-α; VILI, ventilator-induced lung injury.

NECROPTOSIS IN BACTERIAL LUNG INFECTION AND PNEUMONIA

Pneumonia is the most common cause of ARDS (6, 45), and bacterial infection is a well-described trigger of necroptosis. Programmed cell death in response to bacterial infection can serve a beneficial role in host defense, limiting reservoirs of infection and containing pathogens (58), but may also have counterproductive effects caused by excessive tissue damage (34, 51, 58). One example of this is necroptosis induced by Staphylococcus aureus (S. aureus), a major cause of pneumonia.

S. aureus can efficiently induce necroptosis of immune and pulmonary parenchymal cells to deter bacterial clearance. In neutrophils, phagocytosis of S. aureus initially upregulates apoptotic signals that promote efferocytosis by macrophages. However, S. aureus that survives phagocytosis suppresses these apoptotic signals, leading to lytic PMN cell death in a RIPK1-dependent manner suggestive of necroptosis (18). Low levels of S. aureus infection preferentially triggered apoptosis in this study, while high levels of S. aureus infection triggered necroptosis independent of exogenous caspase inhibition, suggesting that during mild infection apoptosis may be the default cell death pathway unless uncontrolled infection requires a more aggressive and immunogenic response (18). Subsequent studies have also demonstrated the ability of S. aureus to induce RIPK3-independent necrotic cell death (18). Lung parenchymal cells also undergo necroptosis; incubation of S. aureus in lung epithelial cells increased RIPK3, while RIPK3 siRNA knockdown reduced RIPK3 expression and necrotic cell death (74).

S. aureus pore-forming toxins are a major mechanism by which S. aureus induces necroptosis. S. aureus pore-forming toxins induce alveolar macrophage necroptosis, which can be rescued by RIPK1, RIPK3, and MLKL inhibition and genetic ablation of RIPK3 and MLKL (34). S. aureus toxin PSMα1 also induces neutrophil necroptosis in vitro and in vivo (83) in a CA-MRSA pneumonia model, in which mice treated with Nec-1 or infected with PSMα1 knockout strains had higher survival and decreased colony-forming units, necrotic cell burden, and pMLKL. As noted above, use of Nec-1 does not exclude a potential role of apoptosis in cell death caused by S. aureus. S. aureus pore-forming toxins induce alveolar and bronchial epithelial cell necroptosis in vitro and in vivo, without the need for exogenous caspase inhibition (16), thus showing the ability of S. aureus to precipitate necroptosis in all major lung cell types.

Other bacteria also initiate necroptosis in lung cells. Serratia and Streptococcus pneumonia pore-forming toxins induce necroptosis in alveolar macrophages (17), in alveolar and bronchial epithelial cells (16) in vitro, and in mice and nonhuman primates (30), without exogenous caspase inhibition or bacterial internalization. Multiple inhibitors of necroptosis rescued alveolar macrophages (17), while RIPK3 knockout mice had improved survival in Serratia pneumonia. Neutrophil phagocytosis of Klebsiella pneumonia induced macrophages to shift from apoptosis to necroptosis, similarly to mechanisms observed in S. aureus infection (27), impairing efferocytosis and increasing inflammation. Treatment with Nec-1s decreased neutrophil accumulation and bacterial titers in mouse lungs; however, effects on animal survival were not reported.

As the preceding examples have demonstrated, inhibiting necroptosis in vivo during bacterial pneumonia has generally shown benefit. Kitur et al. (34) showed that both Ripk3 knockout and Nec-1s-treated mice had improved S. aureus clearance, less histologic injury, and improved barrier function indicated by protein content in BAL fluid. This effect may have been mediated by decreasing necroptotic death of alveolar macrophages, as selective depletion experiments demonstrate alveolar macrophages to be crucial for S. aureus clearance. Ripk3 knockout and Mlkl knockout mice were protected against death in Serratia pneumonia, and lung histology confirmed decreased parenchymal tissue damage, improved barrier function, and less alveolar consolidation, confirming a detrimental role of alveolar macrophage necroptosis in bacterial infection (16, 17). In contrast to these findings, in models of systemic infection, the effects of necroptosis inhibition on survival are considerably mixed, as will be discussed subsequently. Therefore, these findings, while intriguing, leave unanswered the question of whether tissue-specific necroptosis inhibition is needed to prevent localized tissue damage while allowing systemic control of infection.

NECROPTOSIS IN VIRAL PULMONARY INFECTIONS

Programmed necrosis plays a complex role in host defense against viral infections. Cells have developed multiple pathways for sensing viruses, predominantly through detection of nucleic acids, and use necroptosis to prevent the cell from becoming a reservoir for sustained viral replication. In response to this, viruses have evolved their own mechanisms to inhibit host necroptosis. Nonpulmonary viral infections, including herpes simplex virus (HSV) and murine cytomegalovirus (MCMV), have been the most extensively characterized, with both employing proteins with RHIM domains that associate with RIPK3 to inhibit infected cell necroptosis (20). Subsequent work has shown that pulmonary viruses have similar means to disrupt necroptosis and thus maintain a cellular reservoir for replication.

Because the role of necroptosis in viral infections was recently detailed in another review (70), this review will focus on those viruses known to cause ARDS. Influenza virus (IAV and IBV) has been associated with the development of severe ARDS and necrotizing pneumonia. Recent studies have revealed that cells detect influenza and trigger necroptosis through multiple mechanisms. These include the intracellular nucleic acid receptor DAI, which can be activated by IAV viral proteins and ssRNA (37, 69). DAI binds cytosolic nucleic acids and stimulates production of type 1 interferons (30). DAI also contains a RHIM domain, allowing for activation of RIPK3. In two independent studies, deletion of DAI led to higher viral titers and dissemination of virus; however, in one study, Dai knockout mice demonstrated lower mortality following viral infection, while Dai knockout mice had higher mortality in the other study (37, 69). This divergence may have been related to differences in age of mice or other changes in their local environments. In both studies, Dai knockout mice exhibited higher viral titers than wild-type (WT) mice, supporting the hypothesis that nucleic acid signaling through DAI (and possibly necroptosis initiation) acts as a defense mechanism against persistent viral replication. Notably, DAI interacts with diverse signaling pathways in addition to necroptosis, so the effects of the Dai knockout mouse may be less reflective of necroptosis than the effects of the MLKL knockout mouse. IAV can also directly induce necroptosis through the IAV NS1 protein, which triggers MLKL trimerization and membrane localization (12). Collectively, these data suggest that both IAV nucleic acids and proteins trigger necroptotic signaling through discrete mechanisms.

Subsequent studies have confirmed the centrality of necroptosis to influenza host defense, showing significant increases in lethality and tissue destruction when necroptosis is inhibited. At a low multiplicity of infection (MOI) with H7N9 influenza, Ripk3 knockout mice had improved survival over WT (77). This is consistent with bacterial infection studies that necroptosis may be dispensable for host defense during low-virulence or sublethal dose infection (18). However, higher-dose models of H1N1 influenza have underscored the importance of necroptosis during more overwhelming infection. RIPK3 inhibition resulted in excess lethality and high titers of disseminated IAV in H1N1 IAV-infected mice (50), possibly by impacting recruitment of T cells. This finding highlights the interaction of innate and adaptive immunity in response to programmed necrotic cell death and provides a potential mechanism for the protection afforded by necroptosis during influenza infection. A further insight from this study was that RIPK3 mediates both apoptotic and necroptotic cell death and that blockade of each pathway increased cell death by the unopposed pathway. While Mlkl knockout mice survived at normal ratios in IAV infection, perhaps because of a preserved capacity of apoptosis, Mlkl/Fadd knockout mice, deficient in apoptosis and necroptosis, had higher mortality and viremia than even RIPK3 knockout mice (50). Therefore, although inhibition of necroptosis promoted fibroblast survival of IAV infection in vitro, the effects of necroptosis inhibition in vivo in this model remain unclear, as both apoptosis and necroptosis are inhibited in the Mlkl/Fadd knockout mice. The discrepancy between improved fibroblast survival in vitro with worsened survival of the organism following inhibition of cell death in vivo highlights the tension inherent in inflammatory cell death programs. The death of local cells and tissues is intended to promote survival of the organism by removing havens for viral replication, alarming the host to danger, and augmenting host defense. However, induction of this form of cell death is not without consequences, as the intense inflammation generated by necroptosis may overwhelm the host organism itself. These findings highlight the pleiotropic effects of RIPK3 and underscore the need for further study of the necroptotic pathway proteins in a cell- and tissue-specific manner.

NECROPTOSIS IN THE PULMONARY RESPONSE DURING SEPSIS

Sepsis, including nonpulmonary sepsis, is a leading trigger of ARDS, and preclinical models of sepsis commonly result in histologic and pathophysiologic lung injury (6). Accumulating evidence from sepsis models, including sepsis-associated molecule administration and polymicrobial systemic infection induced by cecal ligation and puncture (CLP), suggests that the dysregulated systemic response to pathogens characteristic of sepsis can lead to pulmonary necroptosis.

Several models of sepsis using intrapulmonary or systemic lipopolysaccharide (LPS) administration have demonstrated necroptosis in lung tissue (Fig. 2). Wang et al. (72) found that while low-dose intratracheal LPS induced apoptosis, high-dose LPS caused necrotic cell death of alveolar epithelial cells and alveolar macrophages with increased phospho-RIPK3 and phospho-MLKL. Ripk3 knockout mice were resistant to hypothermia and mortality from both low- and high-dose LPS. Using systemic LPS administration, Gandhirajan et al. (13) confirmed earlier findings of RIPK3 upregulation and RIPK3-dependent cell death via a pathway mediated by stromal interaction molecule 1 (STIM-1) on lung endothelial cells. However, the morphology of cell death and phosphorylation of MLKL were not described, thus leaving the precise modality of cell death unanswered.

Fig. 2.

Triggers and cellular consequences of necroptosis in lung injury. The normal alveolus (center) is characterized by preserved epithelial and endothelial barriers and a paucity of inflammatory cells. Sepsis-associated acute respiratory distress syndrome (ARDS; top left) is characterized by necroptosis of endothelial, type 1, and type II epithelial cells and alveolar macrophages, which may contribute to the pathologic findings in ARDS of decreased endothelial barrier function, microthrombosis, neutrophil extravasation, alveolar flooding, and inflammatory mediator release. Necroptosis of lung endothelial cells is observed in sterile lung injury (top right) in response to intact red blood cells and hemin. Bacterial pneumonia (bottom left) induces necroptosis in all relevant pulmonary cell types through both direct infection of cells and through pore-forming toxins. Pulmonary viruses (bottom right) use multiple mechanisms to induce necroptosis, particularly of alveolar macrophages, enabling the virus to evade host immune responses. However, necroptosis is a crucial host response to viral infection by removal of potential reservoirs for viral replication.

Cecal ligation and puncture (CLP) models of systemic sepsis also induce lung injury and mortality in murine models. Sharma et al. (61) showed that RIPK3 knockout mice were protected from death in CLP-induced sepsis, exhibiting decreased lung injury, reduced neutrophil accumulation in the lungs, and decreased levels of proinflammatory cytokines. In a similar model of intraperitoneal injection of cecal slurry, Hansen et al. (23) showed improved lung injury scores in Ripk3 knockout mice but did not comment on the specific morphology of cell death. Based on these studies, inhibition of necroptosis may show promise as a potential therapy for ARDS triggered by extrapulmonary infections.

However, contradictory findings in sepsis models have complicated interpretation of necroptosis’s role in sepsis. Kitur et al. (35) demonstrated worse outcomes in Ripk3 knockout, Mlkl knockout, and necroptosis inhibitor-treated mice in the context of systemic and cutaneous S. aureus infection. In a CLP sepsis model, Mlkl knockout mice were not protected against mortality despite protection from cerulean-induced pancreatitis (75). Effects on lung injury in both studies were not reported. This conclusion was also reached by Newton et al. (49), who found no protection of Ripk3 knockout in an LPS-induced sepsis model. Interestingly, this group found that Ripk3 knockout alone, but not Mlkl knockout, was protective against hypothermia and weight loss in a low-dose TNF-induced model of systemic inflammation, but that Ripk3- and Mlkl knockout were both protective at higher doses. Ripk3- or Mlkl knockout combined with caspase 8 knockout produced mice that were almost entirely resistant to the effects of TNF-induced inflammation. Given RIPK3’s pleotropic roles in cell death pathways, this again suggests that less catastrophic injury is predominantly apoptotic, while more overwhelming injury triggers an immunogenic necroptotic pathway as a last resort for host defense. An alternative hypothesis is that inhibition of necroptosis in sepsis may have unpredictable effects, depending on whether the benefit of improving host defense outweighs the harm of widespread uncontrolled tissue damage.

Evidence of necroptosis in the human pulmonary response to systemic sepsis is more limited. A recent study of five cohorts of medical critical care patients showed a correlation between plasma RIPK3 levels and mortality, as well as with the respiratory components of the Sequential Organ Failure Assessment score (43). This built on prior associations of RIPK3 levels with mortality in sepsis patients and acute kidney injury in trauma patients, which did not report pulmonary outcomes (43, 62). Nevertheless, the degree to which elevated RIPK3 levels reflect necroptosis specifically, given the pleiotropic pathways through which it is active, remains to be clarified.

NECROPTOSIS IN STERILE LUNG INJURY

Necroptosis as a response to insult is not limited to infections and has been observed following multiple sterile injuries that trigger ARDS. The pattern of unresolving inflammation after sterile injuries has been attributed to release of DAMPs, such as mitochondrial DNA, ATP, uric acid, hemin, formyl peptides, HMGB1, and histones. DAMPs initiate inflammation by ligating pattern recognition receptors (PRRs), activating an intense innate immune cascade. Until recently, evidence that DAMPs, particularly in the extracellular space, directly incite necroptosis has been minimal. Hemin, a DAMP consisting of an oxidized form of heme released during hemolytic anemias such as sickle cell disease, was known to increase endothelial cell activation and barrier dysfunction through a TLR4-dependent but previously uncharacterized pathway (3, 11) This was recently characterized to be necroptosis of pulmonary endothelial cells (64), revealing a potential mechanism by which sickle cell vaso-occlusive crises lead to acute chest syndrome. The discovery that a DAMP can mediate necroptosis is particularly interesting because necroptosis also generates DAMP release through cell lysis, suggesting a possible mechanism for a feed-forward cycle of uncontrolled inflammation and cell death that is often observed in multiple-organ dysfunction after an initial insult.

Remote organ damage that triggers ARDS may also be mediated by necroptosis. The role of necroptosis has been best defined in ARDS following renal ischemia-reperfusion and transplant models (10). Necrosis and specifically necroptosis on electron microscopy were noted to be present in death of type II alveolar and interstitial cells following renal ischemia in a rat model (80). Subsequent work implicated both necroptosis and parthanatos, an alternative form of programmed necrosis, in a similar animal model (82). Renal ischemia-reperfusion induced expression of RIPK1 and RIPK3 in lung epithelial cells, and cell death was reduced by Nec-1 both in vitro and in vivo. This work suggests that, in the controlled setting of organ transplant, necroptosis may be a promising therapeutic target to prevent ischemia-reperfusion injury (16).

Red blood cell (RBC) transfusion is the most common modifiable risk factor for ARDS. We have recently found that red cells can induce necroptosis of lung endothelial cells (57). In vitro, incubation of RBCs with human lung microvascular endothelial cells (ECs) in a static culture model and in a perfused microfluidics model resulted in EC necroptosis. ECs exposed to RBCs released RIPK3 and HMGB1; Nec-1 reduced EC death and release of both RIPK3 and HMGB1. In a murine in vivo transfusion model, transfusion increased lung necrosome formation and HMGB1 release, priming the host to subsequent LPS-induced injury, thus suggesting that one mechanism of transfusion-associated ARDS is the induction of necroptosis that primes the host to subsequent injury (57). Given that transfusions are a planned event, pulmonary complications from transfusion may be attenuated or prevented with simultaneous administration of a necroptosis inhibitor.

Necroptosis has been detected in ventilator-induced lung injury and hyperoxia-mediated lung injury. Increased RIPK3 and MLKL phosphorylation were detected in a hyperoxia mouse model and in human neonates affected by respiratory distress syndrome, a condition linked to hyperoxia (67). Ripk3 knockout mice and those treated with the RIPK3 inhibitors GW440139B and GW’39B decreased RIPK3 and MLKL activation, lung inflammatory cell infiltrate, and alveolar damage. Similarly, in another mouse model, Han et al. (22) detected increases in RIPK1 and RIPK3 by immunohistochemistry, immunoprecipitation, and mRNA after hyperoxia, which was also diminished by Nec-1. RIPK3, but not MLKL, was recently implicated in the development of VILI in a large ICU cohort and mouse model (63). Ripk3 knockout but not Mlkl knockout mice were protected from VILI. This perhaps indicates the importance of nonnecroptotic functions of RIPK3, such as inflammasome activation, which has been previously been shown to be important in ventilator-induced lung injury (36, 48).

Accumulating evidence indicates that necroptosis may play an important pathophysiological role in toxin-induced lung injury. For instance, oleic acid, a fatty acid used in experimental models of lipid embolism-induced ARDS, has been noted to cause both apoptosis and necrotic cell death, particularly of lung endothelium (15) The precise receptors engaged by oleic acid remain unclear, but Pan et al. (52) demonstrated increased RIPK1, RIPK3, MLKL, and necrosome assembly in a rat model of systemic oleic acid-induced lung injury. The same group later demonstrated that Nec-1 successfully abrogated oleic acid-induced declines in pulmonary oxygen exchange, as measured by P/F ratio, barrier function as measured by lung wet/dry ratio, BAL protein and leukocyte count, and histological evidence of injury (53).

CONCLUSIONS AND REMAINING QUESTIONS

Accumulating evidence demonstrates that necroptosis occurs in lung cells in response to a wide range of triggers, and preliminary evidence suggests that inhibition may benefit some subgroups. Necroptosis appears to be vital to host defense in viral infection and possibly in systemic infections such as sepsis, but inhibition in sterile injuries and localized infections may reduce tissue destruction and improve outcomes (16, 17, 35, 49, 50, 61, 73). While the ability to clinically distinguish the presence of viral or bacterial infection and to separate these from sterile inflammation is still imperfect, improvements in clinical diagnostics may allow for early identification of patients in whom necroptosis inhibition could be lifesaving. Furthermore, for patients who may require treatments known to generate adverse effects through necroptosis, such as blood transfusion or mechanical ventilation, specific targeting of necroptosis may prevent lung injury in these settings. Remaining questions, however, will require answers before a clinical benefit can be realized.

Factors currently limiting the therapeutic utility of targeting necroptosis therapeutically hinge on several major knowledge gaps. One is the redundancy inherent to cell death pathways. Evidence demonstrates that inhibition of one pathway can augment others (50). The relationships between apoptosis, necroptosis, and pyroptosis in particular, mediated by the different functionalities of RIPK1 and RIPK3, make it difficult to predict whether inhibiting one pathway will result in less overall cell death or less destructive cell death (82). While inflammatory cell death may be diminished if necroptosis gives way to apoptosis, a switch to pyroptosis or other inflammatory cell death pathways may produce neutral or harmful outcomes. Clarification of the molecular switches determining which modality of cell death will occur and the benefits and harms of necroptosis in specific tissues is essential before translating these findings to clinical therapies. Given the complex interactions between cell death pathways, potential interventions inhibiting necroptosis will require an abundance of caution to prevent off-target and unintended effects.

Second, the beneficial or harmful effects of necroptosis may depend on the insult, with differing responses seen in viral versus bacterial, localized versus systemic, and infectious versus sterile injuries. While the inciting insult is known in experimental settings, timely exclusion of conditions in humans for which necroptosis inhibition is harmful is challenging given current diagnostic capabilities. Even more fundamentally, it remains unclear how to best determine whether necroptosis is occurring in critically ill human subjects. Biomarker selection is controversial given the diversity of triggers and flexibility of pathways that culminate in necroptosis. Plasma RIPK3 and MLKL correlate with survival in critically ill patients (43, 62, 71), but whether plasma RIPK3 reflects necroptosis given the multiple cell death and inflammatory pathways it participates in is unclear. This leaves significant questions, including whether plasma RIPK3 marks necroptosis specifically or cell stress more generally, whether the tissue source of RIPK3 has significance, and whether plasma elevation of RIPK3 precedes widespread cell damage early enough to offer an opportunity for intervention, and these biomarkers have not been validated widely in human populations. Promising new technologies, including the use of imaging flow cytometry to distinguish apoptosis from necroptosis and micro-engineered models of human disease such as microfluidic “lung on a chip” models where the response to patient plasma can be assessed, may significantly aid in detection of necroptosis in human subjects (56, 60).

Despite the myriad of challenges in the field, identifying ways in which necroptosis can be interrupted or exploited holds significant promise for treatment of lung injury and other human diseases. Improvements in clinical detection of necroptosis are on the horizon, and blockade of multiple therapeutic targets of the necroptosis pathway is now possible, with inhibitors available for RIPK1, RIPK3, and MLKL (56). Blockade of multiple cell death pathways simultaneously may improve outcomes, particularly in cases in which cell death can be anticipated, such as transplantation-induced ischemia-reperfusion injury, before transfusions, or in critically ill patients who have not yet developed organ failure (40). While much remains to be understood about the role of necroptosis in human ARDS, there is now an opportunity to gain a deeper understanding of cell death pathways in the development of lung injury and develop strategies to identify and target these pathways with the ultimate goal of preventing lung injury in susceptible patients.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants T32 HL007775-21 and R01 HL126788 (N. S. Mangalmurti) and Department of Defense Grant W81XWH-15-1-0363 (N. S. Mangalmurti).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.F. and N.S.M. prepared figures; H.F. and N.S.M. drafted manuscript; H.F. and N.S.M. edited and revised manuscript; H.F. and N.S.M. approved final version of manuscript.