Eisinger Gregory J, Osman Wissam, Prather Evan R, Julian Mark W, Gavrilin Mikhail A, Crouser Elliott D, Wewers Mark D
Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, The Ohio State University, Wexner Medical Center, Columbus, OH.
Crit Care Explor. 2022 Feb 1;4(2):e0631. doi: 10.1097/CCE.0000000000000631. eCollection 2022 Feb.
Increased monocyte distribution width (MDW) has recently been shown to be a reliable indicator of early sepsis detection. This study therefore sought to determine if inflammasome activation can be linked to monocyte size changes in sepsis.
An in vitro sepsis model using bacterial endotoxin (lipopolysaccharide [LPS]) to study the effect of inflammasome activation on monocyte cell size distribution by microscopy and MDW measurements using a standard clinical hematology analyzer.
University research laboratory.
Healthy adult volunteers and cultured human monocyte cells in wild-type state and after clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 knockout of key inflammasome components (apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, gasdermin-D).
In vitro treatment of specimens with bacterial LPS.
Wild-type THP1 cells demonstrated a significant increase in cell area (207 μm [159-400 μm] vs 160 μm [134-198 μm]; < 0.001) and distribution width (198 vs 55 μm; < 0.0001) by microscopy following treatment with LPS. Increased MDW correlated with inflammasome activation as demonstrated by release of interleukin (IL)-1β and with the presence of large distended pyroptotic cells by microscopy. All of these effects were blocked in the inflammasome knockout cells. Whole blood samples treated similarly also demonstrated IL-1β release and increased MDW (median 24.7 U [22.2-27.2 U] vs 16.3 U [15.1-17.6 U]; = 0.008) as measured using the Beckman-Coulter Unicel DxH900 analyzer. When peripheral blood mononuclear cells were isolated prior to treatment with LPS, microscopy confirmed the presence of large pyroptotic cells correlating to IL-1β release in the human subject samples as well.
The increased MDW seen in patients with sepsis can be reproduced in an in vitro sepsis model and blocked using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology to inactivate the inflammasome. These findings suggest that pyroptotic cellular swelling underlies changes in MDW in septic patients and connect MDW to early events in the inflammatory cascade of sepsis.
近期研究表明，单核细胞分布宽度（MDW）增加是早期脓毒症检测的可靠指标。因此，本研究旨在确定炎性小体激活是否与脓毒症中单核细胞大小变化有关。
采用细菌内毒素（脂多糖[LPS]）建立体外脓毒症模型，通过显微镜观察炎性小体激活对单核细胞大小分布的影响，并使用标准临床血液分析仪测量MDW。
大学研究实验室。
健康成年志愿者以及野生型状态和关键炎性小体成分（含半胱天冬酶募集结构域的凋亡相关斑点样蛋白、半胱天冬酶-1、gasdermin-D）经成簇规律间隔短回文重复序列/CRISPR相关蛋白9敲除后的培养人单核细胞。
用细菌LPS对标本进行体外处理。
显微镜观察显示，野生型THP1细胞经LPS处理后，细胞面积显著增加（207μm[159 - 400μm]对160μm[134 - 198μm]；P<0.001），分布宽度增加（198对55μm；P<0.0001）。MDW增加与炎性小体激活相关，表现为白细胞介素（IL）-1β释放，且显微镜下可见大量肿胀的焦亡细胞。所有这些效应在炎性小体敲除细胞中均被阻断。使用贝克曼库尔特Unicel DxH900分析仪测量，同样处理的全血样本也显示IL-1β释放和MDW增加（中位数24.7 U[22.
