文献学习103–Mitochondria 是活化单核细胞释放的 a Subset of Extracellular Vesicles 可以诱导内皮细胞 Type I IFN 和 TNF 反应

19年的CR

1. LPS-Stimulated THP-1 Monocytic Cells Release Proinflammatory Microvesicles Enriched in Mitochondrial Content and Free Mitochondria

为了探究monocyte-derived microvesicles对内皮细胞的激活能力，作者评估了它们引起免疫反应的能力。与vehicle刺激相比(MV_Co)，LPS刺激下，THP1单核释放了更多的微泡(MV_Stress)(a, b)。(MV_Co)和(MV_Stress) were obtained after 18000g centrifugation of conditioned media and exposed phosphatidylserine(b), but only contained minute amounts of the exosomal marker ALIX (ALG-2-interacting protein X) as compared with extracellular vesicles (exosomes) obtained after 100 000g centrifugation (c)。The mean size of (MV_Stress) obtained after 18000g was 206.6 nm (SD±89.8)(d)。

Fig 1a： MV_Stress可以剂量依赖性的诱导内皮细胞和THP1细胞IL8的表达。
Fig 1b： Importantly, MV_Stress were able to activate endothelial cells in the absence of serum, while the response to LPS was serum-dependent. This excludes a significant contribution of LPS-carryover to the effect of MV_Stress as endothelial cells require serum-derived soluble CD14 (cluster of differentiation 14) for the recognition of LPS.
此前有文献报道，激活的白细胞可以释放mitochondria-derived DAMPs with robust proinflammatory properties，因此作者假设mitochondria contribute to the content and activity of MV_Stress.
Fig 1c： Indeed, THP-1 monocytic cells, prelabeled with mitochondria- and cytoplasm- specific dyes, released microvesicles particularly enriched in mitochondria-specific dye following stimulation with LPS.
Fig 1d： Furthermore, MVStress were particularly enriched in mitochondrial 16S rRNA over cytosolic 18S rRNA as compared with MV_Co.
Fig 1e： Consistent with this, high amounts of COXIV and mitochondria-associated protein Bcl-2 were detected in MV_Stress compared with MV_Co。
Fig 1f： Electron-microscopy identified the presence of free mitochondria and mitochondria within MV_Stress.
Fig 1g： Additionally, LPS-stimulation of THP-1 monocytic cells resulted in an increased release of vesicles presenting the mitochondria outer membrane protein TOM22.
Thus, LPS induces the release of free mitochondria and microvesicles enriched in mitochondrial content by THP-1 monocytic cells. Of note, 即使在高浓度下，MV_Co并没有诱导靶细胞IL8的表达。尽管TOM22+ vesicle确实存在。Thus the biological activity of MV_Stress cannot be explained simply by an increased presence of mitochondrial content.

2. Mitochondrial Activity of Parental Cells Defines the Proinflammatory Potential of Microvesicles

Fig 2a： 随后作者探究了MV_Stress的vesicular integrity是否影响它们的促炎状态。使用声波降解法disintegration of MV_Stress 并没有影响其对IL8表达的诱导能力，也没有影响内皮细胞ICAM-1 (intercellular adhesion molecule) 或 VCAM (vascular cell adhesion molecule) mRNA的表达。

有报道显示LPS刺激会改变线粒体活性。因此作者推测this defines the proinflammatory potential of MV_Stress。
Fig 2b： 首先作者使用extended low-dose Ethidium-Bromide treatment得到了impaired oxidative phosphorylation的THP1 (ρ0 cells)。LPS 刺激 ρ0THP-1 引起了类似数量的 microvesicles 的释放（附件）。但是ρ0 cell来源的 MV_Stress 诱导 IL-8 表达的能力显著下降，内皮细胞 ICAM-1 和 VCAM mRNA 的表达也显著下降。
Fig 2c： 随后，作者在pyruvate丙酮酸盐(which has been reported to preserve mitochondrial membrane potential)存在的情况下给予了THP1 LPS刺激。丙酮酸盐并没有影响LPS刺激下THP1释放的microvesicles的数量(附件)，但是MV_Stress对内皮细胞促炎性诱导的能力却下降了。
Thus, mitochondrial activity of parental cells contributes to the proinflammatory activity of MV_Stress released by them.
Fig 2d： 因为线粒体ROS在固有免疫反应中非常重要，作者检测了给予parental细胞线粒体特异性 ROS scavenger MitoTEMPO 是否可以影响MV_Stress的促炎能力。作者发现，MitoTEMPO并没有影响LPS刺激下THP1释放的microvesicles的数量(附件)，但是MV_Stress对内皮细胞促炎性诱导的能力也下降了。
Moreover, pyruvate supplementation and mitoTEMPO both reduced LPS-induced mitochondrial ROS production in THP-1 cells(附件)
这些数据显示 LPS- induced changes in mitochondrial activity of parental cells determine specific proinflammatory constituents of MV_Stress released by them.

3. Mitochondria Released by or Isolated From LPS-Activated THP-1 Monocytic Cells Activate Endothelial Cells

Fig 3a： 为了探究分离的MV_Stress中游离线粒体的贡献，作者使用TOM22的中和抗体清除了MV_Stress of TOM22+ vesicles (free mitochondria). 处理后TOM22+ vesicles几乎被完全清除，MV_Stress中线粒体16S rRNA显著下降(附件)。MV_Stress depleted of the TOM22+ vesicles (free mitochondria)之后诱导内皮细胞IL-8, ICAM-1, 和 VCAM mRNA表达的能力 显著下降。
Fig 3b： 为了探究LPS诱导的细胞活化是否影响线粒体促炎能力，作者直接比较了the ability of mitochondria isolated from LPS-activated (Mito_Stress) and vehicle-stimulated cells (Mito_Co) to activate endothelial cells. 和MV_Stress一致，只有Mito_Stress而不是Mito_Co可以诱导内皮细胞表达IL-8 , ICAM-1 和 VCAM。
Fig 3c： 此外，分离自ρ0 THP-1的Mito_Stress 更强的降低了促炎能力。
Fig 3d： 重要的是，disintegration of Mito_Stress sonication 并没有改变其诱导内皮细胞 IL-8, ICAM-1/ VCAM mRNA表达的的能力。
These results further support that the presence of proinflammatory constituents in extracellular mitochondria and mitochondria-containing microvesicles is influenced by activation of the parental cells and predetermined before their release.

4. Isolated Mitochondria and Microvesicles From LPS-Activated THP-1 Monocytic Cells Induce Type I IFN and TNF Signaling Pathways in Human Umbilical Vein Endothelial Cells

Fig 4a： 为了探究Mito_Stress的促炎功能，作者对Mito_Stress和Mito_Co 刺激的内皮进行了RNAseq检测。KEGG和GO富集分析结果显示和Mito_Co相比，Mito_Stress刺激的内皮显著富集到I型 IFN 和 TNF 信号通路。
Fig 4b： 从144个差异基因中，作者选择了10个此前报道的这两个pathway中的基因。包括OAS2, MX1, IFIT1, RSAD2, CCL2, IL-8, VCAM, ICAM-1, CXCL10, CXCL11. 这些基因在诱导后上调，与前面的qpcr结果一致。
Fig 4c： 为了探究IFN和TNF信号通路在这些基因表达上的作用，作者在用Mito_Stress干预内皮时使用了type I IFN decoy receptor (B18R) 或 TNFα- blocking antibody (Infliximab)。结果显示B18R下调了Mito_Stress诱导的内皮细胞IFN基因的表达。而Infliximab降低了Mito_Stress诱导的内皮细胞TNF基因的表达。CXCL10和CXCL11的表达只在阻断TNFa后下调。
Fig 4d： 随后作者探究了是否mitochondrial activity可以影响Mito_Stress诱导type I IFN 和 TNF 信号通路的。分离自ρ0 THP-1 monocytic 或 THP-1 的Mito_Stress在预先给 pyruvate 或 MitoTEMPO 后 had a reduced potential to induce the expression of IFN-signaling– and TNF-signaling–dependent genes in endothelial cells。
Fig 4e： To confirm that MV_Stress also induce IFN and TNF signaling, we analyzed the expression of the same set of genes in microvesicle-stimulated endothelial cells. Stimulation with MV_Stress resulted in a similar gene expression profile as incubation with Mito_Stress.
Fig 4f： Preparations of MV_Stress, which were immunodepleted of free mitochondria, had a significantly decreased potential to activate IFN- and TNF-dependent genes compared with sham- treated MV_Stress.
Fig 4g： 最后作者做了一下 in vivo 的验证。作者使用了 a low-grade endotoxemia model in humans。15个 healthy volunteers 接受了 2 ng/kg LPS 静脉注射。在注射前和注射后的对应时间点作者取了血液进行检测。LPS注射引起了TOM22+ vesicles 水平的增加，在注射后2h达到高峰。
Fig 4h： 有趣的是，血浆TNFα的高峰与TOM22+ vesicles的相一致。
Fig 4i： 而 sVCAM-1（反应内皮细胞活化），在LPS注射8h后升高。