17th International Congress on Toxoplasmosis

Inducible nitric oxide synthase (iNOS) is necessary for GBP-mediated T. gondii restriction in murine macrophages via vacuole nitration and intravacuolar network collapse

Innate Immunity and Immune Evasion

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, the precise mechanism of T. gondii restriction is not known. Using automated spatially targeted optical micro proteomics we determined that inducible nitric oxide synthetase (iNOS) was highly enriched at GBP-positive PV in murine macrophages. iNOS expression in myeloid cells was necessary to control T. gondii growth in vivo and in vitro. iNOS activity was dispensable for GBP2 recruitment and PV membrane ruffling, however, these vacuoles contained dividing parasites. T. gondii restriction by iNOS required nitric oxide, nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with the chromosome 3 GBPs to target distinct biology of the PV that are necessary for optimal parasite clearance in murine myeloid cells.

Oral presentation

Caspase-8-mediated cell death of CD8+ T cells is required for control of Toxoplasma gondii in the brain

Immunology of Acute & Chronic Infection

Host cell death represents a major mechanism of limiting intracellular pathogens. Toxoplasma gondii is an intracellular protozoan parasite that establishes a chronic infection of the brain and other immune-privileged tissues. We sought to determine whether cell death is a mediator of parasite control in the brain. Numerous types of programmed cell death have been defined and are regulated by distinct molecular pathways. We focused on the role of caspase-8, which is associated with the extrinsic pathway of apoptosis. The deletion of Casp8 alone in mice results in embryonic lethality associated with widespread necrosis. The additional deletion of Ripk3 results in mice that are viable. We began by infecting mice that lacked Casp8 and Ripk3, Ripk3 alone, and WT C57BL/6 controls with 10 cysts of Me49 parasites. We found that Casp8-/-Ripk3-/- mice were susceptible to T. gondii infection and survived approximately 50 days with overwhelming parasite burdens in the brain. We found that mice lacking Ripk3 and the ability to trigger necroptosis were resistant to infection and had parasite burdens comparable to WT mice. Intriguingly, Casp8-/-Ripk3-/- mice had equivalent or stronger immune responses to T. gondii in the brain with the recruitment of IFN-γ-producing T cells and iNOS-producing macrophages. These results suggested that specific cell types in the brain may be particularly susceptible to T. gondii infection in the absence of Casp8. To this end, we infected WT and Casp8-/-Ripk3-/- mice on a cre-reporter background (flox-stop-flox ZsGreen) with a cre-secreting parasite (Pru-cre-mCherry). As previously reported, we found that primarily neurons are infected in wildtype mice. In Casp8-/-Ripk3-/- mice we found that many more neurons, astrocytes, and surprisingly CD8+ T cells had interacted with parasite. Infection of CD8+ T cells by T. gondii in WT mice has been previously reported by several groups. Given that CD8+ T cells are highly migratory in the brain, cell death may be required to restrict pathogen spread throughout the brain. To test the role of Casp8 specifically in CD8+ T cells, we generated CD8a-cre x Casp8fl/fl x Ripk3-/- x Ai6 mice and Casp8fl/fl x Ripk3-/- x Ai6 controls. We find that mice with Casp8-deficient CD8+ T cells have elevated levels of parasite and CD8+ T cells in the brain at six weeks post-infection. These data suggest that cell death is an additional mechanism by which CD8+ T cells restrict parasite in the brain.

CD8 T memory subset response in encephalitis model of Toxoplasma gondii infection.

CD8 memory subset response in encephalitis model of Toxoplasma gondii infection: Magali Moretto, Keer Chen, Christina Cox, Jie Chen, and Imtiaz A Khan Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, Washington, DC. CD8 T cells play an important role in protection against Toxoplasma gondii, especially during the chronic phase of infection. Due to the ability of these cells to exhibit polyfunctional response including IFNg production and cytotoxic effect, the parasitic replication is kept under control. However, in an encephalitis model of infection, it has been demonstrated that memory CD8 T cells become exhausted due to an increased expression of inhibitory receptors like PD-1 that leads to reactivation of infection and host mortality. While studies performed with chronic viral infections and tumor models have reported that CD8 T cell memory is comprised of a heterogeneous population, in-depth information related to this response against T. gondii infection is scarce. We observed that after T. gondii infection, memory CD8 T cells do not fit into classical effector and memory phenotype but can be distributed into four subsets based on KLRG1 and CD62L expression. KLRG1+CD62L- CD8 T cell subset (pop1) displays the classical attribute of effector T cells while the 3 remaining subsets KLRG1-CD62L– (pop2), KLRG1-CD62L+ (pop3) and KLRG1+CD62L+ (pop4) exhibit higher CD127 expression, a characteristic of memory population signature. Interestingly, pop3 and 4 exhibit increased levels of multiple inhibitory receptors. Adoptive transfer studies suggest that the effector population (pop1), needed for controlling infection, is derived from pop3 that displays stem-like characteristics, with pop4 most likely acting as the intermediate population. These observations suggest that pop3 needs to be efficiently maintained to generate highly polyfunctional pop1 effectors to keep the chronic infection under control.

Professor

Chronic IL-1-induced DNA double-strand break response in hippocampal neurons drives cognitive deficits upon latent T. gondii infection

Chronic inflammation characterized by increased cytokine levels, such as interleukin-1 (IL-1), accompanies many neurological diseases but little is known about IL-1 contribution to cognitive impairment and its interplay with epigenetic processes, including the DNA double-strand break (DSB) response. Here, we demonstrate that H2A.X-dependent DSB signaling in hippocampal neurons drives cognitive deficits upon chronically elevated IL-1. Mice persistently and latently infected with Toxoplasma gondii display impaired spatial memory consolidation along with elevated IL-1β in the hippocampus. We find that neuronal IL-1 signaling in excitatory neurons is required for the spatial memory deficits caused by T. gondii infection and by chronic systemic infusion of IL-1β. In both cases, the deficit in spatial memory was prevented by the abrogation of neuronal H2A.X-dependent signaling. Our results highlight the instrumental role of cytokine-induced DSB-dependent signaling in spatial memory defects. This novel pathological mechanism in inflammation control of neuronal function may extend to several neurological diseases.

Dense Granule Protein 3 of Toxoplasma gondii Plays a Crucial Role in the Capability of the Tissue Cysts of the Parasite to Persist under the Presence of Anti-Cyst CD8+ T Cells during the Chronic Stage of Infection

Toxoplasma gondii establishes chronic infection by forming tissue cysts, and this chronic infection is one of the most common parasitic infections in humans. Our recent studies revealed that whereas CD8+ T cells of genetically resistant BALB/c mice have the capability to remove the tissue cysts of the parasite through their perforin-mediated activities, small portions of the cysts are able to persist under the presence of the anti-cyst CD8+ T cells. It is currently unknown how those small portions of the cysts resist or escape the T cell immunity and persist in the hosts. In the present study, we discovered that the cysts, which persisted under the presence of the perforin-mediated CD8+ T cell immunity, have significantly greater mRNA levels for four dense granule proteins, GRA1, GRA2, GRA3, and GRA7, and one rhoptry protein, ROP35, than the total population of the cysts present in the absence of the T cells. In addition, increased levels of mRNA for GRA1, GRA3, and ROP35 in the cysts significantly correlated with their successful persistence through the condition in which greater degrees of reduction of the cyst burden occurred through anti-cyst CD8+ T cells. In addition, GRA3-deficient T. gondii displayed significantly enhanced elimination of the cysts by anti-cyst CD8+ T cells when compared to the wild-type parasite. These results indicate that GRA3 is a key molecule that mediates the capability of T. gondii cysts to persist by resisting or evading the anti-cyst activity of CD8+ T cells during the later stage of infection.

NLRP3 inflammasome assembly and activation are mediated by dynein-dependent microtubule transport during Toxoplasma gondii infection of human immune cells

IL-1β is a potent pro-inflammatory cytokine that plays a key role in innate immunity against infection. The NLRP3 inflammasome is a multi-protein complex that mediates the processing of pro-IL-1β into mature bioactive IL-1β during T. gondii infection of human peripheral blood monocytes. However, the cellular mechanisms that facilitate the assembly of NLRP3 inflammasome components into a complex in T. gondii-infected cells remain unknown. We utilized super-resolution imaging to visualize the transport of inflammasome components during T. gondii infection of human monocytic cells. NLRP3 co-localized with host cell microtubules and the microtubule-organizing center (MTOC), specifically in infected cells, and proximity-ligation assays (PLA) revealed close interaction between NLRP3 and α-tubulin during infection. Treatment of cells with an inhibitor of cytoplasmic dynein motor proteins to prevent dynein-dependent microtubule transport resulted in reduced NLRP3 interaction with α-tubulin, decreased caspase-1 cleavage, and reduced IL-1β processing and release, suggesting a key role for dynein-mediated transport of NLRP3 on microtubules for assembly and activation of the NLRP3 inflammasome. After inflammasome-mediated cleavage, bioactive IL-1β was released from T. gondii-infected human monocytes in a gasdermin D- and E-independent manner and in the absence of pore formation. To visualize and track IL-1β processing and release during infection, we expressed an IL-1β fluorescence resonance energy transfer (FRET) construct in human monocytic cells, in which mTurquoise and mNeonGreen were fused to the N- and C-termini of pro-IL-1β, respectively. Intact pro-IL-1β exhibited FRET signal, whereas cleavage of pro-IL-1β was detected through the loss of FRET. These studies revealed that T. gondii infection increased processing of IL-1β, which localized proximal to the plasma membrane and in plasma membrane-coated vesicles and protrusions. Collectively, these findings expand our understanding of the mechanisms of NLRP3 inflammasome assembly and IL-1β processing and release during T. gondii infection of human immune cells.

Session IV: Immunology

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