• Therapeutic benefit of Salmonella attributed to LPS and TNF-α is exhaustible and dictated by tumor susceptibility.

      Kocijancic, Dino; Leschner, Sara; Felgner, Sebastian; Komoll, Ronja-Melinda; Frahm, Michael; Pawar, Vinay; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-30)
      The potential of bacteria-mediated tumor therapy (BMTT) is highlighted by more than a century of investigation. Attenuated Salmonella has prevailed as promising therapeutic agents. For BMTT - categorized as an immune therapy - the exact contribution of particular immune reactions to the therapeutic effect remains ambiguous. In addition, one could argue for or against the requirement of bacterial viability and tumor targeting. Herein we evaluate the isolated therapeutic efficacy of purified LPS and TNF-α, which together account for a dominant immunogenic pathway of gram negative bacteria like Salmonella. We show that therapeutic efficacy against CT26 tumors does not require bacterial viability. Analogous to viable Salmonella SL7207, tumor regression by a specific CD8+ T cell response can be induced by purified LPS or recombinant TNF-α (rTNF-α). Conversely, therapeutic effects against RenCa tumors were abrogated upon bacterial avitalization and limited using isolated adjuvants. This argues for an alternative mechanistic explanation for SL7207 against RenCa that depends on viability and persistence. Unable to boost bacterial therapies by co-injection of rTNF-α suggested therapeutic effects along this axis are exhausted by the intrinsic adjuvanticity of bacteria alone. However, the importance of TNF-α for BMTT was highlighted by its support of tumor invasion and colonization in concert with lower infective doses of Salmonella. In consideration, bacterial therapeutic effectiveness along the axis of LPS and TNF-α appears limited, and does not offer the necessary plasticity for different tumors. This emphasizes a need for recombinant strengthening and vehicular exploitation to accommodate potency, plasticity and distinctiveness in BMTT.
    • Therapy of solid tumors using probiotic Symbioflor-2: restraints and potential.

      Kocijancic, Dino; Felgner, Sebastian; Frahm, Michael; Komoll, Ronja-Melinda; Iljazovic, Aida; Pawar, Vinay; Rohde, Manfred; Heise, Ulrike; Zimmermann, Kurt; Gunzer, Florian; Hammer, Juliane; Crull, Katja; Leschner, Sara; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-04-19)
      To date, virulent bacteria remain the basis of most bacteria mediated cancer therapies. For clinical application attenuation is required. However, this might result in a drastically lowered therapeutic capacity. Herein we argue that the E. coli probiotic Symbioflor-2, with a history of safe application may constitute a viable tumor therapeutic candidate. We demonstrate that Symbioflor-2 displays a highly specific tumor targeting ability as determined in murine CT26 and RenCa tumor models. The excellent specificity was ascribed to reduced levels of adverse colonization. A high safety standard was demonstrated in WT and Rag1-/- mice. Thus, Symbioflor-2 may represent an ideal tumor targeting delivery system for therapeutic molecules. Moreover, Symbioflor-2 was capable of inducing CT26 tumor clearance as result of an adjuvant effect on tumor specific CD8+ T cells analogous to the Salmonella variant SL7207. However, lower therapeutic efficacy against RenCa tumors suggested a generally reduced therapeutic potency for probiotics. Interestingly, concurrent depletion of Gr-1+ or Ly6G+ cells installed therapeutic efficacy equal to SL7207, thus highlighting the role of innate effector cells in restraining the anti-tumor effects of Symbioflor-2. Collectively, our findings argue for a strategy of safe strain application and a more sustainable use of bacteria as a delivery system for therapeutic molecules.
    • Topical imiquimod yields systemic effects due to unintended oral uptake.

      Grine, Lynda; Steeland, Sophie; Van Ryckeghem, Sara; Ballegeer, Marlies; Lienenklaus, Stefan; Weiss, Siegfried; Sanders, Niek N; Vandenbroucke, Roosmarijn E; Libert, Claude; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.
    • 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; Weiss, Siegfried (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.
    • Tumour-targeting bacteria-based cancer therapies for increased specificity and improved outcome.

      Felgner, Sebastian; Pawar, Vinay; Kocijancic, Dino; Erhardt, Marc; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-03)
    • 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; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (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 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.
    • 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; Saelens, Xavier; Weiss, Siegfried; Vanham, Guido; Grooten, Johan; De Koker, Stefaan; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (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.