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-1β in macrophages. We find that mice lacking IL-1R1 or IL-1α, but not IL-1β, 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.

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.

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.

SUMMARY 21An intact immune response is critical for survival of hosts chronically infected with 22Toxoplasma gondii. We observe clusters of macrophages surrounding replicating parasite in brain

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.

The discovery of meningeal lymphatic vessels that drain the central nervous system (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.

26Microglia, the resident immune cells of the brain parenchyma, are thought to be first-line defenders 27 against CNS infections. We sought to identify specific roles of microglia in the control of the 28 eukaryotic parasite Toxoplasma gondii, an opportunistic infection that can cause severe 29 neurological disease. In order to identify the specific function of microglia in the brain during 30 infection, we sorted microglia and infiltrating myeloid cells from infected microglia reporter mice. 31Using RNA-sequencing, we find strong NF-kB and inflammatory cytokine signatures 32 overrepresented in blood-derived macrophages versus microglia. Interestingly, we also find that 33 IL-1a is enriched in microglia and IL-1b in macrophages, which was also evident at the protein 34 level. We find that mice lacking IL-1R1 or IL-1a, but not IL-1b, have impaired parasite control 35 and immune cell infiltration specifically within the brain. Further, by sorting purified populations 36 from infected brains, we show that microglia, not peripheral myeloid cells, release IL-1a ex vivo. 37Finally, using knockout mice as well as chemical inhibition, we show that ex vivo IL-1a release is 38 gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in 39 immune infiltration into the brain and parasite control. These results demonstrate that microglia 40 and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii 41 infection, microglia specifically can release the alarmin IL-1a, a cytokine that promotes 42 neuroinflammation and parasite control. 43 44 45 46In this work, we have focused on IL-1, its expression by microglia and macrophages, as 70 well as its role in the brain during chronic T. gondii infection. IL-1 molecules include two main 71 cytokines: IL-1a and IL-1b. IL-1a can function as a canonical alarmin, which is a pre-stored 72 molecule that does not require processing and can be released upon cell death or damage, making 73 it an ideal candidate for an early initiator of inflammation. 19,20 In contrast, IL-1b is produced first 74 as a pro-form that requires cleavage by caspase-1 in order for it to be biologically active, rendering 75 IL-1b dependent on the inflammasome as a platform for caspase-1 activation. [21][22][23] Both of these 76 cytokines signal through the same receptor (IL-1R), a heterodimer of IL-1R1 and IL-1RAcP, with 77 similar affinity. 24 They also lack signal sequences and thus require a loss of membrane integrity to 78 be released. Caspase-mediate cleavage of gasdermin molecules has been identified as a major 79 pathway leading to pore formation and IL-1 release. 80The role of IL-1b and inflammasome pathways in T. gondii infection has been studied in 81 vitro as well as in rodent models of acute infection. In sum, these studies suggest roles for IL-1b, 82 IL18, IL-1R, NLRP1 and/or NLPR3 inflammasome sensors, the inflammasome adaptor protein 83 ASC, and inflammatory caspases-1 and -11. 25-28 However, the role of IL-1 signaling in the brain ...

25Control of chronic CNS infection with the parasite Toxoplasma gondii requires an ongoing T cell 26 response in the brain. Immunosuppressive cytokines are also important for preventing lethal 27 immunopathology during chronic infection. To explore the loss of suppressive cytokine exclusively 28 during the chronic phase of infection we blocked IL-10 receptor (IL-10R). Blockade was associated with 29 widespread changes in the inflammatory response, including increased antigen presenting cell (APC) 30 activation, expansion of CD4+ T cells, and increased neutrophil recruitment to the brain, consistent with 31 previous reports. We then sought to identify regulatory mechanisms contributing to IL-10 production, 32 focusing on ICOS (inducible T cell costimulator), a molecule that promotes IL-10 production in many 33 systems. Unexpectedly, ICOS-ligand (ICOSL) blockade led to a local expansion of effector T cells in the 34 inflamed brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL 35 blockade led to changes in T cells associated with their proliferation and survival. Specifically, we 36 observed increased expression of IL-2 associated signaling molecules, including CD25, STAT5 37 phosphorylation, Ki67, and Bcl-2 in T cells in the brain. Interestingly, increases in CD25 and Bcl-2 were 38 not observed following IL-10R blockade. Also unlike IL-10R blockade, ICOSL blockade led to an 39 expansion of both CD8+ and CD4+ T cells in the brain, with no expansion of peripheral T cell 40 populations or neutrophil recruitment to the brain Overall, these results suggest that IL-10 and ICOS 41 differentially regulate T cell responses in the brain during chronic T. gondii infection. 42 45 themselves. The importance of a balanced immune response is apparent in models of infection, where 46 inflammation is required for pathogen control and survival, yet amplified immune responses observed 47 after depletion of regulatory T cells or immunosuppressive cytokines often leads to exacerbated tissue 48 pathology and increased mortality [3-8]. One such immunosuppressive cytokine, IL-10, has been broadly 49 studied in the context of both tissue homeostasis and during infection, and has been shown to play a key 50 role in suppressing many aspects of an immune response. Production of IL-10 during immune responses 51 to infection has been attributed to a wide variety of cell types, including T cells, dendritic cells, 52 macrophages, NK cells, and B cells [9]. IL-10 also acts on a wide range of cell types, with one of its main 53 roles being the downregulation of MHC and costimulatory molecules in antigen presenting cells (APCs), 54 thereby preventing their full activation capacity and limiting T cell responses [10-12]. IL-10 also has 55 direct effects on T cells, limiting IFNγ and IL-2 production, as well as T cell proliferation in vitro [13, 56 14]. 57Infection with the eukaryotic parasite Toxoplasma gondii leads to widespread activation of the 58 immune system and systemic inflammation that is required for h...