News

group leader: Prof. Schughart

Recent Submissions

  • Long-Term Consequence of Non-neurotropic H3N2 Influenza A Virus Infection for the Progression of Alzheimer's Disease Symptoms.

    Hosseini, Shirin; Michaelsen-Preusse, Kristin; Schughart, Klaus; Korte, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-04-28)
    Influenza viruses until today are a leading cause of worldwide severe pandemics and represent a major threat to human and animal health. Although the primary target of influenza viruses is the lung, infection may manifest with acute and even chronic neurological complications (e.g., status epilepticus, encephalopathies, and encephalitis) potentially increasing the long-term risk for neurodegenerative diseases. We previously described that a peripheral influenza A virus (IAV) infection caused by non-neurotropic H3N2 (maHK68) variant leads to long-term neuroinflammation and synapse loss together with impaired memory formation in young adult mice. Processes of neuroinflammation have been associated with neurodegenerative diseases such as Alzheimer's disease (AD) and prolonged or excessive innate immune responses are considered a risk factor for AD. Here, the role of purely peripheral IAV infection for the development and progression of AD in a transgenic mouse model (APP/PS1) was investigated. At 2 months of age, mice were infected with H3N2 IAV and the detailed analysis of microglia morphology revealed neuroinflammation in the hippocampus already of 6 months old non-infected APP/PS1 mice together with impaired spatial learning, however, microglia activation, amyloid-β plaques load and cognitive impairments were even more pronounced in APP/PS1 mice upon H3N2 infection. Moreover, CA1 hippocampal dendritic spine density was reduced even at 120 dpi compared to wild-type and also to non-infected APP/PS1 mice, whereas neuronal cells number was not altered. These findings demonstrate that non-neurotropic H3N2 IAV infection as a peripheral immune stimulation may exacerbate AD symptoms possibly by triggering microglial hyperactivation.
  • Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

    Hein, Marc D; Arora, Prerna; Marichal-Gallardo, Pavel; Winkler, Michael; Genzel, Yvonne; Pöhlmann, Stefan; Schughart, Klaus; Kupke, Sascha Y; Reichl, Udo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2021-05-03)
    In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.
  • Blood transcriptome analysis of patients with uncomplicated bacterial infection and sepsis.

    Herwanto, Velma; Tang, Benjamin; Wang, Ya; Shojaei, Maryam; Nalos, Marek; Shetty, Amith; Lai, Kevin; McLean, Anthony S; Schughart, Klaus; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BioMedCentral, 2021-02-27)
    The data presented here consists of raw and processed transcriptome data obtained by next generation RNA sequencing from 105 peripheral blood samples from patients with uncomplicated infections, patients who developed sepsis, septic shock patients, and healthy controls. It is provided as raw sequenced reads and as normalized log2 transformed relative expression levels. This data will allow performing detailed analyses of gene expression changes between uncomplicated infections and sepsis patients, such as identification of differentially expressed genes, co-regulated modules as well as pathway activation studies.
  • Improved Functionality of Exhausted Intrahepatic CXCR5+ CD8+ T Cells Contributes to Chronic Antigen Clearance Upon Immunomodulation.

    Kumashie, Kingsley Gideon; Cebula, Marcin; Hagedorn, Claudia; Kreppel, Florian; Pils, Marina C; Koch-Nolte, Friedrich; Rissiek, Björn; Wirth, Dagmar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-02-03)
    Chronic hepatotropic viral infections are characterized by exhausted CD8+ T cells in the presence of cognate antigen in the liver. The impairment of T cell response limits the control of chronic hepatotropic viruses. Immune-modulatory strategies are attractive options to re-invigorate exhausted T cells. However, in hepatotropic viral infections, the knowledge about immune-modulatory effects on the in-situ regulation of exhausted intrahepatic CD8+ T cells is limited. In this study, we elucidated the functional heterogeneity in the pool of exhausted CD8+ T cells in the liver of mice expressing the model antigen Ova in a fraction of hepatocytes. We found a subpopulation of intrahepatic CXCR5+ Ova-specific CD8+ T cells, which are profoundly cytotoxic, exhibiting efficient metabolic functions as well as improved memory recall and self-maintenance. The intrahepatic Ova-specific CXCR5+ CD8+ T cells are possibly tissue resident cells, which may rely largely on OXPHOS and glycolysis to fuel their cellular processes. Importantly, host conditioning with CpG oligonucleotide reinvigorates and promotes exhausted T cell expansion, facilitating complete antigen eradication. The CpG oligonucleotide-mediated reinvigoration may support resident memory T cell formation and the maintenance of CXCR5+ Ova-specific CD8+ T cells in the liver. These findings suggest that CpG oligodinucleotide may preferentially target CXCR5+ CD8+ T cells for expansion to facilitate the revival of exhausted T cells. Thus, therapeutic strategies aiming to expand CXCR5+ CD8+ T cells might provide a novel approach against chronic liver infection.
  • Highly dampened blood transcriptome response in HIV patients after influenza infection.

    Sellers, Subhashini A; Fischer, William A; Heise, Mark T; Schughart, Klaus; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature research, 2021-02-24)
    Respiratory viral (RV) infections represent a major threat for human health worldwide. Persons with HIV (PWH) have a compromised immune response and are thought to be at higher risk for severe RV disease. However, very little is known about the host immune response to RV infection in PWH. Here, we investigated gene expression changes in the peripheral blood of PWH co-infected with RV. Only very few differentially expressed genes could be detected between PWH with and without RV infection, suggesting that the immune response to RV in PWH is strongly dampened. Our data provides important insights into the host response to RV infections in HIV patients.
  • Genetic Dissection of the Regulatory Mechanisms of Ace2 in the Infected Mouse Lung.

    Xu, Fuyi; Gao, Jun; Bergmann, Silke; Sims, Amy C; Ashbrook, David G; Baric, Ralph S; Cui, Yan; Jonsson, Colleen B; Li, Kui; Williams, Robert W; et al. (Frontiers, 2021-01-08)
    Acute lung injury (ALI) is an important cause of morbidity and mortality after viral infections, including influenza A virus H1N1, SARS-CoV, MERS-CoV, and SARS-CoV-2. The angiotensin I converting enzyme 2 (ACE2) is a key host membrane-bound protein that modulates ALI induced by viral infection, pulmonary acid aspiration, and sepsis. However, the contributions of ACE2 sequence variants to individual differences in disease risk and severity after viral infection are not understood. In this study, we quantified H1N1 influenza-infected lung transcriptomes across a family of 41 BXD recombinant inbred strains of mice and both parents-C57BL/6J and DBA/2J. In response to infection Ace2 mRNA levels decreased significantly for both parental strains and the expression levels was associated with disease severity (body weight loss) and viral load (expression levels of viral NA segment) across the BXD family members. Pulmonary RNA-seq for 43 lines was analyzed using weighted gene co-expression network analysis (WGCNA) and Bayesian network approaches. Ace2 not only participated in virus-induced ALI by interacting with TNF, MAPK, and NOTCH signaling pathways, but was also linked with high confidence to gene products that have important functions in the pulmonary epithelium, including Rnf128, Muc5b, and Tmprss2. Comparable sets of transcripts were also highlighted in parallel studies of human SARS-CoV-infected primary human airway epithelial cells. Using conventional mapping methods, we determined that weight loss at two and three days after viral infection maps to chromosome X-the location of Ace2. This finding motivated the hierarchical Bayesian network analysis, which defined molecular endophenotypes of lung infection linked to Ace2 expression and to a key disease outcome. Core members of this Bayesian network include Ace2, Atf4, Csf2, Cxcl2, Lif, Maml3, Muc5b, Reg3g, Ripk3, and Traf3. Collectively, these findings define a causally-rooted Ace2 modulatory network relevant to host response to viral infection and identify potential therapeutic targets for virus-induced respiratory diseases, including those caused by influenza and coronaviruses.
  • Prospective validation study of prognostic biomarkers to predict adverse outcomes in patients with COVID-19: a study protocol.

    Tang, Benjamin; Shojaei, Maryam; Wang, Ya; Nalos, Marek; McLean, Anthony; Afrasiabi, Ali; Kwan, Tim N; Kuan, Win Sen; Zerbib, Yoann; Herwanto, Velma; et al. (BMJ publishing group, 2021-01-06)
    Introduction: Accurate triage is an important first step to effectively manage the clinical treatment of severe cases in a pandemic outbreak. In the current COVID-19 global pandemic, there is a lack of reliable clinical tools to assist clinicians to perform accurate triage. Host response biomarkers have recently shown promise in risk stratification of disease progression; however, the role of these biomarkers in predicting disease progression in patients with COVID-19 is unknown. Here, we present a protocol outlining a prospective validation study to evaluate the biomarkers' performance in predicting clinical outcomes of patients with COVID-19. Methods and analysis: This prospective validation study assesses patients infected with COVID-19, in whom blood samples are prospectively collected. Recruited patients include a range of infection severity from asymptomatic to critically ill patients, recruited from the community, outpatient clinics, emergency departments and hospitals. Study samples consist of peripheral blood samples collected into RNA-preserving (PAXgene/Tempus) tubes on patient presentation or immediately on study enrolment. Real-time PCR (RT-PCR) will be performed on total RNA extracted from collected blood samples using primers specific to host response gene expression biomarkers that have been previously identified in studies of respiratory viral infections. The RT-PCR data will be analysed to assess the diagnostic performance of individual biomarkers in predicting COVID-19-related outcomes, such as viral pneumonia, acute respiratory distress syndrome or bacterial pneumonia. Biomarker performance will be evaluated using sensitivity, specificity, positive and negative predictive values, likelihood ratios and area under the receiver operating characteristic curve. Ethics and dissemination: This research protocol aims to study the host response gene expression biomarkers in severe respiratory viral infections with a pandemic potential (COVID-19). It has been approved by the local ethics committee with approval number 2020/ETH00886. The results of this project will be disseminated in international peer-reviewed scientific journals.
  • Pituitary Tumor Transforming Gene 1 Orchestrates Gene Regulatory Variation in Mouse Ventral Midbrain During Aging

    Gui, Yujuan; Thomas, Mélanie H.; Garcia, Pierre; Karout, Mona; Halder, Rashi; Michelucci, Alessandro; Kollmus, Heike; Zhou, Cuiqi; Melmed, Shlomo; Schughart, Klaus; et al. (Frontiers Media SA, 2020-09-23)
    Dopaminergic neurons in the midbrain are of particular interest due to their role in diseases such as Parkinson’s disease and schizophrenia. Genetic variation between individuals can affect the integrity and function of dopaminergic neurons but the DNA variants and molecular cascades modulating dopaminergic neurons and other cells types of ventral midbrain remain poorly defined. Three genetically diverse inbred mouse strains – C57BL/6J, A/J, and DBA/2J – differ significantly in their genomes (∼7 million variants), motor and cognitive behavior, and susceptibility to neurotoxins. To further dissect the underlying molecular networks responsible for these variable phenotypes, we generated RNA-seq and ChIP-seq data from ventral midbrains of the 3 mouse strains. We defined 1000–1200 transcripts that are differentially expressed among them. These widespread differences may be due to altered activity or expression of upstream transcription factors. Interestingly, transcription factors were significantly underrepresented among the differentially expressed genes, and only one transcription factor, Pttg1, showed significant differences between all three strains. The changes in Pttg1 expression were accompanied by consistent alterations in histone H3 lysine 4 trimethylation at Pttg1 transcription start site. The ventral midbrain transcriptome of 3-month-old C57BL/6J congenic Pttg1−=− mutants was only modestly altered, but shifted toward that of A/J and DBA/2J in 9-month-old mice. Principle component analysis (PCA) identified the genes underlying the transcriptome shift and deconvolution of these bulk RNA-seq changes using midbrain single cell RNA-seq data suggested that the changes were occurring in several different cell types, including neurons, oligodendrocytes, and astrocytes. Taken together, our results show that Pttg1 contributes to gene regulatory variation between mouse strains and influences mouse midbrain transcriptome during aging
  • A New Synuclein-Transgenic Mouse Model for Early Parkinson's Reveals Molecular Features of Preclinical Disease.

    Hendrickx, Diana M; Garcia, Pierre; Ashrafi, Amer; Sciortino, Alessia; Schmit, Kristopher J; Kollmus, Heike; Nicot, Nathalie; Kaoma, Tony; Vallar, Laurent; Buttini, Manuel; et al. (Springer, 2020-09-30)
    Understanding Parkinson's disease (PD), in particular in its earliest phases, is important for diagnosis and treatment. However, human brain samples are collected post-mortem, reflecting mainly end-stage disease. Because brain samples of mouse models can be collected at any stage of the disease process, they are useful in investigating PD progression. Here, we compare ventral midbrain transcriptomics profiles from α-synuclein transgenic mice with a progressive, early PD-like striatal neurodegeneration across different ages using pathway, gene set, and network analysis methods. Our study uncovers statistically significant altered genes across ages and between genotypes with known, suspected, or unknown function in PD pathogenesis and key pathways associated with disease progression. Among those are genotype-dependent alterations associated with synaptic plasticity and neurotransmission, as well as mitochondria-related genes and dysregulation of lipid metabolism. Age-dependent changes were among others observed in neuronal and synaptic activity, calcium homeostasis, and membrane receptor signaling pathways, many of which linked to G-protein coupled receptors. Most importantly, most changes occurred before neurodegeneration was detected in this model, which points to a sequence of gene expression events that may be relevant for disease initiation and progression. It is tempting to speculate that molecular changes similar to those changes observed in our model happen in midbrain dopaminergic neurons before they start to degenerate. In other words, we believe we have uncovered molecular changes that accompany the progression from preclinical to early PD.
  • Long-Term Neuroinflammation Induced by Influenza A Virus Infection and the Impact on Hippocampal Neuron Morphology and Function.

    Hosseini, Shirin; Wilk, Esther; Michaelsen-Preusse, Kristin; Gerhauser, Ingo; Baumgärtner, Wolfgang; Geffers, Robert; Schughart, Klaus; Korte, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Society for Neuroscience, 2018-02-27)
    Acute influenza infection has been reported to be associated with neurological symptoms. However, the long-term consequences of an infection with neurotropic and non-neurotropic influenza A virus (IAV) variants for the CNS remain elusive. We can show that spine loss in the hippocampus after infection with neurotropic H7N7 (rSC35M) and non-neurotropic H3N2 (maHK68) in female C57BL/6 mice persists well beyond the acute phase of the disease. Although spine number was significantly reduced at 30 d postinfection (dpi) with H7N7 or H3N2, full recovery could only be observed much later at 120 dpi. Infection with H1N1 virus, which was shown previously to affect spine number and hippocampus-dependent learning acutely, had no significant long-term effects. Spine loss was associated with an increase in the number of activated microglia, reduced long-term potentiation in the hippocampus, and impairment in spatial memory formation, indicating that IAV-associated inflammation induced functional and structural alterations in hippocampal networks. Transcriptome analyses revealed regulation of many inflammatory and neuron- and glia-specific genes in H3N2- and H7N7-infected mice at day 18 and in H7N7-infected mice at day 30 pi that related to the structural and functional alterations. Our data provide evidence that neuroinflammation induced by neurotropic H7N7 and infection of the lung with a non-neurotropic H3N2 IAV result in long-term impairments in the CNS. IAV infection in humans may therefore not only lead to short-term responses in infected organs, but may also trigger neuroinflammation and associated chronic alterations in the CNS.SIGNIFICANCE STATEMENT In the acute phase of influenza infection, neuroinflammation can lead to alterations in hippocampal neuronal morphology and cognitive deficits. The results of this study now also provide evidence that neuroinflammation induced by influenza A virus (IAV) infection can induce longer-lasting, virus-specific alterations in neuronal connectivity that are still detectable 1 month after infection and are associated with impairments in spatial memory formation. IAV infection in humans may therefore not only lead to short-term responses in infected organs, but may also trigger neuroinflammation and associated chronic alterations in the CNS.
  • Pathway mapping of leukocyte transcriptome in influenza patients reveals distinct pathogenic mechanisms associated with progression to severe infection.

    Zerbib, Yoann; Jenkins, Emily K; Shojaei, Maryam; Meyers, Adrienne F A; Ho, John; Ball, T Blake; Keynan, Yoav; Pisipati, Amarnath; Kumar, Aseem; Kumar, Anand; et al. (BioMedCentral, 2020-02-17)
    BACKGROUND: Influenza infections produce a spectrum of disease severity, ranging from a mild respiratory illness to respiratory failure and death. The host-response pathways associated with the progression to severe influenza disease are not well understood. METHODS: To gain insight into the disease mechanisms associated with progression to severe infection, we analyzed the leukocyte transcriptome in severe and moderate influenza patients and healthy control subjects. Pathway analysis on differentially expressed genes was performed using a topology-based pathway analysis tool that takes into account the interaction between multiple cellular pathways. The pathway profiles between moderate and severe influenza were then compared to delineate the biological mechanisms underpinning the progression from moderate to severe influenza. RESULTS: 107 patients (44 severe and 63 moderate influenza patients) and 52 healthy control subjects were included in the study. Severe influenza was associated with upregulation in several neutrophil-related pathways, including pathways involved in neutrophil differentiation, migration, degranulation and neutrophil extracellular trap (NET) formation. The degree of upregulation in neutrophil-related pathways were significantly higher in severely infected patients compared to moderately infected patients. Severe influenza was also associated with downregulation in immune response pathways, including pathways involved in antigen presentation such as CD4+ T-cell co-stimulation, CD8+ T cell and Natural Killer (NK) cells effector functions. Apoptosis pathways were also downregulated in severe influenza patients compare to moderate and healthy controls. CONCLUSIONS: These findings showed that there are changes in gene expression profile that may highlight distinct pathogenic mechanisms associated with progression from moderate to severe influenza infection.
  • A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes.

    Kollmus, Heike; Fuchs, Helmut; Lengger, Christoph; Haselimashhadi, Hamed; Bogue, Molly A; Östereicher, Manuela A; Horsch, Marion; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Amarie, Oana Veronica; et al. (Springer, 2020-02-14)
    Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias. This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.
  • Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

    Matos, Aline da Rocha; Wunderlich, Katharina; Schloer, Sebastian; Schughart, Klaus; Geffers, Robert; Seders, Martine; Witt, Marlous de; Christersson, Anmari; Wiewrodt, Rainer; Wiebe, Karsten; et al. (Taylor & Francis Open, 2019-01-01)
    Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV in vitro and in vivo. However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.
  • Multiplex profiling of inflammation-related bioactive lipid mediators in Toxocara canis- and Toxocara cati-induced neurotoxocarosis.

    Waindok, Patrick; Janecek-Erfurth, Elisabeth; Lindenwald, Dimitri; Wilk, Esther; Schughart, Klaus; Geffers, Robert; Balas, Laurence; Durand, Thierry; Rund, Katharina Maria; Schebb, Nils Helge; et al. (PLOS, 2019-09-01)
    BACKGROUND: Somatic migration of Toxocara canis- and T. cati-larvae in humans may cause neurotoxocarosis (NT) when larvae accumulate and persist in the central nervous system (CNS). Host- or parasite-induced immunoregulatory processes contribute to the pathogenesis; however, detailed data on involvement of bioactive lipid mediators, e.g. oxylipins or eico-/docosanoids, which are involved in the complex molecular signalling network during infection and inflammation, are lacking. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate if T. canis- and T. cati-induced NT affects the homeostasis of oxylipins during the course of infection, a comprehensive lipidomic profiling in brains (cerebra and cerebella) of experimentally infected C57BL/6J mice was conducted at six different time points post infection (pi) by liquid-chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS). Only minor changes were detected regarding pro-inflammatory prostaglandins (cyclooxygenase pathway). In contrast, a significant increase of metabolites resulting from lipoxygenase pathways was observed for both infection groups and brain regions, implicating a predominantly anti-inflammatory driven immune response. This observation was supported by a significantly increased 13-hydroxyoctadecadienoic acid (HODE)/9-HODE ratio during the subacute phase of infection, indicating an anti-inflammatory response to neuroinfection. Except for the specialised pro-resolving mediator (SPM) neuroprotectin D1 (NPD1), which was detected in mice infected with both pathogens during the subacute phase of infection, no other SPMs were detected. CONCLUSIONS/SIGNIFICANCE: The obtained results demonstrate the influence of Toxocara spp. on oxylipins as part of the immune response of the paratenic hosts. Furthermore, this study shows differences in the alteration of the oxylipin composition between T. canis- and T. cati-brain infection. Results contribute to a further understanding of the largely unknown pathogenesis and mechanisms of host-parasite interactions during NT.
  • The endosomal Toll-like receptors 7 and 9 cooperate in detection of MHV68 infection.

    Bussey, Kendra A; Murthy, Sripriya; Reimer, Elisa; Chan, Baca; Hatesuer, Bastian; Schughart, Klaus; Glaunsinger, Britt; Adler, Heiko; Brinkmann, Melanie M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Amercan Society of Microbiology, 2018-11-14)
    Murine gammaherpesvirus 68 (MHV68) is an amenable small animal model for study of the human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal TLR escort protein UNC93B, endosomal TLR7, 9, and 13, and cell surface TLR2 in MHV68 detection. We found that the interferon α (IFNα) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to wildtype (WT), but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7/Tlr9-/- double knockout pDC, the IFNα response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFNα response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7/Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7/Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7/Tlr9-/- splenocytes, compared to WT. Our data show that UNC93B- dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.
  • Of mice and men: the host response to influenza virus infection.

    Kollmus, Heike; Pilzner, Carolin; Leist, Sarah R; Heise, Mark; Geffers, Robert; Schughart, Klaus; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-08-01)
    Influenza virus (IV) infections represent a very serious public health problem. At present, no established biomarkers exist to support diagnosis for respiratory viral infections and more importantly for severe IV disease. Studies in animal models are extremely important to understand the biological, genetic, and environmental factors that contribute to severe IV disease and to validate biomarker candidates from human studies. However, mouse human cross-species comparisons are often compromised by the fact that animal studies concentrate on the infected lungs, whereas in humans almost all studies use peripheral blood from patients. In addition, human studies do not consider genetic background as variable although human populations are genetically very diverse. Therefore, in this study, we performed a cross-species gene expression study of the peripheral blood from human patients and from the highly genetically diverse Collaborative Cross (CC) mouse population after IV infection. Our results demonstrate that changes of gene expression in individual genes are highly similar in mice and humans. The top-regulated genes in humans were also differentially regulated in mice. We conclude that the mouse is a highly valuable in vivo model system to validate and to discover gene candidates which can be used as biomarkers in humans. Furthermore, mouse studies allow confirmation of findings in humans in a well-controlled experimental system adding enormous value to the understanding of expression and function of human candidate genes.
  • Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice.

    Lambertz, Ruth L O; Pippel, Jan; Gerhauser, Ingo; Kollmus, Heike; Anhlan, Darisuren; Hrincius, Eike R; Krausze, Joern; Kühn, Nora; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-01)
    The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2/ knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2/ as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2/ knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2/ knockout mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.
  • TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.

    Zmora, Pawel; Hoffmann, Markus; Kollmus, Heike; Moldenhauer, Anna-Sophie; Danov, Olga; Braun, Armin; Winkler, Michael; Schughart, Klaus; Pöhlmann, Stefan; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-07)
    The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.
  • Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

    Yang, W; Punyadarsaniya, D; Lambertz, R L O; Lee, D C C; Liang, C H; Höper, D; Leist, S R; Hernández-Cáceres, A; Stech, J; Beer, M; et al. (2017-04-15)
    The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCESwine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptive mutation enhances the replication kinetics, but more mutations are required for IAV of the H9N2 subtype to become virulent.
  • Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection.

    Hatesuer, Bastian; Hoang, Hang Thi Thu; Riese, Peggy; Trittel, Stephanie; Gerhauser, Ingo; Elbahesh, Husni; Geffers, Robert; Wilk, Esther; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
    The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3-/-, Irf7-/-, and Irf3-/-Irf7-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNβ after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.

View more