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Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance
Critical Care Medicine ( IF 8.8 ) Pub Date : 2022-06-01 , DOI: 10.1097/ccm.0000000000005437
Riddhita Mukherjee 1, 2 , Carly A Tompkins 1, 2 , Nicolai P Ostberg 2 , Amit U Joshi 2 , Liliana M Massis 3 , Vijith Vijayan 1 , Kanika Gera 1 , Denise Monack 3 , Timothy T Cornell 1 , Mark W Hall 4 , Daria Mochly-Rosen 2 , Bereketeab Haileselassie 1, 2
Affiliation  

OBJECTIVES: 

Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis.

DESIGN: 

Laboratory investigation.

SETTING: 

University laboratory.

SUBJECTS: 

C57BL/6 and BALB/C mice.

INTERVENTIONS: 

Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge.

MEASUREMENTS AND MAIN RESULTS: 

When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 μL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture.

CONCLUSIONS: 

Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.



中文翻译:

Drp1/Fis1 依赖性病理分裂和相关的细胞外线粒体受损导致巨噬细胞内毒素耐受功能障碍

目标: 

最近的出版物表明,线粒体动力学可以控制细胞外线粒体的质量和数量,从而影响免疫表型。本研究旨在确定 Drp1/Fis1 相互作用介导的病理性线粒体裂变是否会影响脓毒症诱导的免疫麻痹中的细胞外线粒体含量和巨噬细胞功能。

设计: 

实验室调查。

环境: 

大学实验室。

科目: 

C57BL/6 和 BALB/C 小鼠。

干预措施: 

利用内毒素耐受 (ET) 的体外和小鼠模型,我们评估了 Drp1/Fis1 依赖性病理裂变的变化,并同时测量了细胞外线粒体的数量和质量。接下来,通过用分离的健康线粒体(M C)和受损线粒体启动小鼠巨噬细胞,我们确定受损的细胞外线粒体是否能够诱导对随后内毒素挑战的耐受性。最后,我们确定了 Drp1/Fis1 介导的病理性裂变的抑制是否会消除受损细胞外线粒体的释放并改善巨噬细胞对随后内毒素攻击的反应。

测量和主要结果: 

与幼稚巨噬细胞 (NM) 相比,耐内毒素巨噬细胞 (ETM) 表现出 Drp1/Fis1 依赖性线粒体功能障碍和更高水平的受损细胞外线粒体(Mitotracker-Green + 事件/50 μL:ETM = 2.42 × 10 6 ± 4,391 vs NM = 5.69 × 10 5 ± 2,478;p < 0.001)。NM 暴露于受损的细胞外线粒体 (M H ) 会诱导对随后内毒素攻击的交叉耐受,而MC的影响最小(肿瘤坏死因子 [TNF]-α [pg/mL]:NM = 668 ± 3,NM + M H = 221 ± 15,NM + Mc = 881 ± 15;p < 0.0001)。使用 Drp1/Fis1 相互作用的选择性抑制剂七肽 (P110) 抑制 Drp1/Fis1 依赖性线粒体裂变,改善细胞外线粒体功能(细胞外线粒体膜电位,JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0;p < 0.001),随后 ETM 中的免疫反应得到改善(TNF-α [pg/mL];ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4;p < 0.0001 。同样,经 P110 处理的耐内毒素小鼠血浆中受损的细胞外线粒体数量较低(以较高的细胞外线粒体膜电位表示,TMRM/MT-G:内毒素耐受 [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02;p = 0.03)并改善对后续内毒素治疗以及盲肠结扎和穿刺的免疫反应。

结论: 

在 ET 的体外和体内模型中,抑制 Drp1/Fis1 依赖性线粒体断裂可改善巨噬细胞功能和免疫反应。这种益处至少部分是通过减少受损细胞外线粒体的释放来介导的,这有助于内毒素交叉耐受。总而言之,这些数据表明线粒体动力学的改变可能在脓毒症诱导的免疫麻痹中发挥重要作用。

更新日期:2022-05-31
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