Molecular mechanisms and roles of pyroptosis in acute lung injury

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which are characterized by excessive inflammation and accompanied by diffuse injury of alveoli, can result in severe respiratory failures. The morbidity and mortality of patients remain high because the major treatments for ALI/ARDS are mainly supportive due to the lack of effective therapies. Numerous studies have demonstrated that the aggravation of coronavirus disease 2019 (COVID-19) leads to severe pneumonia and even ARDS. Pyroptosis, a biological process identified as a type of programed cell death, is mainly triggered by inflammatory caspase activation and is directly meditated by the gasdermin protein family, as well as being associated with the secretion and release of pro-inflammatory cytokines. Clinical and experimental evidence corroborates that pyroptosis of various cells in the lung, such as immune cells and structural cells, may play an important role in the pathogenesis of “cytokine storms” in ALI/ARDS, including those induced by COVID-19. Here, with a focus on ALI/ARDS and COVID-19, we summarized the recent advances in this field and proposed the theory of an inflammatory cascade in pyroptosis to identify new targets and pave the way for new approaches to treat these diseases.

Introduction

The concept of acute respiratory distress syndrome (ARDS) was first proposed in 1967 based on a series of case reports.^[1] ARDS, triggered by various endogenous and exogenous risk factors, is characterized by significant hypoxemia, diffuse pulmonary infiltrates in radiographs, and diffuse destruction of alveolar epithelial cells and capillary endothelial cells in pathology, as well as increased dead-space ventilation and decreased lung compliance in pathophysiology.^[2,3] Among the various risk factors inside and outside the lung, pneumonia and sepsis resulting from viral or bacterial infection are major issues, and acute lung injury (ALI)/ARDS induced by these factors is probably lethal. For example, ALI/ARDS is the main complication and cause of death in patients with coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2).^[4] In a cohort study conducted in the USA, the crude incidence of ALI/ARDS was 78.9 per 100,000 people/year and the in-hospital mortality was 38.5%, perhaps because the treatments are mainly supportive and are protective strategies rather than causative factor-centered.^[5,6]

Pyroptosis is a newly discovered form of programed cell death that depends on caspase-triggered cleavage of gasdermin proteins following recognition of the ligand in the cytoplasm.^[7] The oligomerization of the N-terminal domain of activated gasdermin on the cell membrane initiates the formation of pores, which results in ion influx, H₂O influx, and pro-inflammatory cytokine efflux, ultimately leading to cell swelling and rupture as well as ampliative inflammatory responses. Bacteria, viruses, toxins, and drugs can all cause pyroptosis, which contributes to maintaining the stability of the internal environment and antagonizing external dangerous factors. However, it may participate in the occurrence and development of various diseases such as infectious diseases, as well as tumors with their collapsed defenses.^[8] Further studies on pyroptosis are helpful in understanding its roles in the occurrence, development, and outcome of related diseases, and will provide new solutions for clinical treatments.

Increasing evidence suggests that pyroptosis of different lung cell types and their related inflammatory reactions play a significant role in the development of ALI. For example, the inflammatory mediators released by pyroptotic macrophages destroy the stability of the lung tissue microenvironment, leading to the sequential pyroptosis of other lung cells to form a cascade reaction. The relationship between pyroptosis and the development of ARDS has been shown by clinical evidence, while blocking the pyroptosis pathway can improve the results of ALI/ARDS. In general, it is necessary to further elucidate the relationship between pyroptosis and ALI/ARDS to better understand the fundamental mechanism, direct further research, and establish essential therapy.

Mechanism of pyroptosis

Pathways of pyroptosis

Previously, Shi et al^[9] divided the pyroptotic pathway into a canonical inflammasome-dependent pathway mediated by caspase-1 and non-canonical inflammasome-dependent pathways dependent on caspase-4/-5 in humans and caspase-11 in mice. These caspases can cleave gasdermin D (GSDMD) upon activation by interacting with intracellular ligands to mediate pyroptosis. Additionally, several recent studies have suggested that caspase-3 and caspase-8, which are traditionally regarded as apoptotic proteins, can also mediate pyroptosis by cleaving gasdermin E (GSDME) and GSDMD, respectively.^[10,11] To summarize, pyroptotic pathways are commonly divided into inflammasome-dependent pathways and non-inflammasome-dependent pathways [Figure 1].

Figure 1.

The mechanisms of pyroptosis. PAMPs and DAMPs stimulate cells and activate canonical inflammasomes such as NLRP3. Then mature caspase-1 cleaves GSDMD into GSDMD-N and promotes secretion of IL-1β and IL-18. GSDMD-N binds to the membrane and forms a pore, which mediates K⁺ efflux, Na⁺ influx, water influx, and cytokine release. LPS can directly activate caspase-4/-5/-11, which can promote the cleavage of GSDMD. Coincidently, caspase-3 cleaves GSDME and acquires GSDME-N, which plays the same role as GSDMD-N. Caspase-8 acts as a positive upstream regulator of caspase-1 and caspase-3. When caspase-1 is inhibited, caspase-8 can directly cleave GSDMD. There also exists a rescue mechanism, whereby ESCRT-III can be recruited to the pores on the membrane to repair them, induced by Ca²⁺ inflow. The figure was created with BioRender.com. AIM2: Absent in melanoma-2; ASC: Apoptosis-associated speck-like protein; CLR: C-type lectin receptor; DAMPs: Damage-associated molecular patterns; ESCRT-III: Endosomal sorting complexes required for transport-III; GSDMD: Gasdermin D; GSDME: Gasdermin E; IL-1β: Interleukin-1β; IL-18: Interleukin-18; LPS: Lipopolysaccharide; NLRC: Nucleotide-binding oligomerization domain, leucine-rich repeat and caspase recruitment domain-containing; NLRP: Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing; PAMPs: Pathogen-associated molecular patterns; TLR: Toll-like receptor.

The canonical inflammasome is a protein complex composed of three parts: a cytoplasmic sensor, an adaptor protein (apoptosis-associated speck-like protein, ASC), and pro-caspase-1.^[12] According to recent studies, the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the absent in melanoma-2 (AIM2)-like receptors are the most common cytoplasmic sensors involved in the formation of canonical inflammasomes.^[12] The ASC protein contains the N-terminal pyrin domain and the C-terminal caspase activation and recruitment domain, which can connect the sensor and the effector, respectively.^[13] NLR family pyrin domain-containing (NLRP) 1, NLRP3, NOD leucine-rich repeat and caspase recruitment domain-containing 4 (NLRC4), and AIM2 are the most studied canonical inflammasomes at present. The NLRP1 inflammasome recognizes a variety of bacterial exotoxins, while the NLRP3 inflammasome can recognize adenosine triphosphate (ATP), Nigeria bacteria, and other pathogen-associated molecular patterns (PAMPs) as well as damage-associated molecular patterns (DAMPs) such as ion beams and lysosomal lysates.^[14,15] The NLRC4 inflammasome mainly recognizes bacterial flagellin, and the Type III secretion system.^[16,17] Additionally, the AIM2 inflammasome responds to endogenous and exogenous DNA molecules and can be activated by viruses that invade cells.^[18] When the PAMPs or DAMPs are recognized by the cytoplasmic sensors, the signal can be transduced through ASC to activate caspase-1, which can cleave GSDMD, pro-interleukin-1β (IL-1β), and pro-interleukin-18 (IL-18).^[19] The N-terminal fragment of GSDMD cleaved by caspase-1 oligomerizes and forms pores on the cell membrane that release the inflammatory cytokines and induce pyroptosis, while activated IL-1β and IL-18 are released extracellularly through the pores and are involved in amplifying the inflammatory response.^[20]

In addition, the non-canonical inflammasome-dependent pathway can also mediate pyroptosis and the release of cytokines. Unlike canonical inflammasomes that rely on cytoplasmic sensors for signal recognition and ASC for transmitting signals, non-canonical inflammasomes, which contain pro-caspase-4/-5/-11 and lipid-A, directly respond to the intracellular lipopolysaccharide (LPS).^[21–25] A recent study showed that the activation of caspase-4, -5, and -11 depends on their own oligomerization and proteolysis, directly cleaving GSDMD to trigger pyroptosis. Meanwhile, caspase-11 and caspase-1 function synergistically in many cases. It is reported that cleavage of pannexin-1 by caspase-11 results in K⁺ efflux through the pannexin-1 channel, which in turn causes NLRP3 activation.^[26] Similarly, it has been shown that caspase-4 can activate the NLRP3 inflammasome through GSDMD-N terminal-guided K⁺ efflux, which plays a synergistic role with caspase-1 in pyroptosis, thereby promoting IL-1β maturation.^[23,27,28] As for the release of cytokines, caspase-4 can cleave pro-IL-18 rather than pro-IL-1β, while caspase-5 has a negligible impact on pro-IL-18 and pro-IL-1β, whose mechanisms and roles during pyroptosis require further elucidation.^[29,30]

The non-inflammasome-dependent pathway of pyroptosis mainly focuses on caspase-3 and caspase-8. Caspase-3, a critical regulator of apoptosis, can also mediate pyroptosis. When stimulated by chemotherapeutic drugs or reactive oxygen species, GSDME, also known as DFNA5, can be directly cleaved by caspase-3 and generate the same N-terminus as GSDMD.^[31–34] The role of caspase-8 is more complicated.^[35] Sarhan et al^[36] demonstrated that upon inhibition of transforming growth factor β-activated kinase following Yersinia infection, activated caspase-8 directly cleaved GSDMD and GSDME indirectly via caspase-3, leading to pyroptosis. In addition to direct cleavage of GSDMD, caspase-8 can also mediate pyroptosis by serving as a positive regulator of the NLRP3-dependent caspase-1 signaling cascade in the presence of caspase-1.^[37,38] Moreover, studies have revealed that caspase-8 can compensate for caspase-1 by cleaving GSDMD at the same site when caspase-1 is inhibited.^[39] The specific mechanisms of caspase-3- and caspase-8-mediated pyroptosis have not been fully elucidated and more research is needed.

Gasdermin family

Gasdermin, the excutor of pyroptosis, is a pore-forming protein that conventionally exists in an autoinhibited state. The gasdermin family is reported to consist of six members (GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and PJVK) in humans and 10 members (GSDMA1, GSDMA2, GSDMA3, GSDMC1, GSDMC2, GSDMC3, GSDMC4, GSDMD, GSDME, and PVJK) in mice.^[40] The reported members that mediate pyroptosis are GSDMA, GSDMB, GSDMC, GSDMD, and GSDME. GSDMA is expressed by the epithelial cells of the skin, tongue, esophagus, stomach, mammary gland, and umbilical cord, and GSDMB expression is detected in lymphocytes, esophagus, stomach, liver, and colon. GSDMC is expressed in the lung, esophagus, stomach, trachea, spleen, intestine, bladder, and skin. GSDMD expression is detected in the esophagus, stomach, intestine, and in lymphocytes, while GSDME is expressed in the lung, placenta, brain, heart, kidney, cochlea, intestine, and IgE-primed mast cells.^[41] Current studies revealed that GSDMD and GSDME can be cleaved by caspase-1/-4/-5/-11 and caspase-8, respectively, to mediate pyroptosis.^[42–44] After cleavage, the N-terminal (P30 domain) of GSDMD/GSDME, the lipophilic end, is released from the autoinhibitory GSDMD-C/GSDME-C domain (P20 domain).^[45,46] The cleaved N-terminus oligomerizes on the membrane, efficiently dissolving the phospholipid bilayer and forming pores in membranes, leading to membrane rupture, chromatin condensation, and DNA fragmentation.^[47–49] The pores formed by the N-terminus on the membrane will result in the outflow of potassium ions and the influx of sodium ions, thereby changing the osmolyte pressure of the membrane and destabilizing the membrane. Meanwhile, sodium ions drawn into the cell through the concentration and electrical gradient will bring in plenty of water at the same time, leading to the swelling and rupture of cells.^[41] Additionally, the extracellular release of mature IL-1β and IL-18 through pores after cell rupture increases significantly.^[41]

Self-rescue mechanism of cells in pyroptosis

Not all cells with gasdermin activation are doomed to die. If the damage to the membrane is not severe, the cell can prevent damage through self-rescue mechanisms, such as using organelle membranes, especially lysosomes, to repair damaged membranes, and using endosomal sorting complexes required for transport (ESCRT) machinery to remove the ruptured cell membrane. ESCRT, a protein complex with a membrane fission function, is mainly involved in the formation of outer vesicles and plasma membrane repair. Among the members of ESCRT, ESCRT-III is essential for repair.^[50] Calcium influx through N-terminus pores, acting as a signal to initiate membrane repair, will recruit ESCRT-III to damaged areas of membrane, and ESCRT-III removes N-terminus pores by forming extracellular vesicles.^[50–52] Scheffer et al^[53] demonstrated that inhibition of the ESCRT-III machinery strongly enhances pyroptosis and IL-1β release in human and murine cells following canonical or non-canonical inflammasome activation. However, because of the complex function of ESCRT-III, the role of ESCRT-III-mediated membrane repair in pyroptosis and anti-inflammatory functions requires more research.

Cells can also be protected from pyroptosis-induced death by other mechanisms. Mg²⁺ restricts oligomerization and membrane localization of GSDMD-N to restrain LPS-induced pyroptosis, which is based on the inhibition of the ATP-gated Ca²⁺ channel P2X7 due to Mg²⁺.^[54] Muendlein et al^[55] revealed an important role of cellular Fas-associated death domain (FADD)-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (cFLIP) in the regulation of pyroptosis. Long isoform cFLIP (cFLIP_L) plays a role in regulating caspase-8 activation and protecting macrophages from LPS-induced pyroptosis, while cFLIP_L deficiency promotes activation of caspase-8 and GSDMD as well as the formation of complex II upon LPS activation, leading to pyroptosis. To date, most studies have focused on drugs and molecules that can inhibit pyroptosis, but the self-rescue mechanisms of cells remain unclear.

Pyroptosis in acute lung injury

Pyroptosis of lung macrophages

Monocytes and macrophages are the innate immune cells of the lungs and are the first-line defensive cells for various infections. In addition, at least two main types of macrophages reside in the lung: interstitial macrophages (IMs) and alveolar macrophages (AMs). Resident pulmonary macrophages differentiate from peripheral monocytes in the blood after they migrate to the lung. AMs are essential for maintaining the stability of lung homeostasis and resisting invasion from the extrapulmonary environment, while IMs have an irreplaceable function in homeostasis regulation.^[56] ALI resulting from infectious factors, such as bacteria, fungi, and viruses, has been demonstrated to be associated with AM pyroptosis.^[57,58] On the one hand, pyroptosis of AMs is beneficial for resisting the invasion of different pathogens, and on the other hand, the release of plentiful inflammatory cytokines and the imbalance in the inflammatory response both amplify the inflammatory response, leading to the deterioration of ALI.^[59]

Thus far, it has been demonstrated that the specific mechanisms behind the induction of pyroptosis in macrophages vary from pathogen to pathogen. For example, Gram-negative bacteria such as Pseudomonas aeruginosa and Legionella pneumophila can initiate NLRC4- and caspase-11-dependent pyroptosis via flagellin and LPS, respectively, while coronavirus and certain fungi such as Candida albicans and Aspergillus fumigatus induce pyroptosis mainly through activating NLRP3 and AIM2 inflammasomes.^[57,58,60] However, the stimulators that lead to macrophage pyroptosis are not limited to pathogens, and many other factors such as ion beams and lysosomal lysates, which are members of DAMPs, can also generate pyroptosis in macrophages.^[61] These studies indicate that macrophages are most prone to pyroptosis.

Several clinical studies have demonstrated that there is a compelling link between the pyroptosis of macrophages and ALI. After analyzing blood samples from 145 sepsis patients, clinical investigators found an increased pyroptotic fraction of peripheral blood mononuclear cells (PBMCs), and the percentage of pyroptotic PBMCs was related to sepsis severity and 28-day mortality.^[62] Zhou et al^[63] demonstrated that during pulmonary ischemia-reperfusion injury, IL-1β from monocyte pyroptosis induced human pulmonary microvascular endothelial cell pyroptosis, resulting in cytokine production, pyroptosis activation, and pulmonary edema. Meanwhile, recent studies have delineated that the pyroptosis-related cytokines IL-1β and IL-18 are significantly elevated in the bronchoalveolar lavage fluid of ARDS patients, and specific inhibition of IL-1β and IL-18 has previously been shown to significantly alleviate lung injury.^[64] Additionally, Homsy et al^[65] detected mononuclear-macrophages-derived exosomes containing activated caspase-1 and GSDMD in the serum of ARDS patients, which may further trigger pyroptotic cascades of other cells. Our study demonstrated that pro-inflammatory factors produced by pyroptotic macrophages could further lead to pyroptosis of mesenchymal stem cells, which might disrupt the ability of lung stem cells to repair lung damage.^[66] Briefly, AM pyroptosis is more common in ALI, while both AMs and peripheral mononuclear macrophages can release cytokines to form an inflammatory cascade.

Based on the relationship between AM pyroptosis and ALI, there are many new approaches to alleviate ALI, such as inhibiting the activity of caspase-1, caspase-11/-4/-5, and NLRP3 inflammasome in AMs. Wu et al^[67] demonstrated that inhibition of AM pyroptosis could reduce LPS-induced lung injury because they observed significantly elevated expression of GSDMD-N, NLRP3, and ASC in AMs by LPS-induced ALI, and the caspase-1 inhibitor acetyl-tyrosyl-valyl-alanyl-aspartyl-chloromethyl ketone could inhibit this process. It is reported that tetramethylpyrazine can decrease the polarization of macrophages, reduce the maturation and secretion of inflammatory factors such as IL-18 and IL-1β, and provide a theoretical basis for the treatment of ALI.^[68,69] Zeng et al^[70] also discovered the potential of inhibiting double-stranded RNA-dependent kinase to relieve pyroptosis and alleviate LPS-induced ALI by inhibiting NLRP3. Another agent, pirfenidone, was also reported to block the activation of the NLRP3 inflammasome to attenuate LPS-induced ALI.^[71] In conclusion, we propose that regulating AM pyroptosis and developing new caspase-1 or NLRP3 inhibitors are very promising strategies in mitigating the development of ALI and widening the horizon of clinical therapy by exploiting more effective therapeutic avenues.

Pyroptosis of other immune cells

Neutrophils are the most abundant cells in innate immunity and are mainly responsible for reducing bacterial-induced alveolar inflammation. Studies have shown that LPS-induced pyroptosis of neutrophils results in the mass release of cytokines and tissue-damaging factors in ALI.^[72] At the same time, pyroptosis of neutrophils also promotes the expression of chemokines such as chemokine (C-X-C motif) ligand 12 (CXCL12) and chemokine (C-C motif) ligand 7 (CCL7), recruiting more neutrophils, which might further aggravate the damage in lung tissues.^[72,73] After the infiltration of neutrophils from the blood circulation to the alveoli, neutrophil extracellular traps (NETs) form via two ways. One is a slow cell death pathway termed NETosis, which goes through nuclear delobulation, disassembly of the nuclear envelope, and then cellular polarization, chromatin decondensation, and plasma membrane rupture. The other is a rapid release form of NETs, which undergo degranulation and expulsion of nuclear chromatin. They can trap, neutralize, and kill invading pathogens.^[72,74,75] However, Li et al^[76] proposed that NETs can activate the AIM2 inflammasome and caspase-1 in AMs, promoting the pyroptosis of AMs. Therefore, we believe that NET formation and macrophage pyroptosis may constitute a vicious cycle in the lung, exacerbating cytokine storms and lung injury [Figure 2]. In addition, NETs can also induce the pyroptosis of epithelial and endothelial cells, leading to tissue damage.^[77]

Figure 2.

Pyroptosis in different cell types of the lung and inflammatory cascade. When pathogens invade the lung, monocytes and macrophages are activated and pyroptosis occurs. The released inflammatory cytokines destroy the stability of the lung tissue microenvironment and cause pyroptosis in other lung immune cells and structural cells, forming a cascade reaction and aggravating lung injury. NETs released from neutrophils can also activate macrophages to induce pyroptosis. Pulmonary epithelial and endothelial cells suffer from pyroptosis, dysfunction, and impaired blood–gas barrier, leading to aggravation of ALI. The figure was created with BioRender.com. ALI: Acute lung injury; CCL7: Chemokine (C-C motif) ligand 7; CXCL12: Chemokine (C-X-C motif) ligand 12; LPS: Lipopolysaccharide; IL: Interleukin; NETosis: Neutrophil extracellular traps release; TNF-α: Tumor necrosis factor-α.

As a result, a great strategy is to alleviate ALI through reducing neutrophil pyroptosis and excessive accumulation of NETs. A study identified a novel target, miR-495, the overexpression of which not only downregulated the pyroptosis of neutrophils, but also reduced neutrophil infiltration, protecting AMs against pyroptosis and mitigating ALI.^[78] Peptidylarginine deiminase 2 deficiency directly inhibits the formation of NETs and promotes improvements in the survival of mice with a cecal ligation and puncture-induced (CLP-induced) lethal sepsis.^[79] To summarize, neutrophil pyroptosis and NET-mediated pyroptosis are also potential targets to treat ALI.

Evidence for lymphocyte pyroptosis has also recently been found. Shao et al^[80] found that the granzyme A in cytotoxic lymphocytes (such as cytotoxic T lymphocytes and natural killer cells) can enter target cells through perforin and lead to pyroptosis by cleaving GSDMB at Lys229/Lys244. At present, there have been few studies on lymphocyte pyroptosis in ALI; thus, the mechanism of lymphocyte pyroptosis and its relationship with ALI needs further research.

Pyroptosis of lung structural cells

The dysfunction of lung epithelial and endothelial cells caused by extensive damage is a major characteristic of ALI. The monolayer pulmonary endothelial cells and epithelial cells constitute the main components of the air–blood barrier.^[81] It was shown that PAMPs and DAMPs in the surrounding environment mediate the pyroptosis of epithelial cells and endothelial cells, which may be a pathological mechanism of air–blood barrier damage. Under the circumstances in which epithelial and endothelial cells are damaged, barrier permeability increases and alveolar edema starts, leading to aggravation of ALI.

Several studies suggest that inhibition of pyroptosis will help to protect epithelial and endothelial cells from damage. Caspase-11-mediated pyroptosis occurs in lung epithelial cells during Burkholderia thailandensis infection, and Casp11 knock-out attenuates infection-induced lung injury.^[82] Meanwhile, in a mouse model of ischemia-reperfusion (IR)-induced sterile lung injury, which results from ventilated lung IR (unilateral left pulmonary artery occlusion) surgery, inhibition of NLRP3 and IL-1β expression in alveolar type 2 epithelial cells can reduce IR inflammation and thus alleviate sterile lung injury.^[83] In addition, Cheng et al^[84] discovered that LPS transfection can lead to caspase-11-mediated pyroptosis of pulmonary vascular endothelial cells, and the pyroptosis was weakened with caspase-11 knockout, suggesting that caspase-11 may be a potential therapeutic target for ALI. Patients with hemorrhagic shock (HS) are prone to ALI, because HS promotes the release of high mobility group box protein 1, which can activate caspase-1 after entering lung endothelial cells, resulting in pyroptosis and aggravating lung injury.^[85] Therefore, inhibiting the pyroptosis of pulmonary epithelial cells and pulmonary vascular endothelial cells can directly block the lung tissue damage. We propose that the regulation of various targets in the pyroptotic pathways of epithelial and endothelial cells can prevent the occurrence and development of lung injury.

Pyroptosis in COVID-19

COVID-19 has so far infected millions of people since 2019. Many patients are mildly infected and asymptomatic, but 10% to 15% of patients have severe disease.^[4,86,87] Unfortunately, there is still no effective, precise treatment for COVID-19, and most patients can only resort to primary supportive care.^[88,89] Multiple research findings suggest that pyroptosis is involved in mediating lung injury, and it may be one of the driving forces behind the occurrence of severe lung injury following COVID-19 infection and the aggravation of severe pneumonia or even ARDS [Figure 3].

Figure 3.

SARS-CoV-2-induced mechanisms of inflammasome activation. SARS-CoV-2 enters cells via ACE2 and TLR-4. After SARS-CoV-2 interacts with ACE2 and TLR-4, NLRP3 inflammasomes are activated, leading to pyroptosis. Then numerous inflammatory cytokines are released and aggravate lung injury. The figure was created with BioRender.com. ACE2: Angiotensin-converting enzyme 2; GSDMD: Gasdermin; IL: Interleukin; NLRP3: Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; TLR-4: Toll-like receptor 4; TMPRSS2: Transmembrane protease serine 2; TNF-α: Tumor necrosis factor-α.

It has been reported that SARS-CoV-2 can enter cells by binding to angiotensin-converting enzyme 2 (ACE2) through spike protein (SP), which is then used for adhesion and subsequent internalization, while the cleavage of SP by transmembrane protease serine 2 (TMPRSS2) enhances the invasion of the virus.^[90] In addition, toll-like receptor 4 (TLR-4) is also thought to help the virus enter cells.^[90] After SARS-CoV-2 infection, the interactions between SP and ACE2 as well as TLR-4 could induce NLRP3 inflammasome activation, leading to pyroptosis. Sun et al^[91] also corroborated that non-structural protein 6 (NSP6) of SARS-CoV-2 can bring about NLRP3 inflammasome activation and pyroptosis by autophagic flux stagnation. Furthermore, a study found that the nucleocapsid protein of SARS-CoV-2 inhibits pyroptosis by binding to GSDMD in monocytes and subsequently blocking the cleavage of GSDMD by caspase-1 during the early stages of infection, which may partially explain asymptomatic infections.^[92] However, once the SARS-CoV-2 virus begins to lyse in epithelial and endothelial cells, it may trigger the pyroptosis of multiple inflammatory cells, mainly macrophages, and cause the release of a large number of pro-inflammatory cytokines from pyroptotic cells, contributing to the formation of a cytokine storm.^[93,94] Studies have shown that IL-1β, IL-8, IL-6, tumor necrosis factor-α (TNF-α), and other pro-inflammatory cytokines in the serum of severe patients are overreactive and are associated with disease progression.^[95] A study in Cell revealed that the serum levels of various pro-inflammatory cytokines were significantly increased in patients with COVID-19, and further experiments in mice confirmed that TNF-α and interferon-γ (IFN-γ) could augment the pyroptosis of macrophages.^[96] Likewise, the abundant inflammatory cytokines diffuse into the vessels and damage the endothelial cells. Related research showed that the pyroptosis of endothelial cells is closely related to vascular dysfunction and thrombosis.^[97] In addition, cytokines infiltrate alveoli and induce pyroptosis of the type 2 epithelial cells, which may not only aggravate lung injury but also impair the self-healing potential of lung tissue.^[98,99] In conclusion, when infected by SARS-CoV-2, inflammation further spreads with the release of cytokines, while monocytes, macrophages, endothelial cells, and epithelial cells undergo pyroptosis, aggravating lung injury.

Collectively, these studies suggest that COVID-19 patients may benefit from blocking the viral components or cytokine-mediated inflammatory cell death. Xiong et al^[100] suggested that IL-1 receptor antagonist (IL-1RA) anakinra selectively blocks IL-1 receptor signaling and can alleviate the inflammatory response during pyroptosis. Moreover, there are other new ideas for interventions in COVID-19, such as inhibiting the NLRP3 inflammasome, blocking GSDMD cleavage to reduce pyroptosis and cytokine release, and targeting NETs to intervene in cell pyroptosis.^[92,101,102] Considering the present situation, it may be worth expanding the exploration of available inhibitors of pyroptosis and assessing their therapeutic effect in COVID-19 patients.

Conclusion

In this review, we described the latest research progress on the mechanisms of pyroptosis, and summarized pyroptosis of different cell types in the lung and the relationship among pyroptosis, ALI, and COVID-19 disease. ALI is characterized by massive infiltration of inflammatory cells in the lung tissue, which can undergo pyroptosis with the stimulation of PAMPs or DAMPs. Pyroptosis is a newly discovered form of programed cell necrosis, and much remains to be understood. The pyroptotic pathways discovered in recent studies mainly include caspase-1/-4/-5/-11-mediated inflammasome-dependent pathways and caspase-3/-8-related inflammasome-independent pathways.^[103] The gasdermin protein family is the direct executor of pyroptosis, but effective and specific blockers for it have not been elucidated thus far. Upon pathogen invasion, monocytes, macrophages, and neutrophils are activated and undergo pyroptosis to release cytokines, which form the cytokine storm. In the microenviroment, cytokine storms not only amplify the inflammatory response, but also cause the pyroptosis of epithelial cells and endothelial cells, leading to an inflammatory cascade and ALI. Pulmonary epithelial and endothelial cells also directly suffer from pyroptosis, dysfunction, and an impaired blood–gas barrier, leading to the aggravation of ALI. Many studies have found that the pyroptosis of lung tissue infected by SARS-CoV-2 is also mediated by the inflammatory cascade reactively in patients with severe lung injury, such as in cases of COVID-19.^[104] This highlights the necessity to identify specific intervention targets to suspend the inflammatory cascade in lung cells and effectively regulate pyroptosis, which may facilitate the development of precision treatment for ALI patients.

Conflicts of interest

None.