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Improving lung allograft function in the early post-operative period through the inhibition of pyroptosis

  • Doug A. Gouchoe ORCID logo , Zhentao Zhang , Jung-Lye Kim , Yong Gyu Lee , Bryan A. Whitson and Hua Zhu EMAIL logo
Published/Copyright: May 16, 2024
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Medical Review
From the journal Medical Review Volume 4 Issue 5

Abstract

Lung transplantation is the only definitive therapy for end-stage pulmonary disease. Less than 20 % of offered lungs are successfully transplanted due to a limited ischemic time window and poor donor lung quality manifested by pulmonary edema, hypoxia, or trauma. Therefore, poor donor organ recovery and utilization are significant barriers to wider implementation of the life-saving therapy of transplantation. While ischemia reperfusion injury (IRI) is often identified as the underlying molecular insult leading to immediate poor lung function in the post-operative period, this injury encompasses several pathways of cellular injury in addition to the recruitment of the innate immune system to the site of injury to propagate this inflammatory cascade. Pyroptosis is a central molecular inflammatory pathway that is the most significant contributor to injury in this early post-operative phase. Pyroptosis is another form of programmed cell death and is often associated with IRI. The mitigation of pyroptosis in the early post-operative period following lung transplantation is a potential novel way to prevent poor allograft function and improve outcomes for all recipients. Here we detail the pyroptotic pathway, its importance in lung transplantation, and several therapeutic modalities that can mitigate this harmful inflammatory pathway.

Keywords: lung transplantation; pyroptosis; ischemia reperfusion injury; IRI; ex vivo lung perfusion; EVLP

Introduction

Lung transplantation is the only definitive therapy for end-stage pulmonary disease [1]. Currently, eight patients are added to the lung transplant waitlist daily, which is a 27.7 % increase since 2010 [2]. Present day, there are ∼4,000 patients on the waitlist, however, the number of transplants performed annually has stagnated at ∼2,000/year since 2010 [3, 4]. Therefore, while the waiting list of patients eligible for transplant grows daily, the ability for providers to find suitable donor allografts to perform lung transplants falls far short of the demand [1, 2, 5]. This is highlighted by the fact that less than 20 % of offered lungs are successfully transplanted due to a limited ischemic time window and poor donor lung quality manifested by pulmonary edema, hypoxia, or trauma [1, 6], [7], [8], [9], [10]. Therefore, poor donor organ quality and utilization are significant barriers to extend the life-saving therapy of transplantation.

Due to the nature of lung transplantation, the prospective allograft will suffer a period of ischemia when procured from the donor, and thereafter suffer a reperfusion injury when re-implanted in the recipient. This pattern of injury is known as ischemia reperfusion injury (IRI). At the cellular level, IRI leads destruction of the endothelial-epithelial barrier and loss of function, which subsequently allows the migration of immune cells into the alveolar space causing increased vascular permeability, which results in increased extravascular water/edema and, ultimately, respiratory failure [11]. Clinically, this contributes to decreased allograft quality and function, which manifests as primary graft dysfunction (PGD). Of those patients fortunate enough to receive a transplant, ∼30 % will develop severe, grade 3 PGD [12]. IRI is often identified as the underlying molecular insult leading to PGD [13], and this injury encompasses several pathways of cellular injury in addition to the recruitment of the innate immune system to the site of injury to propagate this inflammatory cascade [14]. However, pyroptosis is a central molecular inflammatory pathway that is the most significant contributor to injury in this early post-operative phase [15]. Pyroptosis is another form of programmed cell death and is more often associated with IRI than apoptosis [16]. IRI leads to the formation of damage associated molecular patterns (DAMPs), which are recognized by NOD-like receptors (NLRs) and activation results in the formation of inflammasomes. Inflammasomes recruit, and subsequently turn pro-caspase-1 into its active form, caspase-1, which serves two functions within the pyroptotic pathway: turn pro- IL-1β to IL-1β, and cleave Gasdermin-D (GSDMD) into its N-terminus (GSDMD-N) [16]. GSDMD-N interacts with cell-membrane lipids to form transmembrane pores which allow IL-1β, additional cytokines and signals to be released and promote further inflammation and cell death (Figure 1).

Figure 1: Basic overview of pyroptotic pathway. Ischemia reperfusion injury leads to the formation of damage associated molecular patterns (DAMPs), which are recognized by NOD-like receptors (NLRs) and activation results in the formation of inflammasomes. Inflammasomes recruit, and subsequently turn pro-caspase-1 into its active form, caspase-1, which serves two functions within the pyroptotic pathway: turn pro-IL-1β to IL-1β, and cleave Gasdermin-D (GSDMD) into its N-terminus (GSDMD-N). GSDMD-N interacts with cell-membrane lipids to form transmembrane pores which allows IL-1β, additional cytokines and signals to be released and promote further inflammation and cell death.
Figure 1:

Basic overview of pyroptotic pathway. Ischemia reperfusion injury leads to the formation of damage associated molecular patterns (DAMPs), which are recognized by NOD-like receptors (NLRs) and activation results in the formation of inflammasomes. Inflammasomes recruit, and subsequently turn pro-caspase-1 into its active form, caspase-1, which serves two functions within the pyroptotic pathway: turn pro-IL-1β to IL-1β, and cleave Gasdermin-D (GSDMD) into its N-terminus (GSDMD-N). GSDMD-N interacts with cell-membrane lipids to form transmembrane pores which allows IL-1β, additional cytokines and signals to be released and promote further inflammation and cell death.

As pyroptosis can be a quick process, it leads to significant damage early within the peri-operative period and is a significant contributor to PGD and poor allograft function. The objective of this review is to provide: (1) a basic overview of the pyroptotic pathway; (2) the current understandings of pyroptosis as it relates to lung transplantation and pyroptotic mitigation, (3) future directions of novel therapies to improve pyroptotic-related injury.

Discovery of pyroptosis

First described by Zychlinsky et al. in 1992, pyroptosis was initially characterized in macrophages infected by bacteria [17]. However, at that time it was thought to be a subset of apoptosis due to its similarities such as caspase involvement, nuclear condensation and damage [18]. Finally in 2001, the term pyroptosis was coined by D’Souza et al. to describe inflammatory programmed cell death as opposed to non-inflammatory cell death (apoptosis) [19]. As our understanding of the pyroptotic pathway progressed, inflammasome activation was discovered to activate caspases and subsequently lead to the transformation of pro-IL-1β to IL-1β [20]. While initially thought to be caspase-1 dependent, it was soon discovered that caspase-4/5/11 were also involved [21] in addition to caspase-3 [22] and caspase-8 [23]. Furthermore, it was also discovered that caspase activation eventually led to the cleavage of GSDMD and subsequent formation of pores within cell membranes resulting in membrane rupture [21].

Molecular mechanism of pyroptosis

Ultimately, pyroptosis is a form of programmed cell death like apoptosis or necroptosis.

Apoptosis and pyroptosis has some similar end results such as chromatin condensation, direct DNA damage and membrane blebbing [24], [25], [26]. However, the differences in the inflammatory cascade activation and associated local effects are different. Apoptosis is generally considered to be anti-inflammatory - akin to a controlled demolition, whereas pyroptosis is an inflammatory form of programmed cell death more like an ‘explosion’ with associated ‘damage’ effecting nearby cells [27]. Ultimately, pyroptosis differs from apoptosis in that cell death arises from plasma membrane disruption, organelle swelling and mitochondria dysfunction. The result of pyroptosis is that the GSDMD-N forms pores within the cell membrane. These pores serve two functions, to allow water molecules to enter the cell and cause swelling and rupture, and furthermore allow for the escape of IL-1β and IL-18 [28].

There are two main pathways in which pyroptosis are executed: either through the canonical or non-conical pathway. The non-conical pathway is mainly activated by lipopolysaccharide (LPS), and therefore will not be covered in this manuscript [29]. The canonical pathway is directly mediated by inflammasome assembly, which leads to GSDMD cleavage into GSDMD-N and of IL-1β and IL-18 release [30]. Inflammasomes are molecular complexes that are formed in response to injury, which could be from toxins, bacteria, viruses or ischemic injury [31]. The assembly of the inflammasome begins when pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and DAMPs (which are generated during IRI) [32]. Activation of PRRs leads to the assembly of pro-caspase-1, adapter apoptosis-associated speck-like protein containing a caspase recruitment domain (i.e., ASC) and NOD-like receptors (NLRs) to form the inflammasome [33]. Specific to IRI, the NLRs involved in inflammasome formation are mainly NLR family pyrin domain containing 3 (NLRP3). As its name suggests, NLRP3 consists of a leucine-rich repeat, a nucleotide-binding oligomerization domain, and pyrin domain (PYD) [34]. After inflammasome assembly, pro-caspase-1 is cleaved in to its active form, caspase-1 [35]. Caspase-1 serves two functions. First, caspase-1 cleaves the executor protein GSDMD at the Asp275 site to form the 31 kDa GSDMD-N [25], which subsequently interacts with the cell membrane to form pores leading to swelling and pyroptosis [36]. Secondarily, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their active forms, IL-1β and IL-18. These inflammatory cytokines are additionally released through the pores formed by GSDMD-N, resulting in further pyroptosis and damage (Figure 2) [37, 38].

Figure 2: Canonical pathway for pyroptosis. The assembly of the inflammasome begins when pattern recognition receptors (PRRs) recognize damage-associated molecular patterns (DAMPs). Activation of PRRs leads to the assembly of pro-caspase-1, adapter apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NOD-like receptors family pyrin domain containing 3 (NLRP3) to form the inflammasome. After inflammasome assembly, pro-caspase-1 is cleaved into its active form, caspase-1. Caspase-1 cleaves: (1) the executor protein Gasdermin-D (GSDMD) to form its N-terminus (GSDMD-N), which subsequently interacts with the cell membrane to form pores leading to swelling and pyroptosis and; (2) cleaves pro-IL-1β and pro-IL-18 to their activate forms, IL-1β and IL-18.
Figure 2:

Canonical pathway for pyroptosis. The assembly of the inflammasome begins when pattern recognition receptors (PRRs) recognize damage-associated molecular patterns (DAMPs). Activation of PRRs leads to the assembly of pro-caspase-1, adapter apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NOD-like receptors family pyrin domain containing 3 (NLRP3) to form the inflammasome. After inflammasome assembly, pro-caspase-1 is cleaved into its active form, caspase-1. Caspase-1 cleaves: (1) the executor protein Gasdermin-D (GSDMD) to form its N-terminus (GSDMD-N), which subsequently interacts with the cell membrane to form pores leading to swelling and pyroptosis and; (2) cleaves pro-IL-1β and pro-IL-18 to their activate forms, IL-1β and IL-18.

While there has been much research around the NLRP3/GSDMD axis regarding IRI, recently Hou et al. discovered that under hypoxic conditions, gasdermin-C (GSDMC) might play an important role as well. Under hypoxic conditions, the activation of p-Stat3 facilitates the movement of PD-L1 into the cell nucleus. Once inside the nucleus, PD-L1 and p-Stat3 collaborate to enhance the production of GSDMC. Furthermore, they found that caspase-8, which is activated by TNF-α coming from macrophages, is capable of cleaving GSDMC to produce an N-terminal (GSDMC-N) to again form pores within the plasma membrane and trigger pyroptosis [39].

Role of pyroptosis in lung transplantation

No cure exists for patients with end-stage lung disease and lung transplant is the only effective treatment strategy. The transplant waiting list grows each year though the number of transplants performed annually has stagnated at ∼2,000/year since 2010 [5]. The lack of quality lung donors is limiting with only 17 % of donated lungs successfully being transplanted [40]. Of those patients fortunate enough to receive a transplant, ∼30 % will develop severe PGD3, which detrimentally impacts patient outcomes and survival [11, 12]. An evolving strategy to address the critical unmet demand for viable lungs suitable for transplantation involves the utilization of extended criteria donors (ECD), notably through the implementation of donation after circulatory death (DCD) [8]. In the DCD process, a donor has care withdrawn and if the donor passes in an acceptable time (up to 120 min for the lung) [41], an emergent organ recovery is performed. Unfortunately, IRI, which affects all donor organs universally in transplantation, is especially pronounced in these DCD allografts. As one might expect, this uncontrolled death process and variable period of warm ischemia leads to a great deal of variability in donor quality which has the potential to manifest as poor recipient outcomes.

IRI drives the development of PGD whose hallmark is inflammation, leukocyte infiltration, and the activation of cellular immune responses that causes morbidity and mortality [13, 42]. This inflammation leads to cell death, as shown by cell-death related pathways being enriched during transplantation, and during machine perfusion prior to transplantation [43]. As discussed previously, pyroptosis, a lytic form of cell death, is a key pathway triggering inflammation [21, 25, 28, 30, 33, 35, 36, 38]. Pyroptosis is critical for inflammatory response to lung IRI [44, 45], and current studies have shown that IL-1β levels are significantly higher in lung allografts that are deemed unsuitable for transplantation [46]. Specific to lung IRI, monocytes have been shown to promote pyroptosis in endothelial cells in transplant relevant models of injury [45]. The endo-epithelial barrier is of extreme important to the overall integrity of the alveolar space, and pyroptosis mediated damage to the endothelium serves as significant injury that leads to poor graft function and eventually PGD. In addition to endothelial cells, GSDMD can be primarily found within neutrophils, macrophages, and monocytes [44, 45, 47] as well. Specifically important to the pathophysiology of PGD in lung transplantation is the presence of residual donor nonclassical monocytes (NCM) within the pulmonary vasculature, and their ability to recruit recipient neutrophils into the lung allograft.

Donor derived NCMs are retained within the pulmonary vasculature following procurement of a lung allograft, despite adequate measures to rid the allograft of donor immune cells [48]. Upon IRI, these NCMs within the pulmonary vasculature through MyD88 signaling are responsible for CXCL2 production which attracts recipient neutrophils to the newly transplanted allograft upon implantation. These NCMs additionally release IL-1β through pyroptosis (NLRP3 dependent) which activates alveolar macrophages, and which through CCL2 signaling recruits classical monocytes (CM) to the allograft as well. Once recruited to the area of injury, CM produces IL-1β through PKC- and NF-κB–dependent but PI3K-independent pathway [14]. The presence of IL-1β within the pulmonary vasculature activates endothelial cells to downregulate vascular endothelial cadherin (VE-cadherin) transcription, which results in the destruction of tight junctions that are critical to endo-epithelial integrity [49]. Ultimately, the loss of barrier function allows for neutrophil extravasation into the alveolar space, pulmonary edema, loss of pulmonary function and ultimately PGD [14, 48]. While IRI is a trigger for pyroptosis, and ultimately leads to PGD development and adverse reactions in the immediate post-operative period, it can also have detrimental long-term effects which are also pyroptotic involved as well.

PGD can progress to chronic lung allograft dysfunction (CLAD), which exists along a clinical spectrum that ranges from transient dyspnea to hypoxemic respiratory failure needing re-transplantation. The definition of CLAD has evolved over the past 5–8 years and now encompasses the older bronchiolitis obliterans syndrome (BOS) and the newer restrictive allograft syndrome (RAS). CLAD is a spectrum of chronic dysfunction inclusive of RAS and BOS with fibrosis as a significant contributor [50], [51], [52]. Though allograft rejection is mediated by many factors, acquired immunity plays an important role. Type 1 T helper (Th1) and Type 17  Thelper (Th17) cellular response can be modified by both IL-1β and IL-18, thus inhibiting regulatory T-cells, contributing to the development of CLAD [53]. Though current studies surrounding pyroptosis and lung transplant have shown its longitudinal involvement, they are still limitations.

There are many cell types in the pulmonary parenchyma, e.g., alveolar macrophages, interstitial macrophages, AT1 and AT2 pneumocytes, neutrophils and fibroblasts [54]. Though pyroptosis during lung IRI or lung transplant has been implicated in several cell types, further research is needed to see if other cell types are involved as well. Though not transplant specific, there are several other disease pathophysiology that offer key insights. In a disease with a pathophysiology most closely related to PGD, acute respiratory distress syndrome (ARDS), GSDMD was evident in pneumocytes [55]. Within other pulmonary vascular diseases, such as pulmonary artery hypertension, pyroptosis has been found to contribute to endothelial cell dysfunction and breakdown of this important barrier [56]. Specifically, hypoxia caspase-1 mediated damage has been implicated in several studies [57, 58]. Additionally, caspase-4/11 mediated pyroptosis has been implicated as well [59]. While none of these studies were specific to transplant associated IRI, hypoxic injury has been shown to induce pyroptosis within the endothelium, adding to the body of evidence that this injury pattern would be prevalent in peri-operative period during lung transplantation.

Thus, as PGD leads to significant adverse outcomes, agents providing effective mitigation of pyroptosis are novel and of therapeutic benefit. There are no Food and Drug Administration (FDA) approved treatments to heal lung endothelial and epithelial barrier functions, and therefore investigation of therapeutics that inhibit pyroptosis is of scientific value.

Mitigation of pyroptosis and novel therapeutics within lung transplantation

The injury, activation and subsequent dysfunction of the endothelium as well as associated immune cells are central to the pathophysiology of IRI, pyroptosis and PGD. While all lung allografts suffer a degree of IRI, this injury is becoming more prevalent. In solid organ transplantation, there is a critical shortage of adequate donor organs [1, 2]. For all organs, the waitlist is much larger than the number of available organs. In 2020, there were 107,925 total waitlist candidates, but only 39,035 total transplants were performed, leading to an average of 15.5 patients dying on the waitlist daily [2]. Clearly, only a small fraction of potential patients can access transplantation, and increased access could provide a significant public health benefit. To meet the gap in demand and prevent death, transplant teams increasingly turn to marginal or extended criteria donors [2]. In particular, DCD is associated with increased warm ischemic injury [8]. Furthermore, marginal donor organs will make up a larger proportion of transplants in the future as patients in need of life-saving transplants are steadily increasing [1, 6], [7], [8], [9], [10]. A primary factor preventing expanded access is that marginal quality donor organs suffer disproportionately worse IRI and activation of pyroptosis [60].

Currently, organ viability is assessed largely by chest X-ray and arterial blood gases which are crude metrics for PGD. As we are not able to predict the deleterious PGD, as a transplant community, we discard ∼80 % of potential donor lung out of fear of PGD [61]. PGD leads to pulmonary vascular endothelial cell activation and increased vascular permeability, which results in increased extravascular water/edema and, ultimately respiratory failure. Thus, as we are not able to predict PGD and it leads to significant adverse outcomes [62], agents providing effective mitigation of inflammation and PGD are a novel and therapeutic benefit. Utilizing extended criteria donors (i.e., DCD donors), may ultimately help us bridge the gap in available donors. However, there are no current Food and Drug Administration-approved therapies to ameliorate the damage inherent in the DCD donor use and to mitigate the formation of PGD. Thus, improving the function of these organs prior to transplantation is critical to improving transplant outcomes. There are however several potential therapeutic treatments undergoing investigation (Figure 3).

Figure 3: Mitigation of pyroptosis and novel therapeutics. Caspase-1 Inhibitors: VX-740 and VX-765 both reversibly catalyze the active site of caspase-1. NOD-like receptors family pyrin domain containing 3 (NLRP3) Inhibitors: MCC950 is a small molecular inhibitor that inhibits NLRP3 activation in both the canonical and non-canonical pathway by direct inhibition of the NATCH domain and further motifs that prevent oligomerization. Gasdermin (GSDMD) Inhibitors: Disulfiram blocks GSDMD pore formation during pyroptosis. Hu et al. discovered that disulfiram covalently modifies Cys191/192 in GSDMD, thus inhibiting pore formation while still allowing for the formation of IL-1β. Cell Membrane Repair: Endosomal sorting complexes required for transport (ESCRT) machinery can repair the plasma membrane upon GSDMD activation. Mitsugumin 53 (MG53) with tripartite motif-containing (TRIM) family protein also repairs cell membranes.
Figure 3:

Mitigation of pyroptosis and novel therapeutics. Caspase-1 Inhibitors: VX-740 and VX-765 both reversibly catalyze the active site of caspase-1. NOD-like receptors family pyrin domain containing 3 (NLRP3) Inhibitors: MCC950 is a small molecular inhibitor that inhibits NLRP3 activation in both the canonical and non-canonical pathway by direct inhibition of the NATCH domain and further motifs that prevent oligomerization. Gasdermin (GSDMD) Inhibitors: Disulfiram blocks GSDMD pore formation during pyroptosis. Hu et al. discovered that disulfiram covalently modifies Cys191/192 in GSDMD, thus inhibiting pore formation while still allowing for the formation of IL-1β. Cell Membrane Repair: Endosomal sorting complexes required for transport (ESCRT) machinery can repair the plasma membrane upon GSDMD activation. Mitsugumin 53 (MG53) with tripartite motif-containing (TRIM) family protein also repairs cell membranes.

Caspase-1 inhibitors

There are several caspase-1 inhibitors that have been used in the past to combat pyroptosis [63], [64], [65]. VX-740 and VX-765 both reversibly catalyze the active site of caspase-1, which showed decreased release of IL-1β and IL-18 [65], as well as disease mitigation (arthritis) [64]. VX-165 has been studied in IRI, and has been shown to decrease Kupffer cell damage and pyroptosis [66], neuronal ischemic insult [67] as well as pulmonary endothelial damage [68]. In both in vitro and in vivo models, VX-165 was able to decrease markers of pyroptosis, inflammatory and preserve lung tissue architecture. While this study did not involve transplantation as a specific in vivo model, in vivo IRI still offers valuable pre-clinical information that can inform clinicians to create further studies.

NLRP3 inhibitors

MCC950 is perhaps the best studied NLRP3 inhibitor in regard to transplantation in general [69], [70], [71]. MCC950 is a small molecular inhibitor that inhibits NLRP3 activation in both canonical and non-canonical pathway by direct inhibition of the NATCH domain and further motifs that prevent oligomerization [72, 73]. In a large animal model of DCD liver transplantation, the treatment of MCC950 results in improved allograft function as well decreased markers of inflammation [70]. More relevant to pulmonary anatomy and physiology, Xu et al. investigated MCC950 and its ability to preserve function during heart transplantation. The addition of MCC950 either after transplantation or during ex vivo perfusion prior to transplantation resulted in decreased oxidative stress, preservation of function and ultimately ameliorated cardiac IRI [71]. While there are no studies pertaining directly to lung transplantation, MCC950 has been shown to mitigate injury from pneumonia [74].

Gasdermin inhibitors

Disulfiram is an inhibitor of acetaldehyde dehydrogenase and is approved in the treatment of alcohol abuse. Additionally, Disulfiram was found to block GSDMD pore formation during pyroptosis. Hu et al. discovered that Disulfiram covalently modifies Cys191/192 in GSDMD, thus inhibiting pore formation while still allowing for the formation of IL-1β [75]. This is particularly exciting as Disulfiram is already FDA-approved, which could lead to more rapid studies within humans to determine its therapeutic efficacy in decreasing pyroptotic inflammation. Again, no specific lung transplant studies exist focusing on disulfiram mitigation of pyroptosis, and fibrotic injury within lung parenchyma following toxic administration has been shown to be mitigated by its use [76].

There are several pathways that have been shown to directly decrease GSDMD pore formation as well. Endosomal sorting complexes required for transport (ESCRT) machinery have been proposed to repair the plasma membrane upon GSDMD activation [77]. Rühle et al. found that ESCRT machinery was involved in the cellular membrane repair process following GSDMD pore formation, and that lack of ESCRT greatly enhanced pyroptotic effects within a cell. Though there are no current therapeutics that enhance the ESCRT pathway in order to mitigate pyroptosis, cell membrane repair is a promising avenue as well to mitigate GSDMD pore formation. Mitsugumin 53 (MG53) with tripartite motif-containing (TRIM) family protein is also involved in cell membrane repair. MG53 functions by sensing the oxidized extracellular environment upon injury, subsequently undergoing oligomerization, formatting a repair complex via tethering of intracellular vesicles and fusing these vesicles with the plasma membrane to form the repair patch [78, 79]. MG53 has been found in alveolar epithelial cells [80], and administration of an exogenously produced human recombinant MG53 is a potential novel therapeutic for the preservation of the pulmonary parenchyma by decreasing pyroptosis after IRI during transplantation [15, 81].

Limitations with current therapies

Unfortunately, none of these therapies have undergone human testing within the realm of lung transplantation. Therefore, it is difficult to say which class of inhibitor (GSDMD, caspase-1, NLRP3, etc.) would have the most efficacy during lung transplantation. Perhaps the most promising avenue would in fact be GSDMD inhibitors, i.e., disulfiram, as this is already an FDA approved therapy (and has known tolerability/side effects), albeit its use in lung transplantation would be ‘off-label’ [75, 76]. Though a dose for this drug has been established (for its FDA approved use), it would still take additional studies to determine what dose would effectively mitigate pyroptosis, and what additional side effects could potentially arise. Perhaps another promising GSDMD inhibitor could be human recombinant MG53 (rhMG53) as well. As previously mentioned, MG53 is an endogenous protein, and therefore exogenous administration of rhMG53 could be a viable therapy with minimal side effects and tolerable treatment profile [15, 79], [80], [81]. However, again this must be further substantiated in additional pre-clinical and clinical studies.

Caspase-1 inhibitors, though having very favorable pre-clinical results, have not had good long-term results in humans. VX-765 has in fact undergone human testing in several clinical trials for a variety of diseases, and has poor tolerability with an increased rate of adverse side effects [82]. VX-740 has also been tested in humans, showing mitigation of inflammation, though long term has been shown to have increased rates of liver toxicity [82]. Like caspase-1 inhibitors, MCC950 (NLRP3 inhibitor) has also shown liver toxicity as a side effect in human trials [83]. Ultimately, further studies are needed to develop pyroptotic inhibitors which are tolerable, safe, and can effectively mitigate disease.

Ex vivo lung perfusion and the future of allograft repair and modification

While the invention and efficacy of creating novel therapeutics to combat pyroptosis is still a developing field, clinicians should also be focusing on ways to deliver such drugs. The obvious answer is intravenous delivery to either the donor or recipient upon implantation, or a possible oral form drug is given to the recipient over several days following transplantation. However, these delivery strategies pose several issues. The delivery of a drug to a donor poses multiple logistical issues. As these donors are often at community hospitals or potentially several hundreds of miles away at a facility that might not carry novel therapeutics or have the necessary storage equipment, like a quaternary referral center might, it is difficult to imagine this strategy to be the most fruitful in mitigating injury. Additionally, treating the donor immediately upon reperfusion may be able to prevent the reperfusion injury while it cannot mitigate the ischemic insult. Finally, as pyroptotic injury occurs quickly (within 24 h), treating the recipient over several days following transplantation might not be the most feasible way to ameliorate this injury pattern.

One new and aggressive approach to meeting this need for adequate donor organs for transplantation is utilizing normothermic ex vivo lung perfusion (EVLP) as a way to assess the potential allograft function, resuscitate the allograft, and maintain metabolic viability to support a successful transplant [40, 84]. EVLP allows for enhanced evaluation of potential extended criteria donor allografts in a closed circuit outside the body to determine if the organ is in fact suitable for use, thus increasing the number of transplanted lungs [85]. The closed circuit is achieved by creating a ventilatory circuit and vascular circuit. Ventilation is achieved by attaching the trachea to a ventilator, and a vascular circuit is creating by connecting the left atrium to the pulmonary artery with plastic tubing allowing acellular solution to flow with the use of a centrifugal pump [86, 87].

EVLP also allows for the delivery of therapeutics. Currently, EVLP is part of standard clinical practice throughout the world [86, 88, 89], and is used by both transplant programs and external perfusion centers [90] to actively repair and resuscitate potential donor allografts. This practice has not only increased the donor pool to make transplantation more achievable for all but has done so safely with minimal adverse effects in recipients [85, 86, 91]. Currently, EVLP perfusates use either cellular (with red blood cell addition) or acellular perfusates. These perfusates are comprised of low-potassium dextran, glucose, bicarbonate, human serum albumin and additional steroids and antibiotics [92]. Currently, this perfusate is sufficient to rehabilitate prospective lung allografts, mitigate damage and resuscitate them to be suitable for transplantation [93, 94]. However, the next evolution of this technology is to apply innovative therapeutics and strategies within the platform that will be able to better assess, repair and modify organs that are severally damage (i.e., unsuitable to transplantation) and make them safe for transplantation [84, 87].

Currently there are several exciting applications of EVLP as they pertain to modifying and resuscitating potential allografts. Mesaki et al. have demonstrated that through CRISPR modulation with IL-10 gene therapy, rodent lungs were able to overexpress IL-10 leading to protection, decreased inflammation and ultimately proof of concept that gene therapy is possible through this exciting technology [95]. Taking this a step further, Cypel et al. were able to demonstrate similar results in human lungs [96]. Beyond gene therapy, mesenchymal stromal cell therapy is also possible with EVLP as well [97]. In addition to gene and cell therapy, Wang et al. showed that enzymatic additives within the perfusate administered during EVLP could change red blood cell groups during 4-h of clinical EVLP in human lungs [98].

Thus, EVLP is an ideal treatment modality to apply novel therapeutics to repair and resuscitate damaged lung allografts. We envision that the previously described treatment modalities will be able to be added to current perfusates and allow for mitigation of pyroptosis. An added benefit of ex vivo repair is that there are minimal off-target effects, thus potentially mitigating poor tolerability of NLRP3 and caspase-1 inhibitors. Ultimately, developing novel therapeutic regiments to combat pyroptosis to be used during EVLP will increase the donor pool, thus making this life saving therapy achievable for all.

Conclusions

Pyroptosis is a significant pathway that contributes to injury early in the post-operative phase following lung transplantation and can affect the development of CLAD as well. Pyroptosis can be triggered by many stimuli, but during transplantation this is often the result of IRI. Once cells sense DAMPs (as a result from IRI), inflammasome assembly occurs which leads to GSDMD cleavage into GSDMD-N, as well as IL-1β and IL-18 release. IL-1β release is also crucial in the innate cellular response that leads to PGD as well. Long-term, PGD and further activation of the acquire immune response via IL-1β and IL-18 contributes to the development of CLAD. Thus, pyroptosis is implicated in all phases of lung transplantation, though its most harmful effects happen early in the post-operative period. Pyroptosis can be mitigated through several different avenues to include caspase-1, NLRP3, GSDMD inhibition and direct cellular membrane repair. Though many of these inhibitors have been tested in humans, few are promising as of yet, emphasizing the investigation of therapeutics that inhibit pyroptosis is of scientific value. To deliver novel therapeutics there are several methods and timepoints, but considering logistic constraints as well as optimal current modern day therapeutic approaches, EVLP offers an ideal approach. By mitigating the harmful effects of pyroptosis, transplantation will become safer for patients, thus furthering the potential of this life altering treatment.

Corresponding author: Hua Zhu, PhD, Department of Surgery, Division of Cardiac Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA, E-mail:

Funding source: American Heart Association

Award Identifier / Grant number: 23TPA1142638

Funding source: NIH

Award Identifier / Grant number: EY030621, EY032583, HL143000, HL153876

  1. Research ethics: Not applicable.

  2. Informed consent: Not applicable.

  3. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Competing interests: Authors state no conflict of interest.

  5. Research funding: BAW is supported through the National Institute of Health (NIH) grant R01 HL143000 and serves on the Clinical Events Committee of TransMedics OCS. Hua Zhu is supported by the NIH grants: HL153876, EY030621, EY032583 and the American Heart Association grant: 23TPA1142638. This research was also generously supported through the Jewel and Frank Benson Family Endowment and The Jewel and Frank Benson Research Professorship at The Ohio State University.

  6. Data availability: Not applicable.

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Received: 2023-12-07
Accepted: 2024-05-04
Published Online: 2024-05-16
Published in Print: 2024-10-28

© 2024 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Articles in the same Issue

  1. Frontmatter
  2. Reviews
  3. Understanding autoimmune response after SARS-CoV-2 infection and the pathogenesis/mechanisms of long COVID
  4. Improving lung allograft function in the early post-operative period through the inhibition of pyroptosis
  5. The occurrence mechanism, assessment, and non-pharmacological treatment of dyspnea
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https://doi.org/10.1515/mr-2023-0066
Keywords for this article
lung transplantation; pyroptosis; ischemia reperfusion injury; IRI; ex vivo lung perfusion; EVLP
Creative Commons
BY-NC-ND 4.0

Articles in the same Issue

  1. Frontmatter
  2. Reviews
  3. Understanding autoimmune response after SARS-CoV-2 infection and the pathogenesis/mechanisms of long COVID
  4. Improving lung allograft function in the early post-operative period through the inhibition of pyroptosis
  5. The occurrence mechanism, assessment, and non-pharmacological treatment of dyspnea
  6. Regenerative rehabilitation: a novel multidisciplinary field to maximize patient outcomes
  7. Advances in the prerequisite and consequence of STING downstream signalosomes
  8. Complement factor H in molecular regulation of angiogenesis
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