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.

Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopamine neurons. The pathogenesis of PD is poorly understood, though misfolded and/or aggregated forms of the protein α-synuclein have been implicated in several neurodegenerative disease processes, including neuroinflammation and astrocyte activation. Astrocytes in the midbrain play complex roles during PD, initiating both harmful and protective processes that vary over the course of the disease. However, despite their significant regulatory roles during neurodegeneration, the cellular and molecular mechanisms that promote pathogenic astrocyte activity remain mysterious. Here, we show that α-synuclein preformed fibrils (PFFs) induce pathogenic activation of human midbrain astrocytes, marked by inflammatory transcriptional responses, downregulation of phagocytic function, and conferral of neurotoxic activity. These effects required the necroptotic kinases RIPK1 and RIPK3, but were independent of MLKL and necroptosis. Instead, both transcriptional and functional markers of astrocyte activation occurred via RIPK-dependent activation of NF-κB signaling. Our study identifies a previously unknown function for α-synuclein in promoting neurotoxic astrocyte activation, as well as new cell death-independent roles for RIP kinase signaling in the regulation of glial cell biology and neuroinflammation. Together, these findings highlight previously unappreciated molecular mechanisms of pathologic astrocyte activation and neuronal cell death with implications for Parkinsonian neurodegeneration.

RIPK3 Activation Leads to Cytokine Synthesis that Continues after Loss of Cell Membrane Integrity.

Necroptosis is a form of programmed cell death that is defined by activation of the kinase RIPK3 and subsequent cell membrane permeabilization by the effector MLKL. RIPK3 activation can also promote immune responses via production of cytokines and chemokines. How active cytokine production is coordinated with the terminal process of necroptosis is unclear. Here, we report that cytokine production continues within necroptotic cells even after they have lost cell membrane integrity and irreversibly committed to death. This continued cytokine production is dependent on mRNA translation and requires maintenance of endoplasmic reticulum integrity that remains after plasma membrane integrity is lost. The continued translation of cytokines by cellular corpses contributes to necroptotic cell uptake by innate immune cells and priming of adaptive immune responses to antigens associated with necroptotic corpses. These findings imply that cell death and production of inflammatory mediators are coordinated to optimize the immunogenicity of necroptotic cells.

Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity.

Although the signaling events that induce different forms of programmed cell death are well defined, the subsequent immune responses to dying cells in the context of cancer remain relatively unexplored. Necroptosis occurs downstream of the receptor-interacting protein kinases RIPK1 and RIPK3, whose activation leads to lytic cell death accompanied by de novo production of proinflammatory mediators. Here, we show that ectopic introduction of necroptotic cells to the tumor microenvironment promotes BATF3+ cDC1- and CD8+ leukocyte-dependent antitumor immunity accompanied by increased tumor antigen loading by tumor-associated antigen-presenting cells. Furthermore, we report the development of constitutively active forms of the necroptosis-inducing enzyme RIPK3 and show that delivery of a gene encoding this enzyme to tumor cells using adeno-associated viruses induces tumor cell necroptosis, which synergizes with immune checkpoint blockade to promote durable tumor clearance. These findings support a role for RIPK1/RIPK3 activation as a beneficial proximal target in the initiation of tumor immunity. Considering that successful tumor immunotherapy regimens will require the rational application of multiple treatment modalities, we propose that maximizing the immunogenicity of dying cells within the tumor microenvironment through specific activation of the necroptotic pathway represents a beneficial treatment approach that may warrant further clinical development.

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.

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.

The TAM receptor Mertk protects against neuroinvasive viral infection by maintaining blood-brain barrier integrity.

The TAM receptors Tyro3, Axl and Mertk are receptor tyrosine kinases that dampen host innate immune responses following engagement with their ligands Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells. In a form of apoptotic mimicry, many enveloped viruses display phosphatidylserine on the outer leaflet of their membranes, enabling TAM receptor activation and downregulation of antiviral responses. Accordingly, we hypothesized that a deficiency of TAM receptors would enhance antiviral responses and protect against viral infection. Unexpectedly, mice lacking Mertk and/or Axl, but not Tyro3, exhibited greater vulnerability to infection with neuroinvasive West Nile and La Crosse encephalitis viruses. This phenotype was associated with increased blood-brain barrier permeability, which enhanced virus entry into and infection of the brain. Activation of Mertk synergized with interferon-ß to tighten cell junctions and prevent virus transit across brain microvascular endothelial cells. Because TAM receptors restrict pathogenesis of neuroinvasive viruses, these findings have implications for TAM antagonists that are currently in clinical development.

Interferon-λ restricts West Nile virus neuroinvasion by tightening the blood-brain barrier.

Although interferon-λ [also known as type III interferon or interleukin-28 (IL-28)/IL-29] restricts infection by several viruses, its inhibitory mechanism has remained uncertain. We used recombinant interferon-λ and mice lacking the interferon-λ receptor (IFNLR1) to evaluate the effect of interferon-λ on infection with West Nile virus, an encephalitic flavivirus. Cell culture studies in mouse keratinocytes and dendritic cells showed no direct antiviral effect of exogenous interferon-λ, even though expression of interferon-stimulated genes was induced. We observed no differences in West Nile virus burden between wild-type and Ifnlr1(-/-) mice in the draining lymph nodes, spleen, or blood. We detected increased West Nile virus infection in the brain and spinal cord of Ifnlr1(-/-) mice, yet this was not associated with a direct antiviral effect in mouse neurons. Instead, we observed an increase in blood-brain barrier permeability in Ifnlr1(-/-) mice. Treatment of mice with pegylated interferon-λ2 resulted in decreased blood-brain barrier permeability, reduced West Nile virus infection in the brain without affecting viremia, and improved survival against lethal virus challenge. An in vitro model of the blood-brain barrier showed that interferon-λ signaling in mouse brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis- and signal transducer and activator of transcription 1 (STAT1)-independent manner. Our data establish an indirect antiviral function of interferon-λ in which noncanonical signaling through IFNLR1 tightens the blood-brain barrier and restricts viral neuroinvasion and pathogenesis.

An expanded task battery in the Morris water maze reveals effects of Toxoplasma gondii infection on learning and memory in rats.

Infection with the neurotropic parasite Toxoplasma gondii is widespread among human populations; however, the impacts of latent central nervous system (CNS) T. gondii infection have only recently come to light. Epidemiological evidence in humans and experimental studies in rodents have revealed a number of neurological and behavioral sequelae following the establishment of latent CNS toxoplasmosis. Here, we report alterations in learning and memory task performance in latently infected rats using the Morris water maze. While simple spatial reference learning was intact, infected rodents exhibited poor performance compared to controls in probe trials requiring spatial memory recall and progressively poorer performance with increasing time intervals before memory testing, but, surprisingly, enhanced performance in reversal learning tasks. Despite obvious changes to memory task performance, no cysts were detected in the hippocampi of infected rats. Instead, cysts were stochastically distributed across the entire brain, suggesting that behavioral alterations in this study were due to accumulated changes in neurophysiology across multiple anatomical regions. Together, these data provide new evidence that latent toxoplasmosis contributes to neurocognitive symptoms in mammalian hosts, and does so on a broad anatomical scale within the CNS.

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.

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.