• Tcf1 cells are required to maintain the inflationary T cell pool upon MCMV infection.

      Welten, Suzanne P M; Yermanos, Alexander; Baumann, Nicolas S; Wagen, Franziska; Oetiker, Nathalie; Sandu, Ioana; Pedrioli, Alessandro; Oduro, Jennifer D; Reddy, Sai T; Cicin-Sain, Luka; et al. (Springer Nature, 2020-05-08)
      Cytomegalovirus-based vaccine vectors offer interesting opportunities for T cell-based vaccination purposes as CMV infection induces large numbers of functional effector-like cells that accumulate in peripheral tissues, a process termed memory inflation. Maintenance of high numbers of peripheral CD8 T cells requires continuous replenishment of the inflationary T cell pool. Here, we show that the inflationary T cell population contains a small subset of cells expressing the transcription factor Tcf1. These Tcf1+ cells resemble central memory T cells and are proliferation competent. Upon sensing viral reactivation events, Tcf1+ cells feed into the pool of peripheral Tcf1- cells and depletion of Tcf1+ cells hampers memory inflation. TCR repertoires of Tcf1+ and Tcf1- populations largely overlap, with the Tcf1+ population showing higher clonal diversity. These data show that Tcf1+ cells are necessary for sustaining the inflationary T cell response, and upholding this subset is likely critical for the success of CMV-based vaccination approaches.
    • NUDT2 initiates viral RNA degradation by removal of 5'-phosphates.

      Laudenbach, Beatrice T; Krey, Karsten; Emslander, Quirin; Andersen, Line Lykke; Reim, Alexander; Scaturro, Pietro; Mundigl, Sarah; Dächert, Christopher; Manske, Katrin; Moser, Markus; et al. (Springer Nature, 2021-11-25)
      While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5'-triphosphate (PPP-) group that impairs degradation by the canonical 5'-3' degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5'-3' exonuclease XRN1. NUDT2 removes 5'-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5'-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.
    • Exogenous and Endogenous Triggers Differentially Stimulate Pigr Expression and Antibacterial Secretory Immunity in the Murine Respiratory Tract.

      Pausder, Alexander; Fricke, Jennifer; Schughart, Klaus; Schreiber, Jens; Strowig, Till; Bruder, Dunja; Boehme, Julia; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2021-11-26)
      Purpose: Transport of secretory immunoglobulin A (SIgA) through the airway epithelial cell barrier into the mucosal lumen by the polymeric immunoglobulin receptor (pIgR) is an important mechanism of respiratory mucosal host defense. Identification of immunomodulating substances that regulate secretory immunity might have therapeutic implications with regard to an improved immune exclusion. Thus, we sought to analyze secretory immunity under homeostatic and immunomodulating conditions in different compartments of the murine upper and lower respiratory tract (URT&LRT). Methods: Pigr gene expression in lung, trachea, and nasal-associated lymphoid tissue (NALT) of germ-free mice, specific pathogen-free mice, mice with an undefined microbiome, as well as LPS- and IFN-γ-treated mice was determined by quantitative real-time PCR. IgA levels in bronchoalveolar lavage (BAL), nasal lavage (NAL), and serum were determined by ELISA. LPS- and IFN-γ-treated mice were colonized with Streptococcus pneumoniae and bacterial CFUs were determined in URT and LRT. Results: Respiratory Pigr expression and IgA levels were dependent on the degree of exposure to environmental microbial stimuli. While immunostimulation with LPS and IFN-γ differentially impacts respiratory Pigr expression and IgA in URT vs. LRT, only prophylactic IFN-γ treatment reduces nasal colonization with S. pneumoniae. Conclusion: Airway-associated secretory immunity can be partly modulated by exposure to microbial ligands and proinflammatory stimuli. Prophylactic IFN-γ-treatment modestly improves antibacterial immunity in the URT, but this does not appear to be mediated by SIgA or pIgR.
    • Untitled

      Ruck, Tobias; Bock, Stefanie; Pfeuffer, Steffen; Schroeter, Christina B; Cengiz, Derya; Marciniak, Paul; Lindner, Maren; Herrmann, Alexander; Liebmann, Marie; Kovac, Stjepana; et al. (Springer Nature, 2021-10-26)
      It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.
    • Understanding the interaction between cytomegalovirus and tuberculosis in children: The way forward.

      Olbrich, Laura; Stockdale, Lisa; Basu Roy, Robindra; Song, Rinn; Cicin-Sain, Luka; Whittaker, Elizabeth; Prendergast, Andrew J; Fletcher, Helen; Seddon, James A; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2021-12-09)
      Over 1 million children develop tuberculosis (TB) each year, with a quarter dying. Multiple factors impact the risk of a child being exposed to Mycobacterium tuberculosis (Mtb), the risk of progressing to TB disease, and the risk of dying. However, an emerging body of evidence suggests that coinfection with cytomegalovirus (CMV), a ubiquitous herpes virus, impacts the host response to Mtb, potentially influencing the probability of disease progression, type of TB disease, performance of TB diagnostics, and disease outcome. It is also likely that infection with Mtb impacts CMV pathogenesis. Our current understanding of the burden of these 2 diseases in children, their immunological interactions, and the clinical consequence of coinfection is incomplete. It is also unclear how potential interventions might affect disease progression and outcome for TB or CMV. This article reviews the epidemiological, clinical, and immunological literature on CMV and TB in children and explores how the 2 pathogens interact, while also considering the impact of HIV on this relationship. It outlines areas of research uncertainty and makes practical suggestions as to potential studies that might address these gaps. Current research is hampered by inconsistent definitions, study designs, and laboratory practices, and more consistency and collaboration between researchers would lead to greater clarity. The ambitious targets outlined in the World Health Organization End TB Strategy will only be met through a better understanding of all aspects of child TB, including the substantial impact of coinfections.
    • Enhanced Susceptibility of ADAP-Deficient Mice to Infection Is Associated With an Altered Phagocyte Phenotype and Function.

      Böning, Martha A L; Parzmair, Gerald P; Jeron, Andreas; Düsedau, Henning P; Kershaw, Olivia; Xu, Baolin; Relja, Borna; Schlüter, Dirk; Dunay, Ildiko Rita; Reinhold, Annegret; et al. (Frontiers, 2021-09-30)
      The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection.
    • Maternal B Cell-Intrinsic MyD88 Signaling Mediates LPS-Driven Intrauterine Fetal Death.

      Busse, Mandy; Plenagl, Susanne; Campe, Norina Kim Jutta; Müller, Andreas J; Tedford, Kerry; Schumacher, Anne; Zenclussen, Ana Claudia; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-10-08)
      Immunological networks balance tolerance towards paternal alloantigens during pregnancy with normal immune response to pathogens. Subclinical infections can impact this balance and lead to preterm birth or even intrauterine fetal death (IUFD). We recently showed that loss of maternal B cells renders murine fetuses susceptible to IUFD after LPS exposure. Since the signaling pathway involved in this B-cell mediated response remains unclear, we aimed to understand the participation of MyD88 in this response using B-cell-specific MyD88-deficient (BMyD88-/-) mice. B cells isolated from wild-type (WT), BMyD88-/-, CD19-/- and MyD88-/- dams on gestational day (gd) 10 responded differently to LPS concerning cytokine secretion. In vivo LPS challenge on gd 10 provoked IUFD in CD19-/- mothers with functional MyD88, while fetuses from BMyD88-/- and MyD88-/- mice were protected. These outcomes were associated with altered cytokine levels in the maternal serum and changes in CD4+ T-cell responses. Overall, the loss of MyD88 signaling in maternal B cells prevents the activation of cytokine release that leads to IUFD. Thus, while MyD88 signaling in maternal B cells protects the mother from infection, it ultimately kills the fetus. Understanding the cellular mechanisms underlying infection-driven pregnancy complications is the first step to designing powerful therapeutic strategies in the future.
    • Interleukin-11 receptor expression on monocytes is dispensable for their recruitment and pathogen uptake during Leishmania major infection.

      Baars, Iris; Lokau, Juliane; Sauerland, Ina; Müller, Andreas J; Garbers, Christoph; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Academic Press/ Elsevier, 2021-09-13)
      Interleukin-11 (IL-11) is an important member of the IL-6 family of cytokines. IL-11 activates its target cells via binding to a non-signaling α-receptor (IL-11R), which results in recruitment and activation of a gp130 homodimer. The cytokine was initially described as an anti-inflammatory protein, but has recently gained attention as a potent driver in certain types of cancer and different fibrotic conditions. Leishmania spp. are a group of eukaryotic parasites that cause the disease leishmaniasis. They infect phagocytes of their hosts, especially monocytes recruited to the site of infection, and are able to replicate within this rather harsh environment, often resulting in chronic infections of the patient. However, the molecular mechanisms underlying parasite and host cell interactions and factors of the immune cells that are crucial for Leishmania uptake are so far largely unspecified. Recently, increased IL-11 expression in the lesions of patients with cutaneous leishmaniasis has been reported, but the functional relevance is unknown. In this study, we show that monocytes express IL-11R on their cell surface. Furthermore, using an adoptive transfer model of IL-11R-/- monocytes, we analyze the contribution of IL-11 signaling on monocyte recruitment and monocyte infection in a mouse model of cutaneous leishmaniasis and find that IL-11 signaling is dispensable for monocyte recruitment and pathogen uptake during Leishmania major infection.
    • Identification of cell lines CL-14, CL-40 and CAL-51 as suitable models for SARS-CoV-2 infection studies.

      Pommerenke, Claudia; Rand, Ulfert; Uphoff, Cord C; Nagel, Stefan; Zaborski, Margarete; Hauer, Vivien; Kaufmann, Maren; Meyer, Corinna; Denkmann, Sabine A; Riese, Peggy; et al. (PLOS, 2021-08-02)
      The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.
    • Fate mapping of single NK cells identifies a type 1 innate lymphoid-like lineage that bridges innate and adaptive recognition of viral infection.

      Flommersfeld, Sophie; Böttcher, Jan P; Ersching, Jonatan; Flossdorf, Michael; Meiser, Philippa; Pachmayr, Ludwig O; Leube, Justin; Hensel, Inge; Jarosch, Sebastian; Zhang, Qin; et al. (Cell Press, 2021-08-20)
      Upon viral infection, natural killer (NK) cells expressing certain germline-encoded receptors are selected, expanded, and maintained in an adaptive-like manner. Currently, these are thought to differentiate along a common pathway. However, by fate mapping of single NK cells upon murine cytomegalovirus (MCMV) infection, we identified two distinct NK cell lineages that contributed to adaptive-like responses. One was equivalent to conventional NK (cNK) cells while the other was transcriptionally similar to type 1 innate lymphoid cells (ILC1s). ILC1-like NK cells showed splenic residency and strong cytokine production but also recognized and killed MCMV-infected cells, guided by activating receptor Ly49H. Moreover, they induced clustering of conventional type 1 dendritic cells and facilitated antigen-specific T cell priming early during MCMV infection, which depended on Ly49H and the NK cell-intrinsic expression of transcription factor Batf3. Thereby, ILC1-like NK cells bridge innate and adaptive viral recognition and unite critical features of cNK cells and ILC1s.
    • Cell-Free Glycoengineering of the Recombinant SARS-CoV-2 Spike Glycoprotein.

      Ruhnau, Johannes; Grote, Valerian; Juarez-Osorio, Mariana; Bruder, Dunja; Mahour, Reza; Rapp, Erdmann; Rexer, Thomas F T; Reichl, Udo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-08-16)
      The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β-1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the β-1,4-galactosyltransferase (GalTΔTM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.
    • A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

      Bertoglio, Federico; Fühner, Viola; Ruschig, Maximilian; Heine, Philip Alexander; Abassi, Leila; Klünemann, Thomas; Rand, Ulfert; Meier, Doris; Langreder, Nora; Steinke, Stephan; et al. (Cell Press, 2021-07-07)
      The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.
    • Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

      Müller, Thomas R; Jarosch, Sebastian; Hammel, Monika; Leube, Justin; Grassmann, Simon; Bernard, Bettina; Effenberger, Manuel; Andrä, Immanuel; Chaudhry, M Zeeshan; Käuferle, Theresa; et al. (Elsevier, 2021-08-17)
      Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.
    • Tbx21 and foxp3 are Epigenetically Stabilized in T-Bet Tregs That Transiently Accumulate in Influenza A Virus-Infected Lungs.

      Elfaki, Yassin; Yang, Juhao; Boehme, Julia; Schultz, Kristin; Bruder, Dunja; Falk, Christine S; Huehn, Jochen; Floess, Stefan; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-07-14)
      During influenza A virus (IAV) infections, CD4+ T cell responses within infected lungs mainly involve T helper 1 (Th1) and regulatory T cells (Tregs). Th1-mediated responses favor the co-expression of T-box transcription factor 21 (T-bet) in Foxp3+ Tregs, enabling the efficient Treg control of Th1 responses in infected tissues. So far, the exact accumulation kinetics of T cell subsets in the lungs and lung-draining lymph nodes (dLN) of IAV-infected mice is incompletely understood, and the epigenetic signature of Tregs accumulating in infected lungs has not been investigated. Here, we report that the total T cell and the two-step Treg accumulation in IAV-infected lungs is transient, whereas the change in the ratio of CD4+ to CD8+ T cells is more durable. Within lungs, the frequency of Tregs co-expressing T-bet is steadily, yet transiently, increasing with a peak at Day 7 post-infection. Interestingly, T-bet+ Tregs accumulating in IAV-infected lungs displayed a strongly demethylated Tbx21 locus, similarly as in T-bet+ conventional T cells, and a fully demethylated Treg-specific demethylated region (TSDR) within the Foxp3 locus. In summary, our data suggest that T-bet+ but not T-bet- Tregs are epigenetically stabilized during IAV-induced infection in the lung.
    • SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

      Bertoglio, Federico; Meier, Doris; Langreder, Nora; Steinke, Stephan; Rand, Ulfert; Simonelli, Luca; Heine, Philip Alexander; Ballmann, Rico; Schneider, Kai-Thomas; Roth, Kristian Daniel Ralph; et al. (NPG, 2021-03-11)
      COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.
    • OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation.

      Koschel, Josephin; Nishanth, Gopala; Just, Sissy; Harit, Kunjan; Kröger, Andrea; Deckert, Martina; Naumann, Michael; Schlüter, Dirk; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2021-03-12)
      In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.
    • Contact-dependent transmission of Langat and tick-borne encephalitis virus in type I interferon receptor-1 deficient mice.

      Schreier, Sarah; Cebulski, Kristin; Kröger, Andrea; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society for Microbiology, 2021-01-27)
      Tick-borne encephalitis virus (TBEV) is primarily transmitted to humans through tick bites or oral consumption of accordingly contaminated unpasteurized milk or milk products. The detection of TBEV RNA in various body fluids in immunosuppressed human patients is documented. However, the risk of direct contact exposure remains unclear. Interferon-alpha receptor-1 deficient (Ifnar1-/- ) mice, which are lacking the interferon-α/β responses, develop neurologic manifestations after infection with TBEV and Langat virus (LGTV). We showed that subcutaneous, intranasal, and peroral infection of LGTV lead to disease, whereas mice with intragastric application of LGTV showed no disease signs. With LGTV infected mice exhibit seroconversion and significant viral RNA levels was detected in saliva, eye smear, feces and urine. As a result, TBEV and LGTV are transmitted between mice from infected to naïve co-caged sentinel animals. Although intranasal inoculation of LGTV is entirely sufficient to establish the disease in mice, the virus is not transmitted by aerosols. These pooled results from animal models highlight the risks of exposure to TBEV contaminants and the possibility for close contact transmission of TBEV in interferon-alpha receptor-1 deficient laboratory mice.Importance Tick-borne encephalitis is a severe disease of the central nervous system caused by the tick-borne encephalitis virus (TBEV). Every year between 10,000-12,000 people become infected with this flavivirus. The TBEV is usually transmitted to humans via the bite of a tick, but infections due to consumption of infectious milk products are increasingly being reported. Since there is no therapy for an TBEV infection and mechanisms of virus persistence in reservoir animals are unclear, it is important to highlight the risk of exposure to TBEV contaminants and know possible routes of transmission of this virus. The significance of our research is in identifying other infection routes of TBEV and LGTV, and the possibility of close contact transmission.
    • A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

      Rand, Ulfert; Kubsch, Tobias; Kasmapour, Bahram; Cicin-Sain, Luka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2021-01-08)
      Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.
    • Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

      Seelbinder, Bastian; Wallstabe, Julia; Marischen, Lothar; Weiss, Esther; Wurster, Sebastian; Page, Lukas; Löffler, Claudia; Bussemer, Lydia; Schmitt, Anna-Lena; Wolf, Thomas; et al. (Elsevier (Cell Press), 2020-11-17)
      High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.
    • Eosinophilic pulmonary vasculitis as a manifestation of the hyperinflammatory phase of COVID-19.

      Luecke, Eva; Jeron, Andreas; Kroeger, Andrea; Bruder, Dunja; Stegemann-Koniszewski, Sabine; Jechorek, Doerthe; Borucki, Katrin; Reinhold, Dirk; Reinhold, Annegret; Foellner, Sebastian; et al. (Elsevier, 2020-10-26)
      No abstract available