Explore the vast range of titles available.

Background Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. Methods Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR −/− ) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. Results Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. Conclusions These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1-/- mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1-/- mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.

In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1 LPC-KO ) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1 LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1 LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.

Background Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo. Results IBs directed against the polySTs prevented the translocation of the enzymes from the ER to the Golgi-apparatus. Co-immunoprecipitation of ST8SiaII and ST8SiaIV with the corresponding IBs confirmed the intracellular interaction with their cognate antigens. In CHO cells overexpressing ST8SiaII and ST8SiaIV, respectively, the transfection with αST8SiaII-IB or αST8SiaIV-IB inhibited significantly the cell surface expression of polysialylated NCAM. Furthermore stable expression of ST8SiaII-IB, ST8SiaIV-IB and luciferase in the rhabdomyosarcoma cell line TE671 reduced cell surface expression of polySia and delayed tumor growth if cells were xenografted into C57BL/6 J RAG-2 mice. Conclusion Data obtained strongly indicate that αST8SiaII-IB and αST8SiaIV-IB are promising experimental tools to analyze the individual role of the two enzymes during brain development and during migration and proliferation of tumor cells.

Background Toxoplasma gondii ( T. gondii ) is a highly successful parasite being able to cross all biological barriers of the body, finally reaching the central nervous system (CNS). Previous studies have highlighted the critical involvement of the blood–brain barrier (BBB) during T. gondii invasion and development of subsequent neuroinflammation. Still, the potential contribution of the choroid plexus (CP), the main structure forming the blood–cerebrospinal fluid (CSF) barrier (BCSFB) have not been addressed. Methods To investigate T. gondii invasion at the onset of neuroinflammation, the CP and brain microvessels (BMV) were isolated and analyzed for parasite burden. Additionally, immuno-stained brain sections and three-dimensional whole mount preparations were evaluated for parasite localization and morphological alterations. Activation of choroidal and brain endothelial cells were characterized by flow cytometry. To evaluate the impact of early immune responses on CP and BMV, expression levels of inflammatory mediators, tight junctions (TJ) and matrix metalloproteinases (MMPs) were quantified. Additionally, FITC-dextran was applied to determine infection-related changes in BCSFB permeability. Finally, the response of primary CP epithelial cells to T. gondii parasites was tested in vitro. Results Here we revealed that endothelial cells in the CP are initially infected by T. gondii , and become activated prior to BBB endothelial cells indicated by MHCII upregulation. Additionally, CP elicited early local immune response with upregulation of IFN-γ, TNF, IL-6, host-defence factors as well as swift expression of CXCL9 chemokine, when compared to the BMV. Consequently, we uncovered distinct TJ disturbances of claudins, associated with upregulation of MMP-8 and MMP-13 expression in infected CP in vivo, which was confirmed by in vitro infection of primary CP epithelial cells. Notably, we detected early barrier damage and functional loss by increased BCSFB permeability to FITC-dextran in vivo, which was extended over the infection course. Conclusions Altogether, our data reveal a close interaction between T. gondii infection at the CP and the impairment of the BCSFB function indicating that infection-related neuroinflammation is initiated in the CP.

Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Previous experiments using IAV/H1N1 infection models have shown impaired hippocampal neuronal morphology and cognitive abilities, but the underlying pathways have not been fully described. In this study, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes ample peripheral immune response followed by a temporary blood-brain barrier disturbance. Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, detailed multidimensional flow cytometric characterization of immune cells uncovered subtle alterations in the activation status of microglial cells. More specifically, we detected an altered expression pattern of major histocompatibility complex classes I and II, CD80, and F4/80 accompanied by elevated mRNA levels of CD36, CD68, C1QA, and C3, suggesting evolved synaptic pruning. To closer evaluate how these profound changes affect synaptic balance, we established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry. The introduction of this novel technique enabled us to simultaneously quantify the abundance of pre- and postsynapses from distinct brain regions. Our data reveal a significant reduction of VGLUT1 in excitatory presynaptic terminals in the cortex and hippocampus, identifying a subtle dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations. IMPORTANCE Influenza A virus (IAV) causes mainly respiratory tract disease with fever and fatigue but is also associated with behavioral alterations in humans. Here, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes peripheral immune response followed by a temporary blood-brain barrier disturbance. Characterization of immune cells uncovered subtle alterations in the activation status of microglia cells that might reshape neuronal synapses. We established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry to more closely study the synapses. Thus, we detected a specific dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations.

MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10 , IL-1β , CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5′-triphosphate (PPP-) group that impairs degradation by the canonical 5′-3′ degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5′-3′ exonuclease XRN1. NUDT2 removes 5′-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5′-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals. RNA of some viruses is protected from degradation by a 5′ triphosphate group. Here the authors identify nudix hydrolase 2 (NUDT2) as novel antiviral defense protein that dephosphorylates viral RNA and thereby enables its degradation.

The cGAS-STING pathway is a pivotal element of the innate immune system, recognizing cytosolic DNA to initiate the production of type I interferons and pro-inflammatory cytokines. This study investigates the alterations of the cGAS-STING signaling components in the cortex and hippocampus of mice aged 24 and 108 weeks. In the cortex of old mice, an increase in the dsDNA sensor protein cGAS and its product 2′3′-cGAMP was observed, without corresponding activation of downstream signaling, suggesting an uncoupling of cGAS activity from STING activation. This phenomenon may be attributed to increased dsDNA concentrations in the EC neurons, potentially arising from nuclear DNA damage. Contrastingly, the hippocampus did not exhibit increased cGAS activity with aging, but there was a notable elevation in STING levels, particularly in microglia, neurons and astrocytes. This increase in STING did not correlate with enhanced IRF3 activation, indicating that brain inflammation induced by the cGAS-STING pathway may manifest extremely late in the aging process. Furthermore, we highlight the role of autophagy and its interplay with the cGAS-STING pathway, with evidence of autophagy dysfunction in aged hippocampal neurons leading to STING accumulation. These findings underscore the complexity of the cGAS-STING pathway’s involvement in brain aging, with regional variations in activity and potential implications for neurodegenerative diseases.

Background Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. Method Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. Results We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. Conclusions Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.