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The developing brain initially makes more synapses than it needs. With further development, excess synapses are pruned away, leaving mature circuits. Synapses can be eliminated by microglia, which engulf and destroy them. Vainchtein et al. found that the microglia are called into action by astrocytes, supportive cells on which neurons rely. Astrocytes near a redundant synapse release the cytokine interleukin-33 (IL-33), which recruits microglia to the site. In mice, disruptions in this process, as caused by deficiency in IL-33, led to too many excitatory synapses and overactive brain circuitry. Science , this issue p. 1269 Astrocytes use microglia to prune redundant neuronal synapses. Neuronal synapse formation and remodeling are essential to central nervous system (CNS) development and are dysfunctional in neurodevelopmental diseases. Innate immune signals regulate tissue remodeling in the periphery, but how this affects CNS synapses is largely unknown. Here, we show that the interleukin-1 family cytokine interleukin-33 (IL-33) is produced by developing astrocytes and is developmentally required for normal synapse numbers and neural circuit function in the spinal cord and thalamus. We find that IL-33 signals primarily to microglia under physiologic conditions, that it promotes microglial synapse engulfment, and that it can drive microglial-dependent synapse depletion in vivo. These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development.

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish ( Danio rerio ) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions. Microglia remodel synapses and engulf apoptotic cells. The molecular program underlying these distinct functions are unclear. Here, the authors identify distinct microglial subsets associated with synaptic vs. neurogenic regions of the developing zebrafish brain.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we perform single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC. Anti-integrin therapy inhibits lymphocyte trafficking in ulcerative colitis. Here Mennillo et al. use single-cell and spatial -omics to show modulation of mononuclear phagocytes and other networks, identifying gene sets related to treatment response.

The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP’s function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP’s functions during infection by the Kaposi’s sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies.

Traumatic brain injury (TBI) is a leading cause of mortality and disability worldwide and can lead to secondary sequelae such as increased seizure susceptibility. Emerging work suggests that the thalamus, the relay center of the brain that undergoes secondary damage after cortical TBI, is involved with heightened seizure risks after TBI. TBI also induces the recruitment of peripheral immune cells, including T cells, to the site(s) of injury, but it is unclear how these cells impact neurological sequelae post-TBI. Here, we characterize the identities and kinetics of lymphocytic infiltrates into the cortex and thalamus using a mouse model of cortical TBI. We identify a population of IFNγ-producing type 1 lymphocytes that infiltrate specific thalamic subregions over weeks following injury, where they elicit a local IFNγ response in microglia and neuronal subset(s). Depletion of CD4 T cells protects mice from TBI-induced seizure susceptibility by de-repressing other non-CD4 type 1 lymphocytes and disease-associated microglia (DAMs) in the thalamus. Strikingly, we find that a single dose of IFNγ prior to challenge with a proconvulsant agent was sufficient to reduce TBI-induced seizure incidence, severity, and mortality. This work identifies IFNγ as a direct modulator of TBI-associated seizure susceptibility, which could have therapeutic implications for the treatment of TBI patients.

The innate immune system plays essential roles in brain synaptic development, and immune dysregulation is implicated in neurodevelopmental diseases. Here we show that a subset of innate lymphocytes (group 2 innate lymphoid cells, ILC2s) is required for cortical inhibitory synapse maturation and adult social behavior. ILC2s expanded in the developing meninges and produced a surge of their canonical cytokine Interleukin-13 (IL-13) between postnatal days 5-15. Loss of ILC2s decreased cortical inhibitory synapse numbers in the postnatal period where as ILC2 transplant was sufficient to increase inhibitory synapse numbers. Deletion of the IL-4/IL-13 receptor ( ) from inhibitory neurons phenocopied the reduction inhibitory synapses. Both ILC2 deficient and neuronal deficient animals had similar and selective impairments in adult social behavior. These data define a type 2 immune circuit in early life that shapes adult brain function.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we performed single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.Competing Interest StatementS. Lewin has received research support from Takeda. N. El-Nachef is a consultant for Ferring, Federation Bio Grant, and receives funding from Finch Therapeutics, Seres, Freenome, and Assembly Biosciences. U. Mahadevan serves as a consultant for Abbvie, BMS, Boeringher Ingelheim, Gilead, Janssen, Lilly, Pfizer, Prometheus biosciences, Protagonist, Rani Therapeutics, Surrozen, and Takeda. D. Oh has received research support from Merck, PACT Pharma, the Parker Institute for Cancer Immunotherapy, Poseida Therapeutics, TCR2 Therapeutics, Roche/Genentech, and Nutcracker Therapeutics, and travel/accommodations from Roche/Genentech. The Combes lab has received research support from Eli Lilly and Genentech and A. Combes consults for Foundery Innovations. The Kattah lab receives research support from Eli Lilly. M. Kattah has consulted for Sonoma Biotherapeutics and Morphic Therapeutic.Footnotes* We are adding a validation analysis of the CyTOF data, and longitudinal spatial transcriptomics of responders and non-responders to Vedolizumab.* https://github.com/mkattah/UC_VDZ

Microglia are brain resident phagocytes that can engulf synaptic components and extracellular matrix as well as whole neurons. However, whether there are unique molecular mechanisms that regulate these distinct phagocytic states is unknown. Here we define a molecularly distinct microglial subset whose function is to engulf neurons in the developing brain. We transcriptomically identified a cluster of Type I interferon (IFN-I) responsive microglia that expanded 20-fold in the postnatal day 5 somatosensory cortex after partial whisker deprivation, a stressor that accelerates neural circuit remodeling. In situ, IFN-I responsive microglia were highly phagocytic and actively engulfed whole neurons. Conditional deletion of IFN-I signaling (Ifnar1fl/fl) in microglia but not neurons resulted in dysmorphic microglia with stalled phagocytosis and an accumulation of neurons with double strand DNA breaks, a marker of cell stress. Conversely, exogenous IFN-I was sufficient to drive neuronal engulfment by microglia and restrict the accumulation of damaged neurons. IFN-I deficient mice had excess excitatory neurons in the developing somatosensory cortex as well as tactile hypersensitivity to whisker stimulation. These data define a molecular mechanism through which microglia engulf neurons during a critical window of brain development. More broadly, they reveal key homeostatic roles of a canonical antiviral signaling pathway in brain development.Microglia are brain resident phagocytes that can engulf synaptic components and extracellular matrix as well as whole neurons. However, whether there are unique molecular mechanisms that regulate these distinct phagocytic states is unknown. Here we define a molecularly distinct microglial subset whose function is to engulf neurons in the developing brain. We transcriptomically identified a cluster of Type I interferon (IFN-I) responsive microglia that expanded 20-fold in the postnatal day 5 somatosensory cortex after partial whisker deprivation, a stressor that accelerates neural circuit remodeling. In situ, IFN-I responsive microglia were highly phagocytic and actively engulfed whole neurons. Conditional deletion of IFN-I signaling (Ifnar1fl/fl) in microglia but not neurons resulted in dysmorphic microglia with stalled phagocytosis and an accumulation of neurons with double strand DNA breaks, a marker of cell stress. Conversely, exogenous IFN-I was sufficient to drive neuronal engulfment by microglia and restrict the accumulation of damaged neurons. IFN-I deficient mice had excess excitatory neurons in the developing somatosensory cortex as well as tactile hypersensitivity to whisker stimulation. These data define a molecular mechanism through which microglia engulf neurons during a critical window of brain development. More broadly, they reveal key homeostatic roles of a canonical antiviral signaling pathway in brain development.

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct synapse-associated microglial subset in the zebrafish (Danio rerio). We found that ramified microglia populated synapse-rich regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synapse-associated microglia (SAMs) that were highly enriched in the hindbrain, expressed known synapse modulating genes as well as novel candidates, and engulfed synaptic proteins. In contrast, neurogenic-associated microglia (NAMs) were enriched in optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data yielded a functionally annotated atlas of zebrafish microglia (https://www.annamolofskylab.org/microglia-sequencing). Furthermore, they reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish zebrafish hindbrain as a model circuit for investigating microglial-synapse interactions.

Microglia are key remodelers of neuronal synapses during brain development, but the mechanisms that regulate this process and its ultimate impact on neural circuit function are not well defined. We previously identified the IL-1 family cytokine Interleukin-33 (IL-33) as a novel mediator of microglial synapse remodeling. Here we define the phagocytic program induced in microglia in response to IL-33. We find that IL-33 markedly alters the microglial enhancer landscape and exposes AP-1 transcription factor sites that promote target gene expression. We identify the scavenger receptor MARCO and the pattern recognition receptor TLR2 as downstream mediators of IL-33 dependent synapse engulfment. Conditional deletion of IL-33 in the CNS or its receptor on microglia results in increased numbers of excitatory synapses in the corticothalamic circuit and spontaneous epileptiform activity as well as increased seizure susceptibility by early adulthood. These findings define novel mechanisms through which IL-33 coordinates experience-dependent synaptic refinement to restrict hyperexcitability in the developing brain.