Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages

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.Author Summary: Of all intracellular pathogens, Legionella pneumophila show the highest genetic adaptation to the host. They are important human pathogens causing mainly pneumonia, but are also highly successful pathogens of water amoeba. To manipulate host cells for optimal intracellular replication, they express several hundred specific virulence factors, which they apply to their host cells by two different secretion systems. Another intriguing avenue that leads to the transfer of virulence factors to host cells—even over distance—is the transport via outer membrane vesicles (OMV) that are released by Legionella. We report here that after exposure to these vesicles, macrophages become permissive hosts for bacterial replication incapable of efficient pathogen defense. In addition, markers of inflammation that are induced by OMV pre-treatment do not further increase upon subsequent bacterial infection. This goes along with improved survival of infected macrophages despite the increased intracellular bacterial growth. We could attribute this effect to TLR- and NF-κB-dependent mechanisms of microRNA-146a induction and prolonged IRAK-1 depletion.