Human macrophage SCN5A activates an innate immune signaling pathway for antiviral host defense.

Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon ß. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation.

A sodium channel variant in Aedes aegypti as a candidate pathogen sensor for viral-associated molecular patterns.

Recent work demonstrated that a splice variant of a human macrophage voltage-gated sodium channel expressed on endosomes acts as an intracellular sensor for dsRNA, a viral-associated molecular pattern. Here our goal was to identify a candidate gene in a clinically relevant invertebrate model with related cellular and pattern recognition properties. The para gene in drosophila and other insects encodes voltage-gated sodium channels with similar electrophysiological properties to those found in vertebrate excitable membranes. A database search revealed that the AAEL006019 gene in Aedes aegypti, the yellow fever mosquito, encodes a voltage-gated sodium channel that is distinct from genes that encode para-like sodium channels. As compared to para-like channels, the protein products from this gene have deletions in the N-terminus and in the DII-DIII linker region. When over-expressed in an Aedes aegypti cell line, CCL-125, the AAEL006019 channel demonstrated cytoplasmic expression on vesicular-like organelles. Electrophysiologic analysis revealed that the channel mediates small inward currents that are enhanced by synthetic mimics of viral-derived ssRNA, R848 and ORN02, but not the dsRNA mimic, poly I:C. R848 treatment of CCL-125 cells that express high levels of the channels led to increased expression of RelA and Ago2, two mediators of insect innate immunity. These results suggest that the AAEL006019 channel acts as an intracellular pathogen sensor for ssRNA molecular patterns.

The visualization of Orphadata neurology phenotypes.

Disease phenotypes are characterized by signs (what a physician observes during the examination of a patient) and symptoms (the complaints of a patient to a physician). Large repositories of disease phenotypes are accessible through the Online Mendelian Inheritance of Man, Human Phenotype Ontology, and Orphadata initiatives. Many of the diseases in these datasets are neurologic. For each repository, the phenotype of neurologic disease is represented as a list of concepts of variable length where the concepts are selected from a restricted ontology. Visualizations of these concept lists are not provided. We address this limitation by using subsumption to reduce the number of descriptive features from 2,946 classes into thirty superclasses. Phenotype feature lists of variable lengths were converted into fixed-length vectors. Phenotype vectors were aggregated into matrices and visualized as heat maps that allowed side-by-side disease comparisons. Individual diseases (representing a row in the matrix) were visualized as word clouds. We illustrate the utility of this approach by visualizing the neuro-phenotypes of 32 dystonic diseases from Orphadata. Subsumption can collapse phenotype features into superclasses, phenotype lists can be vectorized, and phenotypes vectors can be visualized as heat maps and word clouds.

Inter-rater agreement for the annotation of neurologic signs and symptoms in electronic health records.

The extraction of patient signs and symptoms recorded as free text in electronic health records is critical for precision medicine. Once extracted, signs and symptoms can be made computable by mapping to signs and symptoms in an ontology. Extracting signs and symptoms from free text is tedious and time-consuming. Prior studies have suggested that inter-rater agreement for clinical concept extraction is low. We have examined inter-rater agreement for annotating neurologic concepts in clinical notes from electronic health records. After training on the annotation process, the annotation tool, and the supporting neuro-ontology, three raters annotated 15 clinical notes in three rounds. Inter-rater agreement between the three annotators was high for text span and category label. A machine annotator based on a convolutional neural network had a high level of agreement with the human annotators but one that was lower than human inter-rater agreement. We conclude that high levels of agreement between human annotators are possible with appropriate training and annotation tools. Furthermore, more training examples combined with improvements in neural networks and natural language processing should make machine annotators capable of high throughput automated clinical concept extraction with high levels of agreement with human annotators.

Subtypes of relapsing-remitting multiple sclerosis identified by network analysis.

We used network analysis to identify subtypes of relapsing-remitting multiple sclerosis subjects based on their cumulative signs and symptoms. The electronic medical records of 113 subjects with relapsing-remitting multiple sclerosis were reviewed, signs and symptoms were mapped to classes in a neuro-ontology, and classes were collapsed into sixteen superclasses by subsumption. After normalization and vectorization of the data, bipartite (subject-feature) and unipartite (subject-subject) network graphs were created using NetworkX and visualized in Gephi. Degree and weighted degree were calculated for each node. Graphs were partitioned into communities using the modularity score. Feature maps visualized differences in features by community. Network analysis of the unipartite graph yielded a higher modularity score (0.49) than the bipartite graph (0.25). The bipartite network was partitioned into five communities which were named fatigue, behavioral, hypertonia/weakness, abnormal gait/sphincter, and sensory, based on feature characteristics. The unipartite network was partitioned into five communities which were named fatigue, pain, cognitive, sensory, and gait/weakness/hypertonia based on features. Although we did not identify pure subtypes (e.g., pure motor, pure sensory, etc.) in this cohort of multiple sclerosis subjects, we demonstrated that network analysis could partition these subjects into different subtype communities. Larger datasets and additional partitioning algorithms are needed to confirm these findings and elucidate their significance. This study contributes to the literature investigating subtypes of multiple sclerosis by combining feature reduction by subsumption with network analysis.

Differential DNA methylation associated with multiple sclerosis and disease modifying treatments in an underrepresented minority population.

Black and Hispanic American patients frequently develop earlier onset of multiple sclerosis (MS) and a more severe disease course that can be resistant to disease modifying treatments. The objectives were to identify differential methylation of genomic DNA (gDNA) associated with disease susceptibility and treatment responses in a cohort of MS patients from underrepresented minority populations. Patients with MS and controls with non-inflammatory neurologic conditions were consented and enrolled under an IRB-approved protocol. Approximately 64% of donors identified as Black or African American and 30% as White, Hispanic-Latino. Infinium MethylationEPIC bead arrays were utilized to measure epigenome-wide gDNA methylation of whole blood. Data were analyzed in the presence and absence of adjustments for unknown covariates in the dataset, some of which corresponded to disease modifying treatments. Global patterns of differential methylation associated with MS were strongest for those probes that showed relative demethylation of loci with lower M values. Pathway analysis revealed unexpected associations with shigellosis and amoebiasis. Enrichment analysis revealed an over-representation of probes in enhancer regions and an under-representation in promoters. In the presence of adjustments for covariates that included disease modifying treatments, analysis revealed 10 differentially methylated regions (DMR's) with an FDR <1E-77. Five of these genes (ARID5B, BAZ2B, RABGAP1, SFRP2, WBP1L) are associated with cancer risk and cellular differentiation and have not been previously identified in MS studies. Hierarchical cluster and multi-dimensional scaling analysis of differential DNA methylation at 147 loci within those DMR's was sufficient to differentiate MS donors from controls. In the absence of corrections for disease modifying treatments, differential methylation in patients treated with dimethyl fumarate was associated with immune regulatory pathways that regulate cytokine and chemokine signaling, axon guidance, and adherens junctions. These results demonstrate possible associations of gastrointestinal pathogens and regulation of cellular differentiation with MS susceptibility in our patient cohort. This work further suggests that analyses can be performed in the presence and absence of corrections for immune therapies. Because of their high representation in our patient cohort, these results may be of specific relevance in the regulation of disease susceptibility and treatment responses in Black and Hispanic Americans.

Regulation of expansion of CD11c+ B cells and anti-viral immunity by epithelial V-like antigen.

Prior work demonstrated that epithelial V-like antigen (EVA), a cell surface adhesion molecule, is expressed in B lymphocytes and is necessary for the efficacy of anti-alpha4 integrin treatment of experimental autoimmune encephalomyelitis (EAE), the mouse model of human multiple sclerosis. EVA deficiency is associated with a severe clinical phenotype of EAE in the presence or absence of treatment. Histological analysis revealed enhanced B cell-mediated autoimmunity and deposition of antibody and complement within the brain and spinal cord. Here our goal was to determine the molecular mechanism of EVA regulation of B lymphocyte function. Analysis of bone marrow from MOG-immunized mice revealed increased expansion of CD11c+ B cells in EVA-deficient mice as compared to wild type controls. In vitro studies of mouse bone marrow B lymphocytes revealed enhanced proliferation of the CD11c+ population in response to the Tlr7/8 agonist R848. An increase in R848-induced proliferation of CD11c+ B cells was also seen in vitro in Daudi cells, a human B cell line, following knockdown of the mpzl2 gene that encodes EVA. These mechanisms were characterized further by global expression analysis of bone marrow from immunized EVA-deficient and wild type control mice. These data revealed increased expression of B cell associated genes and decreased expression of the anti-viral oligoadenylate synthase genes, Oas1 and Oas2, in the knockout condition. In Daudi cells, R848 treatment induced an increase in Oas2 expression in control cells that was not observed in EVA-deficient cells. EVA deficiency also was associated with increased transcription of an Epstein-Barr virus gene during lytic replication. These results suggest EVA expression and signaling prevent expansion of CD11c+ B lymphocytes, a cellular phenotype associated with autoimmunity, increase expression of anti-viral oligoadenylate synthase genes, and reduce replication of a DNA virus.

Activation of human macrophage sodium channels regulates RNA processing to increase expression of the DNA repair protein PPP1R10.

Prior work demonstrated that a splice variant of SCN5A, a voltage-gated sodium channel gene, acts as a cytoplasmic sensor for viral dsRNA in human macrophages. Expression of this channel also polarizes macrophages to an anti-inflammatory phenotype in vitro and in vivo. Here we utilized global expression analysis of splice variants to identify novel channel-dependent signaling mechanisms. Pharmacological activation of voltage-gated sodium channels in human macrophages, but not treatment with cytoplasmic poly I:C, was associated with splicing of a retained intron in transcripts of PPP1R10, a regulator of phosphatase activity and DNA repair. Microarray analysis also demonstrated expression of a novel sodium channel splice variant, human macrophage SCN10A, that contains a similar exon deletion as SCN5A. SCN10A localizes to cytoplasmic and nuclear vesicles in human macrophages. Simultaneous expression of human macrophage SCN5A and SCN10A was required to decrease expression of the retained intron and increase protein expression of PPP1R10. Channel activation also increased protein expression of the splicing factor EFTUD2, and knockdown of EFTUD2 prevented channel dependent splicing of the retained PPP1R10 intron. Knockdown of the SCN5A and SCN10A variants in human macrophages reduced the severity of dsDNA breaks induced by treatment with bleomycin and type 1 interferon. These results suggested that human macrophage SCN5A and SCN10A variants mediate an innate immune signaling pathway that limits DNA damage through increased expression of PPP1R10. The functional significance of this pathway is that it may prevent cytotoxicity during inflammatory responses.

Epithelial V-like antigen regulates permeability of the blood-CSF barrier.

Epithelial V-like antigen (EVA), a CD3-binding immunoglobulin-like protein, regulates embryonic thymic development. Here we demonstrate that EVA is expressed in choroid plexus from mature immune competent and lymphocyte-deficient (RAG-/-) mice. Choroid plexus epithelial cells from RAG-/- mice demonstrated reduced junctional integrity and enhanced permeability that was associated with decreased expression of E-cadherin and EVA mRNA as compared to wild-type mice. Following iv infusion of an anti-CD3 antibody (145-2C11) that also binds EVA, expression of E-cadherin and EVA mRNA approached levels seen in wild-type mice. Immuno-fluorescent staining for cadherin also revealed decreased expression in untreated RAG-/- mice that could be increased by 145-2C11 treatment. Expression of mouse EVA in HEK-293 cells followed by challenge with 145-2C11 resulted in increased cytosolic calcium that was not seen in control cells. These results suggest that EVA expressed in choroid plexus cells may regulate the permeability of the blood-CSF barrier.

Characterization of a severe parenchymal phenotype of experimental autoimmune encephalomyelitis in (C57BL6xB10.PL)F1 mice.

We here describe a novel CD4 T cell adoptive transfer model of severe experimental autoimmune encephalomyelitis in (C57BL6xB10.PL)F1 mice. This FI cross developed severe disease characterized by extensive parenchymal spinal cord and brain periventricular white matter infiltrates. In contrast, B10.PL mice developed mild disease characterized by meningeal predominant infiltrates. As determined by cDNA microarray and quantitative real time PCR expression analysis, histologic and flow cytometry analysis of inflammatory infiltrates, and attenuation of disease in class I-deficient and CD8-depleted F1 mice; this severe disease phenotype appears to be regulated by CNS infiltration of CD8 T lymphocytes early in the disease course.

Role of genetic background in P selectin-dependent immune surveillance of the central nervous system.

Although the blood-brain barrier and blood cerebrospinal fluid barrier maintain the central nervous system (CNS) as an immunologically privileged site, T lymphocytes can migrate through unstimulated brain endothelium and epithelium to perform immune surveillance or initiate inflammation. Our prior results suggested that early CNS migration of a CD4 Th1 cell line was facilitated by P selectin (CD62P) in (PL/JxSJL/J)F1 mice. Here, quantitative analysis of migration 2 h following adoptive transfer of fluorescently labeled cells revealed a 53-72% decrease in activated splenocyte, CD4 Th1 and CD8 migration, but not CD4 Th2, in CD62P-deficient C57BL6/J mice. Immunohistochemistry revealed constitutive expression of CD62P within the meninges and choroid plexus epithelia in C57BL6/J and SJL/J, but not BALB/cJ, mice. Activated splenocyte migration was approximately three- to four-fold greater in SJL/J as compared to BALB/cJ mice. Anti-CD62P treatment normalized this difference. Based on these results, we hypothesize that genetically determined kinetics of immune surveillance may regulate the phenotype of subsequent CNS inflammation.

Innate immune viral recognition: relevance to CNS infections.

Innate immune responses mediated by mononuclear phagocytes represent the initial host response to acute viral infection. PRRs, including TLRs, retinoic RLRs,and NOD-like receptors, recognize viral nucleic acid and localized injury signals to initiate proinflammatory responses and activation of adaptive immunity. These responses are host- and viral-dependent. Neurotropic viruses, such as HSV, West Nile virus, and HIV activate and evade innate immune signaling mechanisms by distinct mechanisms. These highly complex pathogen-host interactions determine establishment of infection, severity of clinical disease, development of chronic inflammatory processes, and success of vaccination strategies.

Update on disease-modifying treatments for multiple sclerosis.

PURPOSE: The purpose of this review is to discuss the selection and use of disease- modifying treatments for patients with relapsing forms of multiple sclerosis (MS). METHODS: PubMed was searched (1966-2014) using the terms multiple sclerosis, treatment, interferon, glatiramer acetate, dimethyl fumarate, fingolimod, teriflunomide, natalizumab, rituximab, and alemtuzumab. FINDINGS: MS is a chronic neurological disorder that can cause a substantial degree of disability. Because of its usual onset in young adults, patients may require treatment for several decades. Currently available agents include platform injectable therapies, newer oral agents, and second-line monoclonal antibody treatments. Treatment decisions have become more complex with the introduction of new approaches, and a major goal is to balance perceived efficacy and tolerability in a specific patient with the relative impact of disease activity and adverse events on quality of life. Here the options for disease-modifying treatments for relapsing forms of MS are reviewed, and current and future challenges are discussed. IMPLICATIONS: An evidence-based approach can be used for the selection of disease-modifying treatments based on disease phenotype and severity, adverse events, and perceived efficacy.

Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.

Multiple sclerosis (MS) is the most common nontraumatic cause of neurologic disability in young adults. Despite treatment, progressive tissue injury leads to accumulation of disability in many patients. Here, our goal was to develop an immune-mediated strategy to promote tissue repair and clinical recovery in an MS animal model. We previously demonstrated that a variant of the voltage-gated sodium channel NaV1.5 is expressed intracellularly in human macrophages, and that it regulates cellular signaling. This channel is not expressed in mouse macrophages, which has limited the study of its functions. To overcome this obstacle, we developed a novel transgenic mouse model (C57BL6), in which the human macrophage NaV1.5 splice variant is expressed in vivo in mouse macrophages. These mice were protected from experimental autoimmune encephalomyelitis, the mouse model of MS. During active inflammatory disease, NaV1.5-positive macrophages were found in spinal cord lesions where they formed phagocytic cell clusters; they expressed markers of alternative activation during recovery. NaV1.5-positive macrophages that were adoptively transferred into wild-type recipients with established experimental autoimmune encephalomyelitis homed to lesions and promoted recovery. These results suggest that NaV1.5-positive macrophages enhance recovery from CNS inflammatory disease and could potentially be developed as a cell-based therapy for the treatment of MS.

Epithelial V-like antigen mediates efficacy of anti-alpha4 integrin treatment in a mouse model of multiple sclerosis.

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha4 integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA⁺ lymphocytes following immunization. Following active induction of EAE using the MOG³5⁻55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19⁺, CD21⁺, sIgG⁺), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5⁺IgG⁺ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19⁺, CD21⁺, sIgG⁺ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19⁺, CD21⁺, sIgG⁺ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders.

Current immunotherapy of multiple sclerosis and future challenges: relevance of immune-mediated repair.

Multiple sclerosis is the most common chronic inflammatory disease of the central nervous system and can cause severe neurological disability. Current immune therapy with beta-interferons, glatiramer acetate, immune suppressives, or selective adhesion molecule inhibitors can reduce the frequency and severity of acute attacks in a majority of patients but have little effect on the progressive phase of the disease. Patients who require aggressive immune therapy are also at risk for the development of potentially fatal opportunistic infections. Here current and emerging immune therapy options are discussed, and immune-mediated strategies to preserve normal immune surveillance and mediate tissue repair are proposed.

Current Immunotherapy of Multiple Sclerosis and Future Challenges: Relevance of Immune-mediated Repair.

Multiple sclerosis is the most common chronic inflammatory disease of the central nervous system and can cause severe neurological disability. Current immune therapy with beta-interferons, glatiramer acetate, immune suppressives, or selective adhesion molecule inhibitors can reduce the frequency and severity of acute attacks in a majority of patients but have little effect on the progressive phase of the disease. Patients who require aggressive immune therapy are also at risk for the development of potentially fatal opportunistic infections. Here current and emerging immune therapy options are discussed, and immune-mediated strategies to preserve normal immune surveillance and mediate tissue repair are proposed.

Characterization of epithelial V-like antigen in human choroid plexus epithelial cells: potential role in CNS immune surveillance.

Prior work demonstrated that immune surveillance of the brain occurs primarily through the blood-cerebrospinal (CSF) fluid barrier rather than the blood-brain barrier endothelium. Recently, we identified epithelial V-like antigen (EVA), an immunoglobulin-like adhesion molecule, as a regulator of blood-CSF barrier integrity in a mouse model. Here we characterized EVA expression and function in human choroid plexus epithelial cells and analyzed its role in CD4 T lymphocyte adhesion. In human choroid plexus epithelial cells and a subset of CD4 T lymphocytes, EVA is expressed at high levels. Epithelial adhesion of T lymphocytes is inhibited by a blocking monoclonal antibody that recognizes EVA. T cell adhesion elicits calcium flux in choroid plexus epithelial cells that also can be blocked by an EVA-specific antibody. EVA-positive cell-cell contacts between epithelial and T cells are associated with increased complexity of cytoskeletal epithelial morphology. These results demonstrate that EVA is expressed in human choroid plexus epithelial cells and CD4 T lymphocytes and regulates CD4+ T lymphocyte adhesion to human choroid plexus epithelial cells in vitro. These data suggest a novel mechanism to regulate CNS immune surveillance.

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.