Abstract
Cell death is an integral restriction mechanism of intracellular pathogens in a variety of cell types. In the brain, however, death of long-lived cells can have detrimental effects on cognition, behavior, and survival. We have previously reported extensive cell death in the brain during infection with the intracellular protozoan parasite, Toxoplasma gondii. Here we focus on caspase-8, a regulator of extrinsic apoptosis, during T. gondii brain infection to help elucidate the cell-type-specific role of caspase-8 as a mechanism of pathogen control and tissue preservation. During T. gondii infection, we find that Casp8-/-Ripk3-/- mice have an eight-fold increase in brain parasite burden compared to control mice and succumb to the infection within six weeks. Surprisingly, the Casp8-/-Ripk3-/- mice mount a robust Th1 immune response. These results suggest that specific cell types in the brain may be particularly susceptible to T. gondii infection in the absence of Casp8. To identify these vulnerable cells, we infected control and Casp8-/-Ripk3-/- mice on a cre-reporter background with a cre-secreting parasite. As previously reported, primarily neurons report cre activity in C57BL/6 mice while in Casp8-/-Ripk3-/- mice we found neurons, astrocytes, and CD8+ T cells had interacted with the parasite. Additionally, using Multiplexed Error-Robust Fluorescence in Situ Hybridization (MERFISH) spatial transcriptomics we found Casp8 is highly expressed in brain infiltrating CD8+ and CD4+ T cells, infiltrating macrophages, and microglia with a lower degree of expression in endothelial cells, the choroid plexus, and pericytes during chronic T. gondii infection.
To test the role of Casp8 in individual cell types we crossed Casp8fl/flRipk3-/- mice with cre lines targeting neurons, astrocytes, microglia, myeloid cells, and CD8+ T cells. We found that Casp8 deficiency in neurons, astrocytes, microglia, and myeloid cells did not impact the control of infection in the brain. Surprisingly, in CD8aCreCasp8fl/flRipk3-/-Ai6 mice we found elevated levels of parasite in the brain at six- and eight-weeks post-infection despite comparable Th1 immune responses that typically control infection. Additionally, we identified infected CD8+ T cells suggesting a mechanism of parasite survival in the absence of caspase-8. It has been well-characterized that the role of CD8+ T cells in restricting T. gondii is dependent on cytokine production and cytolytic killing of infected cells. Our data suggest that caspase-8-mediated cell death is an additional mechanism by which CD8+ T cells restrict parasite in the brain and mediate resistance to infection.
Multiple signals can activate caspase-8 activity in T cells. In our spatial transcriptomics we found that CD8+ T cells highly express TNFR, Fas, and ZBP1, all activators of caspase-8. TNFR is well known to play a role in immune response to T. gondii infection, primarily through myeloid cell production of nitric oxide synthase. Fas is a well described activator of extrinsic apoptosis. Fas deficiency leads to development of lymphoproliferation and splenomegaly, driven by increases in T cell populations, as is seen in Casp8-deficient mice. To examine the role of Fas, we used Faslpr mice and found an increase in parasite burden at six weeks post-infection and a strong Th1 immune response suggesting that Fas is also critical to restrict parasite expansion specifically in the brain during chronic T. gondii infection.
ZBP1 is an innate sensor of nucleic acids in the Z-conformation and regulates immune responses via cell death pathways and NF-kB signaling. Previous work has demonstrated an essential role for ZBP1 in viral restriction, however the role ZBP1 plays in protection against other pathogens is in early stages of exploration. ZBP1 is highly expressed in brain infiltrating CD8+ T cells during chronic T. gondii infection and can regulate caspase-8 activation. Here we sought to explore the role of ZBP1 during T. gondii infection. During early infection, we found an increase in parasite burden, a decrease in NK cell, neutrophil, and monocyte recruitment, and impaired NK cell cytokine production in the peritoneum of Zbp1-/- mice. Interestingly, during the early adaptive immune response, the immune cell recruitment defects and impaired parasite control were ameliorated. During the brain stage of infection, ZBP1 regulated parasite restriction and numerous inflammatory responses to infection. Our findings establish ZBP1 as a critical regulator of host defense to T. gondii and highlight the importance of ZBP1 in development of inflammation during both the acute and chronic infection.
