19年的CR

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

為了探究monocyte-derived microvesicles對(duì)內(nèi)皮細(xì)胞的激活能力棚壁，作者評(píng)估了它們引起免疫反應(yīng)的能力在旱。與vehicle刺激相比(MV_Co)，LPS刺激下，THP1單核釋放了更多的微泡(MV_Stress)(a, b)玄坦。(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)玩郊。The mean size of (MV_Stress) obtained after 18000g was 206.6 nm (SD±89.8)(d)。

Fig 1a： MV_Stress可以劑量依賴性的誘導(dǎo)內(nèi)皮細(xì)胞和THP1細(xì)胞IL8的表達(dá)留搔。
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.
此前有文獻(xiàn)報(bào)道，激活的白細(xì)胞可以釋放mitochondria-derived DAMPs with robust proinflammatory properties铛铁，因此作者假設(shè)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隔显。
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, 即使在高濃度下却妨，MV_Co并沒有誘導(dǎo)靶細(xì)胞IL8的表達(dá)。盡管TOM22+ vesicle確實(shí)存在括眠。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： 隨后作者探究了MV_Stress的vesicular integrity是否影響它們的促炎狀態(tài)彪标。使用聲波降解法disintegration of MV_Stress 并沒有影響其對(duì)IL8表達(dá)的誘導(dǎo)能力，也沒有影響內(nèi)皮細(xì)胞ICAM-1 (intercellular adhesion molecule) 或 VCAM (vascular cell adhesion molecule) mRNA的表達(dá)掷豺。

有報(bào)道顯示LPS刺激會(huì)改變線粒體活性捞烟。因此作者推測(cè)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)当船。LPS 刺激 ρ0THP-1 引起了類似數(shù)量的 microvesicles 的釋放（附件）题画。但是ρ0 cell來源的 MV_Stress 誘導(dǎo) IL-8 表達(dá)的能力顯著下降，內(nèi)皮細(xì)胞 ICAM-1 和 VCAM mRNA 的表達(dá)也顯著下降德频。
Fig 2c： 隨后婴程，作者在pyruvate丙酮酸鹽(which has been reported to preserve mitochondrial membrane potential)存在的情況下給予了THP1 LPS刺激。丙酮酸鹽并沒有影響LPS刺激下THP1釋放的microvesicles的數(shù)量(附件)抱婉，但是MV_Stress對(duì)內(nèi)皮細(xì)胞促炎性誘導(dǎo)的能力卻下降了档叔。
Thus, mitochondrial activity of parental cells contributes to the proinflammatory activity of MV_Stress released by them.
Fig 2d： 因?yàn)榫€粒體ROS在固有免疫反應(yīng)中非常重要，作者檢測(cè)了給予parental細(xì)胞線粒體特異性 ROS scavenger MitoTEMPO 是否可以影響MV_Stress的促炎能力蒸绩。作者發(fā)現(xiàn)衙四，MitoTEMPO并沒有影響LPS刺激下THP1釋放的microvesicles的數(shù)量(附件)，但是MV_Stress對(duì)內(nèi)皮細(xì)胞促炎性誘導(dǎo)的能力也下降了患亿。
Moreover, pyruvate supplementation and mitoTEMPO both reduced LPS-induced mitochondrial ROS production in THP-1 cells(附件)
這些數(shù)據(jù)顯示 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： 為了探究分離的MV_Stress中游離線粒體的貢獻(xiàn)传蹈，作者使用TOM22的中和抗體清除了MV_Stress of TOM22+ vesicles (free mitochondria). 處理后TOM22+ vesicles幾乎被完全清除，MV_Stress中線粒體16S rRNA顯著下降(附件)步藕。MV_Stress depleted of the TOM22+ vesicles (free mitochondria)之后誘導(dǎo)內(nèi)皮細(xì)胞IL-8, ICAM-1, 和 VCAM mRNA表達(dá)的能力 顯著下降惦界。
Fig 3b： 為了探究LPS誘導(dǎo)的細(xì)胞活化是否影響線粒體促炎能力，作者直接比較了the ability of mitochondria isolated from LPS-activated (Mito_Stress) and vehicle-stimulated cells (Mito_Co) to activate endothelial cells. 和MV_Stress一致咙冗，只有Mito_Stress而不是Mito_Co可以誘導(dǎo)內(nèi)皮細(xì)胞表達(dá)IL-8 , ICAM-1 和 VCAM沾歪。
Fig 3c： 此外，分離自ρ0 THP-1的Mito_Stress 更強(qiáng)的降低了促炎能力雾消。
Fig 3d： 重要的是灾搏，disintegration of Mito_Stress sonication 并沒有改變其誘導(dǎo)內(nèi)皮細(xì)胞 IL-8, ICAM-1/ VCAM mRNA表達(dá)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： 為了探究Mito_Stress的促炎功能立润，作者對(duì)Mito_Stress和Mito_Co 刺激的內(nèi)皮進(jìn)行了RNAseq檢測(cè)狂窑。KEGG和GO富集分析結(jié)果顯示和Mito_Co相比，Mito_Stress刺激的內(nèi)皮顯著富集到I型 IFN 和 TNF 信號(hào)通路桑腮。
Fig 4b： 從144個(gè)差異基因中泉哈，作者選擇了10個(gè)此前報(bào)道的這兩個(gè)pathway中的基因。包括OAS2, MX1, IFIT1, RSAD2, CCL2, IL-8, VCAM, ICAM-1, CXCL10, CXCL11. 這些基因在誘導(dǎo)后上調(diào)，與前面的qpcr結(jié)果一致丛晦。
Fig 4c： 為了探究IFN和TNF信號(hào)通路在這些基因表達(dá)上的作用奕纫，作者在用Mito_Stress干預(yù)內(nèi)皮時(shí)使用了type I IFN decoy receptor (B18R) 或 TNFα- blocking antibody (Infliximab)。結(jié)果顯示B18R下調(diào)了Mito_Stress誘導(dǎo)的內(nèi)皮細(xì)胞IFN基因的表達(dá)采呐。而Infliximab降低了Mito_Stress誘導(dǎo)的內(nèi)皮細(xì)胞TNF基因的表達(dá)若锁。CXCL10和CXCL11的表達(dá)只在阻斷TNFa后下調(diào)。
Fig 4d： 隨后作者探究了是否mitochondrial activity可以影響Mito_Stress誘導(dǎo)type I IFN 和 TNF 信號(hào)通路的斧吐。分離自ρ0 THP-1 monocytic 或 THP-1 的Mito_Stress在預(yù)先給 pyruvate 或 MitoTEMPO 后 had a reduced potential to induce the expression of IFN-signaling– and TNF-signaling–dependent genes in endothelial cells又固。
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 的驗(yàn)證。作者使用了 a low-grade endotoxemia model in humans煤率。15個(gè) healthy volunteers 接受了 2 ng/kg LPS 靜脈注射仰冠。在注射前和注射后的對(duì)應(yīng)時(shí)間點(diǎn)作者取了血液進(jìn)行檢測(cè)。LPS注射引起了TOM22+ vesicles 水平的增加蝶糯，在注射后2h達(dá)到高峰洋只。
Fig 4h： 有趣的是，血漿TNFα的高峰與TOM22+ vesicles的相一致昼捍。
Fig 4i： 而 sVCAM-1（反應(yīng)內(nèi)皮細(xì)胞活化）识虚，在LPS注射8h后升高。