Necroptosis is one of several programmed lytic cell death processes for which key effector proteins have only been defined and experimentally dissected in the last decade. Unlike apoptotic cell death, where cellular contents undergo membrane-contained caspase-mediated disassembly, necroptosis leads to the release of highly inflammatory intracellular components e.g. histones into the surrounding milieu.¹

Necroptotic cell death is thought to have originally evolved as a pathogen defence mechanism. As such, necroptosis can be induced by a series of cytokine- and pathogen detecting- receptors binding their appropriate ligands. Many of these signaling routes to necroptosis employ the apex kinase receptor-interacting serine/threonine protein kinase 1 (RIPK1). All upstream necroptotic signaling culminates in the activation of the effector protein Mixed Lineage Kinase domain-Like (MLKL) by its obligate activating kinase, receptor interacting protein kinase 3 (RIPK3). Following activation, MLKL associates with phospholipids to promote potassium efflux and eventual membrane bilayer destabilization, oncosis and cell death.¹

This lytic form of programmed cell death has been implicated in the etiology of a range of human pathologies. Specifically, its role in lung disease has recently come to the fore following prominent publications implicating necroptosis in lung epithelial cell damage and inflammatory neutrophil influx. Following a lethal dose of influenza A virus (IAV), the absence of necroptosis reduces mortality, protecting mice from lung epithelial cell damage and influx of Neutrophil Extracellular TRAP (NET)-forming neutrophils.² Mice lacking necroptosis were also seen to be protected from airway remodelling and inflammation in a model of cigarette smoke-induced chronic obstructive pulmonary disease (COPD).³ These and other studies illustrate the contribution of necroptosis to the progression of lung pathologies and present an intriguing basis for recent work by Oikonomou et al.⁴

The relative contribution of necroptosis in specific tissues or cell types can be investigated using whole body or tissue-specific genetic modification of upstream and core necroptotic machinery. Genetic deletion of obligate necroptotic machinery (MLKL or RIPK3), or ablation of RIPK1 kinase activity, enables examination of necroptotic contribution through elimination. Conversely, genetic manipulation of certain upstream pathway components can be used to induce spontaneous activation of necroptosis. The most commonly used models of spontaneous necroptosis in mice are Fas-associated protein with death domain (FADD) and Caspase-8 knockouts. In addition to their adapter and enzymatic roles in apoptosis pathways, FADD and Caspase-8 also function as gate keepers to the necroptosis-inducing signaling platform comprising RIPK1 and RIPK3. The absence of FADD or Caspase-8 makes way for the unimpeded assembly of this necroptosis activation platform, the activation of MLKL and cell death. Whole body FADD or Caspase-8 gene knockout mice die before birth. Oikonomou et al. employ both airway epithelial cell (AEC)-specific ablation of FADD (necroptosis activation) and complementary whole body RIPK3 knockout, MLKL knockout or RIPK1 kinase activity knock-in (necroptosis elimination) to dissect the role of this pathway in a model of asthma induced by house dust mite extract sensitization and challenge.

Regulation of necroptosis in barrier tissues, such as skin and intestines, is known to be an important mechanism in maintaining immune homeostasis. Targeted gene deletion of FADD, Caspase-8, or RIPK1 sensitises epithelial cells to necroptosis. This strategy has been used by the Pasparakis lab and others to demonstrate that dysregulated necroptosis in intestinal epithelial cells or keratinocytes alone is sufficient to induce the spontaneous development of inflammatory gut or skin damage.^5-8 In barrier tissues like the gut or skin, where abundant microbially derived stimuli favour the expression and unmitigated assembly of RIPK1-RIPK3, the specific genetic ablation of FADD alone is sufficient to trigger the cascade of necroptosis and inflammation. Oikonomou et al. conclude that mice with a FADD-deficient airway epithelium (FADD^AEC-KO) do not exhibit this same capacity for spontaneous necroptosis in the lung at steady state. They propose this may be due to a lower microbial load in the lung relative to the gut or skin. However, priming the airway epithelium with a physiologically-relevant stimulus in the form of house dust mite extract unmasks a capacity for an inflammatory response in the lungs of FADD^AEC-KO mice that is over and above that of FADD-sufficient mice (Figure 1).

This augmented inflammatory response in FADD^AEC-KO mice manifests in both enhanced inflammatory cytokines and immune cell infiltrates. Oikonomou et al. used genetic deletion of Ripk3 to assess the contribution of FADD-deficient AEC necroptosis in house dust mite-induced airway inflammation. FADD^AEC-KO, Ripk3^-/- mice exhibited limited immune cell infiltration and similar inflammatory cytokines levels when compared to control mice. Consistent with this finding, FADD^AEC-KO, Ripk1^D138N kinase dead, and FADD^AEC-KO, Mlkl^-/- mice also showed an attenuation of this house dust mite-induced pathology.

FADD^AEC-KO mice were protected from induced airway hyperresponsiveness, resembling mice that had not been treated with house dust mite extract. Despite exaggerated inflammation induced by house dust mite extract, FADD^AEC-KO mice exhibited reduced mucus production stemming from reduced numbers of mucus-producing goblet cells in the lung relative to fadd ^fl/fl controls. This loss of goblet cells was not attributed to enhanced necroptosis in these mice, but to the expression of Cre-recombinase itself. While this confounding experimental artifact precluded examination of the full physiological impact of enhanced airway epithelial cell necroptosis in this model, it still stands that inflammation, mucus production and airway hyperresponsiveness certainly go hand-in-hand in real life. It is thus reasonable to predict that necroptosis-enhanced airway inflammation would also enhance mucus production and airway hyperresponsiveness. These carefully controlled experimental data reveal an important caveat to the future use of the Scgb1a1 (AEC-specific promoter)-Cre transgene for the study of airway epithelial cell death itself, with airway epithelial cell death unrelated to FADD activity being observed on the microscopic level. This study by Oikonomou et al. also lends further credence to previous research that examined the effectiveness of targeting downstream mucus hypersecretion for the amelioration of airway hyperresponsiveness,⁹ but certainly does not detract from the potential benefits of stopping the train further up the etiological line at necroptosis.

An enhanced propensity for airway epithelial cell necroptosis (through conditional FADD ablation) quantitatively intensifies inflammation in the lungs. While unable to directly demonstrate or quantify necroptotic epithelial cell death in situ in FADD^AEC-KO mice, the protein level of the MLKL-activating kinase, RIPK3, is clearly upregulated in the lungs. Detection of dead or near-dead necroptotic cells remains highly challenging in mouse tissues however, improvements in necroptosis-detecting tools and techniques will soon permit a more reliable detection in such scenarios.¹⁰

The work of Oikonomou et al. prompts further questions regarding the potential role of necroptotic cell death in human asthma: Is the propensity for necroptosis what distinguishes patients with severe life-long chronic asthma from those with milder forms? Is it the activation and perpetuation of necroptosis that underpins the role of common respiratory viruses in inducing asthma? Is the enhanced propensity for airway epithelial cell necroptosis mediated purely by infection history and the adaptive immune response, and/or is there a direct genetic influence on propensity for necroptosis that contributes to asthma risk in humans? Etiology aside, this work provides an important precedent for further preclinical exploration of whether asthmatics could benefit from the emerging class of necroptosis-inhibitor drugs currently in development.

坏死性凋亡是几个程序性裂解细胞死亡过程之一，其关键效应蛋白仅在过去十年中才被定义和实验解剖。与凋亡细胞死亡不同，其中细胞内容物经历包含膜的半胱天冬酶介导的分解，坏死性凋亡导致高度炎症的细胞内成分（例如组蛋白）释放到周围环境中。¹

坏死性细胞死亡被认为最初是作为病原体防御机制进化而来的。因此，可以通过一系列细胞因子和病原体检测受体与其适当的配体结合来诱导坏死性凋亡。许多这些导致坏死性凋亡的信号通路采用与顶点激酶受体相互作用的丝氨酸/苏氨酸蛋白激酶 1 (RIPK1)。所有上游的坏死性凋亡信号都以效应蛋白混合谱系激酶结构域样 (MLK​​L) 的专性激活激酶、受体相互作用蛋白激酶 3 (RIPK3) 激活而告终。激活后，MLKL 与磷脂结合以促进钾外流并最终导致膜双层不稳定、溶胀和细胞死亡。¹

这种程序性细胞死亡的裂解形式与一系列人类病理学的病因有关。具体而言，随着有关肺上皮细胞损伤和炎性中性粒细胞流入的坏死性凋亡的重要出版物，其在肺部疾病中的作用最近脱颖而出。注射致死剂量的甲型流感病毒 (IAV) 后，没有坏死性凋亡可降低死亡率，保护小鼠免受肺上皮细胞损伤和中性粒细胞胞外陷阱 (NET) 形成中性粒细胞的流入。²在香烟烟雾诱发的慢性阻塞性肺疾病 (COPD) 模型中，还观察到缺乏坏死性凋亡的小鼠免受气道重塑和炎症的影响。³这些和其他研究说明了坏死性凋亡对肺部病理学进展的贡献，并为 Oikonomou等人最近的工作提供了一个有趣的基础。⁴

可以使用上游和核心 necroptotic 机制的全身或组织特异性遗传修饰来研究 necroptosis 在特定组织或细胞类型中的相对贡献。专性坏死机制（MLKL 或 RIPK3）的遗传缺失，或 RIPK1 激酶活性的消融，可以通过消除来检查坏死性凋亡的贡献。相反，某些上游途径成分的基因操作可用于诱导自发激活坏死性凋亡。最常用的小鼠自发性坏死性凋亡模型是 Fas 相关蛋白与死亡域 (FADD) 和 Caspase-8 敲除。除了它们在细胞凋亡途径中的接头和酶作用外，FADD 和 Caspase-8 还充当由 RIPK1 和 RIPK3 组成的坏死性凋亡诱导信号平台的守门人。FADD 或 Caspase-8 的缺失为这种坏死性凋亡激活平台的无阻碍组装、MLKL 的激活和细胞死亡让路。全身FADD或Caspase-8基因敲除小鼠在出生前死亡。奥科诺穆等人. 采用气道上皮细胞 (AEC) 特异性消融 FADD（坏死性凋亡激活）和互补的全身 RIPK3 敲除、MLKL 敲除或 RIPK1 激酶活性敲除（坏死性凋亡消除）来剖析该途径在哮喘诱导模型中的作用通过屋尘螨提取物致敏和激发。

众所周知，皮肤和肠道等屏障组织中坏死性凋亡的调节是维持免疫稳态的重要机制。FADD、Caspase-8 或 RIPK1 的靶向基因缺失使上皮细胞对坏死性凋亡敏感。Pasparakis 实验室和其他人已经使用这种策略来证明仅在肠上皮细胞或角质形成细胞中失调的坏死性凋亡就足以诱导炎症性肠道或皮肤损伤的自发发展。^5-8在肠道或皮肤等屏障组织中，大量微生物衍生的刺激有利于 RIPK1-RIPK3 的表达和未减轻的组装，仅FADD的特定基因消融就足以触发坏死性凋亡和炎症的级联反应。奥科诺穆等人. 得出的结论是，具有 FADD 缺陷气道上皮 (FADD ^AEC-KO ) 的小鼠在稳态时不会表现出相同的肺自发性坏死性凋亡能力。他们提出这可能是由于肺中相对于肠道或皮肤的微生物负荷较低。然而，用屋尘螨提取物形式的生理相关刺激物启动气道上皮，揭示了 FADD ^AEC-KO小鼠肺部炎症反应的能力，这种反应能力超过了 FADD 充足小鼠的能力（图 1） ）。

这种在 FADD ^AEC-KO小鼠中增强的炎症反应表现为增强的炎症细胞因子和免疫细胞浸润。奥科诺穆等人。使用Ripk3 的基因缺失来评估 FADD 缺陷的 AEC 坏死性凋亡在屋尘螨诱导的气道炎症中的贡献。与对照小鼠相比，FADD ^AEC-KO、Ripk3 ^-/-小鼠表现出有限的免疫细胞浸润和相似的炎性细胞因子水平。与该发现一致，FADD ^AEC-KO、Ripk1 ^D138N激酶死亡，FADD ^AEC-KO、Mlkl ^-/- 小鼠也表现出这种屋尘螨引起的病理学减弱。

FADD ^AEC-KO小鼠免受诱导的气道高反应性，类似于未用屋尘螨提取物处理的小鼠。尽管房尘螨提取物引起的炎症加剧，但 FADD ^AEC-KO小鼠表现出减少的粘液产生，这是由于肺中产生粘液的杯状细胞数量相对于fadd  ^{fl/fl 减少}控件。杯状细胞的这种损失不是由于这些小鼠中增强的坏死性凋亡，而是由于 Cre 重组酶本身的表达。虽然这个令人困惑的实验伪像排除了在这个模型中检查增强的气道上皮细胞坏死的完整生理影响，但炎症、粘液产生和气道高反应性在现实生活中肯定是齐头并进的。因此，预测坏死性凋亡增强的气道炎症也会增加粘液产生和气道高反应性是合理的。这些精心控制的实验数据揭示了未来使用Scgb1a1的重要警告（AEC 特异性启动子）-Cre 转基因，用于研究气道上皮细胞死亡本身，在微观水平上观察到与 FADD 活性无关的气道上皮细胞死亡。Oikonomou等人的这项研究。还进一步证实了先前的研究，该研究检查了针对下游粘液分泌过多对改善气道高反应性的有效性，⁹但当然不会减损在坏死性凋亡时进一步停止列车的潜在益处。

气道上皮细胞坏死性凋亡的增强倾向（通过有条件的 FADD 消融）定量地加剧了肺部的炎症。虽然无法直接证明或量化FADD ^AEC-KO小鼠的原位坏死性上皮细胞死亡，但 MLKL 激活激酶 RIPK3 的蛋白质水平在肺中明显上调。在小鼠组织中检测死亡或接近死亡的 necroptotic 细胞仍然具有很高的挑战性，然而，necroptosis 检测工具和技术的改进将很快允许在这种情况下进行更可靠的检测。¹⁰

Oikonomou等人的工作. 提示关于坏死性细胞死亡在人类哮喘中的潜在作用的进一步问题：坏死性凋亡的倾向是将严重终生慢性哮喘患者与轻度哮喘患者区分开来的原因吗？是否是坏死性凋亡的激活和持续存在支持常见呼吸道病毒在诱发哮喘中的作用？气道上皮细胞坏死性凋亡的增强倾向是否纯粹由感染史和适应性免疫反应介导，和/或是否对导致人类哮喘风险的坏死性凋亡倾向有直接的遗传影响？撇开病因不谈，这项工作为进一步临床前探索哮喘患者是否可以从目前正在开发的新型坏死性凋亡抑制剂药物中获益提供了一个重要的先例。