• Strong interferon-inducing capacity of a highly virulent variant of influenza A virus strain PR8 with deletions in the NS1 gene.

      Kochs, Georg; Martínez-Sobrido, Luis; Lienenklaus, Stefan; Weiss, Siegfried; García-Sastre, Adolfo; Staeheli, Peter; Department of Virology, University of Freiburg, D-79008 Freiburg, Germany. georg.kochs@uniklinik-freiburg.de (2009-12)
      Influenza viruses lacking the interferon (IFN)-antagonistic non-structural NS1 protein are strongly attenuated. Here, we show that mutants of a highly virulent variant of A/PR/8/34 (H1N1) carrying either a complete deletion or C-terminal truncations of NS1 were far more potent inducers of IFN in infected mice than NS1 mutants derived from standard A/PR/8/34. Efficient induction of IFN correlated with successful initial virus replication in mouse lungs, indicating that the IFN response is boosted by enhanced viral activity. As the new NS1 mutants can be handled in standard biosafety laboratories, they represent convenient novel tools for studying virus-induced IFN expression in vivo.
    • Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.

      Osterloh, Anke; Kalinke, Ulrich; Weiss, Siegfried; Fleischer, Bernhard; Breloer, Minka; Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany. osterloh@bni.uni-hamburg.de (2007-02-16)
      Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.
    • Therapeutic Potential of Bacteria against Solid Tumors.

      Hatzikirou, Haralampos; López Alfonso, Juan Carlos; Leschner, Sara; Weiss, Siegfried; Meyer-Hermann, Michael; BRICS, Braunschweiger Zentrum für Systembiologie, Rebenring 56, 38106 Braunschweig, Germany. (2017-04-01)
      Intentional bacterial infections can produce efficacious antitumor responses in mice, rats, dogs, and humans. However, low overall success rates and intense side effects prevent such approaches from being employed clinically. In this work, we titered bacteria and/or the proinflammatory cytokine TNFα in a set of established murine models of cancer. To interpret the experiments conducted, we considered and calibrated a tumor-effector cell recruitment model under the influence of functional tumor-associated vasculature. In this model, bacterial infections and TNFα enhanced immune activity and altered vascularization in the tumor bed. Information to predict bacterial therapy outcomes was provided by pretreatment tumor size and the underlying immune recruitment dynamics. Notably, increasing bacterial loads did not necessarily produce better long-term tumor control, suggesting that tumor sizes affected optimal bacterial loads. Short-term treatment responses were favored by high concentrations of effector cells postinjection, such as induced by higher bacterial loads, but in the longer term did not correlate with an effective restoration of immune surveillance. Overall, our findings suggested that a combination of intermediate bacterial loads with low levels TNFα administration could enable more favorable outcomes elicited by bacterial infections in tumor-bearing subjects. Cancer Res; 77(7); 1553-63. ©2017 AACR.
    • Tumor invasion of Salmonella enterica serovar Typhimurium is accompanied by strong hemorrhage promoted by TNF-alpha.

      Leschner, Sara; Westphal, Kathrin; Dietrich, Nicole; Viegas, Nuno; Jablonska, Jadwiga; Lyszkiewicz, Marcin; Lienenklaus, Stefan; Falk, Werner; Gekara, Nelson; Loessner, Holger; et al. (2009)
      Several facultative anaerobic bacteria with potential therapeutic abilities are known to preferentially colonize solid tumors after systemic administration. How they efficiently find and invade the tumors is still unclear. However, this is an important issue to be clarified when bacteria should be tailored for application in cancer therapy.
    • Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.

      Andzinski, Lisa; Kasnitz, Nadine; Stahnke, Stephanie; Wu, Ching-Fang; Gereke, Marcus; von Köckritz-Blickwede, Maren; Schilling, Bastian; Brandau, Sven; Weiss, Siegfried; Jablonska, Jadwiga; et al. (2016-04-15)
      The importance of tumor associated neutrophils (TANs) in cancer development is in the meantime well established. Numerous of clinical data document the adverse prognostic effects of neutrophil infiltration in solid tumors. However, certain tumor therapies need functional neutrophils to be effective, suggesting altered neutrophil polarization associated with different outcomes for cancer patients. Therefore, modulation of neutrophilic phenotypes represents a potent therapeutic option, but factors mediating neutrophil polarization are still poorly defined. In this manuscript we provide evidence that type I IFNs alter neutrophilic phenotype into anti-tumor, both in mice and human. In the absence of IFN-β, pro-tumor properties, such as reduced tumor cytotoxicity with low neutrophil extracellular traps (NETs) expression, low ICAM1 and TNF-α expression, dominated neutrophil phenotypes in primary lesion and premetastatic lung. Interestingly, such neutrophils have significantly prolonged life-span. Notably, interferon therapy in mice altered TAN polarization towards anti-tumor N1. Similar changes in neutrophil activation could be observed in melanoma patients undergoing type I IFN therapy. Altogether, these data highlight the therapeutic potential of interferons, suggesting optimization of its clinical use as potent anti-tumor agent.
    • Type I Interferons Interfere with the Capacity of mRNA Lipoplex Vaccines to Elicit Cytolytic T Cell Responses.

      De Beuckelaer, Ans; Pollard, Charlotte; Van Lint, Sandra; Roose, Kenny; Van Hoecke, Lien; Naessens, Thomas; Udhayakumar, Vimal Kumar; Smet, Muriel; Sanders, Niek; Lienenklaus, Stefan; et al. (2016-11)
      Given their high potential to evoke cytolytic T cell responses, tumor antigen-encoding messenger RNA (mRNA) vaccines are now being intensively explored as therapeutic cancer vaccines. mRNA vaccines clearly benefit from wrapping the mRNA into nano-sized carriers such as lipoplexes that protect the mRNA from degradation and increase its uptake by dendritic cells in vivo. Nevertheless, the early innate host factors that regulate the induction of cytolytic T cells to mRNA lipoplex vaccines have remained unresolved. Here, we demonstrate that mRNA lipoplexes induce a potent type I interferon (IFN) response upon subcutaneous, intradermal and intranodal injection. Regardless of the route of immunization applied, these type I IFNs interfered with the generation of potent cytolytic T cell responses. Most importantly, blocking type I IFN signaling at the site of immunization through the use of an IFNAR blocking antibody greatly enhanced the prophylactic and therapeutic antitumor efficacy of mRNA lipoplexes in the highly aggressive B16 melanoma model. As type I IFN induction appears to be inherent to the mRNA itself rather than to unique properties of the mRNA lipoplex formulation, preventing type I IFN induction and/or IFNAR signaling at the site of immunization might constitute a widely applicable strategy to improve the potency of mRNA vaccination.
    • Visualizing production of beta interferon by astrocytes and microglia in brain of La Crosse virus-infected mice.

      Kallfass, Carsten; Ackerman, Andreas; Lienenklaus, Stefan; Weiss, Siegfried; Heimrich, Bernd; Staeheli, Peter; Department of Virology, University of Freiburg, Freiburg, Germany. (2012-10)
      Beta interferon (IFN-β) is a major component of innate immunity in mammals, but information on the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell types responsible for IFN-β production during viral encephalitis, we used reporter mice that express firefly luciferase under the control of the IFN-β promoter and stained organ sections with luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double-staining experiments with cell-type-specific markers revealed that similar numbers of astrocytes and microglia of infected brains were luciferase positive, whereas virus-infected neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast majority of the IFN-β-producing cells in infected brains were astrocytes rather than microglia. Similar conclusions were reached in a second series of experiments in which conditional reporter mice expressing the luciferase reporter gene solely in defined cell types were infected with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than infected neurons represent the major source of IFN-β in LACV-infected mouse brains. They further indicate that IFN-β synthesis in astrocytes and microglia is differentially affected by the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two cell types.
    • Visualizing the beta interferon response in mice during infection with influenza A viruses expressing or lacking nonstructural protein 1.

      Kallfass, Carsten; Lienenklaus, Stefan; Weiss, Siegfried; Staeheli, Peter; Department of Virology, University of Freiburg, Freiburg, Germany. (2013-06)
      The innate host defense against influenza virus is largely dependent on the type I interferon (IFN) system. However, surprisingly little is known about the cellular source of IFN in the infected lung. To clarify this question, we employed a reporter mouse that contains the firefly luciferase gene in place of the IFN-β-coding region. IFN-β-producing cells were identified either by simultaneous immunostaining of lungs for luciferase and cellular markers or by generating conditional reporter mice that express luciferase exclusively in defined cell types. Two different strains of influenza A virus were employed that either do or do not code for nonstructural protein 1 (NS1), which strongly suppresses innate immune responses of infected cells. We found that epithelial cells and lung macrophages, which represent the prime host cells for influenza viruses, showed vigorous IFN-β responses which, however, were severely reduced and delayed if the infecting virus was able to produce NS1. Interestingly, CD11c(+) cell populations that were either expressing or lacking macrophage markers produced the bulk of IFN-β at 48 h after infection with wild-type influenza A virus. Our results demonstrate that the virus-encoded IFN-antagonistic factor NS1 disarms specifically epithelial cells and lung macrophages, which otherwise would serve as main mediators of the early response against infection by influenza virus.