Cellular and molecular defence mechanisms against Legionella infection

Abstract

The two most prevalent causes of Legionnaires’ disease are <em>L. pneumophila</em> and <em>L. longbeachae</em>. The rising incidence of the Legionnaires’ disease over the past two decades together with the increasing prevalence of <em>L. longbeachae</em> in the Northern hemisphere highlights the necessity to gain a more detailed insight into the <em>L. longbeachae</em>-induced immune response. The aim of this thesis was to investigate the cellular and molecular mechanisms required for protection, focussing on immune cell responses and intracellular host-bacterial interactions. <br /> By combining a murine mouse model, a novel fluorescent reporter to track <em>L. longbeachae</em>, and cell depletion experiments, I uncovered the differential contribution of tissue-resident alveolar macrophages (AM) and infiltrating neutrophils to the defence against <em>L. longbeachae</em>. Early during infection, AM contained most of the bacteria. AM numbers sharply decreased during infection, which was accompanied by a large influx of neutrophils that also internalized bacteria. Comparative analysis of bacterial viability revealed that neutrophils were more efficient at killing and clearing of <em>L. longbeachae</em> than AM. In contrast, the results presented here indicated that lung-resident AM promoted infection, most likely by serving as a replicative niche. <br /> Defence against <em>L. pneumophila</em> is known to require IFN-gamma but not IL-18, a strong IFN-gamma inducer. However, Il18r1^–/– mice were less able to clear <em>L. longbeachae</em> and had significantly impaired IFN-gamma levels in the lung. Furthermore, IL-18R signalling was critical for the efficient bacterial killing by neutrophils via production of reactive oxygen species. Previous bone marrow chimera experiments in our laboratory suggested that IL-18R expression in epithelial cells was necessary and sufficient for protection against <em>L. longbeachae</em>. However, genetic in vivo experiments using the Cre-lox-system revealed that IL-18R⁺ NK cells and T cells were central players in the IL-18-dependent anti-<em>L. longbeachae</em> defence via IFN-gamma secretion, whereas IL-18R expression by ciliated bronchiolar epithelial cells did not confer protection. <br /> Finally, our results highlighted key mechanisms by which Legionella subverts host macrophages to form an intracellular endoplasmic reticulum (ER)-like vacuole as its intracellular replicative niche. Establishment of the Legionella-containing vacuole (LCV) by recruitment of ER-derived vesicles induces ER stress. Although the relevance of ER stress in this process is unclear, effector proteins secreted by <em>L. pneumophila</em> are known to inhibit onset of the ER stress response. Using the pharmacological ER stress inducer thapsigargin, we showed that ER stress induces a protective host response promoting the secretion of pro-inflammatory cytokines, limiting intracellular <em>L. pneumophila</em> replication, and improving host survival. Mechanistically, ER stress induced a novel non-canonical activation of the transcription factor STAT1 via the IRE1 kinase driving transcription of the IFN-gamma-induced chemokine CXCL10. These results highlighted a potential role of the host ER stress response in the initiation of a protective cellular immune response towards <em>L. pneumophila</em>.