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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cryptococcus strains with different pathogenic potentials have diverse protein secretomes.

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.

Trans-signaling is a dominant mechanism for the pathogenic actions of interleukin-6 in the brain.

IL-6 is implicated in the pathogenesis of various neuroinflammatory and neurodegenerative disorders of the CNS. IL-6 signals via binding to either the membrane bound IL-6Rα (classic signaling) or soluble (s)IL-6Ra (trans-signaling) that then form a complex with gp130 to activate the JAK/STAT signaling pathway. The importance of classic versus trans-signaling in mediating IL-6 actions in the living CNS is relatively unknown and was the focus of this investigation. Bigenic mice (termed GFAP-IL6/sgp130 mice) were generated with CNS-restricted, astrocyte-targeted production of IL-6 and coproduction of the specific inhibitor of IL-6 trans-signaling, human sgp130-Fc. Transgene-encoded IL-6 mRNA levels were similar in the brain of GFAP-IL6 and GFAP-IL6/sgp130 mice. However, GFAP-IL6/sgp130 mice had decreased pY(705)-STAT3 in the brain due to a reduction in the total number of pY(705)-STAT3-positive cells and a marked loss of pY(705)-STAT3 in specific cell types. Blockade of trans-signaling in the brain of the GFAP-IL6 mice significantly attenuated Serpina3n but not SOCS3 gene expression, whereas vascular changes including angiogenesis and blood-brain barrier leakage as well as gliosis were also reduced significantly. Hippocampal neurogenesis which was impaired in GFAP-IL6 mice was rescued in young GFAP-IL6 mice with cerebral sgp130 production. Finally, degenerative changes in the cerebellum characteristic of GFAP-IL6 mice were absent in GFAP-IL6/sgp130 mice. The findings indicate that in the CNS: (1) sgp130 is able to block IL-6 trans-signaling, (2) trans-signaling is important for IL-6 cellular communication with selective cellular and molecular targets, and (3) blocking of trans-signaling alleviates many of the detrimental effects of IL-6.

Analysis of IL-6/gp130 family receptor expression reveals that in contrast to astroglia, microglia lack the oncostatin M receptor and functional responses to oncostatin M.

The interleukin (IL)-6/gp130 family of cytokines (e.g., IL-6, IL-11, leukemia inhibitory factor (LIF) and oncostatin M (OSM)) play important roles in the central nervous system (CNS) during neuroinflammation and neurodevelopment. However, little is known regarding the responses by astroglia and microglia to this family of cytokines. Here the expression of the IL-6/gp130 cytokine receptors and subsequent signal pathway activation was examined in murine astrocytes and microglia in vitro. Astrocytes had high levels of OSMR mRNA while lower levels of IL-6R, LIFR and IL-11R mRNAs were also present. In comparison, in microglia there was no detectable OSMR mRNA, higher levels of IL-6R mRNA and lower levels of the LIFR and IL-11R mRNAs. The OSMR protein was present in astrocytes but was undetectable in microglia. Conversely, the IL-6R protein was present in microglia but not detectable in astrocytes. In astrocytes but not microglia, phosphorylation of STAT1 and STAT3 occurred in response to OSM, whereas both microglia and astrocytes responded to hyper-IL-6 (IL-6 linked to the soluble IL-6 receptor). Finally, in both microglia and astrocytes, OSM failed to activate NFκB or induce iNOS and nitrite production. We conclude: (1) notable differences exist in the expression of receptors utilized by the IL-6/gp130 family of cytokines in astrocytes and microglia, and (2) the findings provide a molecular basis for the differential response to OSM by astrocytes versus microglia and demonstrate a fundamental means for achieving cellular specificity in the response of these glial cells to this cytokine.

Astrocyte-targeted production of IL-10 induces changes in microglial reactivity and reduces motor neuron death after facial nerve axotomy.

Interleukin-10 (IL-10) is a cytokine that plays a crucial role in regulating the inflammatory response and immune reactions. In the central nervous system (CNS), IL-10 is mainly produced by astrocytes and microglia and it is upregulated after various insults, such as experimental autoimmune encephalomyelitis, middle cerebral artery occlusion, excitotoxicity and traumatic brain injury. To better understand the effects of IL-10 in the normal and injured CNS, we generated transgenic mice (termed GFAP-IL-10Tg) that expressed the murine IL-10 gene under the transcriptional control of the glial fibrillary acidic protein (GFAP) promoter. Previous studies demonstrated marked changes in the microglial phenotype in these mice under basal conditions. The objective of the present study was to investigate the effects of local astrocyte-targeted IL-10 production on glial activation, neuronal degeneration and leukocyte recruitment after axotomy. GFAP-IL-10Tg mice had marked changes in the phenotype of activated microglial cells, as well as in the number of microglial clusters and in microglial cell density. These microglial changes are accompanied by a twofold increase in lymphocyte infiltration in GFAP-IL-10Tg mice and around twofold decrease in neuronal cell death at 21 dpi. Altogether, our findings suggested that astrocyte-targeted production of IL-10 impacted the microglial response and lymphocyte recruitment and culminated in a beneficial effect on neuronal survival.

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.

IRF7-dependent type I interferon production induces lethal immune-mediated disease in STAT1 knockout mice infected with lymphocytic choriomeningitis virus.

UNLABELLED: Following systemic infection with lymphocytic choriomeningitis virus (LCMV), STAT1 knockout (KO) mice but not wild-type, STAT2 KO, IRF9 KO, or IFNAR KO mice develop lethal disease perpetrated by CD4(+) T cells. IRF7 is a key transcriptional activator of type I IFN (IFN-I) during LCMV infection. Here, the role of IRF7 in the lethal host response to LCMV infection in STAT1 KO mice was examined. In contrast to STAT1 KO mice, STAT1/IRF7 double KO (DKO) mice survived LCMV infection with a reduced immune pathology in key organs, such as the liver and spleen. However, similar to STAT1 KO mice, STAT1/IRF7 DKO mice failed to control LCMV replication and spread. LCMV infection in STAT1 KO mice was associated with a significant elevation in the levels of a number of cytokines in serum, including IFN-Is, but this was largely absent in STAT1/IRF7 DKO mice, which had a modest increase in the levels of gamma interferon and CCL2 only. Since IRF7 is known to be a key transcriptional regulator of IFN-I gene expression, the possible role of IFN-I in lethal disease was examined further. STAT1/IFNAR DKO mice, in contrast to STAT1 KO mice, all survived infection with LCMV and exhibited little tissue immune pathology. Additionally, STAT1 KO mice that were deficient for either of the two IFN-I signaling molecules, STAT2 or IRF9, also survived LCMV infection. We conclude that the lethal immune-mediated disease resulting from LCMV infection in STAT1 KO mice is (i) dependent on IRF7-induced IFN-I production and (ii) driven by noncanonical IFN-I signaling via STAT2 and IRF9. IMPORTANCE: Here we report on the basis for the novel, fatal immune-mediated disease of STAT1 KO mice infected with LCMV. Our findings show that, surprisingly, the pathogenesis of this disease is dependent on IRF7-mediated type I interferon production. Moreover, our study identifies noncanonical type I interferon signaling via STAT2 and IRF9 to be essential for the type I IFN-driven fatal disease in LCMV-infected STAT1 KO mice. These results further highlight the significance of noncanonical type I IFN signaling in the pathogenesis of host-mediated injury following viral infection.

The bacteriostatic protein lipocalin 2 is induced in the central nervous system of mice with west Nile virus encephalitis.

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.