Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment

Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy

Host strategies against virus entry via the olfactory system.

In mammals, odorants are inhaled through the nose and inside the nasal cavity they trigger olfactory sensory neurons (OSN)  that are located within the olfactory epithelium. OSN project their axons into glomerular structures of the olfactory bulb. There they synapse with dendrites of second-order neurons that project their axons to the olfactory cortex. Thus, olfaction is based on direct interaction of environmental matters with OSN. This poses the question of how neurotropic viruses are prevented from infecting OSN and entering the central nervous system. Recent evidence indicates that upon instillation of neurotropic virus OSN are readily infected. By axonal transport virus reaches the glomerular  layer of the olfactory bulb where it is efficiently curbed by a type I IFN dependent mechanism. In this review local mechanisms limiting virus entry via the olfactory system and virus spread within the CNS are recapitulated in the context of anatomical properties of the olfactory system

Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner.

Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release

Upon intranasal vesicular stomatitis virus infection, astrocytes in the olfactory bulb are important interferon Beta producers that protect from lethal encephalitis.

Previously we found that following intranasal (i.n.) infection with neurotropic vesicular stomatitis virus (VSV) type I interferon receptor (IFNAR) triggering of neuroectodermal cells was critically required to constrain intracerebral virus spread. To address whether locally active IFN-β was induced proximally, we studied spatiotemporal conditions of VSV-mediated IFN-β induction. To this end, we performed infection studies with IFN-β reporter mice. One day after intravenous (i.v.) VSV infection, luciferase induction was detected in lymph nodes. Upon i.n. infection, luciferase induction was discovered at similar sites with delayed kinetics, whereas on days 3 and 4 postinfection enhanced luciferase expression additionally was detected in the foreheads of reporter mice. A detailed analysis of cell type-specific IFN-β reporter mice revealed that within the olfactory bulb IFN-β was expressed by neuroectodermal cells, primarily by astrocytes and to a lesser extent by neurons. Importantly, locally induced type I IFN triggered distal parts of the brain as indicated by the analysis of ISRE-eGFP mice which after i.n. VSV infection showed enhanced green fluorescent protein (eGFP) expression throughout the brain. Compared to wild-type mice, IFN-β(-/-) mice showed increased mortality to i.n. VSV infection, whereas upon i.v. infection no such differences were detected highlighting the biological significance of intracerebrally expressed IFN-β. In conclusion, upon i.n. VSV instillation, IFN-β responses mounted by astrocytes within the olfactory bulb critically contribute to the antiviral defense by stimulating distal IFN-β-negative brain areas and thus arresting virus spread

Oncolytic Rodent Protoparvoviruses Evade a TLR- and RLR-Independent Antiviral Response in Transformed Cells

The oncolytic rodent protoparvoviruses (PVs) minute virus of mice (MVMp) and H-1 parvovirus (H-1PV) are promising cancer viro-immunotherapy candidates capable of both exhibiting direct oncolytic activities and inducing anticancer immune responses (AIRs). Type-I interferon (IFN) production is instrumental for the activation of an efficient AIR. The present study aims at characterizing the molecular mechanisms underlying PV modulation of IFN induction in host cells. MVMp and H-1PV triggered IFN production in semi-permissive normal mouse embryonic fibroblasts (MEFs) and human peripheral blood mononuclear cells (PBMCs), but not in permissive transformed/tumor cells. IFN production triggered by MVMp in primary MEFs required PV replication and was independent of the pattern recognition receptors (PRRs) Toll-like (TLR) and RIG-like (RLR) receptors. PV infection of (semi-)permissive cells, whether transformed or not, led to nuclear translocation of the transcription factors NFĸB and IRF3, hallmarks of PRR signaling activation. Further evidence showed that PV replication in (semi-)permissive cells resulted in nuclear accumulation of dsRNAs capable of activating mitochondrial antiviral signaling (MAVS)-dependent cytosolic RLR signaling upon transfection into naïve cells. This PRR signaling was aborted in PV-infected neoplastic cells, in which no IFN production was detected. Furthermore, MEF immortalization was sufficient to strongly reduce PV-induced IFN production. Pre-infection of transformed/tumor but not of normal cells with MVMp or H-1PV prevented IFN production by classical RLR ligands. Altogether, our data indicate that natural rodent PVs regulate the antiviral innate immune machinery in infected host cells through a complex mechanism. In particular, while rodent PV replication in (semi-)permissive cells engages a TLR-/RLR-independent PRR pathway, in transformed/tumor cells this process is arrested prior to IFN production. This virus-triggered evasion mechanism involves a viral factor(s), which exert(s) an inhibitory action on IFN production, particularly in transformed/tumor cells. These findings pave the way for the development of second-generation PVs that are defective in this evasion mechanism and therefore endowed with increased immunostimulatory potential through their ability to induce IFN production in infected tumor cells

In the Flt3-L derived DC cultures pDC, CD11b-like DC and CD8α-like DC show enhanced CD69 expression after coculture with HCV transfected hepatoma cells.

<p>Flt3-L derived DC were co-cultivated with HCV transfected hepatoma cells or stimulated with VSV-M2 at a MOI 1 for 18 h and analyzed by flow cytometry. Cells were gated on (A) SiglecH⁺ CD11c⁺ pDC, (B) SIRPα⁺ CD11b⁺ CD11b-like DC or (C) Clec9A⁺ CD24⁺ CD8α-like DC and analyzed for the up-regulation of CD69 expression. (D) Quantification of the mean fluorescent intensity (MFI) of CD69 expression by Flt3-L derived pDC, CD11b-like DC and CD8α-like DC (n = 3) (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; 2-way ANOVA, means + SD; n.s. not significant).</p

Type I IFN production by Flt3-L DC cultures is dependent on HCV RNA replication and independent of cell-to-cell contact.

<p>(A) Huh7.5 cells were mock transfected or transfected with SGR, Jc1 or Jc1ΔGDD (ΔGDD) RNA, co-cultivated with Flt3-L derived DC cultures and the amount of IFN-α in the supernatant was determined (n = 3). (B) Mock or HCV RNA transfected hepatoma cells were treated with 0.5 μg/mL RNAse or 1 unit DNAse before Flt3-L DC were added in a coculture. After 18 h, IFN-α was detected in the cell-free supernatants (n = 3). Flt3-L derived DC were seeded and stimulated with Jc1 (C) or 5 μL concentrated SN from Mock or HCV SGR transfected cells (D) (n = 3). (E) Extracellular vesicles were isolated from concentrated SN from Mock, pUCΔGDD (ΔGDD) or HCV SGR transfected cells. 5 μL of isolated vesicles were used to stimulate Flt3-L DC for 18 h and IFN-α was quantified in the cell-free supernatant by ELISA (n = 6). (F) Protein content of isolated extracellular vesicles was analyzed using antibodies against polypeptides typically enriched in exosomes (Hsp70, AnxII, CD81, CD63 and actin). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test and 2-way ANOVA, means + SD; n.s. not significant).</p

HCV-replicating cells induce IFN-β induction <i>in vivo</i>.

<p>(A) IFN-β^+/Δβluc mice were s.c. injected with 5x10⁶ mock transfected (left flank) or HCV subgenomic RNA transfected (SGR2; right flank) Huh7.5 cells. At the indicated time points post s.c. injection, luciferin was injected i.v. and luciferase activity was measured using the IVIS Spectrum <i>in vivo</i> imaging system. (B) Quantification of luciferase activity. Each symbol represents a region of interest (ROI) analysis of an individual animal (n = 6). (C) IFN-β^+/Δβluc mice were s.c. injected with 5x10⁶ pUCΔGDD (ΔGDD) transfected or HCV SGR RNA transfected Huh7.5 cells treated with or without the HCV protease inhibitor telaprevir. At the indicated time points post s.c. injection, luciferin was injected i.v., the luciferase activity measured using the IVIS Spectrum <i>in vivo</i> imaging system and the signal quantified. Each symbol represents an individual animal (n = 3–4). (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; 2-way ANOVA, means + SD; n.s. not significant).</p