Date Published: April 22, 2016
Publisher: Public Library of Science
Author(s): Anna Lena Jung, Cornelia Stoiber, Christina E. Herkt, Christine Schulz, Wilhelm Bertrams, Bernd Schmeck, Zhao-Qing Luo.
The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.
Bacteria have developed numerous strategies to deliver virulence factors into their eukaryotic host cells. In close proximity, the transfer of virulence factors can take place by direct translocation into the host cytosol. Distant cells can be reached by the secretion of soluble proteases, lipases or toxins to the extracellular environment . Additionally, Gram-negative bacteria developed the strategy of outer membrane vesicle (OMV) formation. OMVs are small, spheroid membrane vesicles of 10–300 nm in diameter, secreted during all phases of growth as well as in a variety of growth environments (liquid culture, solid culture, biofilms) [2, 3]. They transport diverse virulence factors, including proteins, adhesins, toxins and enzymes as well as non-protein antigens such as lipopolysaccharide (LPS), which is present on the outer leaflet of the OMV membrane . They serve as a means of communication among bacteria, but can also be recognized and taken up by eukaryotic cells . OMVs may influence the course of infection and the host immune response by presenting pathogen-associated molecular patters (PAMPs) and antigens to their respective host receptors . For example OMVs derived from Clostridium perfringens induce cytokine secretion in macrophages, Borellia burgdorferi OMVs activate B cells, and vesicles secreted by Helicobacter pylori act on gastric epithelial cells [6–8]. In addition, OMVs transport active virulence factors of Gram-negative bacteria which gain access to the extracellular environment and can act over long distances, since the vesicular membrane protects the luminal cargo from extracellular host proteases and facilitates penetration into tissue [9–11]. OMVs have been found not only in close proximity to the site of bacterial colonization, but also in body fluids and distant organs . Furthermore, they can mediate bacterial binding and invasion into host cells and cause cytotoxicity .
OMVs are potent pro-inflammatory stimulators of different cell types, carrying endotoxin and additional bacterial antigens . OMVs from Acinetobacter baumannii induce cytokine secretion in epithelial cells , Clostridium perfringens OMVs stimulate the murine macrophage cell line RAW264.7 to produce G-CSF, TNF-α, and IL-6 . L. pneumophila also produces OMVs that can be taken up by macrophages and furthermore induce tissue damage in human lung tissue explants . Until now, the majority of OMV studies focused on direct OMV-mediated changes in their target cell/tissue. In this study, we investigated the impact of OMV pre-treatment on a following encounter of macrophages with L. pneumophila. THP-1 cells responded upwards of 0.01 μg/mL OMV with IL-8 secretion in a time- and dose-dependent manner. Additionally, the cells secreted IL-1β, IL-6, IL-10, and TNF-α dose-dependently. In contrast to THP-1 cells, stimulation of an alveolar type II epithelial cell line with L. pneumophila OMVs was reported to require much higher doses to induce pro-inflammatory activation (50 μg in contrast to 0.01 μg/mL) . Human primary macrophages induce TNF-α release when exposed to 0.3 μg/mL L. pneumophila OMVs . Here, we observed a response intensity to OMVs similar to what others observed when administering whole L. pneumophila . Based on LPS-measurements, we assume that the experimentally used OMV doses resemble those that occur under infection conditions.