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.

Astrocyte reactivity in a mouse model of SCN8A epileptic encephalopathy.

OBJECTIVE: SCN8A epileptic encephalopathy is caused predominantly by de novo gain-of-function mutations in the voltage-gated sodium channel Nav 1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti-seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav 1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy. METHODS: Using immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Nav 1.6, and Kir 4.1 channel currents were assessed in astrocytes in wild-type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+). RESULTS: Astrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium-sensitive Kir 4.1 currents compared to age-matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures. SIGNIFICANCE: Astrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir 4.1-mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression.

Somatostatin-Positive Interneurons Contribute to Seizures in SCN8A Epileptic Encephalopathy.

SCN8A epileptic encephalopathy is a devastating epilepsy syndrome caused by mutant SCN8A, which encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. Using both sexes of a transgenic mouse model of SCN8A epileptic encephalopathy, we found that selective expression of the R1872W SCN8A mutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. Patch-clamp electrophysiology experiments revealed that SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of WT SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark of SCN8A mutations, were observed and were found to contribute directly to aberrant SST physiology in computational modeling and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only in SCN8A epileptic encephalopathy, but epilepsy in general.SIGNIFICANCE STATEMENTSCN8A epileptic encephalopathy is a devastating neurological disorder that results from de novo mutations in the sodium channel isoform Nav1.6. Inhibitory neurons express NaV1.6, yet their contribution to seizure generation in SCN8A epileptic encephalopathy has not been determined. We show that mice expressing a human-derived SCN8A variant (R1872W) selectively in somatostatin (SST) interneurons have audiogenic seizures. Physiological recordings from SST interneurons show that SCN8A mutations lead to an elevated persistent sodium current which drives initial hyperexcitability, followed by premature action potential failure because of depolarization block. Furthermore, chemogenetic activation of WT SST interneurons leads to audiogenic seizure activity. These findings provide new insight into the importance of SST inhibitory interneurons in seizure initiation, not only in SCN8A epileptic encephalopathy, but for epilepsy broadly.

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.

Neuronal integrity and complement control synaptic material clearance by microglia after CNS injury.

Phagocytosis of synaptic material by microglia is critical for central nervous system development. Less well understood is this microglial function in the injured adult brain. Assay of microglial phagocytosis is challenging, because peripheral myeloid cells engraft the site of injury, which could obscure interpretation of microglial roles. The model used here, optic nerve crush injury, results in degeneration of synapses in the dorsal lateral geniculate nucleus (dLGN), which stimulates rapid activation and engulfment of synaptic material by resident microglia without myeloid cell engraftment. Pharmacological depletion of microglia causes postinjury accumulation of synaptic debris, suggesting that microglia are the dominant postinjury phagocytes. Genetic or pharmacological manipulations revealed that neuronal activity does not trigger microglia phagocytosis after injury. RNA sequencing reveals C1q and CD11b/CR3 involvement in clearance of debris by dLGN-resident microglia. Indeed, C1qa-/- and Itgam-/- mice exhibit impaired postinjury debris clearance. Our results show how neurodegenerative debris is cleared by microglia and offers a model for studying its mechanisms and physiological roles.