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.

Phenotypes associated with genetic determinants of type I interferon regulation in the UK Biobank: a protocol.

Background: Type I interferons are cytokines involved in innate immunity against viruses. Genetic disorders of type I interferon regulation are associated with a range of autoimmune and cerebrovascular phenotypes. Carriers of pathogenic variants involved in genetic disorders of type I interferons are generally considered asymptomatic. Preliminary data suggests, however, that genetically determined dysregulation of type I interferon responses is associated with autoimmunity, and may also be relevant to sporadic cerebrovascular disease and dementia. We aim to determine whether functional variants in genes involved in type I interferon regulation and signalling are associated with the risk of autoimmunity, stroke, and dementia in a population cohort. Methods: We will perform a hypothesis-driven candidate pathway association study of type I interferon-related genes using rare variants in the UK Biobank (UKB). We will manually curate type I interferon regulation and signalling genes from a literature review and Gene Ontology, followed by clinical and functional filtering. Variants of interest will be included based on pre-defined clinical relevance and functional annotations (using LOFTEE, M-CAP and a minor allele frequency <0.1%). The association of variants with 15 clinical and three neuroradiological phenotypes will be assessed with a rare variant genetic risk score and gene-level tests, using a Bonferroni-corrected p-value threshold from the number of genetic units and phenotypes tested. We will explore the association of significant genetic units with 196 additional health-related outcomes to help interpret their relevance and explore the clinical spectrum of genetic perturbations of type I interferon. Ethics and dissemination: The UKB has received ethical approval from the North West Multicentre Research Ethics Committee, and all participants provided written informed consent at recruitment. This research will be conducted using the UKB Resource under application number 93160. We expect to disseminate our results in a peer-reviewed journal and at an international cardiovascular conference.

Cerebrospinal fluid neurofilament light chain in multiple sclerosis and its subtypes: a meta-analysis of case-control studies.

OBJECTIVE: Neurofilament is a biomarker of axonal injury proposed as a useful adjunct in the monitoring of patients with multiple sclerosis (MS). We conducted a systematic review and meta-analysis of case-control studies that have measured neurofilament light chain (NfL) levels in cerebrospinal fluid (CSF) of people with MS (pwMS), in order to determine whether, and to what degree, CSF NfL levels differentiate MS from controls, or the subtypes or stages of MS from each other. METHODS: Guidelines on Preferred Reporting Items for Systematic Reviews and Meta-Analyses were followed. Electronic databases were searched for published and 'grey' literature, with 151 hits. Of 51 full articles screened, 20 were included in qualitative analysis, and 14 in meta-analysis. RESULTS: CSF NfL was higher in 746 pwMS than 435 (healthy and disease) controls, with a moderate effect size of 0.61 (p < 0.00001). Mean CSF NfL levels were significantly higher in 176 pwMS with relapsing disease than 92 with progressive disease (2124.8 ng/L, SD 3348.9 vs 1121.4 ng/L, SD 947.7, p = 0.0108). CSF NfL in 138 pwMS in relapse (irrespective of MS subtype) was double that seen in 268 pwMS in remission (3080.6 ng/L, SD 4715.9 vs 1541.7 ng/L, SD 2406.5, p < 0.0001). CONCLUSIONS: CSF NfL correlates with MS activity throughout the course of MS, reflecting the axonal damage in pwMS. Relapse is more strongly associated with elevated CSF NfL levels than the development of progression, and NfL may be most useful as a marker of disease 'activity' rather than as a marker of disability or disease stage.

Neurological Disease in Lupus: Toward a Personalized Medicine Approach.

The brain and nervous system are important targets for immune-mediated damage in systemic lupus erythematosus (SLE), resulting in a complex spectrum of neurological syndromes. Defining nervous system disease in lupus poses significant challenges. Among the difficulties to be addressed are a diversity of clinical manifestations and a lack of understanding of their mechanistic basis. However, despite these challenges, progress has been made in the identification of pathways which contribute to neurological disease in SLE. Understanding the molecular pathogenesis of neurological disease in lupus will inform both classification and approaches to clinical trials.

Detection of interferon alpha protein reveals differential levels and cellular sources in disease.

Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation.

Human ß-defensin 3 increases the TLR9-dependent response to bacterial DNA.

Human ß-defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self-DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt-3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self-DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9-dependent response. In contrast to hBD3, lipofection of self-DNA enhances inflammatory signaling, but the response is predominantly TLR9-independent. Together, these data show that hBD3 has a role in the innate immune-mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy-number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA-based vaccines.

Rare variants of the 3'-5' DNA exonuclease TREX1 in early onset small vessel stroke.

Background: Monoallelic and biallelic mutations in the exonuclease TREX1 cause monogenic small vessel diseases (SVD). Given recent evidence for genetic and pathophysiological overlap between monogenic and polygenic forms of SVD, evaluation of TREX1 in small vessel stroke is warranted. Methods: We sequenced the TREX1 gene in an exploratory cohort of patients with lacunar stroke (Edinburgh Stroke Study, n=290 lacunar stroke cases). We subsequently performed a fully blinded case-control study of early onset MRI-confirmed small vessel stroke within the UK Young Lacunar Stroke Resource (990 cases, 939 controls). Results: No patients with canonical disease-causing mutations of TREX1 were identified in cases or controls. Analysis of an exploratory cohort identified a potential association between rare variants of TREX1 and patients with lacunar stroke. However, subsequent controlled and blinded evaluation of TREX1 in a larger and MRI-confirmed patient cohort, the UK Young Lacunar Stroke Resource, identified heterozygous rare variants in 2.1% of cases and 2.3% of controls. No association was observed with stroke risk (odds ratio = 0.90; 95% confidence interval, 0.49-1.65 p=0.74). Similarly no association was seen with rare TREX1 variants with predicted deleterious effects on enzyme function (odds ratio = 1.05; 95% confidence interval, 0.43-2.61 p=0.91). Conclusions: No patients with early-onset lacunar stroke had genetic evidence of a TREX1-associated monogenic microangiopathy. These results show no evidence of association between rare variants of TREX1 and early onset lacunar stroke. This includes rare variants that significantly affect protein and enzyme function. Routine sequencing of the TREX1 gene in patients with early onset lacunar stroke is therefore unlikely to be of diagnostic utility, in the absence of syndromic features or family history.

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.

Correction: Human ß-D-3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid.

[This corrects the article DOI: 10.1371/journal.pgen.1005673.].

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Human ß-Defensin 3 [corrected] Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid.

Human ß-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six ß-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-ß in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-ß. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-ß, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans.

Type I interferon dysregulation and neurological disease.

Type I interferon is an essential component of the brain's innate immune defence, conferring protection against viral infection. Recently, dysregulation of the type I interferon pathway has been implicated in the pathogenesis of a spectrum of neuroinfectious and neuroinflammatory disorders. Underactivity of the type I interferon response is associated with a predisposition to herpes simplex encephalitis. Conversely, a group of 'interferonopathic' disorders, characterized by severe neuroinflammation and overactivity of type I interferon, has been described. Elucidation of the genetic basis of these Mendelian neuroinflammatory diseases has uncovered important links between nucleic acid sensors, innate immune activation and neuroinflammatory disease. These mechanisms have an important role in the pathogenesis of more common polygenic diseases that can affect the brain, such as lupus and cerebral small vessel disease. In this article, we review the spectrum of neurological disease associated with type I interferon dysregulation, as well as advances in our understanding of the molecular and cellular pathogenesis of these conditions. We highlight the potential utility of type I interferon as both a biomarker and a therapeutic target in neuroinflammatory disease.