The brain microvasculature is a primary mediator of interferon-α neurotoxicity in human cerebral interferonopathies.

Aicardi-Goutières syndrome (AGS) is an autoinflammatory disease characterized by aberrant interferon (IFN)-α production. The major cause of morbidity in AGS is brain disease, yet the primary source and target of neurotoxic IFN-α remain unclear. Here, we demonstrated that the brain was the primary source of neurotoxic IFN-α in AGS and confirmed the neurotoxicity of intracerebral IFN-α using astrocyte-driven Ifna1 misexpression in mice. Using single-cell RNA sequencing, we demonstrated that intracerebral IFN-α-activated receptor (IFNAR) signaling within cerebral endothelial cells caused a distinctive cerebral small vessel disease similar to that observed in individuals with AGS. Magnetic resonance imaging (MRI) and single-molecule ELISA revealed that central and not peripheral IFN-α was the primary determinant of microvascular disease in humans. Ablation of endothelial Ifnar1 in mice rescued microvascular disease, stopped the development of diffuse brain disease, and prolonged lifespan. These results identify the cerebral microvasculature as a primary mediator of IFN-α neurotoxicity in AGS, representing an accessible target for therapeutic intervention.

Interferon-α receptor antisense oligonucleotides reduce neuroinflammation and neuropathology in a mouse model of cerebral interferonopathy.

Chronic and elevated levels of the antiviral cytokine IFN-α in the brain are neurotoxic. This is best observed in patients with genetic cerebral interferonopathies such as Aicardi-Goutières syndrome. Cerebral interferonopathies typically manifest in early childhood and lead to debilitating disease and premature death. There is no cure for these diseases with existing treatments largely aimed at managing symptoms. Thus, an effective therapeutic strategy is urgently needed. Here, we investigated the effect of antisense oligonucleotides targeting the murine IFN-α receptor (Ifnar1 ASOs) in a transgenic mouse model of cerebral interferonopathy. Intracerebroventricular injection of Ifnar1 ASOs into transgenic mice with brain-targeted chronic IFN-α production resulted in a blunted cerebral interferon signature, reduced neuroinflammation, restoration of blood-brain barrier integrity, absence of tissue destruction, and lessened neuronal damage. Remarkably, Ifnar1 ASO treatment was also effective when given after the onset of neuropathological changes, as it reversed such disease-related features. We conclude that ASOs targeting the IFN-α receptor halt and reverse progression of IFN-α-mediated neuroinflammation and neurotoxicity, opening what we believe to be a new and promising approach for the treatment of patients with cerebral interferonopathies.

Microglia shield the murine brain from damage mediated by the cytokines IL-6 and IFN-α.

Sustained production of elevated levels of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is detrimental and directly contributes to the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Using transgenic mice with CNS-targeted production of IL-6 (GFAP-IL6) or IFN-α (GFAP-IFN), we have recently demonstrated that microglia are prominent target and effector cells and mount stimulus-specific responses to these cytokines. In order to further clarify the phenotype and function of these cells, we treated GFAP-IL6 and GFAP-IFN mice with the CSF1R inhibitor PLX5622 to deplete microglia. We examined their ability to recover from acute microglia depletion, as well as the impact of chronic microglia depletion on the progression of disease. Following acute depletion in the brains of GFAP-IL6 mice, microglia repopulation was enhanced, while in GFAP-IFN mice, microglia did not repopulate the brain. Furthermore, chronic CSF1R inhibition was detrimental to the brain of GFAP-IL6 and GFAP-IFN mice and gave rise to severe CNS calcification which strongly correlated with the absence of microglia. In addition, PLX5622-treated GFAP-IFN mice had markedly reduced survival. Our findings provide evidence for novel microglia functions to protect against IFN-α-mediated neurotoxicity and neuronal dysregulation, as well as restrain calcification as a result of both IL-6- and IFN-α-induced neuroinflammation. Taken together, we demonstrate that CSF1R inhibition may be an undesirable target for therapeutic treatment of neuroinflammatory diseases that are driven by elevated IL-6 and IFN-α production.

Strawberry notch homolog 2 regulates the response to interleukin-6 in the central nervous system.

BACKGROUND: The cytokine interleukin-6 (IL-6) modulates a variety of inflammatory processes and, context depending, can mediate either pro- or anti-inflammatory effects. Excessive IL-6 signalling in the brain is associated with chronic inflammation resulting in neurodegeneration. Strawberry notch homolog 2 (Sbno2) is an IL-6-regulated gene whose function is largely unknown. Here we aimed to address this issue by investigating the impact of Sbno2 disruption in mice with IL-6-mediated neuroinflammation. METHODS: Mice with germline disruption of Sbno2 (Sbno2-/-) were generated and crossed with transgenic mice with chronic astrocyte production of IL-6 (GFAP-IL6). Phenotypic, molecular and transcriptomic analyses were performed on tissues and primary cell cultures to clarify the role of SBNO2 in IL-6-mediated neuroinflammation. RESULTS: We found Sbno2-/- mice to be viable and overtly normal. By contrast GFAP-IL6 × Sbno2-/- mice had more severe disease compared with GFAP-IL6 mice. This was evidenced by exacerbated neuroinflammation and neurodegeneration and enhanced IL-6-responsive gene expression. Cell culture experiments on primary astrocytes from Sbno2-/- mice further showed elevated and sustained transcript levels of a number of IL-6 stimulated genes. Notably, despite enhanced disease in vivo and gene expression both in vivo and in vitro, IL-6-stimulated gp130 pathway activation was reduced when Sbno2 is disrupted. CONCLUSION: Based on these results, we propose a role for SBNO2 as a novel negative feedback regulator of IL-6 that restrains the excessive inflammatory actions of this cytokine in the brain.

PLX5622 Reduces Disease Severity in Lethal CNS Infection by Off-Target Inhibition of Peripheral Inflammatory Monocyte Production.

PLX5622 is a CSF-1R inhibitor and microglia-depleting reagent, widely used to investigate the biology of this central nervous system (CNS)-resident myeloid population, but the indirect or off-target effects of this agent remain largely unexplored. In a murine model of severe neuroinflammation induced by West Nile virus encephalitis (WNE), we showed PLX5622 efficiently depleted both microglia and a sub-population of border-associated macrophages in the CNS. However, PLX5622 also significantly depleted mature Ly6Chi monocytes in the bone marrow (BM), inhibiting their proliferation and lethal recruitment into the infected brain, reducing neuroinflammation and clinical disease scores. Notably, in addition, BM dendritic cell subsets, plasmacytoid DC and classical DC, were depleted differentially in infected and uninfected mice. Confirming its protective effect in WNE, cessation of PLX5622 treatment exacerbated disease scores and was associated with robust repopulation of microglia, rebound BM monopoiesis and markedly increased inflammatory monocyte infiltration into the CNS. Monoclonal anti-CSF-1R antibody blockade late in WNE also impeded BM monocyte proliferation and recruitment to the brain, suggesting that the protective effect of PLX5622 is via the inhibition of CSF-1R, rather than other kinase targets. Importantly, BrdU incorporation in PLX5622-treated mice, suggest remaining microglia proliferate independently of CSF-1 in WNE. Our study uncovers significantly broader effects of PLX5622 on the myeloid lineage beyond microglia depletion, advising caution in the interpretation of PLX5622 data as microglia-specific. However, this work also strikingly demonstrates the unexpected therapeutic potential of this molecule in CNS viral infection, as well as other monocyte-mediated diseases.

The cytokines interleukin-6 and interferon-α induce distinct microglia phenotypes.

BACKGROUND: Elevated production of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is implicated in the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Transgenic mice with CNS-targeted chronic production of IL-6 (GFAP-IL6) or IFN-α (GFAP-IFN) recapitulate important clinical and pathological features of these human diseases. The activation of microglia is a prominent manifestation found both in the human diseases and in the transgenic mice, yet little is known about how this contributes to disease pathology. METHODS: Here, we used a combination of ex vivo and in situ techniques to characterize the molecular, cellular and transcriptomic phenotypes of microglia in GFAP-IL6 versus GFAP-IFN mice. In addition, a transcriptomic meta-analysis was performed to compare the microglia response from GFAP-IL6 and GFAP-IFN mice to the response of microglia in a range of neurodegenerative and neuroinflammatory disorders. RESULTS: We demonstrated that microglia show stimulus-specific responses to IL-6 versus IFN-α in the brain resulting in unique and extensive molecular and cellular adaptations. In GFAP-IL6 mice, microglia proliferated, had shortened, less branched processes and elicited transcriptomic and molecular changes associated with phagocytosis and lipid processing. In comparison, microglia in the brain of GFAP-IFN mice exhibited increased proliferation and apoptosis, had larger, hyper-ramified processes and showed transcriptomic and surface marker changes associated with antigen presentation and antiviral response. Further, a transcriptomic meta-analysis revealed that IL-6 and IFN-α both contribute to the formation of a core microglia response in animal models of neurodegenerative and neuroinflammatory disorders, such as Alzheimer's disease, tauopathy, multiple sclerosis and lipopolysaccharide-induced endotoxemia. CONCLUSIONS: Our findings demonstrate that microglia responses to IL-6 and IFN-α are highly stimulus-specific, wide-ranging and give rise to divergent phenotypes that modulate microglia responses in neuroinflammatory and neurodegenerative diseases.

A novel phosphoproteomic landscape evoked in response to type I interferon in the brain and in glial cells.

BACKGROUND: Type I interferons (IFN-I) are key responders to central nervous system infection and injury and are also increased in common neurodegenerative diseases. Their effects are primarily mediated via transcriptional regulation of several hundred interferon-regulated genes. In addition, IFN-I activate several kinases including members of the MAPK and PI3K families. Yet, how changes to the global protein phosphoproteome contribute to the cellular response to IFN-I is unknown. METHODS: The cerebral phosphoproteome of mice with brain-targeted chronic production of the IFN-I, IFN-α, was obtained. Changes in phosphorylation were analyzed by ontology and pathway analysis and kinase enrichment predictions. These were verified by phenotypic analysis, immunohistochemistry and immunoblots. In addition, primary murine microglia and astrocytes, the brain's primary IFN-I-responding cells, were acutely treated with IFN-α and the global phosphoproteome was similarly analyzed. RESULTS: We identified widespread protein phosphorylation as a novel mechanism by which IFN-I mediate their effects. In our mouse model for IFN-I-induced neurodegeneration, protein phosphorylation, rather than the proteome, aligned with the clinical hallmarks and pathological outcome, including impaired development, motor dysfunction and seizures. In vitro experiments revealed extensive and rapid IFN-I-induced protein phosphorylation in microglia and astrocytes. Response to acute IFN-I stimulation was independent of gene expression and mediated by a small number of kinase families. The changes in the phosphoproteome affected a diverse range of cellular processes and functional analysis suggested that this response induced an immediate reactive state and prepared cells for subsequent transcriptional responses. CONCLUSIONS: Our studies reveal a hitherto unappreciated role for changes in the protein phosphorylation landscape in cellular responses to IFN-I and thus provide insights for novel diagnostic and therapeutic strategies for neurological diseases caused by IFN-I.

High-parameter cytometry unmasks microglial cell spatio-temporal response kinetics in severe neuroinflammatory disease.

BACKGROUND: Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a 'microglia-like' phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is important to understand their temporal contribution to disease and inform effective intervention in its pathogenesis. During West Nile virus (WNV) encephalitis, widespread neuronal infection drives substantial CNS infiltration of inflammatory monocytes, causing severe immunopathology and/or death, but the role of microglia in this remains unclear. METHODS: Using high-parameter cytometry and dimensionality-reduction, we devised a simple, novel gating strategy to identify microglia and infiltrating myeloid cells during WNV-infection. Validating our strategy, we (1) blocked the entry of infiltrating myeloid populations from peripheral blood using monoclonal blocking antibodies, (2) adoptively transferred BM-derived monocytes and tracked their phenotypic changes after infiltration and (3) labelled peripheral leukocytes that infiltrate into the brain with an intravenous dye. We demonstrated that myeloid immigrants populated only the identified macrophage gates, while PLX5622 depletion reduced all 4 subsets defined by the microglial gates. RESULTS: Using this gating approach, we identified four consistent microglia subsets in the homeostatic and WNV-infected brain. These were P2RY12hi CD86-, P2RY12hi CD86+ and P2RY12lo CD86- P2RY12lo CD86+. During infection, 2 further populations were identified as 'inflammatory' and 'microglia-like' macrophages, recruited from the bone marrow. Detailed kinetic analysis showed significant increases in the proportions of both P2RY12lo microglia subsets in all anatomical areas, largely at the expense of the P2RY12hi CD86- subset, with the latter undergoing compensatory proliferation, suggesting replenishment of, and differentiation from this subset in response to infection. Microglia altered their morphology early in infection, with all cells adopting temporal and regional disease-specific phenotypes. Late in disease, microglia produced IL-12, downregulated CX3CR1, F4/80 and TMEM119 and underwent apoptosis. Infiltrating macrophages expressed both TMEM119 and P2RY12 de novo, with the microglia-like subset notably exhibiting the highest proportional myeloid population death. CONCLUSIONS: Our approach enables detailed kinetic analysis of resident vs infiltrating myeloid cells in a wide range of neuroinflammatory models without non-physiological manipulation. This will more clearly inform potential therapeutic approaches that specifically modulate these cells.

Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

BACKGROUND: When the homeostasis of the central nervous system (CNS) is altered, microglial cells become activated displaying a wide range of phenotypes that depend on the specific site, the nature of the activator, and particularly the microenvironment generated by the lesion. Cytokines are important signals involved in the modulation of the molecular microenvironment and hence play a pivotal role in orchestrating microglial activation. Among them, interleukin-6 (IL-6) is a pleiotropic cytokine described in a wide range of pathological conditions as a potent inducer and modulator of microglial activation, but with contradictory results regarding its detrimental or beneficial functions. The objective of the present study was to evaluate the effects of chronic IL-6 production on the immune response associated with CNS-axonal anterograde degeneration. METHODS: The perforant pathway transection (PPT) paradigm was used in transgenic mice with astrocyte-targeted IL6-production (GFAP-IL6Tg). At 2, 3, 7, 14, and 21 days post-lesion, the hippocampal areas were processed for immunohistochemistry, flow cytometry, and protein microarray. RESULTS: An increase in the microglia/macrophage density was observed in GFAP-IL6Tg animals in non-lesion conditions and at later time-points after PPT, associated with higher microglial proliferation and a major monocyte/macrophage cell infiltration. Besides, in homeostasis, GFAP-IL6Tg showed an environment usually linked with an innate immune response, with more perivascular CD11b+/CD45high/MHCII+/CD86+ macrophages, higher T cell infiltration, and higher IL-10, IL-13, IL-17, and IL-6 production. After PPT, WT animals show a change in microglia phenotype expressing MHCII and co-stimulatory molecules, whereas transgenic mice lack this shift. This lack of response in the GFAP-IL6Tg was associated with lower axonal sprouting. CONCLUSIONS: Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

Contribution of STAT1 to innate and adaptive immunity during type I interferon-mediated lethal virus infection.

Signal transducers and activators of transcription (STAT) 1 is critical for cellular responses to type I interferons (IFN-Is), with the capacity to determine the outcome of viral infection. We previously showed that while wildtype (WT) mice develop mild disease and survive infection with lymphocytic choriomeningitis virus (LCMV), LCMV infection of STAT1-deficient mice results in a lethal wasting disease that is dependent on IFN-I and CD4+ cells. IFN-Is are considered to act as a bridge between innate and adaptive immunity. Here, we determined the relative contribution of STAT1 on innate and adaptive immunity during LCMV infection. We show that STAT1 deficiency results in a biphasic disease following LCMV infection. The initial, innate immunity-driven phase of disease was characterized by rapid weight loss, thrombocytopenia, systemic cytokine and chemokine responses and leukocyte infiltration of infected organs. In the absence of an adaptive immune response, this first phase of disease largely resolved resulting in survival of the infected host. However, in the presence of adaptive immunity, the disease progressed into a second phase with continued cytokine and chemokine production, persistent leukocyte extravasation into infected tissues and ultimately, host death. Overall, our findings demonstrate the key contribution of STAT1 in modulating innate and adaptive immunity during type I interferon-mediated lethal virus infection.

CNS-Specific Synthesis of Interleukin 23 Induces a Progressive Cerebellar Ataxia and the Accumulation of Both T and B Cells in the Brain: Characterization of a Novel Transgenic Mouse Model.

Interleukin 23 (IL-23) is a key mediator in neuroinflammation in numerous autoimmune diseases including multiple sclerosis (MS). However, the pathophysiology of IL-23 and how it contributes to neuroinflammation is poorly defined. To further clarify the role of IL-23 in CNS inflammation, we generated a transgenic mouse model (GF-IL23) with astrocyte-targeted expression of both IL-23 subunits, IL-23p19, and IL-23p40. These GF-IL23 mice spontaneously develop a progressive ataxic phenotype, which corresponds to cerebellar tissue destruction, and inflammatory infiltrates most prominent in the subarachnoidal and perivascular space. The CNS-cytokine milieu was characterized by numerous inflammatory mediators such as IL-17a and IFNγ. However, the leukocytic infiltrates were surprisingly predominated by B cells. To further examine the impact of the CNS-specific IL-23 synthesis on an additional systemic inflammatory stimulus, we applied the LPS-induced endotoxemia model. Administration of LPS in GF-IL23 mice resulted in early and pronounced microglial activation, enhanced cytokine production and, in sharp contrast to control animals, in the formation of lymphocytic infiltrates. Our model confirms a critical role for IL-23 in the induction of inflammation in the CNS, in particular facilitating the accumulation of lymphocytes in and around the brain. Thereby, CNS-specific synthesis of IL-23 is able to induce a cascade of inflammatory cytokines leading to microglia activation, astrocytosis, and ultimately tissue damage. The presented transgenic model will be a useful tool to further dissect the role of IL-23 in neuroinflammation.

Microglia responses to interleukin-6 and type I interferons in neuroinflammatory disease.

Microglia are the resident macrophages of the central nervous system (CNS). They are a heterogenous, exquisitely responsive, and highly plastic cell population, which enables them to perform diverse roles. They sense and respond to the local production of many different signals, including an assorted range of cytokines. Microglia respond strongly to interleukin-6 (IL-6) and members of the type I interferon (IFN-I) family, IFN-alpha (IFN-α), and IFN-beta (IFN-ß). Although these cytokines are essential in maintaining homeostasis and for activating and regulating immune responses, their chronic production has been linked to the development of distinct human neurological diseases, termed "cerebral cytokinopathies." IL-6 and IFN-α have been identified as key mediators in the pathogenesis of neuroinflammatory disorders including neuromyelitis optica and Aicardi-Goutières syndrome, respectively, whereas IFN-ß has an emerging role as a causal factor in age-associated cognitive decline. One of the key features that unites these diseases is the presence of highly reactive microglia. The current understanding is that microglia contribute to the development of cerebral cytokinopathies and represent an important therapeutic target. However, it remains to be resolved whether microglia have beneficial or detrimental effects. Here we review and discuss what is currently known about the microglial response to IL-6 and IFN-I, based on both animal models and clinical studies. Foundational knowledge regarding the microglial response to IL-6 and IFN-I is now being used to devise therapeutic strategies to ameliorate neuroinflammation and promote repair: either through targeting microglia, or by targeting the reduction of CNS levels or downstream biological pathways of IL-6 or IFN-I.

Microglia have a more extensive and divergent response to interferon-α compared with astrocytes.

Type I interferons (IFN-I) are crucial for effective antimicrobial defense in the central nervous system (CNS) but also can cause severe neurological disease (termed cerebral interferonopathy) as exemplified by Aicardi-Goutières Syndrome. In the CNS, microglia and astrocytes have essential roles in host responses to infection and injury, with both cell types responding to IFN-I. While the IFN-I signaling pathways are the same in astrocytes and microglia, the extent to which the IFN-I responses of these cells differ, if at all, is unknown. Here we determined the global transcriptional responses of astrocytes and microglia to the IFN-I, IFN-α. We found that under basal conditions, each cell type has a unique gene expression pattern reflective of its developmental origin and biological function. Following stimulation with IFN-α, astrocytes and microglia also displayed a common core response that was characterized by the increased expression of genes required for pathogen detection and elimination. Compared with astrocytes, microglia had a more extensive and diverse response to IFN-α with significantly more genes with expression upregulated (282 vs. 141) and downregulated (81 vs. 3). Further validation was documented for selected IFN-I-regulated genes in a murine model of cerebral interferonopathy. In all, the findings highlight not only overlapping but importantly divergent responses to IFN-I by astrocytes versus microglia. This suggests specialized roles for these cells in host defense and in the development of cerebral interferonopathy.

IL-17A Promotes Granulocyte Infiltration, Myelin Loss, Microglia Activation, and Behavioral Deficits During Cuprizone-Induced Demyelination.

Recent evidence suggests a pivotal role of the proinflammatory cytokine interleukin - 17A (IL-17) in demyelinating autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS). Nevertheless, it remains unclear if this cytokine exerts direct effects on CNS resident cells during MS or modulates the function of infiltrating immune cells towards a more detrimental phenotype. Here, we investigated the effects of locally produced IL-17 during experimental demyelination of the CNS using the cuprizone (CPZ) model in mice with (GF/IL17) or without transgenic production of IL-17 by astrocytes in the CNS. During early demyelination, GF/IL17 mice demonstrated enhanced activity and decreased anxiety-related behavior in the elevated plus maze suggesting a more severe disease course. Furthermore, in GF/IL17 mice, toxic demyelination was accelerated and synthesis of myelin proteins was reduced. Early demyelination was accompanied by an increased ratio of infiltrating granulocytes in GF/ILl17 mice. The presence of IL-17 during CPZ treatment increased the accumulation of activated microglia and sustained microglial proliferation during myelin loss. Taken together, our results argue for a detrimental role of IL-17 during demyelinating diseases.

IRF9 Prevents CD8+ T Cell Exhaustion in an Extrinsic Manner during Acute Lymphocytic Choriomeningitis Virus Infection.

Effective CD8+ T cell responses play an important role in determining the course of a viral infection. Overwhelming antigen exposure can result in suboptimal CD8+ T cell responses, leading to chronic infection. This altered CD8+ T cell differentiation state, termed exhaustion, is characterized by reduced effector function, upregulation of inhibitory receptors, and altered expression of transcription factors. Prevention of overwhelming antigen exposure to limit CD8+ T cell exhaustion is of significant interest for the control of chronic infection. The transcription factor interferon regulatory factor 9 (IRF9) is a component of type I interferon (IFN-I) signaling downstream of the IFN-I receptor (IFNAR). Using acute infection of mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong, we show here that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and IFN-I and by controlling levels of IRF7, a transcription factor essential for IFN-I production. Infection of IRF9- or IFNAR-deficient mice led to a loss of early restriction of viral replication and impaired antiviral responses in dendritic cells, resulting in CD8+ T cell exhaustion and chronic infection. Differences in the antiviral activities of IRF9- and IFNAR-deficient mice and dendritic cells provided further evidence of IRF9-independent IFN-I signaling. Thus, our findings illustrate a CD8+ T cell-extrinsic function for IRF9, as a signaling factor downstream of IFNAR, in preventing overwhelming antigen exposure resulting in CD8+ T cell exhaustion and, ultimately, chronic infection.IMPORTANCE During early viral infection, overwhelming antigen exposure can cause functional exhaustion of CD8+ T cells and lead to chronic infection. Here we show that the transcription factor interferon regulatory factor 9 (IRF9) plays a decisive role in preventing CD8+ T cell exhaustion. Using acute infection of mice with LCMV strain Armstrong, we found that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and Irf7, encoding a transcription factor crucial for type I interferon (IFN-I) production, as well as by controlling the levels of IFN-I. Infection of IRF9-deficient mice led to a chronic infection that was accompanied by CD8+ T cell exhaustion due to defects extrinsic to T cells. Our findings illustrate an essential role for IRF9, as a mediator downstream of IFNAR, in preventing overwhelming antigen exposure causing CD8+ T cell exhaustion and leading to chronic viral infection.

Reduced cuprizone-induced cerebellar demyelination in mice with astrocyte-targeted production of IL-6 is associated with chronically activated, but less responsive microglia.

BACKGROUND: Cerebellar pathology is a frequent feature of multiple sclerosis (MS), a demyelinating and neuroinflammatory disease of the central nervous system (CNS). Interleukin (IL)-6 is a multifunctional cytokine with a potential role in MS. Here we studied cuprizone-induced cerebellar pathology in transgenic mice with astrocyte-targeted production of IL-6 (GFAP-IL6), specifically focusing on demyelination, oligodendrocyte depletion and microglial cell response. RESULTS: Over the course of cuprizone treatment, when compared with WT mice, GFAP-IL6Tg showed a reduced demyelination in the deep lateral cerebellar nuclei (LCN). The oligodendrocyte numbers in the LCN were comparable between WT and GFAP-IL6Tg mice after 4-6weeks of cuprizone treatment, however after the chronic cuprizone treatment (12weeks) we detected higher numbers of oligodendrocytes in GFAP-IL6Tg mice. Contrary to strong cuprizone-induced microglial activation in the LCN of WT mice, GFAP-IL6Tg mice had minimal cuprizone-induced microglial changes, despite an already existing reactive microgliosis in control GFAP-IL6Tg not present in control WT mice. CONCLUSIONS: Our results show that chronic transgenic production of IL-6 reduced cuprizone-induced cerebellar demyelination and induced a specific activation state of the resident microglia population (Iba1+, CD11b+, MHCII+, CD68-), likely rendering them less responsive to subsequent injury signals.

Type I interferon-regulated gene expression and signaling in murine mixed glial cells lacking signal transducers and activators of transcription 1 or 2 or interferon regulatory factor 9.

Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-α-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-α was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-α exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.

Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.

Many drugs have been reported to cause thrombotic microangiopathy (TMA), yet evidence supporting a direct association is often weak. In particular, TMA has been reported in association with recombinant type I interferon (IFN) therapies, with recent concern regarding the use of IFN in multiple sclerosis patients. However, a causal association has yet to be demonstrated. Here, we adopt a combined clinical and experimental approach to provide evidence of such an association between type I IFN and TMA. We show that the clinical phenotype of cases referred to a national center is uniformly consistent with a direct dose-dependent drug-induced TMA. We then show that dose-dependent microvascular disease is seen in a transgenic mouse model of IFN toxicity. This includes specific microvascular pathological changes seen in patient biopsies and is dependent on transcriptional activation of the IFN response through the type I interferon α/ß receptor (IFNAR). Together our clinical and experimental findings provide evidence of a causal link between type I IFN and TMA. As such, recombinant type I IFN therapies should be stopped at the earliest stage in patients who develop this complication, with implications for risk mitigation.

Astrocyte-targeted production of interleukin-6 reduces astroglial and microglial activation in the cuprizone demyelination model: Implications for myelin clearance and oligodendrocyte maturation.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Interleukin (IL)-6 is a pleiotropic cytokine with a potential role in MS. Here we used transgenic mice with astrocyte-targeted production of IL-6 (GFAP-IL6Tg) to study the effect of IL-6 in the cuprizone-induced demyelination paradigm, which is an experimental model of de- and re-myelination, both hallmarks of MS. Our results demonstrated that cuprizone-treated GFAP-IL6Tg mice showed a significant reduction in astroglial and especially microglial activation/accumulation in the corpus callosum in comparison with the corresponding cuprizone-treated wild type (WT). Production of a key microglial attracting chemokine CXCL10, as well as CXCL1 and CCL4 was lower in cuprizone-treated GFAP-IL6Tg mice compared with cuprizone-treated WT. Reduced microglial cell accumulation was associated with inefficient removal of degraded myelin and axonal protection in cuprizone-treated GFAP-IL6Tg mice, compared with WT mice at the peak of demyelination. In addition, transgenic production of IL-6 did not alter initial oligodendrocyte (OL) apoptosis and oligodendrocyte precursor cell recruitment to the lesion site, but it impaired early OL differentiation, possibly due to impaired removal of degraded myelin. Indeed, a microglial receptor involved in myelin phagocytosis, TREM2, as well as the phagolysosomal protein CD68 were lower in cuprizone-treated GFAP-IL6Tg compared with WT mice. Our results show for the first time that astrocyte-targeted production of IL-6 may play a role in modulating experimental demyelination induced by cuprizone. Further understanding of the IL-6-mediated molecular mechanisms involved in the regulation of demyelination is needed, and may have implications for the development of future therapeutic strategies for the treatment of MS. GLIA 2016;64:2104-2119.

Strawberry notch homolog 2 is a novel inflammatory response factor predominantly but not exclusively expressed by astrocytes in the central nervous system.

Interleukin-6 (IL-6) participates in the host response to injury and infection in the central nervous system (CNS). We identified strawberry notch homolog 2 (Sbno2) as an IL-6-stimulated gene in murine astrocytes. Sbno2 is a mouse homolog of the sno gene in Drosophila but little is known about the regulation or function of the mammalian gene. Here we examined the regulation of the Sbno2 gene in astrocytes in vitro and in the murine CNS following systemic endotoxin administration. In murine and human cultured astrocytes, Sbno2 gene expression was significantly upregulated in a dose- and time-dependent fashion by hyper-IL-6 (IL-6 + soluble IL-6 receptor). The level of Sbno2 mRNA was also upregulated significantly in murine astrocytes by other glycoprotein130 cytokine-family members and the pro-inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha. These changes were reflected by corresponding alterations in the level of the SBNO2 protein. Inhibiting protein synthesis resulted in higher Sbno2 mRNA and did not abolish the upregulation of Sbno2 mRNA mediated by hyper-IL-6. Inhibition of transcription led to a rapid reduction in hyper-IL-6-induced Sbno2 mRNA in astrocytes suggesting that the Sbno2 mRNA is quite unstable. Following intra-peritoneal lipopolysaccharide injection in mice, Sbno2 mRNA levels in the brain were significantly increased. Cellular localization studies revealed that this increase in Sbno2 mRNA occurred predominantly in astrocytes and in the choroid plexus and in some microglia, endothelial cells, and neurons. These findings are consistent with SBNO2 functioning as an acute inflammatory response gene in astrocytes as well as other cells in the CNS.