RIPK3 Restricts Viral Pathogenesis via Cell Death-Independent Neuroinflammation.

Receptor-interacting protein kinase-3 (RIPK3) is an activator of necroptotic cell death, but recent work has implicated additional roles for RIPK3 in inflammatory signaling independent of cell death. However, while necroptosis has been shown to contribute to antiviral immunity, death-independent roles for RIPK3 in host defense have not been demonstrated. Using a mouse model of West Nile virus (WNV) encephalitis, we show that RIPK3 restricts WNV pathogenesis independently of cell death. Ripk3-/- mice exhibited enhanced mortality compared to wild-type (WT) controls, while mice lacking the necroptotic effector MLKL, or both MLKL and caspase-8, were unaffected. The enhanced susceptibility of Ripk3-/- mice arose from suppressed neuronal chemokine expression and decreased central nervous system (CNS) recruitment of T lymphocytes and inflammatory myeloid cells, while peripheral immunity remained intact. These data identify pleiotropic functions for RIPK3 in the restriction of viral pathogenesis and implicate RIPK3 as a key coordinator of immune responses within the CNS.

The Nucleotide Sensor ZBP1 and Kinase RIPK3 Induce the Enzyme IRG1 to Promote an Antiviral Metabolic State in Neurons.

As long-lived post-mitotic cells, neurons employ unique strategies to resist pathogen infection while preserving cellular function. Here, using a murine model of Zika virus (ZIKV) infection, we identified an innate immune pathway that restricts ZIKV replication in neurons and is required for survival upon ZIKV infection of the central nervous system (CNS). We found that neuronal ZIKV infection activated the nucleotide sensor ZBP1 and the kinases RIPK1 and RIPK3, core components of virus-induced necroptotic cell death signaling. However, activation of this pathway in ZIKV-infected neurons did not induce cell death. Rather, RIPK signaling restricted viral replication by altering cellular metabolism via upregulation of the enzyme IRG1 and production of the metabolite itaconate. Itaconate inhibited the activity of succinate dehydrogenase, generating a metabolic state in neurons that suppresses replication of viral genomes. These findings demonstrate an immunometabolic mechanism of viral restriction during neuroinvasive infection.

RIPK3 promotes brain region-specific interferon signaling and restriction of tick-borne flavivirus infection.

Innate immune signaling in the central nervous system (CNS) exhibits many remarkable specializations that vary across cell types and CNS regions. In the setting of neuroinvasive flavivirus infection, neurons employ the immunologic kinase receptor-interacting kinase 3 (RIPK3) to promote an antiviral transcriptional program, independently of the traditional function of this enzyme in promoting necroptotic cell death. However, while recent work has established roles for neuronal RIPK3 signaling in controlling mosquito-borne flavivirus infections, including West Nile virus and Zika virus, functions for RIPK3 signaling in the CNS during tick-borne flavivirus infection have not yet been explored. Here, we use a model of Langat virus (LGTV) encephalitis to show that RIPK3 signaling is specifically required in neurons of the cerebellum to control LGTV replication and restrict disease pathogenesis. This effect did not require the necroptotic executioner molecule mixed lineage kinase domain like protein (MLKL), a finding similar to previous observations in models of mosquito-borne flavivirus infection. However, control of LGTV infection required a unique, region-specific dependence on RIPK3 to promote expression of key antiviral interferon-stimulated genes (ISG) in the cerebellum. This RIPK3-mediated potentiation of ISG expression was associated with robust cell-intrinsic restriction of LGTV replication in cerebellar granule cell neurons. These findings further illuminate the complex roles of RIPK3 signaling in the coordination of neuroimmune responses to viral infection, as well as provide new insight into the mechanisms of region-specific innate immune signaling in the CNS.

Copper-Phosphido Catalysis: Enantioselective Addition of Phosphines to Cyclopropenes.

We describe a copper catalyst that promotes the addition of phosphines to cyclopropenes at ambient temperature. A range of cyclopropylphosphines bearing different steric and electronic properties can now be accessed in high yields and enantioselectivities. Enrichment of phosphorus stereocenters is also demonstrated via a Dynamic Kinetic Asymmetric Transformation (DyKAT) process. A combined experimental and theoretical mechanistic study supports an elementary step featuring insertion of a CuI -phosphido into a carbon-carbon double bond. Density functional theory calculations reveal migratory insertion as the rate- and stereo-determining step, followed by a syn-protodemetalation.

Zika virus-induced TNF-α signaling dysregulates expression of neurologic genes associated with psychiatric disorders.

BACKGROUND: Zika virus (ZIKV) is an emerging flavivirus of global concern. ZIKV infection of the central nervous system has been linked to a variety of clinical syndromes, including microcephaly in fetuses and rare but serious neurologic disease in adults. However, the potential for ZIKV to influence brain physiology and host behavior following apparently mild or subclinical infection is less well understood. Furthermore, though deficits in cognitive function are well-documented after recovery from neuroinvasive viral infection, the potential impact of ZIKV on other host behavioral domains has not been thoroughly explored. METHODS: We used transcriptomic profiling, including unbiased gene ontology enrichment analysis, to assess the impact of ZIKV infection on gene expression in primary cortical neuron cultures. These studies were extended with molecular biological analysis of gene expression and inflammatory cytokine signaling. In vitro observations were further confirmed using established in vivo models of ZIKV infection in immunocompetent hosts. RESULTS: Transcriptomic profiling of primary neuron cultures following ZIKV infection revealed altered expression of key genes associated with major psychiatric disorders, such as bipolar disorder and schizophrenia. Gene ontology enrichment analysis also revealed significant changes in gene expression associated with fundamental neurobiological processes, including neuronal development, neurotransmission, and others. These alterations to neurologic gene expression were also observed in the brain in vivo using several immunocompetent mouse models of ZIKV infection. Mechanistic studies identified TNF-α signaling via TNFR1 as a major regulatory mechanism controlling ZIKV-induced changes to neurologic gene expression. CONCLUSIONS: Our studies reveal that cell-intrinsic innate immune responses to ZIKV infection profoundly shape neuronal transcriptional profiles, highlighting the need to further explore associations between ZIKV infection and disordered host behavioral states.

Point-of-care radiology service at the US Open Tennis Championships.

Professional tennis tournaments have onsite sports medicine physicians who oversee the athletes' overall health during competition, including musculoskeletal injury and general illnesses. The medical team is composed of orthopedic and non-operative sports medicine physicians. Frequently, the tournament doctors require imaging to localize and grade musculoskeletal injuries and to make decisions regarding treatment, safe training, and return to match play. The most versatile and readily available imaging modality to evaluate for acute musculoskeletal injury is point-of-care ultrasonography. In 2015, a dedicated radiology service was created at the US Open by bringing in a musculoskeletal radiologist who would perform ultrasounds in a formal and consistent manner. In addition, the radiologist was tasked with onsite radiography as well as review of all MRI examinations done at the imaging center. This article describes how this radiology service was implemented, what types of studies were performed and the advantages of having an onsite musculoskeletal radiologist at the tournament. This service allowed the medical team to provide the comprehensive and efficient medical care required in a major professional tennis event. It also showed the value of having the in-person expertise of the musculoskeletal radiologist in the sports medicine team. This same model could be applied to other professional sporting events.

STING is required for host defense against neuropathological West Nile virus infection.

West Nile Virus (WNV), an emerging and re-emerging RNA virus, is the leading source of arboviral encephalitic morbidity and mortality in the United States. WNV infections are acutely controlled by innate immunity in peripheral tissues outside of the central nervous system (CNS) but WNV can evade the actions of interferon (IFN) to facilitate CNS invasion, causing encephalitis, encephalomyelitis, and death. Recent studies indicate that STimulator of INterferon Gene (STING), canonically known for initiating a type I IFN production and innate immune response to cytosolic DNA, is required for host defense against neurotropic RNA viruses. We evaluated the role of STING in host defense to control WNV infection and pathology in a murine model of infection. When challenged with WNV, STING knock out (-/-) mice displayed increased morbidity and mortality compared to wild type (WT) mice. Virologic analysis and assessment of STING activation revealed that STING signaling was not required for control of WNV in the spleen nor was WNV sufficient to mediate canonical STING activation in vitro. However, STING-/- mice exhibited a clear trend of increased viral load and virus dissemination in the CNS. We found that STING-/- mice exhibited increased and prolonged neurological signs compared to WT mice. Pathological examination revealed increased lesions, mononuclear cellular infiltration and neuronal death in the CNS of STING-/- mice, with sustained pathology after viral clearance. We found that STING was required in bone marrow derived macrophages for early control of WNV replication and innate immune activation. In vivo, STING-/- mice developed an aberrant T cell response in both the spleen and brain during WNV infection that linked with increased and sustained CNS pathology compared to WT mice. Our findings demonstrate that STING plays a critical role in immune programming for the control of neurotropic WNV infection and CNS disease.

Outcomes of RIP Kinase Signaling During Neuroinvasive Viral Infection.

Neuroinvasive viral diseases are a considerable and growing burden on global public health. Despite this, these infections remain poorly understood, and the molecular mechanisms that govern protective versus pathological neuroinflammatory responses to infection are a matter of intense investigation. Recent evidence suggests that necroptosis, an immunogenic form of programmed cell death, may contribute to the pathogenesis of viral encephalitis. However, the receptor-interacting protein (RIP) kinases that coordinate necroptosis, RIPK1 and RIPK3, also appear to have unexpected, cell death-independent functions in the central nervous system (CNS) that promote beneficial neuroinflammation during neuroinvasive infection. Here, we review the emerging evidence in this field, with additional discussion of recent work examining roles for RIPK signaling and necroptosis during noninfectious pathologies of the CNS, as these studies provide important additional insight into the potential for specialized neuroimmune functions for the RIP kinases.

Astrocyte-T cell crosstalk regulates region-specific neuroinflammation.

During multiple sclerosis (MS), an inflammatory and neurodegenerative disease of the central nervous system (CNS), symptoms, and outcomes are determined by the location of inflammatory lesions. While we and others have shown that T cell cytokines differentially regulate leukocyte entry into perivascular spaces and regional parenchymal localization in murine models of MS, the molecular mechanisms of this latter process are poorly understood. Here, we demonstrate that astrocytes exhibit region-specific responses to T cell cytokines that promote hindbrain versus spinal cord neuroinflammation. Analysis of cytokine receptor expression in human astrocytes showed region-specific responsiveness to Th1 and Th17 inflammatory cytokines. Consistent with this, human and murine astrocytes treated with these cytokines exhibit differential expression of the T cell localizing molecules VCAM-1 and CXCR7 that is both cytokine and CNS region-specific. Using in vivo models of spinal cord versus brain stem trafficking of myelin-specific T cells and astrocyte-specific deletion strategies, we confirmed that Th1 and Th17 cytokines differentially regulate astrocyte expression of VCAM-1 and CXCR7 in these locations. Finally, stereotaxic injection of individual cytokines into the hindbrain or spinal cord revealed region- and cytokine-specific modulation of localizing cue expression by astrocytes. These findings identify a role for inflammatory cytokines in mediating local astrocyte-dependent mechanisms of immune cell trafficking within the CNS during neuroinflammation.

Dissecting Oligomeric States with Photoactivated Localization Microscopy: A Numerical Model.

Although photoactivated localization microscopy offers the potential to interrogate protein interactions in the physiological environment of a cell, uncertainties in the detection efficiency of photoactivatable proteins lead to complications with data interpretation. Here, we present a numerical model that provides probabilities to detect neighboring molecules dependent on their oligomerization status, density, detection efficiency, and radius, and can be used to assess oligomeric states or detection efficiencies of two molecular species. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1ß Independently of Gasdermin-D.

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1ß. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1ß released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1ß independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1ß independently of gasdermin-D.

IL-1R1 signaling regulates CXCL12-mediated T cell localization and fate within the central nervous system during West Nile Virus encephalitis.

Immune cell entry into the virally infected CNS is vital for promoting viral clearance yet may contribute to neuropathology if not rigorously regulated. We previously showed that signaling through IL-1R1 is critical for effector T cell reactivation and virologic control within the CNS during murine West Nile virus (WNV) encephalitis. WNV-infected IL-1R1(-/-) mice also display increased parenchymal penetration of CD8(+) T cells despite lack of CD4-mediated full activation, suggesting dysregulation of molecular components of CNS immune privilege. In this study, we show that IL-1 signaling regulates the CNS entry of virus-specific lymphocytes, promoting protective immune responses to CNS viral infections that limit immunopathology. Analysis of blood-brain barrier function in the WNV-infected IL-1R1(-/-) mice revealed no alterations in permeability. However, parenchymal proinflammatory chemokine expression, including CCL2, CCL5, and CXCL10, was significantly upregulated, whereas microvasculature CXCL12 expression was significantly decreased in the absence of IL-1 signaling. We show that during WNV infection, CD11b(+)CD45(hi) infiltrating cells (macrophages) are the primary producers of IL-1ß within the CNS and, through the use of an in vitro blood-brain barrier model, that IL-1ß promotes CXCR4-mediated T cell adhesion to brain microvasculature endothelial cells. Of interest, IFNγ(+) and CD69(+) WNV-primed T cells were able to overcome CXCL12-mediated adhesion via downregulation of CXCR4. These data indicate that infiltrating IL-1ß-producing leukocytes contribute to cellular interactions at endothelial barriers that impart protective CNS inflammation by regulating the parenchymal entry of CXCR4(+) virus-specific T cells during WNV infection.

A test of current models for the mechanism of milk-lipid droplet secretion.

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced green fluorescent protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5-0.7% (w/w) of the total protein, i.e. over 50-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk.

CCR5 limits cortical viral loads during West Nile virus infection of the central nervous system.

BACKGROUND: Cell-mediated immunity is critical for clearance of central nervous system (CNS) infection with the encephalitic flavivirus, West Nile virus (WNV). Prior studies from our laboratory have shown that WNV-infected neurons express chemoattractants that mediate recruitment of antiviral leukocytes into the CNS. Although the chemokine receptor, CCR5, has been shown to play an important role in CNS host defense during WNV infection, regional effects of its activity within the infected brain have not been defined. METHODS: We used CCR5-deficient mice and an established murine model of WNV encephalitis to determine whether CCR5 activity impacts on WNV levels within the CNS in a region-specific fashion. Statistical comparisons between groups were made with one- or two-way analysis of variance; Bonferroni's post hoc test was subsequently used to compare individual means. Survival was analyzed by the log-rank test. Analyses were conducted using Prism software (GraphPad Prism). All data were expressed as means ± SEM. Differences were considered significant if P ≤ 0.05. RESULTS: As previously shown, lack of CCR5 activity led to increased symptomatic disease and mortality in mice after subcutaneous infection with WNV. Evaluation of viral burden in the footpad, draining lymph nodes, spleen, olfactory bulb, and cerebellum derived from WNV-infected wild-type, and CCR5(-/-) mice showed no differences between the genotypes. In contrast, WNV-infected, CCR5(-/-) mice exhibited significantly increased viral burden in cortical tissues, including the hippocampus, at day 8 post-infection. CNS regional studies of chemokine expression via luminex analysis revealed significantly increased expression of CCR5 ligands, CCL4 and CCL5, within the cortices of WNV-infected, CCR5(-/-) mice compared with those of similarly infected WT animals. Cortical elevations in viral loads and CCR5 ligands in WNV-infected, CCR5(-/-) mice, however, were associated with decreased numbers of infiltrating mononuclear cells and increased permeability of the blood-brain barrier. CONCLUSIONS: These data indicate that regional differences in chemokine expression occur in response to WNV infection of the CNS, and that cortical neurons require CCR5 activity to limit viral burden in this brain region.

Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm.

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional "compartmentalization" has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

2'-O methylation of the viral mRNA cap by West Nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo.

Prior studies have shown that 2'-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2'-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2'-O methylation of the 5' viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1⁻/⁻ mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD(50) values in Ifit1⁻/⁻ compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1⁻/⁻ brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⁺ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2'-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.

Arterial thoracic outlet syndrome.

Arterial thoracic outlet syndrome is a rare cause of shoulder pain due to compression of the subclavian or axillary artery within the thoracic outlet. It is the least common form of thoracic outlet syndrome but is potentially dangerous as it can result in significant morbidity. An athlete initially may present with exertional pain, early fatigability, a dull ache, or discomfort in the affected arm. History and physical examination are paramount in diagnosis, and imaging confirms the anatomy. Surgical repair or resection alleviates the compression of the affected structure and allows for a safe return to participation. Familiarity with this condition aids in the prompt diagnosis and treatment of this disorder.

RIPK3 promotes neuronal survival by suppressing excitatory neurotransmission during CNS viral infection.

While recent work has identified roles for immune mediators in the regulation of neural activity, the capacity for cell intrinsic innate immune signaling within neurons to influence neurotransmission remains poorly understood. However, the existing evidence linking immune signaling with neuronal function suggests that modulation of neurotransmission may serve previously undefined roles in host protection during infection of the central nervous system. Here, we identify a specialized function for RIPK3, a kinase traditionally associated with necroptotic cell death, in preserving neuronal survival during neurotropic flavivirus infection through the suppression of excitatory neurotransmission. We show that RIPK3 coordinates transcriptomic changes in neurons that suppress neuronal glutamate signaling, thereby desensitizing neurons to excitotoxic cell death. These effects occur independently of the traditional functions of RIPK3 in promoting necroptosis and inflammatory transcription. Instead, RIPK3 promotes phosphorylation of the key neuronal regulatory kinase CaMKII, which in turn activates the transcription factor CREB to drive a neuroprotective transcriptional program and suppress deleterious glutamatergic signaling. These findings identify an unexpected function for a canonical cell death protein in promoting neuronal survival during viral infection through the modulation of neuronal activity, highlighting new mechanisms of neuroimmune crosstalk.

Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors.

Flaviviruses pose a significant threat to public health due to their ability to infect the central nervous system (CNS) and cause severe neurologic disease. Astrocytes play a crucial role in the pathogenesis of flavivirus encephalitis through their maintenance of blood-brain barrier (BBB) integrity and their modulation of immune cell recruitment and activation within the CNS. We have previously shown that receptor interacting protein kinase-3 (RIPK3) is a central coordinator of neuroinflammation during CNS viral infection, a function that occurs independently of its canonical function in inducing necroptotic cell death. To date, however, roles for necroptosis-independent RIPK3 signaling in astrocytes are poorly understood. Here, we use mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an unexpected anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes was required for host survival in multiple models of flavivirus encephalitis, where it restricted neuropathogenesis by limiting immune cell recruitment to the CNS. Transcriptomic analysis revealed that, despite inducing a traditional pro-inflammatory transcriptional program, astrocytic RIPK3 paradoxically promoted neuroprotection through the upregulation of serpins, endogenous protease inhibitors with broad immunomodulatory activity. Notably, intracerebroventricular administration of SerpinA3N in infected mice preserved BBB integrity, reduced leukocyte infiltration, and improved survival outcomes in mice lacking astrocytic RIPK3. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation and suggests new therapeutic targets for the treatment of flavivirus encephalitis.