Immunological checkpoints in the control of murine Salmonella enterica infection: IFN-γ pathways and early dendritic cell death

Abstract

Salmonella enterica is a Gram-negative intracellular pathogen, which can cause typhoid fever and non-typhoidal salmonellosis. Every year ~22 million cases and ~200,000 deaths are reported for typhoid fever and ~93 million cases and ~155,000 deaths for non-typhoidal salmonellosis. Innate immunity provides the very early protection against Salmonella, a better understanding of which may lead to a progress in treatment and prevention to Salmonella infection. Dendritic cells (DC) are one of the first cells to sense Salmonella in vivo, and play an important role in initiating a cascade of innate immune control, including phagocytising bacteria and the activation of inflammasomes, which further induces cell death and the production of pro- inflammatory cytokines, such as IFN-γ. In addition, dendritic cells are potent antigen presenting cells (APC) that induce the development of protective adaptive immunity against Salmonella. However, it has been reported that Salmonella possesses various mechanisms, including regulating phagolysosomal fusion and delaying vacuole acidification, and down-regulating flagellin expression to prevent antigen presentation, highlighting the dynamic and complex nature of DC-Salmonella interactions. In recent years, the critical role of DCs in immunity against Salmonella has gained increased attention, however the cellular and molecular mechanisms of the DC-Salmonella interactions are not fully understood.

The first aim of this study was to study the survival and death in infected DCs during Salmonella infection, utilising murine bone marrow-derived DCs (BMDCs), which are sensitive to Salmonella-induced cell death within hours of infection. It is found that several virulence factors such as lipopolysaccharide (LPS), Type III secretion system 1 (SPI-1) and flagellin contribute to, and in combination maximise, death in BMDCs. Intriguingly, BMDCs that were not directly infected with Salmonella were killed upon infection of neighbouring cells in culture. An apparently similar ‘bystander’ cell death was induced by co-culturing with filtered supernatant from infected BMDCs, suggesting a role for contact-independent mechanisms. Infected BMDCs released several cytokines, including IL-6, MCP-1 and TNF-α. However, blockade of intracellular protein transport and secretion of cytokines by monesin did not alter Salmonella-induced cell death in uninfected bystanders, suggesting that the bystander effect is not dependent on mediators released from infected BMDCs. BMDCs from mice with gene knockouts in key pathways that are involved in DC immune responses against Salmonella were also tested, and decreased death was observed in ICE-/- BMDCs, suggesting that caspase-1/caspase-11-mediated pyroptosis could be responsible for direct as well as bystander BMDC death.

The second aim was to determine the contribution of IFN-γ and the IFN-γ induction pathways in Salmonella infection. Previous studies in our lab have shown that flagellin-induced NLRC4 inflammasome activation in splenic DCs triggers non- cognate memory CD8+ T cells to produce IFN-γ, a critical mediator of innate immunity against Salmonella. It was shown in the present study that deletion of individual components of the NLRC4 inflammasome pathway, e.g. caspase-1 or IL- 18, can lead to a moderate reduction of IFN-γ production, but the impact on the control of Salmonella in infected mice is minimal, suggesting that NLRC4 pathway is not the only source of IFN-γ and that low level of IFN-γ may be sufficient for full protection against Salmonella. In the studies presented here, it was shown that LPS- induced activation of the TLR4 pathway is also an important source of IFN-γ and that mice deficient in components of TLR4 pathway has poor early control of bacterial load during S. Typhimurium BRD509 infection. Interestingly, deficiency in the TLR4 pathway led to an increase rather than reduction of IFN-γ, suggesting that IFN-γ is regulated by different pathways and that TLR4 pathway may be involved in other immune responses that are important for early control of Salmonella.