Type‐I interferons suppress microglial production of the lymphoid chemokine, CXCL13

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108063/1/glia22692.pd

Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections

Astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis

The matricellular proteins, secreted protein acidic and rich in cysteine (SPARC) and SPARC-like 1 (SPARCL1), are produced by astrocytes and control excitatory synaptogenesis in the central nervous system. While SPARCL1 directly promotes excitatory synapse formation in vitro and in the developing nervous system in vivo, SPARC specifically antagonizes the synaptogenic actions of SPARCL1. We hypothesized these proteins also help maintain existing excitatory synapses in adult hosts, and that local inflammation in the spinal cord alters their production in a way that dynamically modulates motor synapses and impacts the severity of paralysis during experimental autoimmune encephalomyelitis (EAE) in mice. Using a spontaneously remitting EAE model, paralysis severity correlated inversely with both expression of synaptic proteins and the number of synapses in direct contact with the perikarya of motor neurons in spinal grey matter. In both remitting and non-remitting EAE models, paralysis severity also correlated inversely with sparcl1:sparc transcript and SPARCL1:SPARC protein ratios directly in lumbar spinal cord tissue. In vitro, astrocyte production of both SPARCL1 and SPARC was regulated by T cell-derived cytokines, causing dynamic modulation of the SPARCL1:SPARC expression ratio. Taken together, these data support a model whereby proinflammatory cytokines inhibit SPARCL1 and/or augment SPARC expression by astrocytes in spinal grey matter that, in turn, cause either transient or sustained synaptic retraction from lumbar spinal motor neurons thereby regulating hind limb paralysis during EAE. Ongoing studies seek ways to alter this SPARCL1:SPARC expression ratio in favor of synapse reformation/maintenance and thus help to modulate neurologic deficits during times of inflammation. This could identify new astrocyte-targeted therapies for diseases such as multiple sclerosis

Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds

Disruption of mitochondrial electron transport suppresses WEEV replication.

<div><p>(A) Schematic of mETC enzyme complexes. The known targets for the inhibitors shown in italics are indicated by the cross bars. Cyt c, cytochrome C; CoQ, coenzyme Q.</p> <p>(B) Antiviral activity and toxicity of mETC inhibitors. Cells were treated with increasing concentrations of the indicated inhibitors, and replicon inhibition, total cellular ATP production, and cytotoxicity were measured in separate assays. Results are presented as IC₅₀ or CC₅₀ values for the indicated parameter, and represent the mean ± SEM from at least three independent experiments. The numerical values on the graph indicate fold-differences in IC₅₀ values between replicon inhibition and ATP production suppression for the indicated select compounds. For rotenone, the comparison was made with CC₅₀ values, since we were unable to calculate reliable IC₅₀ values for ATP production suppression.</p> <p>(C) Complementation assays with select mETC inhibitors and WEEV replicons. Cells were treated with 100 μM of the indicated supplement or antioxidant and antimycin A (AA), CCCP, or mycophenolic acid (MPA) at 2X or 5X replicon IC₅₀ concentrations, and replicon activity was measured 16-20 h later. Results represent the mean ± SEM from four independent experiments. <i>p</i>-value < 0.05* or 0.005** compared to supplement- or antioxidant-only treated controls. 2-MPG, <i>N</i>-(2-mercaptopropionyl)glycine.</p> <p>(D) Complementation assay with antimycin A and infectious virus. BE(2)-C cells were infected with FMV at an MOI = 1, treated simultaneously with 100 μM of the indicated supplement or antioxidant and control DMSO or antimycin A at 5X replicon IC₅₀ concentration, and viral titers in tissue culture supernatants were measured at 24 hpi. Results represent the mean ± SEM from four independent experiments. **<i>p</i>-value < 0.005 compared to inhibitor-treated controls without supplementation (open bars).</p></div

Antimycin A derivatives produced by <i>Streptomyces</i> have potent antiviral activity against WEEV serogroup alphaviruses.

<div><p>(A) Molecular structure of antimycin A. Core structure is shown at the top, and the individual R1 and R2 constituents of derivatives A1a, A2a, A3a, A4a, and A10a are shown below the core structure. Specific atom designations correspond to the NMR results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone.0082318.s005" target="_blank">Table S1</a>.</p> <p>(B) Antiviral activity of commercial antimycin A (AA) and mycophenolic acid (MPA) analyzed with WEEV replicons. Dose titration results for both replicon activity (closed symbols) and viability (open symbols) are presented as the percent untreated control cells and represent the mean ± SEM from at least five independent experiments. Calculated IC₅₀ values for anti-replicon activity are shown on the graph for both compounds, and an average MW of 550 g/mol was used to estimate molar concentrations for commercial antimycin A.</p> <p>(C and D) Antiviral activity of commercial AA and MPA analyzed with infectious WEEV (C) or FMV (D) in BE(2)-C neuronal cells. Cells were infected with WEEV (MOI = 0.1) or FMV (MOI = 1), treated simultaneously with compounds at the indicated concentrations, and virus production was measured by plaque assay at 24 hpi. Results are presented as infectious virion concentration in tissue culture supernatants and represent the mean ± SEM from at least three independent experiments. Calculated IC₅₀ values are shown on the graph for both compounds, and for commercial antimycin A these values were determined as described above in (B). The dashed reference lines represent results from infected cells treated with DMSO control.</p> <p>(E) HPLC separation of individual antimycin A derivatives from commercial stock compound. Only the select portion of an HPLC tracing that contained the four most prominent peaks is shown, and the various grey scale tracings represent different absorbance wavelengths. The identification of individual antimycin A derivatives represented by the four most prominent peaks is shown, where structures were determined by NMR analysis of purified fractions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone.0082318.s005" target="_blank">Table S1</a>).</p> <p>(F) Antiviral activity of individual antimycin A derivatives analyzed with WEEV replicons. Dose titration results are presented as the percent untreated control cells and represent the mean ± SEM from at least four independent experiments. Calculated IC₅₀ values for individual derivatives are shown on the graph, and were calculated using MWs of 548.63, 534.61, 520.58, and 506.55 g/mol for antimycins A1a, A2a, A3a, and A4a, respectively. The methoxy group in 2-methoxyantimycin A3 (MeO-AA3) is located at the 6’ position in the core antimycin structure shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone-0082318-g005" target="_blank">Figure 5A</a>. ND, not determined.</p></div