Macrophages are professional phagocytes that efficiently clear microbes, dying cells, and debris. Nonetheless, some pathogenic bacteria and parasites can subvert the macrophage phagosome into a vacuolar replicative niche. Exogenous macrophage activation by the cytokine interferon gamma (IFNγ) tips the equilibrium toward pathogen restriction, host survival, and subsequent adaptive immune responses. The relevance of IFNγ-induced immunity to human health has been demonstrated in patients with genetic defects in IFNγ signaling, who are profoundly susceptible to vacuolar pathogens such as Mycobacterium tuberculosis. Still, much remains to be discovered about IFNγ effector functions, and about their co-regulation by signaling downstream of the many innate immune sensors in macrophages. First, we asked whether IFNγ-induced vesicle trafficking mechanisms affect the maturation of phagosomes containing the bacterium Legionella pneumophila, the causative agent of Legionnaire's disease. We used functional genetic screening to discover candidate genes involved. From 380 genes in a curated vesicle trafficking-related set, 15 were selected as candidate IFNγ pathway members by RNAi screening in cell line and primary mouse macrophages. Functional validation of top candidates was inconclusive, but revealed potential roles for membrane tetraspanins and the AP3 complex in IFNγ-induced microbial restriction. Our second goal was to determine whether innate immune sensing affects IFNγ-induced bacterial restriction. Using macrophages from mice deficient in key elements of innate immune sensing pathways, we discovered that the antiviral transcription factor IRF3, which functions downstream of many nucleic acid sensing pathways, suppresses IFNγ-induced restriction of L. pneumophila and the protozoan parasite Trypanosoma cruzi. While activated IRF3 localizes to the nuclei in resting macrophages infected with L. pneumophila, it is mostly excluded from nuclei in macrophages activated with IFNγ prior to infection. This suggests a cascade of suppression in which IFNγ responses inhibit IRF3 activation, but residual IRF3 activity antagonizes IFNγ effectors. IRF3-mediated inhibition of IFNγ-inducible nitric oxide synthase was partially, but incompletely responsible for the phenotype observed; further candidate effectors were identified by gene expression profiling. We speculate that antagonism between IFNγ and IRF3-mediated mechanisms may facilitate a balance of vacuolar pathogen immunity with viral defense, or with protection of tissue damage by nitric oxide and other IFNγ-dependent responses.

Listeria monocytogenes (LM) and Salmonella enterica serovar Typhimurium (ST) are intracellular bacterial pathogens that cause invasive disease in immune-altered individuals, including the immunocompromised and pregnant women. The mechanisms that modulate innate immunity to intracellular infection, particularly during pregnancy, are not well-understood. Functional selectin ligands play critical roles in leukocyte recruitment during inflammation. Increased control of LM infection in functional selectin ligand-deficient (FtDKO) mice is associated with increased levels of circulating innate immune cells, despite defective leukocyte migration compared to WT mice. Adoptive transfer of WT and FtDKO bone marrow (BM) cells to irradiated WT and FtDKO recipients demonstrates that BM reconstitution and the increased neutrophil phenotype of FtDKO mice is independent of functional selectin ligand expression within the host environment. Thus, functional selectin ligand deficiency enhances inherent innate immune resistance to intracellular infection. We then examined the impact of pregnancy-associated immunological changes on maternal susceptibility to intracellular infections. ST infection in pregnant mice results in profound systemic infection, increased fetal loss and enhanced serum and placental expression of pro-inflammatory cytokines. Pregnant mice showed decreased ratios of pro-inflammatory Th17 cells relative to anti-inflammatory regulatory T cells (Tregs) when compared to non-pregnant mice during infection. Functional inactivation of Tregs in vivo restored control of infection and normal Th17-to-Treg ratios, and reduced fetal loss. These indicate that modulation of Th17 and Treg responses impacts maternal and fetal protection from ST infection. Lastly, we examined the roles of type I interferons (IFNs) in modulating innate immunity to intracellular infections during pregnancy. Type I IFN receptor deficiency (IFNAR-/-) enhances immunity to LM and ST in the non-pregnant state by limiting pathogen-induced leukocyte death. We show that pregnant IFNAR-/- mice infected with LM retain increased protection from infection relative to WT controls. In contrast, protection conferred by IFNAR deficiency against ST infection in the non-pregnant state is abrogated during pregnancy. Distinctive maternal responses to LM and ST are associated with differential regulation of leukocyte distribution and cytokine expression in maternal systemic and/or placental compartments. Taken together, modulation of key mechanisms involved in leukocyte recruitment, immune-regulation and cytokine signaling impact host susceptibility to intracellular infections.

Staphylococcus aureus est capable d’être internalisé par les cellules eucaryotes, notamment au cours des infections osseuses, puis d’induire la mort de la cellule. Deux principaux mécanismes ont été décrits comme associés à la cytotoxicité de S. aureus : l’échappement phagosomal et le détournement de l’autophagie. Nous avons exploré ces deux mécanismes et le rôle de plusieurs toxines staphylococciques majeures (alpha-toxine (Hla), phenol-soluble modulins (PSM), bêta-toxine (Hlb)) dans la mort cellulaire, grâce à un modèle in vitro d’infection intracellulaire et des mutants isogéniques. L’échappement phagosomal nécessitant l’expression d’Hlb, inactivée par l’insertion d’un phage chez la majorité des souches cliniques, nous avons testé l’hypothèse d’une excision de ce phage induite par le stress intracellulaire. Nous avons montré que la restauration de l’expression d’Hlb par excision du phage existe de manière spontanée mais n’est pas induite par le passage intracellulaire et n’est pas associée à une hausse de la cytotoxicité. Dans un second temps, nous avons exploré la cytotoxicité d’une souche de S. aureus hypervirulente et avons montré qu’elle est associée à un détournement de l’autophagie via l’inhibition de la fusion autophagosome-lysosome, ceci étant associé à l’expressions des PSMa. Au contraire, Hla, les PSMß et la d-toxine ne semblent jouer aucun rôle intracellulaire chez les S. aureus hypervirulents. Enfin, nous avons mis en évidence que, dans un autre fond génétique de S. aureus, associé au pied diabétique, la cytotoxicité est principalement liée à la capacité de multiplication intracellulaire de la souche, modulée par la présence d’un phage / Staphylococcus aureus is able to invade eukaryotic cells, in particular during bone infections, and induce cell death. Two mechanisms have been described as associated with S. aureus cytotoxicity: phagosomal escape and autophagy subversion. We investigated these two mechanisms and the respective roles of several staphylococcal toxins, alpha-toxin (Hla), phenol-soluble modulins (PSMs) and beta-toxin (Hlb), in cell death, using an in vitro intracellular infection model and isogenic mutants of S. aureus. Because Hlb is required for phagosomal escape but this toxin is inactivated by a prophage inserted into the hlb gene in most of clinical isolates of S. aureus, we tested the hypothesis of an excision of this phage induced by intracellular stress. We showed that restoration of Hlb expression due to the excision of hlb-converting phage exists spontaneously but is not induced by intracellular environment and does not increase the cytotoxicity of the strain. In a second part, we explored the cytotoxicity of an hypervirulent strain of S. aureus and demonstrated that it is associated with a subversion of host cell autophagy via an inhibition of autophagosome-lysosome fusion, in a PSMa-dependent manner. Conversely, Hla, PSMß and d-toxin appear to have no intracellular role in the cytotoxicity of hypervirulent S. aureus strains. Finally, we showed that, in another genetic background of S. aureus associated with diabetic foot ulcer, cytotoxicity was linked to the ability of intracellular replication of the strain, which was modulated by the presence of a phage