head of the department: Prof. Kalinke

Recent Submissions

  • Application of light sheet microscopy for qualitative and quantitative analysis of bronchus-associated lymphoid tissue in mice.

    Mzinza, David Twapokera; Fleige, Henrike; Laarmann, Kristin; Willenzon, Stefanie; Ristenpart, Jasmin; Spanier, Julia; Sutter, Gerd; Kalinke, Ulrich; Valentin-Weigand, Peter; Förster, Reinhold; TWINCORE, Zentrum für experimentelle uns klinische Ifektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-02-12)
    Bronchus-associated lymphoid tissue (BALT) develops at unpredictable locations around lung bronchi following pulmonary inflammation. The formation and composition of BALT have primarily been investigated by immunohistology that, due to the size of the invested organ, is usually restricted to a limited number of histological sections. To assess the entire BALT of the lung, other approaches are urgently needed. Here, we introduce a novel light sheet microscopy-based approach for assessing lymphoid tissue in the lung. Using antibody staining of whole lung lobes and optical clearing by organic solvents, we present a method that allows in-depth visualization of the entire bronchial tree, the lymphatic vasculature and the immune cell composition of the induced BALT. Furthermore, three-dimensional analysis of the entire lung allows the qualitative and quantitative enumeration of the induced BALT. Using this approach, we show that a single intranasal application of the replication-deficient poxvirus MVA induces BALT that constitutes up to 8% of the entire lung volume in mice deficient in CCR7, in contrast to wild type mice (WT). Furthermore, BALT induced by heat-inactivated E. coli is dominated by a pronounced T cell infiltration in Cxcr5-deficient mice, in contrast to WT mice.Cellular and Molecular Immunology advance online publication, 12 February 2018; doi:10.1038/cmi.2017.150.
  • Type I Interferon Signaling Is Required for CpG-Oligodesoxynucleotide-Induced Control of Leishmania major, but Not for Spontaneous Cure of Subcutaneous Primary or Secondary L. major Infection.

    Schleicher, Ulrike; Liese, Jan; Justies, Nicole; Mischke, Thomas; Haeberlein, Simone; Sebald, Heidi; Kalinke, Ulrich; Weiss, Siegfried; Bogdan, Christian; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018)
    We previously showed that in mice infected with Leishmania major type I interferons (IFNs) initiate the innate immune response to the parasite at day 1 and 2 of infection. Here, we investigated which type I IFN subtypes are expressed during the first 8 weeks of L. major infection and whether type I IFNs are essential for a protective immune response and clinical cure of the disease. In self-healing C57BL/6 mice infected with a high dose of L. major, IFN-α4, IFN-α5, IFN-α11, IFN-α13, and IFN-β mRNA were most prominently regulated during the course of infection. In C57BL/6 mice deficient for IFN-β or the IFN-α/β-receptor chain 1 (IFNAR1), development of skin lesions and parasite loads in skin, draining lymph node, and spleen was indistinguishable from wild-type (WT) mice. In line with the clinical findings, C57BL/6 IFN-β-/-, IFNAR1-/-, and WT mice exhibited similar mRNA expression levels of IFN-γ, interleukin (IL)-4, IL-12, IL-13, inducible nitric oxide synthase, and arginase 1 during the acute and late phase of the infection. Also, myeloid dendritic cells from WT and IFNAR1-/- mice produced comparable amounts of IL-12p40/p70 protein upon exposure to L. major in vitro. In non-healing BALB/c WT mice, the mRNAs of IFN-α subtypes (α2, α4, α5, α6, and α9) were rapidly induced after high-dose L. major infection. However, genetic deletion of IFNAR1 or IFN-β did not alter the progressive course of infection seen in WT BALB/c mice. Finally, we tested whether type I IFNs and/or IL-12 are required for the prophylactic effect of CpG-oligodesoxynucleotides (ODN) in BALB/c mice. Local and systemic administration of CpG-ODN 1668 protected WT and IFN-β-/- mice equally well from progressive leishmaniasis. By contrast, the protective effect of CpG-ODN 1668 was lost in BALB/c IFNAR1-/- (despite a sustained suppression of IL-4) and in BALB/c IL-12p35-/- mice. From these data, we conclude that IFN-β and IFNAR1 signaling are dispensable for a curative immune response to L. major in C57BL/6 mice and irrelevant for disease development in BALB/c mice, whereas IL-12 and IFN-α subtypes are essential for the disease prevention by CpG-ODNs in this mouse strain.
  • Tolerogenic Transcriptional Signatures of Steady-State and Pathogen-Induced Dendritic Cells.

    Vendelova, Emilia; Ashour, Diyaaeldin; Blank, Patrick; Erhard, Florian; Saliba, Antoine-Emmanuel; Kalinke, Ulrich; Lutz, Manfred B; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018)
    Dendritic cells (DCs) are key directors of tolerogenic and immunogenic immune responses. During the steady state, DCs maintain T cell tolerance to self-antigens by multiple mechanisms including inducing anergy, deletion, and Treg activity. All of these mechanisms help to prevent autoimmune diseases or other hyperreactivities. Different DC subsets contribute to pathogen recognition by expression of different subsets of pattern recognition receptors, including Toll-like receptors or C-type lectins. In addition to the triggering of immune responses in infected hosts, most pathogens have evolved mechanisms for evasion of targeted responses. One such strategy is characterized by adopting the host's T cell tolerance mechanisms. Understanding these tolerogenic mechanisms is of utmost importance for therapeutic approaches to treat immune pathologies, tumors and infections. Transcriptional profiling has developed into a potent tool for DC subset identification. Here, we review and compile pathogen-induced tolerogenic transcriptional signatures from mRNA profiling data of currently available bacterial- or helminth-induced transcriptional signatures. We compare them with signatures of tolerogenic steady-state DC subtypes to identify common and divergent strategies of pathogen induced immune evasion. Candidate molecules are discussed in detail. Our analysis provides further insights into tolerogenic DC signatures and their exploitation by different pathogens.
  • Macrophage depletion by liposome-encapsulated clodronate suppresses seizures but not hippocampal damage after acute viral encephalitis.

    Waltl, Inken; Käufer, Christopher; Bröer, Sonja; Chhatbar, Chintan; Ghita, Luca; Gerhauser, Ingo; Anjum, Muneeb; Kalinke, Ulrich; Löscher, Wolfgang; TWINCORE, Zentrum für experimentelle uns klinische Ifektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-02)
    Viral encephalitis is a major risk factor for the development of seizures and epilepsy, but the underlying mechanisms are only poorly understood. Mouse models such as viral encephalitis induced by intracerebral infection with Theiler's virus in C57BL/6 (B6) mice allow advancing our understanding of the immunological and virological aspects of infection-induced seizures and their treatment. Previous studies using the Theiler's virus model in B6 mice have indicated that brain-infiltrating inflammatory macrophages and the cytokines released by these cells are key to the development of acute seizures and hippocampal damage in this model. However, approaches used to prevent or reduce macrophage infiltration were not specific, so contribution of other mechanisms could not be excluded. In the present study, we used a more selective and widely used approach for macrophage depletion, i.e., systemic administration of clodronate liposomes, to study the contribution of macrophage infiltration to development of seizures and hippocampal damage. By this approach, almost complete depletion of monocytic cells was achieved in spleen and blood of Theiler's virus infected B6 mice, which was associated with a 70% decrease in the number of brain infiltrating macrophages as assessed by flow cytometry. Significantly less clodronate liposome-treated mice exhibited seizures than liposome controls (P<0.01), but the development of hippocampal damage was not prevented or reduced. Clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage. The unexpected mismatch between occurrence of seizures and hippocampal damage is thought-provoking and suggests that the mechanisms involved in degeneration of specific populations of hippocampal neurons in encephalitis-induced epilepsy are more complex than previously thought.
  • Hematopoietic stem cell gene therapy for IFNγR1 deficiency protects mice from mycobacterial infections.

    Hetzel, Miriam; Mucci, Adele; Blank, Patrick; Nguyen, Ariane Hai Ha; Schiller, Jan; Halle, Olga; Kühnel, Mark-Philipp; Billig, Sandra; Meineke, Robert; Brand, Daniel; Herder, Vanessa; Baumgärtner, Wolfgang; Bange, Franz-Christoph; Goethe, Ralph; Jonigk, Danny; Förster, Reinhold; Gentner, Bernhard; Casanova, Jean-Laurent; Bustamante, Jacinta; Schambach, Axel; Kalinke, Ulrich; Lachmann, Nico; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-02-01)
    Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1orIFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that expressIfnγr1either constitutively or myeloid specifically. Transduction of mouseIfnγr1 -/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity againstMycobacterium aviumandMycobacterium bovisBacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs intoIfnγr1-/-mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.
  • Personalized adoptive immunotherapy for patients with EBV-associated tumors and complications: Evaluation of novel naturally processed and presented EBV-derived T-cell epitopes.

    Bieling, Maren; Tischer, Sabine; Kalinke, Ulrich; Blasczyk, Rainer; Buus, Søren; Maecker-Kolhoff, Britta; Eiz-Vesper, Britta; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-01-12)
    Morbidity and mortality of immunocompromised patients are increased by primary infection with or reactivation of Epstein-Barr virus (EBV), possibly triggering EBV+post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) promises a non-toxic immunotherapy to effectively prevent or treat these complications. To improve immunotherapy and immunomonitoring this study aimed at identifying and evaluating naturally processed and presented HLA-A*03:01-restricted EBV-CTL epitopes as immunodominant targets. More than 15000 peptides were sequenced from EBV-immortalized B cells transduced with soluble HLA-A*03:01, sorted using different epitope prediction tools and eleven candidates were preselected. T2 and Flex-T peptide-binding and dissociation assays confirmed the stability of peptide-MHC complexes. Their immunogenicity and clinical relevance were evaluated by assessing the frequencies and functionality of EBV-CTLs in healthy donors (n> 10) and EBV+PTLD-patients (n= 5) by multimer staining, Eli- and FluoroSpot assays. All eleven peptides elicited EBV-CTL responses in the donors. Their clinical applicability was determined by small-scale T-cell enrichment using Cytokine Secretion Assay and immunophenotyping. Mixtures of these peptides when added to the EBV Consensus pool revealed enhanced stimulation and enrichment efficacy. These EBV-specific epitopes broadening the repertoire of known targets will improve manufacturing of clinically applicable EBV-CTLs and monitoring of EBV-specific T-cell responses in patients.
  • Type I interferon promotes alveolar epithelial type II cell survival during pulmonary Streptococcus pneumoniae infection and sterile lung injury in mice.

    Maier, Barbara B; Hladik, Anastasiya; Lakovits, Karin; Korosec, Ana; Martins, Rui; Kral, Julia B; Mesteri, Ildiko; Strobl, Birgit; Müller, Mathias; Kalinke, Ulrich; Merad, Miriam; Knapp, Sylvia; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2016)
    Protecting the integrity of the lung epithelial barrier is essential to ensure respiration and proper oxygenation in patients suffering from various types of lung inflammation. Type I interferon (IFN-I) has been associated with pulmonary epithelial barrier function, however, the mechanisms and involved cell types remain unknown. We aimed to investigate the importance of IFN-I with respect to its epithelial barrier strengthening function to better understand immune-modulating effects in the lung with potential medical implications. Using a mouse model of pneumococcal pneumonia, we revealed that IFN-I selectively protects alveolar epithelial type II cells (AECII) from inflammation-induced cell death. Mechanistically, signaling via the IFN-I receptor on AECII is sufficient to promote AECII survival. The net effects of IFN-I are barrier protection, together with diminished tissue damage, inflammation, and bacterial loads. Importantly, we found that the protective role of IFN-I can also apply to sterile acute lung injury, in which loss of IFN-I signaling leads to a significant reduction in barrier function caused by AECII cell death. Our data suggest that IFN-I is an important mediator in lung inflammation that plays a protective role by antagonizing inflammation-associated cell obstruction, thereby strengthening the integrity of the epithelial barrier.
  • Coadministration of a Plasmid Encoding HIV-1 Gag Enhances the Efficacy of Cancer DNA Vaccines.

    Lambricht, Laure; Vanvarenberg, Kevin; De Beuckelaer, Ans; Van Hoecke, Lien; Grooten, Johan; Ucakar, Bernard; Lipnik, Pascale; Sanders, Niek N; Lienenklaus, Stefan; Préat, Véronique; Vandermeulen, Gaëlle; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2016)
    DNA vaccination holds great promise for the prevention and treatment of cancer and infectious diseases. However, the clinical ability of DNA vaccines is still controversial due to the limited immune response initially observed in humans. We hypothesized that electroporation of a plasmid encoding the HIV-1 Gag viral capsid protein would enhance cancer DNA vaccine potency. DNA electroporation used to deliver plasmids in vivo, induced type I interferons, thereby supporting the activation of innate immunity. The coadministration of ovalbumin (OVA) and HIV-1 Gag encoding plasmids modulated the adaptive immune response. This strategy favored antigen-specific Th1 immunity, delayed B16F10-OVA tumor growth and improved mouse survival in both prophylactic and therapeutic vaccination approaches. Similarly, a prophylactic DNA immunization against the melanoma-associated antigen gp100 was enhanced by the codelivery of the HIV-1 Gag plasmid. The adjuvant effect was not driven by the formation of HIV-1 Gag virus-like particles. This work highlights the ability of both electroporation and the HIV-1 Gag plasmid to stimulate innate immunity for enhancing cancer DNA vaccine immunogenicity and demonstrates interesting tracks for the design of new translational genetic adjuvants to overcome the current limitations of DNA vaccines in humans.
  • Impaired IFNγ-Signaling and Mycobacterial Clearance in IFNγR1-Deficient Human iPSC-Derived Macrophages.

    Neehus, Anna-Lena; Lam, Jenny; Haake, Kathrin; Merkert, Sylvia; Schmidt, Nico; Mucci, Adele; Ackermann, Mania; Schubert, Madline; Happle, Christine; Kühnel, Mark Philipp; Blank, Patrick; Philipp, Friederike; Goethe, Ralph; Jonigk, Danny; Martin, Ulrich; Kalinke, Ulrich; Baumann, Ulrich; Schambach, Axel; Roesler, Joachim; Lachmann, Nico; TWiNCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str.7, 30625 Hannover, Germany. (2018-01-09)
    Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages.
  • cGAS-STING-TBK1-IRF3/7 induced interferon-β contributes to the clearing of non tuberculous mycobacterial infection in mice.

    Ruangkiattikul, Nanthapon; Nerlich, Andreas; Abdissa, Ketema; Lienenklaus, Stefan; Suwandi, Abdulhadi; Janze, Nina; Laarmann, Kristin; Spanier, Julia; Kalinke, Ulrich; Weiss, Siegfried; Goethe, Ralph; TWNCORe, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynnen-Str. 7, 30625 Hannover, Germany. (2017-10-03)
    Type I interferons (IFN-I), such as IFN-α and IFN-β are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-β than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-β in macrophages. Both bacteria induced IFN-β via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-β activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-β impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.
  • Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.

    Volckmar, Julia; Gereke, Marcus; Ebensen, Thomas; Riese, Peggy; Philipsen, Lars; Lienenklaus, Stefan; Wohlleber, Dirk; Klopfleisch, Robert; Stegemann-Koniszewski, Sabine; Müller, Andreas J; Gruber, Achim D; Knolle, Percy; Guzman, Carlos A; Bruder, Dunja; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-03-07)
    Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection.
  • RIG-I/MAVS and STING signaling promote gut integrity during irradiation- and immune-mediated tissue injury.

    Fischer, Julius C; Bscheider, Michael; Eisenkolb, Gabriel; Lin, Chia-Ching; Wintges, Alexander; Otten, Vera; Lindemans, Caroline A; Heidegger, Simon; Rudelius, Martina; Monette, Sébastien; Porosnicu Rodriguez, Kori A; Calafiore, Marco; Liebermann, Sophie; Liu, Chen; Lienenklaus, Stefan; Weiss, Siegfried; Kalinke, Ulrich; Ruland, Jürgen; Peschel, Christian; Shono, Yusuke; Docampo, Melissa; Velardi, Enrico; Jenq, Robert R; Hanash, Alan M; Dudakov, Jarrod A; Haas, Tobias; van den Brink, Marcel R M; Poeck, Hendrik; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2017-04-19)
    The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation- and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet-derived protein 3 γ (RegIIIγ). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation.
  • Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection.

    Ivin, Masa; Dumigan, Amy; de Vasconcelos, Filipe N; Ebner, Florian; Borroni, Martina; Kavirayani, Anoop; Przybyszewska, Kornelia N; Ingram, Rebecca J; Lienenklaus, Stefan; Kalinke, Ulrich; Stoiber, Dagmar; Bengoechea, Jose A; Kovarik, Pavel; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str.7 30625 Hannover, Germany. (2017-11)
    Klebsiella pneumoniae is a significant cause of nosocomial pneumonia and an alarming pathogen owing to the recent isolation of multidrug resistant strains. Understanding of immune responses orchestrating K. pneumoniae clearance by the host is of utmost importance. Here we show that type I interferon (IFN) signaling protects against lung infection with K. pneumoniae by launching bacterial growth-controlling interactions between alveolar macrophages and natural killer (NK) cells. Type I IFNs are important but disparate and incompletely understood regulators of defense against bacterial infections. Type I IFN receptor 1 (Ifnar1)-deficient mice infected with K. pneumoniae failed to activate NK cell-derived IFN-γ production. IFN-γ was required for bactericidal action and the production of the NK cell response-amplifying IL-12 and CXCL10 by alveolar macrophages. Bacterial clearance and NK cell IFN-γ were rescued in Ifnar1-deficient hosts by Ifnar1-proficient NK cells. Consistently, type I IFN signaling in myeloid cells including alveolar macrophages, monocytes and neutrophils was dispensable for host defense and IFN-γ activation. The failure of Ifnar1-deficient hosts to initiate a defense-promoting crosstalk between alveolar macrophages and NK cell was circumvented by administration of exogenous IFN-γ which restored endogenous IFN-γ production and restricted bacterial growth. These data identify NK cell-intrinsic type I IFN signaling as essential driver of K. pneumoniae clearance, and reveal specific targets for future therapeutic exploitations.
  • Development of the First World Health Organization Lentiviral Vector Standard: Toward the Production Control and Standardization of Lentivirus-Based Gene Therapy Products.

    Zhao, Yuan; Stepto, Hannah; Schneider, Christian K; TwinCore, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2017-08)
    Gene therapy is a rapidly evolving field. So far, there have been >2,400 gene therapy products in clinical trials and four products on the market. A prerequisite for producing gene therapy products is ensuring their quality and safety. This requires appropriately controlled and standardized production and testing procedures that result in consistent safety and efficacy. Assuring the quality and safety of lentivirus-based gene therapy products in particular presents a great challenge because they are cell-based multigene products that include viral and therapeutic proteins as well as modified cells. In addition to the continuous refinement of a product, changes in production sites and manufacturing processes have become more and more common, posing challenges to developers regarding reproducibility and comparability of results. This paper discusses the concept of developing a first World Health Organization International Standard, suitable for the standardization of assays and enabling comparison of cross-trial and cross-manufacturing results for this important vector platform. The standard will be expected to optimize the development of gene therapy medicinal products, which is especially important, given the usually orphan nature of the diseases to be treated, naturally hampering reproducibility and comparability of results.
  • Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway.

    Lirussi, Darío; Ebensen, Thomas; Schulze, Kai; Trittel, Stephanie; Duran, Veronica; Liebich, Ines; Kalinke, Ulrich; Guzmán, Carlos Alberto; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-07-19)
    Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8(+) cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation.
  • Microbiota Normalization Reveals that Canonical Caspase-1 Activation Exacerbates Chemically Induced Intestinal Inflammation.

    Błażejewski, Adrian J; Thiemann, Sophie; Schenk, Alexander; Pils, Marina C; Gálvez, Eric J C; Roy, Urmi; Heise, Ulrike; de Zoete, Marcel R; Flavell, Richard A; Strowig, Till; Helmholtz Centre for infection research, Inhoffenstr. 7. 38124 Braunschweig, Germany. (2017-06-13)
    Inflammasomes play a central role in regulating intestinal barrier function and immunity during steady state and disease. Because the discoveries of a passenger mutation and a colitogenic microbiota in the widely used caspase-1-deficient mouse strain have cast doubt on previously identified direct functions of caspase-1, we reassessed the role of caspase-1 in the intestine. To this end, we generated Casp1(-/-) and Casp11(-/-) mice and rederived them into an enhanced barrier facility to standardize the microbiota. We found that caspase-11 does not influence caspase-1-dependent processing of IL-18 in homeostasis and during DSS colitis. Deficiency of caspase-1, but not caspase-11, ameliorated the severity of DSS colitis independent of microbiota composition. Ablation of caspase-1 in intestinal epithelial cells was sufficient to protect mice against DSS colitis. Moreover, Casp1(-/-) mice developed fewer inflammation-induced intestinal tumors than control mice. These data show that canonical inflammasome activation controls caspase-1 activity, contributing to exacerbation of chemical-induced colitis.
  • Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo.

    Hirche, Christoph; Frenz, Theresa; Haas, Simon F; Döring, Marius; Borst, Katharina; Tegtmeyer, Pia-K; Brizic, Ilija; Jordan, Stefan; Keyser, Kirsten; Chhatbar, Chintan; Pronk, Eline; Lin, Shuiping; Messerle, Martin; Jonjic, Stipan; Falk, Christine S; Trumpp, Andreas; Essers, Marieke A G; Kalinke, Ulrich; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynnen Str. 7, 30625 Hannover, Germany. (2017-06-13)
    Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.
  • Immune protection against reinfection with nonprimate hepacivirus.

    Pfaender, Stephanie; Walter, Stephanie; Grabski, Elena; Todt, Daniel; Bruening, Janina; Romero-Brey, Inés; Gather, Theresa; Brown, Richard J P; Hahn, Kerstin; Puff, Christina; Pfankuche, Vanessa M; Hansmann, Florian; Postel, Alexander; Becher, Paul; Thiel, Volker; Kalinke, Ulrich; Wagner, Bettina; Bartenschlager, Ralf; Baumgärtner, Wolfgang; Feige, Karsten; Pietschmann, Thomas; Cavalleri, Jessika M V; Steinmann, Eike; TWINCORE, Zentrum für experimentelle und klinische Infectionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2017-03-21)
    Hepatitis C virus (HCV) displays a restricted host species tropism and only humans and chimpanzees are susceptible to infection. A robust immunocompetent animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control, and prophylactic vaccine development. The closest homolog of HCV is the equine nonprimate hepacivirus (NPHV), which shares similar features with HCV and thus represents an animal model to study hepacivirus infections in their natural hosts. We aimed to dissect equine immune responses after experimental NPHV infection and conducted challenge experiments to investigate immune protection against secondary NPHV infections. Horses were i.v. injected with NPHV containing plasma. Flow cytometric analysis was used to monitor immune cell frequencies and activation status. All infected horses became viremic after 1 or 2 wk and viremia could be detected in two horses for several weeks followed by a delayed seroconversion and viral clearance. Histopathological examinations of liver biopsies revealed mild, periportally accentuated infiltrations of lymphocytes, macrophages, and plasma cells with some horses displaying subclinical signs of hepatitis. Following viral challenge, an activation of equine immune responses was observed. Importantly, after a primary NPHV infection, horses were protected against rechallenge with the homologous as well as a distinct isolate with only minute amounts of circulating virus being detectable.
  • Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H; Züst, Roland; Hwang, Mihyun; V'kovski, Philip; Stalder, Hanspeter; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; van Kuppeveld, Frank J M; Silverman, Robert H; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C; Ziebuhr, John; Weiss, Susan R; Kalinke, Ulrich; Thiel, Volker; TWINCORE, Zentrum für experimentelle und klinische Infectionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2017-02)
    Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.
  • A highly conserved sequence of the viral TAP inhibitor ICP47 is required for freezing of the peptide transport cycle.

    Matschulla, Tony; Berry, Richard; Gerke, Carolin; Döring, Marius; Busch, Julia; Paijo, Jennifer; Kalinke, Ulrich; Momburg, Frank; Hengel, Hartmut; Halenius, Anne; TWINCORE, Zentrum für experimentelle und klinische Infectionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2017-06-07)
    The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1-35). Here we show that mutation of the highly conserved 50PLL52 motif within the central region of ICP47 attenuates its inhibitory capacity. Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation.

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