Division of Infectious Diseases, Department of Medicine, Stanford University, Stanford, CA, United States.
Immunology Program, Stanford University, Stanford, CA, United States.
Front Cell Infect Microbiol. 2023 Oct 26;13:1250339. doi: 10.3389/fcimb.2023.1250339. eCollection 2023.
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 , modifies the innate immune response to 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 and . These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection.
是一种主要的人类病原体，特别擅长定植囊性纤维化患者的气道。噬菌体在感染部位高度丰富，但它们对哺乳动物免疫的影响尚不清楚。我们之前曾表明，由 产生的温和丝状噬菌体 Pf4 通过 TLR3 信号改变对 的先天免疫反应，但潜在机制尚不清楚。值得注意的是，Pf4 是单链 DNA 和溶原噬菌体，其产生通常不会导致其细菌宿主裂解。我们之前发现，人类或鼠类免疫细胞内化 Pf4 会触发适应性病毒模式识别受体，并基于噬菌体 RNA 的存在导致细菌持续存在。我们现在报告 Pf4 噬菌体减弱了对细菌内毒素的炎症反应，部分原因是噬菌体颗粒上附着的细菌囊泡介导的。外膜囊泡 (OMV) 由革兰氏阴性菌产生，在宿主病原体相互作用中起关键作用。最近，有证据表明 OMV 可以不同地包装小 RNA。在这项研究中，我们表明 Pf4 被 OMV 装饰，尽管经过纯化步骤，但 Pf4 仍附着在 Pf4 上。这些噬菌体被人细胞内吞，并递送至内体囊泡。我们证明，OMV 内的短 RNA 形成发夹结构，触发 TLR3 依赖性 I 型干扰素产生，并拮抗抗菌细胞因子和趋化因子的产生。特别是，Pf4 噬菌体抑制 CXCL5，从而阻止内毒素引起的中性粒细胞趋化。此外，阻断 IFNAR 或 TLR3 信号会消除 Pf4 与 OMV 结合对巨噬细胞激活的影响。在小鼠急性肺炎模型中，与 OMV 结合的 Pf4 处理的小鼠在 BAL 液中的中性粒细胞浸润明显少于用纯化的 Pf4 处理的小鼠。这些巨噬细胞表型的变化在功能上是相关的：暴露于 Pf4 与 OMV 共装饰的细胞的条件培养基在诱导中性粒细胞迁移方面明显效果较差 和 。这些结果表明 Pf4 噬菌体改变了对细菌内毒素和 OMV 的先天免疫，可能在细菌定植或感染部位减轻炎症。
