Immune control of Legionella pneumophila lung infection
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2018
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Melbourne Authors
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Doherty Institute
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PhD thesis
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Open Access
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© 2018 Dr. Chao Yang
Abstract
Legionnaire’s Disease is a life-threatening lung infection caused by the bacterium Legionella pneumophila. Recently, studies have revealed that cytokines such as IL-1α, TNF and IFNγ secreted by innate immune cells facilitate the restriction of intracellular replication of L. pneumophila. However, the exact mechanisms by which innate immune cells and cytokines contribute to the control of L. pneumophila lung infection are still unclear. In this study, we demonstrated that the bactericidal activity of monocyte-derived cells (MCs) was IFNγ-dependent while alveolar macrophages (AMs) did not respond to IFNγ during L. pneumophila infection. To understand IFNγ-mediated killing machinery in MCs, we sorted MCs from the lungs of wild type C57BL/6 (WT) mice and Ifng-/- mice 3 days after L. pneumophila infection and performed RNA-sequencing. We found that a set of genes belonging to the Interferon-stimulated GTPase (ISGase) superfamily that functions in cell-autonomous immunity was highly expressed in WT MCs compared to Ifng-/- MCs. Using Real-time quantitative reverse transcription-PCR (qRT-PCR), we confirmed that expression of these genes in MCs was induced by IFNγ during L. pneumophila infection. Further, we observed that Guanylate-binding protein 1 (Gbp1) was predominantly expressed in MCs compared to AMs during L. pneumophila lung infection. These findings suggested that Gbp1 might have an important role in the clearance of L. pneumophila by MCs in vivo.
Mechanistically, we observed that IFNγ could disrupt the translocation of bacterial Dot/Icm effector proteins by L. pneumophila in macrophages. Dot/Icm effectors are required for biogenesis of the intracellular replicative Legnionella-containing vacuole (LCV). Interestingly, MCs were refractory to the translocation of Dot/Icm effectors even in the absence of IFNγ in vivo. This suggested that L. pneumophila could not establish the LCV and replicate in MCs. One possible explanation for increased bacterial numbers in Ifng-/- MCs might be lack of induction of the killing and degradation machinery mediated by ISGases. We observed that Gbp1, Gbp2 and Irga6 promoted LCV lysis and lysosomal clearance of intracellular L. pneumophila in the presence of IFNγ.
In addition, we observed that the IFNγ receptor 1 (IFNGR1) was dramatically downregulated in AMs but not MCs during L. pneumophila infection in vivo, which might explain why AMs showed little response to IFNγ in the control of L. pneumophila intracellular replication in vivo. Further studies in vivo and in vitro, suggested that the downregulation of IFNGR1 in macrophages was dependent on both MyD88 and Trif signalling and NF-κB activation. Constitutive expression of IFNGR1 in AMs significantly improved the capacity of AMs to restrict intracellular replication of L. pneumophila in the presence of IFNγ in vivo.
Overall this study demonstrated that MCs are critical innate immune cells that respond to IFNγ stimulation to control intracellular replication of L. pneumophila in vivo and that ISGases might contribute to the lysosome mediated clearance of intracellular L. pneumophila in MCs. In addition, the downregulation of IFNGR1 in AMs dampened their response to IFNγ stimulation, which may partially explain the susceptibility of AMs to L. pneumophila infection in vivo. Hence, this study has uncovered potential mechanisms underpinning IFNγ-mediated cell-autonomous immunity against L. pneumophila. This permits future study into understanding the detailed molecular and cellular mechanisms that contribute to the control of L. pneumophila intracellular replication.