Legionella pneumophila infection activates bystander cells differentially by bacterial and host cell vesicles

Anna Lena Jung,Christine Schulz,Alexandra Sittka-Stark,Nicoletta Scheller,Kathrin Bolte,Wilhelm Bertrams,Kerstin Seidel,Bernd Schmeck,Christina Elena Herkt

doi:10.1038/s41598-017-06443-1

Abstract

Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1β in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or – partially – RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.

Highlights

Legionella pneumophila (L. pneumophila), a gram-negative bacterium, is described as a causative pathogen of pneumonia[1]
THP-1 cells respond to L. pneumophila infection with increased secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and MCP-1, Supplementary Fig. 1) and notably extracellular vesicles (EVs) (Fig. 1a)
The amount of particles/mL in this so called 100 k pellet increased with multiplicity of infection, which could be observed upon administration of the sterile stimulant IL-1β

Summary

Introduction

Legionella pneumophila (L. pneumophila), a gram-negative bacterium, is described as a causative pathogen of pneumonia[1]. L. pneumophila are phagocytosed and contained in vesicular cytosolic bodies, the phagosomes, that are bound for lysosomal degradation Legionella actively blocks this mechanism of cellular defence by transmembrane secretion of effector proteins into the host cell via the dot/Icm type IV secretion system[4]. These factors are instrumental to formation of a replication niche, the Legionella-containing vacuole (LCV), which is recruited from ER-derived vesicles. L. pneumophila OMVs are known to be pro-inflammatory activators of epithelial cells and macrophages[20, 21] They can modulate the course of infection in vitro. We sought to better understand the impact of intercellular communication via EVs during the course of L. pneumophila infection and to distinguish the role of different EV subsets, namely OMVs and exosomes, on alveolar recipient cells

Objectives

Methods

Results

Conclusion