group leader: Prof. Wirth

Recent Submissions

  • Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth.

    Laske, Tanja; Bachmann, Mandy; Dostert, Melanie; Karlas, Alexander; Wirth, Dagmar; Frensing, Timo; Meyer, Thomas F; Hauser, Hansjörg; Reichl, Udo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-01-01)
    The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.
  • ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

    Büssow, Konrad; Themann, Philipp; Luu, Sabine; Pentrowski, Paul; Harting, Claudia; Majewski, Mira; Vollmer, Veith; Köster, Mario; Grashoff, Martina; Zawatzky, Rainer; et al. (Plos, 2019-01-01)
    Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
  • An endothelial cell line infected by Kaposi's sarcoma-associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo.

    Dubich, Tatyana; Lieske, Anna; Santag, Susann; Beauclair, Guillaume; Rückert, Jessica; Herrmann, Jennifer; Gorges, Jan; Büsche, Guntram; Kazmaier, Uli; Hauser, Hansjörg; et al. (2019-01-04)
    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi's sarcoma. KEY MESSAGES: Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection. KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus. A 3D invasiveness assay is predictive for tumor reduction in vivo. Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo.
  • Advances and Challenges of Biodegradable Implant Materials with a Focus on Magnesium-Alloys and Bacterial Infections

    Rahim, Muhammad; Ullah, Sami; Mueller, Peter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MPDI, 2018-07-10)
    Medical implants made of biodegradable materials could be advantageous for temporary applications, such as mechanical support during bone-healing or as vascular stents to keep blood vessels open. After completion of the healing process, the implant would disappear, avoiding long-term side effects or the need for surgical removal. Various corrodible metal alloys based on magnesium, iron or zinc have been proposed as sturdier and potentially less inflammatory alternatives to degradable organic polymers, in particular for load-bearing applications. Despite the recent introduction of magnesium-based screws, the remaining hurdles to routine clinical applications are still challenging. These include limitations such as mechanical material characteristics or unsuitable corrosion characteristics. In this article, the salient features and clinical prospects of currently-investigated biodegradable implant materials are summarized, with a main focus on magnesium alloys. A mechanism of action for the stimulation of bone growth due to the exertion of mechanical force by magnesium corrosion products is discussed. To explain divergent in vitro and in vivo effects of magnesium, a novel model for bacterial biofilm infections is proposed which predicts crucial consequences for antibacterial implant strategies.
  • Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.

    Ullah, Sami; Seidel, Katja; Türkkan, Sibel; Warwas, Dawid Peter; Dubich, Tatyana; Rohde, Manfred; Hauser, Hansjörg; Behrens, Peter; Kirschning, Andreas; Köster, Mario; et al. (2018-12-23)
    Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release.
  • Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.

    Becker, Jennifer; Kinast, Volker; Döring, Marius; Lipps, Christoph; Duran, Veronica; Spanier, Julia; Tegtmeyer, Pia-Katharina; Wirth, Dagmar; Cicin-Sain, Luka; Alcamí, Antonio; et al. (2018-01-01)
    Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-β. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.
  • Biocompatible Coatings from Smart Biopolymer Nanoparticles for Enzymatically Induced Drug Release.

    Tolle, Christian; Riedel, Jan; Mikolai, Carina; Winkel, Andreas; Stiesch, Meike; Wirth, Dagmar; Menzel, Henning; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-28)
    Nanoparticles can be used as a smart drug delivery system, when they release the drug only upon degradation by specific enzymes. A method to create such responsive materials is the formation of hydrogel nanoparticles, which have enzymatically degradable crosslinkers. Such hydrogel nanoparticles were prepared by ionotropic gelation sodium alginate with lysine-rich peptide sequences-either α-poly-L-lysine (PLL) or the aggrecanase-labile sequence KKKK-GRD-ARGSV↓NITEGE-DRG-KKKK. The nanoparticle suspensions obtained were analyzed by means of dynamic light scattering and nanoparticle tracking analysis. Degradation experiments carried out with the nanoparticles in suspension revealed enzyme-induced lability. Drugs present in the polymer solution during the ionotropic gelation can be encapsulated in the nanoparticles. Drug loading was investigated for interferon-β (IFN-β) as a model, using a bioluminescence assay with MX2Luc2 cells. The encapsulation efficiency for IFN-β was found to be approximately 25%. The nanoparticles suspension can be used to spray-coat titanium alloys (Ti-6Al-4V) as a common implant material. The coatings were proven by ellipsometry, reflection-absorption infrared spectroscopy, and X-ray photoelectron spectroscopy. An enzyme-responsive decrease in layer thickness is observed due to the degradation of the coatings. The Alg/peptide coatings were cytocompatible for human gingival fibroblasts (HGFIB), which was investigated by CellTiterBlue and lactate dehydrogenase (LDH) assay. However, HGFIBs showed poor adhesion and proliferation on the Alg/peptide coatings, but these could be improved by modification of the alginate with a RGD-peptide sequence. The smart drug release system presented can be further tailored to have the right release kinetics and cell adhesion properties.
  • Identification of a Predominantly Interferon-λ-Induced Transcriptional Profile in Murine Intestinal Epithelial Cells.

    Selvakumar, Tharini A; Bhushal, Sudeep; Kalinke, Ulrich; Wirth, Dagmar; Hauser, Hansjörg; Köster, Mario; Hornef, Mathias W; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-01-01)
    Type I (α and β) and type III (λ) interferons (IFNs) induce the expression of a large set of antiviral effector molecules
  • Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis.

    Borst, Katharina; Frenz, Theresa; Spanier, Julia; Tegtmeyer, Pia-Katharina; Chhatbar, Chintan; Skerra, Jennifer; Ghita, Luca; Namineni, Sukumar; Lienenklaus, Stefan; Köster, Mario; et al. (2017-12-21)
    Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation. Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-β induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1 VACV infection induced local IFN-β responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution. Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis.
  • Memory CD8 T cells support the maintenance of hematopoietic stem cells in the bone marrow

    Geerman, Sulima; Brasser, Giso; Bhushal, Sudeep; Salerno, Fiamma; Kragten, Natasja A.; Hoogenboezem, Mark; de Haan, Gerald; Wolkers, Monika C.; Pascutti, María Fernanda; Nolte, Martijn A.; et al.
  • Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

    Stichling, Nicole; Suomalainen, Maarit; Flatt, Justin W; Schmid, Markus; Pacesa, Martin; Hemmi, Silvio; Jungraithmayr, Wolfgang; Maler, Mareike D; Freudenberg, Marina A; Plückthun, Andreas; et al. (2018-03)
    Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.
  • A mathematical model of the impact of insulin secretion dynamics on selective hepatic insulin resistance.

    Zhao, Gang; Wirth, Dagmar; Schmitz, Ingo; Meyer-Hermann, Michael; Braunschweiger Zentrum für Systembiologie, Rebenring 56, 38106, Germany. (2017-11-08)
    Physiological insulin secretion exhibits various temporal patterns, the dysregulation of which is involved in diabetes development. We analyzed the impact of first-phase and pulsatile insulin release on glucose and lipid control with various hepatic insulin signaling networks. The mathematical model suggests that atypical protein kinase C (aPKC) undergoes a bistable switch-on and switch-off, under the control of insulin receptor substrate 2 (IRS2). The activation of IRS1 and IRS2 is temporally separated due to the inhibition of IRS1 by aPKC. The model further shows that the timing of aPKC switch-off is delayed by reduced first-phase insulin and reduced amplitude of insulin pulses. Based on these findings, we propose a sequential model of postprandial hepatic control of glucose and lipid by insulin, according to which delayed aPKC switch-off contributes to selective hepatic insulin resistance, which is a long-standing paradox in the field.
  • CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells.

    Riehn, Mathias; Cebula, Marcin; Hauser, Hansjörg; Wirth, Dagmar; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
    Intrapulmonary immune reactions are impaired by the tolerogenic environment of the lung. This is manifested by the absence of effective endogenous T cell responses upon neoantigen expression. This tolerance is considered to contribute to lung cancer and inefficient immune therapeutic interventions. To investigate the mechanisms contributing to lung tolerance and to overcome these restrictions, we developed a transgenic mouse model with induction of a neoantigen (OVA) exclusively in alveolar type II epithelial cells. This model is characterized by the absence of functional endogenous T cell responses upon OVA neoantigen induction. Standard DNA and protein vaccination protocols resulted in the accumulation of high numbers of antigen-specific CD8 T cells in the lung. However, clearance of antigen-expressing cells was not achieved. To overcome this tolerance, we induced inflammatory conditions by coapplication of the TLR ligands LPS and CpG-ODN during intrapulmonary vaccinations. Both ligands induced high numbers of neoantigen-specific T cells in the lung. However, only coapplication of CpG-ODN was sufficient to establish functional cytotoxic responses resulting in the elimination of neoantigen presenting target cells. Remarkably, CpG-ODN was also crucial for functional memory responses upon re-induction of the neoantigen. The results highlight the need of TLR9 co-stimulation for overcoming tolerization, which might be a key factor for therapeutic interventions.
  • Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

    Gödecke, Natascha; Zha, Lisha; Spencer, Shawal; Behme, Sara; Riemer, Pamela; Rehli, Michael; Hauser, Hansjörg; Wirth, Dagmar; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, D38124 Braunschweig, Germany. (2017-09-19)
    Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
  • TLR9-Mediated Conditioning of Liver Environment Is Essential for Successful Intrahepatic Immunotherapy and Effective Memory Recall.

    Cebula, Marcin; Riehn, Mathias; Hillebrand, Upneet; Kratzer, Ramona F; Kreppel, Florian; Koutsoumpli, Georgia; Daemen, Toos; Hauser, Hansjoerg; Wirth, Dagmar; Helmholtz -Zentrum für Infektionsforschung GmbH. Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-07-14)
    Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination-induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response.
  • Cell Polarization and Epigenetic Status Shape the Heterogeneous Response to Type III Interferons in Intestinal Epithelial Cells.

    Bhushal, Sudeep; Wolfsmüller, Markus; Selvakumar, Tharini A; Kemper, Lucas; Wirth, Dagmar; Hornef, Mathias W; Hauser, Hansjörg; Köster, Mario; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
    Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist, intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression, IFN-λ responsiveness is restricted mainly to epithelial cells. Here, we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially, we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently, HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second, in contrast to the type I IFN system, polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally, we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity.
  • The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function.

    Kunkiel, Jessica; Gödecke, Natascha; Ackermann, Mania; Hoffmann, Dirk; Schambach, Axel; Lachmann, Nico; Wirth, Dagmar; Moritz, Thomas; Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-11)
    Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.
  • Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.

    Marecic, Valentina; Shevchuk, Olga; Ozanic, Mateja; Mihelcic, Mirna; Steinert, Michael; Jurak Begonja, Antonija; Abu Kwaik, Yousef; Santic, Marina; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
    Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.
  • Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci

    Nehlsen, Kristina; da Gama-Norton, Leonor; Schucht, Roland; Hauser, Hansjörg; Wirth, Dagmar (2011-11-22)
  • Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes.

    Ochel, Aaron; Cebula, Marcin; Riehn, Mathias; Hillebrand, Upneet; Lipps, Christoph; Schirmbeck, Reinhold; Hauser, Hansjoerg; Wirth, Dagmar; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-11)
    Liver infections with hepatotropic viruses, such as hepatitis B virus and hepatitis C virus are accompanied by viral persistence and immune failure. CD8+ T cells are crucial mediators of the intrahepatic antiviral immune response. Chronic infections of the liver and other organs correlate with T-cell exhaustion. It was previously suggested that high antigen load could result in T-cell exhaustion. We aimed at elucidating the impact of different intrahepatic antigen loads on the quality of CD8+ T-cell-mediated immunity by employing an infection-free transgenic mouse model expressing ovalbumin (Ova) as the target antigen. Adoptive transfer of OT-I cells induced a transient intrahepatic immune response toward both high and low Ova levels. However, antigen clearance was achieved only in mice expressing low antigen levels. In contrast, T cells exposed to high antigen levels underwent exhaustion and became depleted, causing antigen persistence. Moreover, when functional T cells were exposed to high intrahepatic antigen levels, a complete transition toward exhaustion was observed. Thus, this study shows that the antigen expression level in the liver correlates inversely with T-cell immunity in vivo and governs the efficiency of immune responses upon antigen presentation.

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