A mouse model-based screening platform for the identification of immune activating compounds such as natural products for novel cancer immunotherapies.

The therapy of cancer continues to be a challenge aggravated by the evolution of resistance against current medications. As an alternative for the traditional tripartite treatment options of surgery, radiation and chemotherapy, immunotherapy is gaining increasing attention due to the opportunity of more targeted approaches. Promising targets are antigen-presenting cells which drive innate and adaptive immune responses. The discovery and emergence of new drugs and lead structures can be inspired by natural products which comprise many highly bioactive molecules. The development of new drugs based on natural products is hampered by the current lack of guidelines for screening these structures for immune activating compounds. In this work, we describe a phenotypic preclinical screening pipeline for first-line identification of promising natural products using the mouse as a model system. Favorable compounds are defined to be non-toxic to immune target cells, to show direct anti-tumor effects and to be immunostimulatory at the same time. The presented screening pipeline constitutes a useful tool and aims to integrate immune activation in experimental approaches early on in drug discovery. It supports the selection of natural products for later chemical optimization, direct application in in vivo mouse models and clinical trials and promotes the emergence of new innovative drugs for cancer treatment.

Production of IFNß by Conventional Dendritic Cells after Stimulation with Viral Compounds and IFNß-Independent IFNAR1-Signaling Pathways are Associated with Aggravation of Polymicrobial Sepsis.

Viral infections are associated with increased incidence of severe sepsis. Particularly during the early stages, type I interferons (IFNs) are known mediators of detrimental effects. However, the functional role of early interferon ß (IFNß) and its cellular source during sepsis in the context of preexisting viral infections has not been defined. Using the colon ascendens stent peritonitis (CASP) model, we demonstrate that IFNß-/- and type I IFN receptor (IFNAR1)-/- mice were less susceptible to sepsis after pre-stimulation with the viral mimetic poly(I:C). Wild type (WT) mice treated with poly(I:C) exhibited altered expression patterns of TNF and IL-12p40 during CASP which were dependent on IFNß or IFNAR1, suggesting a mechanism for the increased sepsis susceptibility of WT mice. Using a double cytokine reporter mouse model, we present novel data on the simultaneous expression of IFNß and IL-12p40 on a single cell level during polymicrobial sepsis in vivo. Conventional dendritic cells (cDCs) were identified as primary source of IFNß and the protective cytokine IL-12p40 after CASP surgery irrespective of poly(I:C) pre-stimulation. These data demonstrated that if polymicrobial sepsis is preceded by a viral infection, IFNß and IL-12p40 are expressed by polyfunctional cDCs suggesting that these cells can play both detrimental and beneficial roles during sepsis development.

IFNß secreted by microglia mediates clearance of myelin debris in CNS autoimmunity.

INTRODUCTION: Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological disability. Interferon ß (IFNß) represents a standard treatment for relapsing-remitting MS and exogenous administration of IFNß exhibits protective effects in experimentally induced CNS autoimmunity. Also, genetic deletion of IFNß in mice leads to an aggravation of disease symptoms in the MS model of experimental autoimmune encephalomyelitis (EAE). However, neither the underlying mechanisms mediating the beneficial effects nor the cellular source of IFNß have been fully elucidated. RESULTS: In this report, a subpopulation of activated microglia was identified as the major producers of IFNß in the CNS at the peak of EAE using an IFNß-fluorescence reporter mouse model. These IFNß expressing microglia specifically localized to active CNS lesions and were associated with myelin debris in demyelinated cerebellar organotypic slice cultures (OSCs). In response to IFNß microglia showed an enhanced capacity to phagocytose myelin in vitro and up-regulated the expression of phagocytosis-associated genes. IFNß treatment was further sufficient to stimulate association of microglia with myelin debris in OSCs. Moreover, IFNß-producing microglia mediated an enhanced removal of myelin debris when co-transplanted onto demyelinated OSCs as compared to IFNß non-producing microglia. CONCLUSIONS: These data identify activated microglia as the major producers of protective IFNß at the peak of EAE and as orchestrators of IFNß-induced clearance of myelin debris.

The TGF-ß signaling modulators TRAP1/TGFBRAP1 and VPS39/Vam6/TLP are essential for early embryonic development.

The pleiotropic cytokine transforming growth factor-ß (TGF-ß) signals through different pathways among which the Smad- and the MAP-Kinase pathways are already well characterized. Both pathways utilize adaptor/chaperone molecules that facilitate or modulate the intracellular signaling events. Two of the proteins shown in vitro to play a role in Smad-dependent signaling are the TGF-ß Receptor Associated Protein-1 (TRAP1, also TGFBRAP1) and its homologue VPS39, also known as Vam6 and TRAP1-Like-Protein (TLP). We generated mice deficient for TRAP1 and VPS39/TLP, respectively. Absence of TRAP1 protein results in death at either of two defined timepoints during embryogenesis, before the blastula stage or during gastrulation, whereas most of the VPS39 deficient mice die before E6.5. Heterozygous mice show no overt phenotype. In summary, our data indicate that TRAP1 and VPS39 are nonredundant and essentially required for early embryonic development.

A fluorescence reporter model defines "Tip-DCs" as the cellular source of interferon ß in murine listeriosis.

Production of type I interferons, consisting mainly of multiple IFNα subtypes and IFNß, represents an essential part of the innate immune defense against invading pathogens. While in most situations, namely viral infections, this class of cytokines is indispensable for host survival they mediate a detrimental effect during infection with L. monocytogenes by rendering macrophages insensitive towards IFNγ signalling which leads to a lethal bacterial pathology in mice. Due to a lack of suitable analytic tools the precise identity of the cell population responsible for type I IFN production remains ill-defined and so far these cells have been described to be macrophages. As in general IFNß is the first type I interferon to be produced, we took advantage of an IFNß fluorescence reporter-knockin mouse model in which YFP is expressed from a bicistronic mRNA linked by an IRES to the endogenous ifnb mRNA to assess the IFNß production on a single cell level in situ. Our results showed highest frequencies and absolute numbers of IFNß+ cells in the spleen 24 h after infection with L. monocytogenes where they were located predominately in the white pulp within the foci of infection. Detailed FACS surface marker analyses, intracellular cytokine stainings and T cell proliferation assays revealed that the IFNß+ cells were a phenotypically and functionally further specialized subpopulation of TNF and iNOS producing DCs (Tip-DCs) which are known to be essential for the early containment of L. monocytogenes infection. We proved that the IFNß+ cells exhibited the hallmark characteristics of Tip-DCs as they produced iNOS and TNF and possessed T cell priming abilities. These results point to a yet unappreciated ambiguous role for a multi-effector, IFNß producing subpopulation of Tip-DCs in controlling the balance between containment of L. monocytogenes infection and effects detrimental to the host driven by IFNß.