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Title: The role of IFN-γ in cell-autonomous immune responses against Salmonella Typhimurium and Toxoplasma gondii
Author: Rolland, Solene
ISNI:       0000 0004 9359 9728
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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The research presented in this thesis investigates the function of interferon gamma in vacuoles, namely Salmonella enterica serovar Typhimurium and Toxoplasma gondii. IFN-γ is a cytokine that functions in both the innate and the adaptive immune responses. IFN-γ is mainly secreted by T cells and natural killer (NK) cells and activates macrophages, promotes antigen presentation and enhances antiviral and antibacterial immunity. In the case of bacterial pathogens, such as S.Typhimurium, priming with IFN-γ leads to an inflammatory but controlled cell death mechanism known as pyroptosis, following bacterial invasion of the cytosol or experimental lipopolysaccharide (LPS) transfection. LPS is a major component of the outer membrane of Gram-negative bacteria and is sensed in the cytosol by human Caspase-4. The research described in Chapter 2 used a genome-wide CRISPR-Cas9 screen as an unbiased approach to identify new IFN-γ-induced genes that play a role in the cell death pathway. This viability screen enriched for cells resistant to LPS-mediated cell death after IFN-γ priming. Their sequencing generated a list of potential candidate genes including expected genes such as IFN-γ receptors (Ifngr) and novel genes such as Nckap1, Fip1l1, Srsf10, Safb. However, none of these additional candidates were validated because their deficiency did not provide significant resistance to the cell death phenotype. Overall, these results suggest that, in addition to the Caspase-4-Gasdermin-D axis, the role of IFN-γ-induced genes in the cell death pathway triggered by cytosolic LPS remains to be investigated. This thesis also examined the role of IFN-γ in response to infection by the eukaryotic parasite Toxoplasma gondii, an apicomplexan that actively invades host cells and resides within a unique parasitophorous vacuole. The work presented in Chapter 3 showed that with type II T.gondii (PRU strain), IFN-γ inhibits replication in an autophagy-dependent manner. This strain is coated by ubiquitin and by the individual subunits (HOIP, HOIL-1, and Sharpin) of the linear-ubiquitin assembly complex (LUBAC). Autophagy cargo receptors, such as NDP52, p62, and Optineurin, are also recruited to the type II, intracellular parasites. Although all these recruitments were IFN-γ-dependent, the role of LUBAC and the mechanisms for its recruitment remain unknown. This research further showed that PRU changes cytokine expression levels of infected cells: IL-1α, IL-6, and CCL5 expressions are induced upon infection and potentially in a LUBAC-dependent fashion. Overall, the work presented in this thesis provides new insights into the role of IFN-γ in antibacterial and antiparasitic cell-autonomous innate immunity. Further experiments could validate other candidate genes from the screen or investigate the role of LUBAC in Toxoplasmosis.
Supervisor: Randow, Felix Sponsor: MRC-LMB
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: immunity ; cell-autonomous defence ; S.Typhimurium ; T.gondii ; LUBAC ; non-canonical inflammasome ; pyroptosis ; CASP4