Lorey Martina B, Rossi Katriina, Eklund Kari K, Nyman Tuula A, Matikainen Sampsa
From the ‡Rheumatology, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, Finland.
§Department of Immunology, Institute of Clinical Medicine, University of Oslo and Rikshospitalet Oslo, Oslo 0424, Norway
Mol Cell Proteomics. 2017 Apr;16(4 suppl 1):S187-S199. doi: 10.1074/mcp.M116.064840. Epub 2017 Feb 14.
Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, isintegrin nd etalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.
革兰氏阴性菌与人类多种传染病相关。炎性小体是细胞溶质蛋白复合物，当细胞遇到病原体或其他有害因子时组装而成。非经典半胱天冬酶-4/5炎性小体由革兰氏阴性菌衍生的脂多糖（LPS）和内源性氧化磷脂激活。蛋白质分泌是先天免疫反应的关键组成部分。在此，我们使用无标记定量蛋白质组学来表征人类原代巨噬细胞内LPS识别后非经典炎性小体激活时的整体蛋白质分泌情况。在进行蛋白质组学分析之前，使用尺寸排阻离心法将总分泌蛋白分离为两个部分，即富集的细胞外囊泡（EV）部分和剩余分泌蛋白（RS）部分。我们从EV部分鉴定出1048种蛋白质，从RS部分鉴定出1223种蛋白质。其中，有640种在两个部分中都被鉴定出来，这表明非经典炎性小体激活了多个部分重叠的蛋白质分泌途径。我们鉴定出了几种在宿主抵抗严重革兰氏阴性菌感染的反应中起关键作用的分泌蛋白。细胞内LPS识别后，可溶性分泌蛋白（RS部分）高度富集与炎症相关的蛋白质。感染后，几种核糖体蛋白在EV部分中高度丰富，我们的数据强烈表明，翻译机制的分泌和伴随的翻译抑制是宿主针对革兰氏阴性菌感知半胱天冬酶-4/5炎性小体反应的重要组成部分。细胞内LPS识别导致在富集的EV部分中分泌两种金属蛋白酶，即含整合素和金属蛋白酶结构域的蛋白10（ADAM10）和基质金属蛋白酶14（MMP14）。ADAM10的释放可能通过这些细胞因子的ADAM10依赖性膜脱落与关键炎性细胞因子肿瘤坏死因子（TNF）和髓系细胞重要生长因子巨噬细胞集落刺激因子（M-CSF）的分泌相关。半胱天冬酶-4/5炎性小体激活还导致在富集的EV部分中分泌危险相关分子S100A8和胸腺素α原。S100A8和胸腺素α原都是识别细胞外LPS的Toll样受体4的配体，它们可能在非经典炎性小体激活期间导致内毒素休克。
