研究目的：

作为先天免疫系统的“前线卫士”，中性粒细胞以促炎作用和病原体清除能力著称，通过吞噬病原体、脱颗粒、产生活性氧 (ROS) 以及形成中性粒细胞胞外诱捕网 (NETs) 等多种方式发挥免疫防御功能。作为终末分化细胞，中性粒细胞寿命短暂，半衰期通常不足24小时，会快速经历衰老死亡，正常成人每日每公斤体重生成并清除约10⁹个中性粒细胞。中性粒细胞的短暂寿命和快速清除引发了学界对其在超出正常生命周期之外可能发挥的功能的猜测。随着中性粒细胞胞外捕网 (Neutrophil Extracellular Traps，NETs) 的发现，这一猜测得到了有力的佐证。在NETs形成过程中，激活的中性粒细胞膜穿孔，释放出染色质、颗粒蛋白和组蛋白的组合，形成网状结构以捕获和杀死细菌。这种机制不仅延长了中性粒细胞功能的持续时间，还展现了超越其有限生命周期的功能潜力。基于以上背景，本研究将探索是否存在其他源自中性粒细胞的结构或机制，也能超越其正常生命周期发挥作用。

研究方法：

采用梯度离心技术分离衰老中性粒细胞释放的细胞外囊泡，通过共聚焦荧光显微镜、透射电镜、纳米颗粒分析技术以及流式细胞术系统表征其超微结构、生物发生特征、表面标志物及分子组成，基于其独特的特征，该囊泡被命名为衰老中性粒细胞源性大型细胞外囊泡 (Large Aging Neutrophil-Derived Vesicles，LAND-Vs)。为明确LAND-Vs的生物学功能，本研究建立金黄色葡萄球菌诱导的小鼠急性肺炎模型，并通过气管内注射途径给予LAND-Vs干预，系统评估其在肺部炎症调控中的作用。同时，采用中性粒细胞特异性表达EGFP (绿色荧光蛋白) ，而其他细胞表达tdTomato (红色荧光蛋白) 的双荧光报告小鼠构建肺炎模型，通过肺组织冰冻切片联合荧光显微成像技术，特异性追踪中性粒细胞来源(EGFP⁺)的LAND-Vs在炎症部位的分布特征；为解析LAND-Vs的功能性蛋白组分，整合质谱流式细胞术和高通量蛋白质组学技术，系统构建其蛋白质表达谱并解析亚群异质性特征，鉴定出CD55高表达的功能性亚群。鉴于CD55作为补体调控关键节点，本研究采用补体C3基因敲除小鼠模型，证实LAND-Vs通过CD55依赖性途径调控C3转化酶活性，进而介导其抗炎效应。采用免疫荧光染色联合活细胞动态成像技术，结合成像流式细胞分析，定量解析中性粒细胞衰老过程中CD55的时空表达动态及其调控LAND-Vs生成的分子机制。最终通过构建中性粒细胞特异性CD55条件敲除小鼠模型，在体内水平确证内源性LAND-Vs通过CD55介导的补体调控通路在肺炎转归中的关键作用。

研究结果：

中性粒细胞在衰老过程中，可产生直径约1 μm的独特细胞外囊泡群体。基于其独特的粒径分布、超微结构、生物发生模式等特征，该细胞外囊泡群体被命名为衰老中性粒细胞源性大型细胞外囊泡 (Large Aging Neutrophil-Derived Vesicles, LAND-Vs)。

为明确LAND-Vs的生物学功能，本研究将LAND-Vs气管内注射到金黄色葡萄球菌诱导的小鼠急性肺炎模型中。定量分析显示，LAND-Vs治疗组较对照组显著降低支气管肺泡灌洗液中炎症细胞募集和促炎细胞因子分泌，减轻肺炎组织损伤，并提高肺炎小鼠的生存率。值得注意的是，LAND-Vs干预未影响细菌载量及肺泡巨噬细胞数量。

采用双荧光报告小鼠肺炎模型，我们在肺炎部位直接观察到中性粒细胞来源的EGFP⁺LAND-Vs结构。临床验证显示，肺炎患者肺组织样本中LAND-Vs检出率高于健康对照组，揭示其在体内病理条件性的生物学相关性。

为解析LAND-Vs的功能性蛋白组分，我们构建了LAND-Vs的蛋白质谱并解析亚群异质性，发现显著高表达CD55和CD47的亚群，占比超过60%。功能验证显示：CD55综合抗体处理组，CD55基因敲除组以及CD55⁻分选亚群的 LAND-Vs抗炎活性显著下降，而CD55⁺ LAND-Vs抗炎活性显著。

机制研究表明，CD55通过结合C3b/C4b抑制C3转化酶形成从而抑制补体C3剪切活化，进而阻断补体级联反应。在C3基因敲除小鼠模型中，CD55⁺ LAND-Vs未能改善肺炎病理进程，证实其抗炎效应依赖补体C3调控通路。

中性粒细胞衰老（CXCR4表达上升）过程中，膜上CD55发生脂筏依赖性极性重分布，并通过RhoA/ROCK信号通路介导的膜出芽机制，在极化区域形成LAND-Vs。

为了研究LAND-Vs在肺炎中的动态作用，我们监测了LAND-Vs和补体活化片段 C3a 和 C3b 的水平。结果显示补体片段在感染后 6 小时开始升高，48小时骤降。相反，LAND-Vs在24至48小时持续累积，其丰度与补体活性呈现显著负相关，而其他补体调控因子 (Factor H/ I/B) 丰度与补体活性没有相关性。这一结果表明，内源性LAND-Vs是炎症消退阶段调控补体的关键因素。

由于LAND-Vs表面高表达“don’t eat me”信号分子 (如CD47) ，其被吞噬细胞吞噬清除显著降低，使得LAND-Vs在炎症消退阶段维持高水平，从而发挥抗炎功能并促进炎症消退。

为了进一步验证CD55介导LAND-Vs抗炎功能的作用，在中性粒细胞特异性敲除CD55的肺炎小鼠中，过继转移WT LAND-Vs将敲除小鼠炎症反应降低到基线水平，而CD55 KO LAND-Vs没有抗炎作用，证明CD55是LAND-Vs调控炎症的核心效应分子。

临床队列研究显示，轻症COVID-19患者血浆中LAND-Vs数量显著高于健康对照，并且明显高于重症COVID-2019患者组。这一结果提示LAND-Vs的水平可能与病情缓解密切相关。

研究结论：

本研究发现中性粒细胞在衰老的过程中产生新定义的抗炎性细胞外囊泡——LAND-Vs，通过其膜表面CD55分子抑制补体系统级联激活，发挥出强大且持久的抗炎作用，促进炎症消退。总之，本研究首次揭示了LAND-Vs作为衰老中性粒细胞分泌的关键炎症调控载体，是衰老中性粒细胞主动产生的产物。其发现不仅表明中性粒细胞可以通过LAND-Vs发挥超越其有限生命周期的功能，也为炎症和感染性疾病提供了新的潜在的治疗靶点。

Objective:

As uncomplicated front-line troopers of the innate immune system, neutrophils are renowned for their pro-inflammatory functions and pathogen-clearing capabilities. They exert immune defense through a variety of mechanisms including phagocytosis, degranulation, release of reactive oxygen species (ROS), and the formation of neutrophil extracellular traps (NETs). As terminally differentiated cells, neutrophils have a remarkably short lifespan, with a half-life typically less than 24 hours. Approximately 1 billion neutrophils are generated and cleared daily as they rapidly undergo senescence and cell death. The short lifespan and rapid clearance of neutrophils have raised questions about whether they might exert functions beyond their expected lifespan. The discovery of neutrophil extracellular traps (NETs), intricate mesh-like structures formed when neutrophils expel their DNA and associated proteins into the extracellular matrix, has dramatically broadened the scope of neutrophil functionality and pushed the boundaries of neutrophil functionality beyond their ephemeral lifespan. Nonetheless, the extent to which other structures derived from neutrophils share this parallel attribute has not been fully elucidated. Therefore, this study aims to explore whether other structures or mechanisms derived from neutrophils may also function beyond their normal lifespan. 

Methods:

Using gradient centrifugation, extracellular vesicles (EVs) released by aging neutrophils were isolated and systematically characterized by confocal fluorescence microscopy, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry to determine their ultrastructure, biogenesis, surface markers, and molecular composition. Based on their distinct characteristics, these vesicles were named Large Aging Neutrophil-Derived Vesicles (LAND-Vs). To elucidate the biological function of LAND-Vs, a mouse model of acute pneumonia induced by Staphylococcus aureus was established. LAND-Vs were administered via intratracheal injection, and their role in modulating pulmonary inflammation was comprehensively evaluated. To track the distribution of neutrophil-derived (EGFP⁺) LAND-Vs at sites of lung inflammation, a dual-fluorescent reporter mouse model was generated, in which neutrophils specifically expressed EGFP while other cell types expressed tdTomato. To dissect the functional protein components of LAND-Vs, mass cytometry and high-throughput proteomics were integrated to construct a comprehensive protein expression profile and analyze their subpopulation heterogeneity. A functional subpopulation of LAND-Vs with high CD55 expression was identified. Given that CD55 is a critical regulator of the complement system, a complement C3 knockout mouse model was employed to demonstrate that LAND-Vs exert their anti-inflammatory effects through a CD55-dependent pathway that modulates C3 convertase activity. The spatiotemporal dynamics of CD55 expression during neutrophil aging and its role in LAND-Vs biogenesis were quantitatively analyzed using immunofluorescence staining, live-cell imaging, and imaging flow cytometry. Finally, a neutrophil-specific CD55 conditional knockout mouse model was generated to confirm, at the in vivo level, the essential role of endogenous LAND-Vs in regulating complement activity via CD55-mediated pathways during the resolution of pneumonia.

Results:

1) During the aging process, neutrophils produce a unique population of extracellular vesicles (EVs) with a diameter of approximately 1 μm. Based on their distinct size distribution, ultrastructure, and biogenesis patterns, this population of EVs has been designated as Large Aging Neutrophil-Derived Vesicles (LAND-Vs).

2) To investigate the biological function of LAND-Vs, they were administered intratracheally into a Staphylococcus aureus-induced mouse model of acute pneumonia. Quantitative analyses revealed that mice treated with LAND-Vs exhibited a significant reduction in inflammatory cell recruitment and pro-inflammatory cytokine secretion in bronchoalveolar lavage fluid (BALF), attenuated lung tissue damage, and improved survival rates compared to control groups. Notably, LAND-Vs intervention did not alter bacterial burden or the number of alveolar macrophages.

3) Using a dual-fluorescent reporter mouse model of pneumonia, we directly observed EGFP⁺ LAND-Vs structures derived from neutrophils at sites of pulmonary inflammation. Clinical validation further revealed that LAND-Vs were detected at a higher frequency in lung tissue samples from pneumonia patients compared to healthy controls, underscoring their pathological relevance under in vivo conditions.

4) To characterize the functional protein components of LAND-Vs, we constructed their proteomic profile and analyzed subpopulation heterogeneity. A subpopulation expressing high levels of CD55 and CD47 was identified, comprising over 60% of total LAND-Vs. Functional validation demonstrated that the anti-inflammatory activity of LAND-Vs was significantly attenuated in groups treated with a CD55 neutralizing antibody, in CD55 knockout models, or when using CD55⁻ LAND-Vs subpopulations. In contrast, CD55⁺ LAND-Vs exhibited robust anti-inflammatory effects.

5) Mechanistic studies revealed that CD55 exerts its function by binding to C3b/C4b, thereby inhibiting the formation of C3 convertase and preventing complement C3 cleavage and activation, ultimately blocking the complement cascade. In C3 knockout mouse models, CD55⁺ LAND-Vs failed to ameliorate pneumonia pathology, confirming that their anti-inflammatory effects are dependent on the complement C3 regulatory pathway.

6) During neutrophil aging (characterized by increased CXCR4 expression), CD55 undergoes lipid raft-dependent polarized redistribution on the membrane. Through a RhoA/ROCK signaling pathway-mediated membrane budding mechanism, CD55 accumulates in the polarized regions to facilitate the formation of LAND-Vs.

7) To investigate the dynamic role of LAND-Vs during pneumonia, we monitored the levels of complement activation fragments C3a and C3b. These fragments began to rise at 6 hours post-infection and sharply declined at 48 hours. In contrast, LAND-Vs steadily accumulated between 24 and 48 hours, with their abundance showing a significant negative correlation with complement activity. Other complement regulatory factors (e.g., Factor H, I, and B) demonstrated no such correlation with complement activity. These findings suggest that endogenous LAND-Vs are critical regulators of complement activity during the resolution phase of inflammation.

8) Due to the high expression of “don’t eat me” signals (e.g., CD47) on the surface of LAND-Vs, their clearance by phagocytes is markedly reduced. This enables LAND-Vs to persist at elevated levels during the late phase of inflammation, exerting anti-inflammatory effects and promoting the resolution of inflammation.

9) To further validate the role of CD55 in mediating the anti-inflammatory function of LAND-Vs, neutrophil-specific CD55 knockout mice were used in a pneumonia model. Adoptive transfer of wild-type (WT) LAND-Vs reduced the inflammatory response in CD55-deficient mice to baseline levels, whereas CD55-deficient (CD55 KO) LAND-Vs failed to exert any anti-inflammatory effects. These findings confirm that CD55 is the key effector molecule driving the inflammation-regulating function of LAND-Vs.

10) Clinical cohort studies revealed that plasma levels of LAND-Vs were significantly higher in mild COVID-19 patients compared to healthy controls and markedly higher than those in severe COVID-19 patients. This observation suggests that LAND-V levels may be closely associated with disease resolution and clinical recovery.

Conclusion: 

This study identifies a newly defined anti-inflammatory extracellular vesicle, termed LAND-Vs (Large Aging Neutrophil-Derived Vesicles), which are produced during neutrophil aging. LAND-Vs exert potent and sustained anti-inflammatory effects by inhibiting complement cascade activation via their membrane-bound CD55, thereby promoting inflammation resolution. In summary, this study is the first to reveal that LAND-Vs act as critical inflammatory regulatory carriers secreted by aging neutrophils and represent an active product of neutrophil aging. Their discovery not only demonstrates that neutrophils can extend their functional roles beyond their limited lifespan through LAND-Vs but also provides a novel potential therapeutic target for the treatment of inflammatory and infectious diseases.