Meningeal lymphatic drainage promotes T cell responses against Toxoplasma gondii but is dispensable for parasite control in the brain.

The discovery of meningeal lymphatic vessels that drain the CNS has prompted new insights into how immune responses develop in the brain. In this study, we examined how T cell responses against CNS-derived antigen develop in the context of infection. We found that meningeal lymphatic drainage promotes CD4+ and CD8+ T cell responses against the neurotropic parasite Toxoplasma gondii in mice, and we observed changes in the dendritic cell compartment of the dural meninges that may support this process. Indeed, we found that mice chronically, but not acutely, infected with T. gondii exhibited a significant expansion and activation of type 1 and type 2 conventional dendritic cells (cDC) in the dural meninges. cDC1s and cDC2s were both capable of sampling cerebrospinal fluid (CSF)-derived protein and were found to harbor processed CSF-derived protein in the draining deep cervical lymph nodes. Disrupting meningeal lymphatic drainage via ligation surgery led to a reduction in CD103+ cDC1 and cDC2 number in the deep cervical lymph nodes and caused an impairment in cDC1 and cDC2 maturation. Concomitantly, lymphatic vessel ligation impaired CD4+ and CD8+ T cell activation, proliferation, and IFN-γ production at this site. Surprisingly, however, parasite-specific T cell responses in the brain remained intact following ligation, which may be due to concurrent activation of T cells at non-CNS-draining sites during chronic infection. Collectively, our work reveals that CNS lymphatic drainage supports the development of peripheral T cell responses against T. gondii but remains dispensable for immune protection of the brain.

Microglial STAT1-sufficiency is required for resistance to toxoplasmic encephalitis.

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that establishes a life-long chronic infection largely restricted to the central nervous system (CNS). Constant immune pressure, notably IFN-γ-STAT1 signaling, is required for preventing fatal pathology during T. gondii infection. Here, we report that abrogation of STAT1 signaling in microglia, the resident immune cells of the CNS, is sufficient to induce a loss of parasite control in the CNS and susceptibility to toxoplasmic encephalitis during the early stages of chronic infection. Using a microglia-specific genetic labeling and targeting system that discriminates microglia from blood-derived myeloid cells that infiltrate the brain during infection, we find that, contrary to previous in vitro reports, microglia do not express inducible nitric-oxide synthase (iNOS) during T. gondii infection in vivo. Instead, transcriptomic analyses of microglia reveal that STAT1 regulates both (i) a transcriptional shift from homeostatic to "disease-associated microglia" (DAM) phenotype conserved across several neuroinflammatory models, including T. gondii infection, and (ii) the expression of anti-parasitic cytosolic molecules that are required for eliminating T. gondii in a cell-intrinsic manner. Further, genetic deletion of Stat1 from microglia during T. gondii challenge leads to fatal pathology despite largely equivalent or enhanced immune effector functions displayed by brain-infiltrating immune populations. Finally, we show that microglial STAT1-deficiency results in the overrepresentation of the highly replicative, lytic tachyzoite form of T. gondii, relative to its quiescent, semi-dormant bradyzoite form typical of chronic CNS infection. Our data suggest an overall protective role of CNS-resident microglia against T. gondii infection, illuminating (i) general mechanisms of CNS-specific immunity to infection (ii) and a clear role for IFN-STAT1 signaling in regulating a microglial activation phenotype observed across diverse neuroinflammatory disease states.

Astrocytes promote a protective immune response to brain Toxoplasma gondii infection via IL-33-ST2 signaling.

It is of great interest to understand how invading pathogens are sensed within the brain, a tissue with unique challenges to mounting an immune response. The eukaryotic parasite Toxoplasma gondii colonizes the brain of its hosts, and initiates robust immune cell recruitment, but little is known about pattern recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but, like many other pattern recognition pathways, IL-33 can signal peripherally, and the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released locally into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment and/or maintenance of blood-derived anti-parasitic immune cells, including proliferating, IFN-γ-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33 receptor-deficient astrocytes fail to mount an adequate adaptive immune response in the CNS to control parasite burden-demonstrating, genetically, that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released locally, and sensed locally, to engage the peripheral immune system in controlling a pathogen.

Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection.

Microglia, resident immune cells of the CNS, are thought to defend against infections. Toxoplasma gondii is an opportunistic infection that can cause severe neurological disease. Here we report that during T. gondii infection a strong NF-κB and inflammatory cytokine transcriptional signature is overrepresented in blood-derived macrophages versus microglia. Interestingly, IL-1α is enriched in microglia and IL-1ß in macrophages. We find that mice lacking IL-1R1 or IL-1α, but not IL-1ß, have impaired parasite control and immune cell infiltration within the brain. Further, we show that microglia, not peripheral myeloid cells, release IL-1α ex vivo. Finally, we show that ex vivo IL-1α release is gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in brain inflammation and parasite control. These results demonstrate that microglia and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii infection, microglia can release the alarmin IL-1α, promoting neuroinflammation and parasite control.

IL-10 and ICOS Differentially Regulate T Cell Responses in the Brain during Chronic Toxoplasma gondii Infection.

Control of chronic CNS infection with the parasite Toxoplasma gondii requires ongoing T cell responses in the brain. Immunosuppressive cytokines are also important for preventing lethal immunopathology during chronic infection. To explore the loss of suppressive cytokines exclusively during the chronic phase of infection, we blocked IL-10R in chronically infected mice. Consistent with previous reports, IL-10R blockade led to severe, fatal tissue destruction associated with widespread changes in the inflammatory response, including increased APC activation, expansion of CD4+ T cells, and neutrophil recruitment to the brain. We then sought to identify regulatory mechanisms contributing to IL-10 production, focusing on ICOS, a molecule implicated in IL-10 production. Unexpectedly, ICOS ligand (ICOSL) blockade led to a local expansion of effector T cells in the brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL blockade led to changes in T cells associated with their proliferation and survival. We observed increased expression of IL-2-associated signaling molecules CD25, STAT5 phosphorylation, Ki67, and Bcl-2 in T cells in the brain, along with decreased apoptosis. Interestingly, increases in CD25 and Bcl-2 were not observed following IL-10R blockade. Also, unlike IL-10R blockade, ICOSL blockade led to an expansion of both CD8+ and CD4+ T cells in the brain, with no expansion of peripheral T cells or neutrophil recruitment to the brain and no severe tissue destruction. Overall, these results suggest that IL-10 and ICOS differentially regulate T cell responses in the brain during chronic T. gondii infection.