Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.
Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.
复制标题
DOI：
10.3389/fcimb.2023.1250339
复制
发表时间：
2023
影响因子：
5.7
通讯作者：
中科院分区：
文献类型：
--
作者：
铜绿假单胞菌是一种主要的人类病原体，在囊性纤维化患者的气道中特别有效。噬菌体在感染部位非常丰富，但它们对哺乳动物免疫力的影响仍不清楚。我们先前表明，Pf 4，一种由铜绿假单胞菌产生的温和丝状噬菌体，通过TLR 3信号转导改变对铜绿假单胞菌感染的先天免疫应答，但其潜在机制仍不清楚。值得注意的是，Pf 4是单链DNA和溶原性噬菌体，并且其产生通常不会导致其细菌宿主的裂解。我们以前确定，Pf 4的内化由人类或小鼠免疫细胞触发适应不良的病毒模式识别受体，并导致基于噬菌体RNA的存在的细菌持久性。我们现在报告说，Pf 4噬菌体抑制炎症反应的细菌内毒素，这是介导的部分通过细菌囊泡连接到噬菌体颗粒。外膜囊泡（OMV）是革兰氏阴性菌产生的一种囊泡，在宿主与病原菌的相互作用中起着重要作用。最近，有证据表明OMV差异包装小RNA。在这项研究中，我们表明，Pf 4的装饰与OMV保持附着到Pf 4，尽管纯化步骤。这些蛋白质被人细胞内吞并递送至内体囊泡。我们证明，短RNA内的OMV形成发夹结构，触发TLR 3依赖的I型干扰素的生产和拮抗生产的抗菌细胞因子和趋化因子。特别是，Pf 4可抑制CXCL 5，阻止响应内毒素的有效嗜中性粒细胞趋化性。此外，阻断IFNAR或TLR 3信号传导消除了与OMV结合的Pf 4对巨噬细胞活化的作用。在鼠急性肺炎模型中，用与OMV相关的Pf 4处理的小鼠在BAL液中显示出比用纯化的Pf 4处理的小鼠显著更少的嗜中性粒细胞浸润。巨噬细胞表型的这些变化在功能上是相关的：暴露于Pf 4修饰的OMV的细胞的条件培养基在体外和体内诱导中性粒细胞迁移的有效性显着降低。这些结果表明，Pf 4可改变对细菌内毒素和OMV的先天免疫，潜在地抑制细菌定植或感染部位的炎症。图形摘要：OMV/Pf 4复合物介导的骨髓细胞对细菌刺激反应的修饰模型。当从其宿主铜绿假单胞菌中挤出时，Pf 4与OMV结合。OMV/Pf 4复合物被肺驻留的骨髓细胞如巨噬细胞内吞。OMV内携带的降解保护发夹结构RNA触发TLR 3激活。TLR 3信号传导诱导I型IFN产生，I型IFN产生反过来抑制响应于其它细菌产物的促炎细胞因子产生。被抑制的细胞因子包括中性粒细胞化学引诱物，如CXCL 1和CXCL 5，导致中性粒细胞流入铜绿假单胞菌感染部位并最终导致细菌持续存在。
Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection. Graphical abstract—a model for OMV/Pf4 complex-mediated modification of the myeloid cell response to bacterial stimulation. While extruding from its host P. aeruginosa, Pf4 gets associated with OMVs. OMV/Pf4 complexes are endocytosed by lung-resident myeloid cells such as macrophages. Degradation-protected hairpin-structured RNA carried within OMVs triggers TLR3 activation. TLR3 signaling induces type I IFN production which in turn inhibits pro-inflammatory cytokine production in response to other bacterial products. Among the cytokines inhibited are neutrophil chemoattractants such as CXCL1 and CXCL5 resulting in abrogated neutrophil influx to the site of P. aeruginosa infection and eventually to the persistence of the bacteria.