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In an experimental viral encephalitis mouse model in which mice are infected with reovirus, we show that IFN-γ induces blood-brain barrier leakage. We show that IFN-γ promotes Rho kinase activity, resulting in actin cytoskeletal contractions in the brain endothelium that lead to vascular junctional disorganization and cell-cell separations. These studies now provide insight into a previously unknown mechanism for how blood-brain barrier breakdown occurs in viral encephalitis and implicates IFN-γ-Rho kinase activity as major contributor to this phenomenon. By identifying this mechanism of blood-brain barrier breakdown, we now provide potential therapeutic targets in treating patients with viral causes of encephalitis with the hope of limiting damage to the central nervous system. Blood-brain barrier (BBB) breakdown is a hallmark of many diseases of the central nervous system (CNS). Loss of BBB integrity in CNS diseases such as viral encephalitis results in the loss of nutrient/oxygen delivery, rapid infiltration of immune cells, and brain swelling that can exacerbate neuronal injury. Despite this, the cellular and molecular mechanisms that underlie BBB breakdown in viral encephalitis are incompletely understood. We undertook a comprehensive analysis of the cellular and molecular signaling events that induce BBB breakdown in an experimental model of virus-induced encephalitis in which neonatal mice are infected with reovirus (serotype 3 strain Abney). We show that BBB leakage during reovirus infection correlates with morphological changes in the vasculature, reductions in pericytes (BBB supporting cells), and disorganization of vascular junctions. Pathway analysis on RNA sequencing from brain endothelial cells identified the activation of interferon (IFN) signaling within the brain vasculature following reovirus infection. Our in vitro and in vivo studies show that type II IFN mediated by IFN-γ, a well known antiviral signal, is a major contributor to BBB leakage during reovirus infection. We show that IFN-γ reduces barrier properties in cultured brain endothelial cells through Rho kinase (ROCK)-mediated cytoskeletal contractions, resulting in junctional disorganization and cell-cell separations. In vivo neutralization of IFN-γ during reovirus infection significantly improved BBB integrity, pericyte coverage, attenuated vascular ROCK activity, and junctional disorganization. Our work supports a model in which IFN-γ acts directly on the brain endothelium to induce BBB breakdown through a mechanism involving ROCK-induced junctional disorganization. IMPORTANCE In an experimental viral encephalitis mouse model in which mice are infected with reovirus, we show that IFN-γ induces blood-brain barrier leakage. We show that IFN-γ promotes Rho kinase activity, resulting in actin cytoskeletal contractions in the brain endothelium that lead to vascular junctional disorganization and cell-cell separations. These studies now provide insight into a previously unknown mechanism for how blood-brain barrier breakdown occurs in viral encephalitis and implicates IFN-γ-Rho kinase activity as major contributor to this phenomenon. By identifying this mechanism of blood-brain barrier breakdown, we now provide potential therapeutic targets in treating patients with viral causes of encephalitis with the hope of limiting damage to the central nervous system.

Variants at the leucine-rich repeat kinase-2 ( LRRK2 ) and α-synuclein ( SNCA ) loci are associated with Parkinson’s disease (PD) risk. Viral infections have also been linked to increased risk of developing PD. In exploring a role for each of the encoded proteins in host response against brain-directed viral infections, we previously demonstrated that two Lrrk2 knock-in variants as well as Snca expression altered survival rates from viral encephalitis following intranasal inoculation of newborn mice with a double-stranded RNA virus: respiratory-enteric-orphan virus, serotype-3 strain Dearing (reovirus T3D). Here, we examined whether outcomes of direct inoculation of the brain by reovirus T3D, which invariably causes lethal encephalitis within 15 days, would also be modified by variants in Lrrk2 and Snca . When we inoculated newborn mice intracerebrally with reovirus T3D, we found that compared to wild-type littermates Lrrk2 p.G2019S kinase-hyperactive and p.D1994S kinase-inactive mutant mice did not show any significant difference in time-to-death or in viral titres in the brain, and revealed no sex difference. In parallel studies, the reduction or absence of endogenous α-synuclein did not alter the course of disease in reovirus T3D-infected mice. Together, these findings suggest that while variants in the PD-linked Lrrk2 and Snca genes influenced disease outcomes of intranasally acquired reovirus T3D encephalitis, they did not affect survival outcomes in the intracerebrally acquired reovirus T3D encephalitis model.

Background: Viral encephalitis (VE) is a common infectious disease of the central nervous system in children. Children with severe disease may have progressive neurological damage and even lead to death. Aims: To assess the serum miR-142-3p levels in children with VE and the correlation between miR-142-3p and the severity and prognosis of VE. Besides, its relationship with nerve injury and inflammatory response was assessed. Methods: Children with VE were regarded as a case group and healthy children served as control. The content of serum miR-142-3p was determined using real-time quantitative PCR. The risk factors associated with severity and prognosis of cases were evaluated using logistic analysis. The discrepancy in miR-142-3p levels, nerve injury-related indicators, and inflammatory cytokines were contrasted among groups. The ROC curve was conducted to assess the diagnostic performance of serum miR-142-3p in predicting prognosis of children with VE. Results: The altered expression of miR-142-3p in serum of children with VE was enhanced in contrast to healthy control. Serum nerve injury indicators MBP, β-EP, and NSE levels and serum inflammatory cytokines IL-6, IL-18, and IFN-γ were high in children with VE in contrast to healthy control, and had positive relevance with serum miR-142-3p. Besides, serum miR-142-3p was a risk factor associated with the severity and prognosis of children with VE. Serum miR-142-3p had diagnostic performance in predicting the prognosis of children with VE. Conclusion: Serum miR-142-3p content is high in children with VE and maybe a diagnosis marker for predicting prognosis. The specific miR-142-3p expression may be directly related to the severity of nerve injury and inflammatory response for VE.

Microglia serve as a front-line defense against neuroinvasive viral infection, however, determination of their actual transcriptional profiles under conditions of health and disease is challenging. Here, we used various experimental approaches to delineate the transcriptional landscape of microglia during viral infection. Intriguingly, multiple activation genes were found to be artificially induced in sorted microglia and we demonstrated that shear stress encountered during cell sorting was one of the key inducers. Post-hoc analysis revealed that publicly available large-scale single-cell RNA sequencing datasets were significantly tainted by aberrant signatures that are associated with cell sorting. By exploiting the ribosomal tagging approach, we developed a strategy to enrich microglia-specific transcripts by comparing immunoprecipitated RNA with total RNA. Such enriched transcripts were instrumental in defining bona fide signatures of microglia under conditions of health and virus infection. These unified microglial signatures may serve as a benchmark to retrospectively assess ex vivo artefacts from available atlases. Leveraging the microglial translatome, we found enrichment of genes implicated in T-cell activation and cytokine production during the course of VSV infection. These data linked microglia with T-cell re-stimulation and further underscored that microglia are involved in shaping antiviral T-cell responses in the brain. Collectively, our study defines the transcriptional landscape of microglia under steady state and during viral encephalitis and highlights cellular interactions between microglia and T cells that contribute to the control of virus dissemination.

Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. For sensitivity testing HIV and HBV positive sera were serially diluted in CSF from an uninfected patient. NGS repeatedly detected HIV and HBV sequences present at concentrations from 10 5 to 10 2 and from 10 5 to 10 viral copies/reaction, respectively. However, when the same protocols were applied to RT-PCR/PCR positive CSF samples from 6 patients with enteroviral encephalitis (median viral load 47 copies/ml) and 15 patients with HSV, CMV or VZV encephalitis (median viral load 148 copies/ml), only 7 (28.6%) were identified as positive. In conclusions, while NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common.

Programmed cell death-1 (PD-1) receptor signaling dampens the functionality of T cells faced with repetitive antigenic stimulation from chronic infections or tumors. Using intracerebral (i.c.) inoculation with mouse polyomavirus (MuPyV), we have shown that CD8 T cells establish a PD-1 , tissue-resident memory population in the brains (bT ) of mice with a low-level persistent infection. In MuPyV encephalitis, PD-L1 was expressed on infiltrating myeloid cells, microglia and astrocytes, but not on oligodendrocytes. Engagement of PD-1 on anti-MuPyV CD8 T cells limited their effector activity. NanoString gene expression analysis showed that neuroinflammation was higher in PD-L1 than wild type mice at day 8 post-infection, the peak of the MuPyV-specific CD8 response. During the persistent phase of infection, however, the absence of PD-1 signaling was found to be associated with a lower inflammatory response than in wild type mice. Genetic disruption and intracerebroventricular blockade of PD-1 signaling resulted in an increase in number of MuPyV-specific CD8 bT and the fraction of these cells expressing CD103, the αE integrin commonly used to define tissue-resident T cells. However, PD-L1 mice persistently infected with MuPyV showed impaired virus control upon i.c. re-infection with MuPyV. Collectively, these data reveal a temporal duality in PD-1-mediated regulation of MuPyV-associated neuroinflammation. PD-1 signaling limited the severity of neuroinflammation during acute infection but sustained a level of inflammation during persistent infection for maintaining control of virus re-infection.

  Human herpesvirus-6 (HHV-6) infection can rarely cause life-threatening conditions, such as encephalitis, in otherwise healthy children, with unclear pathogenesis. We studied a child who presented with acute HHV-6 encephalitis at the age of 10 months and who was homozygous for a novel missense mutation in IRAK4 , encoding interleukin-1 receptor-associated kinase 4, identified by whole-exome sequencing. We tested the damaging impact of this mutation in silico by molecular dynamics simulations and in vitro by biochemical and functional experiments utilizing cell lines and patient’s cells. We found that the mutation is severely hypomorphic, impairing both the expression and function of IRAK-4. Patient’s leukocytes had barely detectable levels of IRAK-4 and diminished anti-viral immune responses to various stimuli inducing different Toll-like receptors and cytosolic nucleic acid sensors. Overall, these findings suggest that acute HHV-6 encephalitis can result from inborn errors of immunity to virus. This study represents the first report of isolated acute HHV-6 infection causing encephalitis in an inherited primary immunodeficiency, notably autosomal recessive (AR) partial IRAK-4 deficiency, and the first report of AR IRAK-4 deficiency presenting with a severe viral disease, notably HHV-6 encephalitis upon an acute infection, thereby expanding the clinical spectrum of IRAK-4 deficiency.

Abstract PurposeEnterovirus A71 (EV71) causes a broad spectrum of childhood diseases, ranging from asymptomatic infection or self-limited hand-foot-and-mouth disease (HFMD) to life-threatening encephalitis. The molecular mechanisms underlying these different clinical presentations remain unknown. We hypothesized that EV71 encephalitis in children might reflect an intrinsic host single-gene defect of antiviral immunity. We searched for mutations in the toll-like receptor 3 (TLR3) gene. Such mutations have already been identified in children with herpes simplex virus encephalitis (HSE).MethodsWe sequenced TLR3 and assessed the impact of the mutations identified. We tested dermal fibroblasts from a patient with EV71 encephalitis and a TLR3 mutation and other patients with known genetic defects of TLR3 or related genes, assessing the response of these cells to TLR3 agonist poly(I:C) stimulation and EV71 infection.ResultsThree children with EV71 encephalitis were heterozygous for rare mutations—TLR3 W769X, E211K, and R867Q—all of which were shown to affect TLR3 function. Furthermore, fibroblasts from the patient heterozygous for the W769X mutation displayed an impaired, but not abolished, response to poly(I:C). We found that TLR3-deficient and TLR3-heterozygous W769X fibroblasts were highly susceptible to EV71 infection.ConclusionsAutosomal dominant TLR3 deficiency may underlie severe EV71 infection with encephalitis. Human TLR3 immunity is essential to protect the central nervous system against HSV-1 and EV71. Children with severe EV71 infections, such as encephalitis in particular, should be tested for inborn errors of TLR3 immunity.

Background Heme oxygenase-1 (HO-1) is a critical cytoprotective enzyme that limits oxidative stress, inflammation, and cellular injury within the central nervous system (CNS) and other tissues. We previously demonstrated that HO-1 protein expression is decreased within the brains of HIV+ subjects and that this HO-1 reduction correlates with CNS immune activation and neurocognitive dysfunction. To define a potential CNS protective role for HO-1 against HIV, we analyzed a well-characterized HIV autopsy cohort for two common HO-1 promoter region polymorphisms that are implicated in regulating HO-1 promoter transcriptional activity, a (GT)n dinucleotide repeat polymorphism and a single nucleotide polymorphism (A(-413)T). Shorter HO-1 (GT)n repeats and the ‘A’ SNP allele associate with higher HO-1 promoter activity. Methods Brain dorsolateral prefrontal cortex tissue samples from an autopsy cohort of HIV−, HIV+, and HIV encephalitis (HIVE) subjects ( n  = 554) were analyzed as follows: HO-1 (GT)n polymorphism allele lengths were determined by PCR and capillary electrophoresis, A(-413)T SNP alleles were determined by PCR with allele specific probes, and RNA expression of selected neuroimmune markers was analyzed by quantitative PCR. Results HIV+ subjects with shorter HO-1 (GT)n alleles had a significantly lower risk of HIVE; however, shorter HO-1 (GT)n alleles did not correlate with CNS or peripheral viral loads. In HIV+ subjects without HIVE, shorter HO-1 (GT)n alleles associated significantly with lower expression of brain type I interferon response markers ( MX1 , ISG15 , and IRF1 ) and T-lymphocyte activation markers ( CD38 and GZMB ). No significant correlations were found between the HO-1 (GT)n repeat length and brain expression of macrophage markers ( CD163 , CD68 ), endothelial markers ( PECAM1 , VWF ), the T-lymphocyte marker CD8A , or the B-lymphocyte maker CD19 . Finally, we found no significant associations between the A(-413)T SNP and HIVE diagnosis, HIV viral loads, or any neuroimmune markers. Conclusion Our data suggest that an individual’s HO-1 promoter region (GT)n polymorphism allele repeat length exerts unique modifying risk effects on HIV-induced CNS neuroinflammation and associated neuropathogenesis. Shorter HO-1 (GT)n alleles increase HO-1 promoter activity, which could provide neuroprotection through decreased neuroimmune activation. Therapeutic strategies that induce HO-1 expression could decrease HIV-associated CNS neuroinflammation and decrease the risk for development of HIV neurological disease.

Emergency hematopoiesis facilitates the rapid expansion of inflammatory immune cells in response to infections by pathogens, a process that must be carefully regulated to prevent potentially life threatening inflammatory responses. Here, we describe a novel regulatory role for the cytokine IFNγ that is critical for preventing fatal encephalitis after viral infection. HSV1 encephalitis (HSE) is triggered by the invasion of the brainstem by inflammatory monocytes and neutrophils. In mice lacking IFNγ (GKO), we observed unrestrained increases in G-CSF levels but not in GM-CSF or IL-17. This resulted in uncontrolled expansion and infiltration of apoptosis-resistant, degranulating neutrophils into the brainstem, causing fatal HSE in GKO but not WT mice. Excessive G-CSF in GKO mice also induced granulocyte derived suppressor cells, which inhibited T-cell proliferation and function, including production of the anti-inflammatory cytokine IL-10. Unexpectedly, we found that IFNγ suppressed G-CSF signaling by increasing SOCS3 expression in neutrophils, resulting in apoptosis. Depletion of G-CSF, but not GM-CSF, in GKO mice induced neutrophil apoptosis and reinstated IL-10 secretion by T cells, which restored their ability to limit innate inflammatory responses resulting in protection from HSE. Our studies reveals a novel, complex interplay among IFNγ, G-CSF and IL-10, which highlights the opposing roles of G-CSF and IFNγ in regulation of innate inflammatory responses in a murine viral encephalitis model and reveals G-CSF as a potential therapeutic target. Thus, the antagonistic G-CSF-IFNγ interactions emerge as a key regulatory node in control of CNS inflammatory responses to virus infection.