• OCTN2-mediated acetyl-l-carnitine transport in human pulmonary epithelial cells in vitro

      Salomon, Johanna J.; Gausterer, Julia C.; Selo, Mohammed Ali; Hosoya, Ken Ichi; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus Michael; Ehrhardt, Carsten; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (MPDI, 2019-08-01)
      The carnitine transporter OCTN2 is associated with asthma and other inflammatory diseases. The aims of this work were (i) to determine carnitine uptake into freshly isolated human alveolar type I (ATI)-like epithelial cells in primary culture, (ii) to compare the kinetics of carnitine uptake between respiratory epithelial in vitro cell models, and (iii) to establish whether any cell line was a suitable model for studies of carnitine transport at the air-blood barrier. Levels of time-dependent [3H]-acetyl-l-carnitine uptake were similar in ATI-like, NCl-H441, and Calu-3 epithelial cells, whereas uptake into A549 cells was ~5 times higher. Uptake inhibition was more pronounced by OCTN2 modulators, such as l-Carnitine and verapamil, in ATI-like primary epithelial cells compared to NCl-H441 and Calu-3 epithelial cells. Our findings suggest that OCTN2 is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium and that none of the tested cell lines are optimal surrogates for primary cells.
    • Preferential uptake of chitosan-coated PLGA nanoparticles by primary human antigen presenting cells.

      Durán, Verónica; Yasar, Hanzey; Becker, Jennifer; Thiyagarajan, Durairaj; Loretz, Brigitta; Kalinke, Ulrich; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Elsevier, 2019-07-31)
      Biodegradable polymeric nanoparticles (NP) made from poly (lactid-co-glycolide) acid (PLGA) and chitosan (CS) hold promise as innovative formulations for targeted delivery. Since interactions of such NP with primary human immune cells have not been characterized, yet, here we assessed the effect of PLGA or CS-PLGA NP treatment on human peripheral blood mononuclear cells (PBMC), as well as on monocyte-derived DC (moDC). Amongst PBMC, antigen presenting cells (APC) showed higher uptake of both NP preparations than lymphocytes. Furthermore, moDC internalized CS-PLGA NP more efficiently than PLGA NP, presumably because of receptor-mediated endocytosis. Consequently, CS-PLGA NP were delivered mostly to endosomal compartments, whereas PLGA NP primarily ended up in lysosomes. Thus, CS-PLGA NP confer enhanced delivery to endosomal compartments of APC, offering new therapeutic options to either induce or modulate APC function and to inhibit pathogens that preferentially infect APC.
    • STING induces early IFN-β in the liver and constrains myeloid cell-mediated dissemination of murine cytomegalovirus.

      Tegtmeyer, Pia-Katharina; Spanier, Julia; Borst, Katharina; Becker, Jennifer; Riedl, André; Hirche, Christoph; Ghita, Luca; Skerra, Jennifer; Baumann, Kira; Lienenklaus, Stefan; et al. (Springer-Nature, 2019-06-27)
      Cytomegalovirus is a DNA-encoded β-herpesvirus that induces STING-dependent type 1 interferon responses in macrophages and uses myeloid cells as a vehicle for dissemination. Here we report that STING knockout mice are as resistant to murine cytomegalovirus (MCMV) infection as wild-type controls, whereas mice with a combined Toll-like receptor/RIG-I-like receptor/STING signaling deficiency do not mount type 1 interferon responses and succumb to the infection. Although STING alone is dispensable for survival, early IFN-β induction in Kupffer cells is STING-dependent and controls early hepatic virus propagation. Infection experiments with an inducible reporter MCMV show that STING constrains MCMV replication in myeloid cells and limits viral dissemination via these cells. By contrast, restriction of viral dissemination from hepatocytes to other organs is independent of STING. Thus, during MCMV infection STING is involved in early IFN-β induction in Kupffer cells and the restriction of viral dissemination via myeloid cells, whereas it is dispensable for survival.
    • Modulation of TAP-dependent antigen compartmentalization during human monocyte-to-DC differentiation.

      Döring, Marius; Blees, Hanna; Koller, Nicole; Tischer-Zimmermann, Sabine; Müsken, Mathias; Henrich, Frederik; Becker, Jennifer; Grabski, Elena; Wang, Junxi; Janssen, Hans; et al. (American Society of Hematology, 2019-03-26)
      Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.
    • RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade.

      Heidegger, Simon; Kreppel, Diana; Bscheider, Michael; Stritzke, Florian; Nedelko, Tatiana; Wintges, Alexander; Bek, Sarah; Fischer, Julius C; Graalmann, Theresa; Kalinke, Ulrich; et al. (Elsevier, 2019-03-06)
      Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 enhances antitumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. Here we established a vaccination strategy that combines engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4 blockade. We used in vitro transcribed 5'-triphosphorylated RNA (3pRNA) to therapeutically target the RIG-I pathway. We performed in vitro functional analysis in bone-marrow derived dendritic cells and investigated RIG-I-enhanced vaccines in different murine melanoma models. We found that protein vaccination together with RIG-I ligation via 3pRNA strongly synergizes with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8 Overall, our data demonstrate the potency of a novel combinatorial vaccination strategy combining RIG-I-driven immunization with CTLA-4 blockade to prevent and treat experimental melanoma. FUND: German Research Foundation (SFB 1335, SFB 1371), EMBO, Else Kröner-Fresenius-Foundation, German Cancer Aid, European Hematology Association, DKMS Foundation for Giving Life, Dres. Carl Maximilian and Carl Manfred Bayer-Foundation.
    • Interferon-γ Receptor Signaling in Dendritic Cells Restrains Spontaneous Proliferation of CD4 T Cells in Chronic Lymphopenic Mice.

      Knop, Laura; Frommer, Charlotte; Stoycheva, Diana; Deiser, Katrin; Kalinke, Ulrich; Blankenstein, Thomas; Kammertoens, Thomas; Dunay, Ildiko Rita; Schüler, Thomas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen-Str.7,30625 Hannover, Germany. (Frontiers, 2019-01-01)
      In lymphopenic mice, T cells become activated and undergo lymphopenia-induced proliferation (LIP). However, not all T cells are equally sensitive to lymphopenia. Several lymphopenia-insensitive T cell clones were described and their non-responsiveness was mainly attributed to clone-specific properties. Here, we provide evidence for an additional, host-dependent mechanism restraining LIP of lymphopenia-insensitive CD4+ T cells. We show that such cells undergo LIP in lymphopenic mice lacking IFN-γ receptor (IFN-γR) expression, a process, which is promoted by the autocrine action of T cell-derived IFN-γ. Additionally, LIP of lymphopenia-insensitive CD4+ T cells requires an intact microflora and is accompanied by the massive accumulation of IL-6 and dendritic cells (DCs). Consistent with these results, IL-6 neutralization and the DC-specific restoration of IFN-γR expression are both sufficient to restrict LIP. Hence, the insensitivity of CD4+ T cells to lymphopenia relies on cell-intrinsic properties and a complex interplay between the commensal microflora, IL-6, IFN-γR+ DCs, and T cell-derived IFN-γ.
    • Myeloid Cells Restrict MCMV and Drive Stress-Induced Extramedullary Hematopoiesis through STAT1.

      Gawish, Riem; Bulat, Tanja; Biaggio, Mario; Lassnig, Caroline; Bago-Horvath, Zsuzsanna; Macho-Maschler, Sabine; Poelzl, Andrea; Simonović, Natalija; Prchal-Murphy, Michaela; Rom, Rita; et al. (Elsevier, 2019-02-26)
      Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection-associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen.
    • Cell therapy products: focus on issues with manufacturing and quality control of chimeric antigen receptor T-cell therapies

      Eyles, Jim E; Vessillier, Sandrine; Jones, Anika; Stacey, Glyn; Schneider, Christian K; Price, Jack; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.
      Recent accelerated approvals of Chimeric Antigen Receptor T‐cell (CAR‐T) therapies targeting refractory haematological malignancies underscore the potential for this novel technology platform to provide new therapeutic options for oncology areas with high unmet medical needs. However, these powerful ‘living drugs’ are markedly different to conventional small molecule and biologic therapies on several levels. The highly complex nature and varied composition of CAR‐T based products still requires considerable investigation to resolve the best approaches to ensure reproducible and cost‐effective manufacture, clinical development, and application. This review will focus on key issues for manufacturing and quality control of these exciting new therapeutic modalities, preceded by a brief description of CAR principals and clinical development considerations. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
    • Microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage.

      Waltl, Inken; Käufer, Christopher; Gerhauser, Ingo; Chhatbar, Chintan; Ghita, Luca; Kalinke, Ulrich; Löscher, Wolfgang; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2018-11-01)
      In the central nervous system (CNS), innate immune surveillance is mainly coordinated by microglia. These CNS resident myeloid cells are assumed to help orchestrate the immune response against infections of the brain. However, their specific role in this process and their interactions with CNS infiltrating immune cells, such as blood-borne monocytes and T cells are only incompletely understood. The recent development of PLX5622, a specific inhibitor of colony-stimulating factor 1 receptor that depletes microglia, allows studying the role of microglia in conditions of brain injury such as viral encephalitis, the most common form of brain infection. Here we used this inhibitor in a model of viral infection-induced epilepsy, in which C57BL/6 mice are infected by a picornavirus (Theiler's murine encephalomyelitis virus) and display seizures and hippocampal damage. Our results show that microglia are required early after infection to limit virus distribution and persistence, most likely by modulating T cell activation. Microglia depletion accelerated the occurrence of seizures, exacerbated hippocampal damage, and led to neurodegeneration in the spinal cord, which is normally not observed in this mouse strain. This study enhances our understanding of the role of microglia in viral encephalitis and adds to the concept of microglia-T cell crosstalk.
    • Regulatory T-Cells Mediate IFN-α-Induced Resistance against Antigen-Induced Arthritis.

      Chenna Narendra, Sudeep; Chalise, Jaya Prakash; Biggs, Sophie; Kalinke, Ulrich; Magnusson, Mattias; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Frontiers, 2018-01-01)
      CD4 Arthritis was triggered by intra-articular injection of methylated bovine serum albumin (mBSA) in wild-type mice, Foxp3DTReGFP Both control mice and mice devoid of IFNAR-signaling in T helper cells were protected from arthritis by IFN-α. Depletion of T By activating IDO during antigen sensitization, IFN-α activates T
    • 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.
    • Pulmonale Immunität bei Tuberkulose

      Herzmann, C.; Dallenga, T.; Kalinke, U.
      Tuberculosis is transmitted by inhalation of Mycobacterium tuberculosis-containing aerosols; 75 % of all patients show pulmonary manifestation. Immune responses after exposure that lead to clinical symptoms occur mainly in the respiratory tract and are only poorly understood. In most cases, cells of the innate immune system are believed to control the growth of or eradicate inhaled mycobacteria. However, this cannot be verified in vivo using standard methods. Subsequently, CD4+ and CD8+ T cell-driven adaptive immune responses are induced that attempt to control bacterial growth. The humoral defence appears to be less important. This article gives an overview of the current understanding of pulmonary immune mechanisms during exposure, latent infection, active disease and therapy of tuberculosis. Übersicht Herzmann C et al. Pulmonale Immunität bei … Pneumologie H
    • Type I Interferon Receptor Signaling of Neurons and Astrocytes Regulates Microglia Activation during Viral Encephalitis.

      Chhatbar, Chintan; Detje, Claudia N; Grabski, Elena; Borst, Katharina; Spanier, Julia; Ghita, Luca; Elliott, David A; Jordão, Marta Joana Costa; Mueller, Nora; Sutton, James; et al. (2018-10-02)
      In sterile neuroinflammation, a pathological role is proposed for microglia, whereas in viral encephalitis, their function is not entirely clear. Many viruses exploit the odorant system and enter the CNS via the olfactory bulb (OB). Upon intranasal vesicular stomatitis virus instillation, we show an accumulation of activated microglia and monocytes in the OB. Depletion of microglia during encephalitis results in enhanced virus spread and increased lethality. Activation, proliferation, and accumulation of microglia are regulated by type I IFN receptor signaling of neurons and astrocytes, but not of microglia. Morphological analysis of myeloid cells shows that type I IFN receptor signaling of neurons has a stronger impact on the activation of myeloid cells than of astrocytes. Thus, in the infected CNS, the cross talk among neurons, astrocytes, and microglia is critical for full microglia activation and protection from lethal encephalitis.
    • The olfactory epithelium as a port of entry in neonatal neurolisteriosis.

      Pägelow, Dennis; Chhatbar, Chintan; Beineke, Andreas; Liu, Xiaokun; Nerlich, Andreas; van Vorst, Kira; Rohde, M; Kalinke, Ulrich; Förster, Reinhold; Halle, Stephan; et al. (2018-10-15)
      Bacterial infections of the central nervous system (CNS) remain a major cause of mortality in the neonatal population. Commonly used parenteral infection models, however, do not reflect the early course of the disease leaving this critical step of the pathogenesis largely unexplored. Here, we analyzed nasal exposure of 1-day-old newborn mice to Listeria monocytogenes (Lm). We found that nasal, but not intragastric administration, led to early CNS infection in neonate mice. In particular, upon bacterial invasion of the olfactory epithelium, Lm subsequently spread along the sensory neurons entering the brain tissue at the cribriform plate and causing a significant influx of monocytes and neutrophils. CNS infection required listeriolysin for penetration of the olfactory epithelium and ActA, a mediator of intracellular mobility, for translocation into the brain tissue. Taken together, we propose an alternative port of entry and route of infection for neonatal neurolisteriosis and present a novel infection model to mimic the clinical features of late-onset disease in human neonates.
    • Chemokine receptors CCR2 and CX3CR1 regulate viral encephalitis-induced hippocampal damage but not seizures.

      Käufer, Christopher; Chhatbar, Chintan; Bröer, Sonja; Waltl, Inken; Ghita, Luca; Gerhauser, Ingo; Kalinke, Ulrich; Löscher, Wolfgang; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-09-18)
      Viral encephalitis is a major risk factor for the development of seizures, epilepsy, and hippocampal damage with associated cognitive impairment, markedly reducing quality of life in survivors. The mechanisms underlying seizures and hippocampal neurodegeneration developing during and after viral encephalitis are only incompletely understood, hampering the development of preventive treatments. Recent findings suggest that brain invasion of blood-born monocytes may be critically involved in both seizures and brain damage in response to encephalitis, whereas the relative role of microglia, the brain's resident immune cells, in these processes is not clear. CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation. We used
    • Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice.

      Koestner, Wolfgang; Spanier, Julia; Klause, Tanja; Tegtmeyer, Pia-K; Becker, Jennifer; Herder, Vanessa; Borst, Katharina; Todt, Daniel; Lienenklaus, Stefan; Gerhauser, Ingo; et al. (2018-08-01)
      During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-β reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-β responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR-/- mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.
    • 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.
    • 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; et al. (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.