Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.
Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.
复制标题
DOI：
10.1002/hep.28252
复制
发表时间：
2016-03
期刊：
影响因子：
--
通讯作者：
Gores GJ
中科院分区：
文献类型：
--
作者：
Ibrahim SH;Hirsova P;Tomita K;Bronk SF;Werneburg NW;Harrison SA;Goodfellow VS;Malhi H;Gores GJ
在非酒精性脂肪性肝炎（NASH）的鼠模型中，混合谱系激酶3（MLK 3）缺陷减少巨噬细胞相关炎症。然而，肝细胞中MLK 3活化与NASH中巨噬细胞驱动的炎症之间的机制联系尚不清楚。在此，我们报告MLK 3介导释放（C-X-C基序）配体10（CXCL 10）-负载细胞外囊泡（EV）从脂毒性肝细胞，诱导巨噬细胞趋化性。原代小鼠肝细胞（PMH）和Huh 7细胞用棕榈酸酯或溶血磷脂酰胆碱（LPC）处理。通过微分超离心分离释放的EV。LPC处理PMH或Huh 7细胞诱导EV的释放，这通过MLK 3的遗传或药理学抑制来防止。质谱鉴定了EV中的有效趋化因子CXCL 10，其在从LPC处理的肝细胞与未处理的细胞分离的EV中显著富集。绿色荧光蛋白（GFP）标记的CXCL 10存在于囊泡结构中，并且在LPC处理共转染的Huh-7细胞后与红色荧光蛋白（RFP）标记的EV标志物分化簇（CD）63共定位。MLK 3的基因缺失或药理学抑制阻止了EV中CXCL 10的富集。用脂毒性肝细胞衍生的EV处理小鼠骨髓衍生的巨噬细胞诱导巨噬细胞趋化性，这种作用可通过与CXCL 10中和抗血清孵育来阻断。与WT小鼠相比，喂食NASH诱导饮食的MLK 3缺陷小鼠具有降低的总血浆EV和含CXCL 10的EV浓度。在肝细胞脂毒性过程中，活化的MLK 3诱导携带CXCL 10的囊泡从肝细胞释放，所述囊泡对巨噬细胞具有趋化性。
Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage associated-inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMH) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and co-localized with the red fluorescent protein (RFP)-tagged EV marker cluster of differentiation (CD) 63 following LPC treatment of co-transfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone marrow-derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antisera. MLK3 deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs, and CXCL10 containing EVs compared to WT mice. during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.