Rodriguez-Rodrigues Nahuel, Castillo Luis A, Landoni Verónica I, Martire-Greco Daiana, Milillo M Ayelén, Barrionuevo Paula, Fernández Gabriela C
Laboratorio de Fisiología de los Procesos Inflamatorios, Instituto de Medicina Experimental CONICET, Academia Nacional de MedicinaBuenos Aires, Argentina.
Front Cell Infect Microbiol. 2017 Jul 6;7:306. doi: 10.3389/fcimb.2017.00306. eCollection 2017.
Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria through recognition of pRNA, and this sensing triggers potent bactericidal mechanisms.
多形核中性粒细胞（PMN）是抗菌宿主防御的第一道细胞防线。它们通过天然模式识别受体（如Toll样受体（TLR））识别病原体相关分子模式（PAMP）来感知病原体。本研究的目的是调查PMN是否能感知细菌的活力，并探索哪种活力因子可能参与这一现象。为此，使用活的大肠杆菌（Ec）和热灭活的大肠杆菌（HK-Ec）对分离出的人PMN的不同功能进行了评估。我们发现，细菌活力对于诱导PMN激活是必不可少的，这通过前向散射（FSC）增加、CD11b表面表达、趋化性、活性氧（ROS）生成和中性粒细胞胞外陷阱（NET）形成来衡量。由于未加帽的非聚腺苷酸化原核mRNA已被巨噬细胞和树突状细胞识别为与细菌活力相关的PAMP，因此从活的Ec中纯化了总原核RNA（pRNA），并将其用作PMN的刺激物。pRNA引发的反应与活细菌观察到的反应相似。无法从HK-Ec中分离出RNA，这解释了死细菌缺乏作用的原因。此外，死细菌的上清液能够诱导PMN激活，这与该上清液中存在pRNA有关，pRNA是在杀灭过程中释放出来的。当用RNA酶处理死细菌的上清液或分离出的pRNA时，pRNA诱导的杀菌功能（ROS和NETosis）被消除。此外，内吞作用对于pRNA诱导的ROS生成和NETosis是必要的，并且在全血中诱导pRNA诱导的ROS需要启动。然而，当pRNA用RNase消化时，与运动和脱颗粒相关的反应（FSC增加、CD11b上调和趋化性）仍然会被触发，并且不依赖于pRNA内吞作用或PMN启动。总之，我们的结果表明，PMN通过识别pRNA来感知活细菌，并且这种感知触发了强大的杀菌机制。
