Siegfried Weiss

NK cell activation in visceral leishmaniasis requires TLR9, myeloid DCs, and IL-12, but is independent of plasmacytoid DCs

Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-α/β and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-α/β. Depletion of pDCs did not impair the activation of NK cells in L. infantum–infected mice. In contrast, L. infantum–induced NK cell cytotoxicity and IFN-γ production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9−/− mice, which lacked IL-12 expression by mDCs, and in IL-12−/− mice, whereas IFN-α/β receptor−/− mice showed only a minor reduction of NK cell IFN-γ expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-γ release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis.

La Crosse Bunyavirus Nonstructural Protein NSs Serves To Suppress the Type I Interferon System of Mammalian Hosts

ABSTRACT La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the hosts IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.

Protective role of beta interferon in host defense against influenza A virus.

ABSTRACT Type I interferon (IFN), which includes the IFN-α and -β subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-β remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-β-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-β-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-β and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-β plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-α.

Modified Vaccinia Virus Ankara Induces Toll-Like Receptor-Independent Type I Interferon Responses

ABSTRACT Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus strain undergoing clinical evaluation as a replication-deficient vaccine vector against various infections and tumor diseases. To analyze the basis of its high immunogenicity, we investigated the mechanism of how MVA induces type I interferon (IFN) responses. MVA stimulation of bone marrow-derived dendritic cells (DC) showed that plasmacytoid DC were main alpha IFN (IFN-α) producers that were triggered independently of productive infection, viral replication, or intermediate and late viral gene expression. Increased IFN-α levels were induced upon treatment with mildly UV-irradiated MVA, suggesting that a virus-encoded immune modulator(s) interfered with the host cytokine response. Mice devoid of Toll-like receptor 9 (TLR9), the receptor for double-stranded DNA, mounted normal IFN-α responses upon MVA treatment. Furthermore, mice devoid of the adaptors of TLR signaling MyD88 and TRIF and mice deficient in protein kinase R (PKR) showed IFN-α responses that were only slightly reduced compared to those of wild-type mice. MVA-induced IFN-α responses were critically dependent on autocrine/paracrine triggering of the IFN-α/β receptor and were independent of IFN-β, thus involving “one-half” of a positive-feedback loop. In conclusion, MVA-mediated type I IFN secretion was primarily triggered by non-TLR molecules, was independent of virus propagation, and critically involved IFN feedback stimulation. These data provide the basis to further improve MVA as a vaccine vector.

Synergistic and Differential Modulation of Immune Responses by Hsp60 and Lipopolysaccharide

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) γ release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNα production in professional APC. Neutralization of IFNα as well as the absence of functional IFNαβ receptor on APC and T cells interfered with Hsp60-mediated IFNγ secretion in antigen-dependent T cell activation, strongly suggesting that IFNα 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 IFNγ 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.

GARP: a key receptor controlling FOXP3 in human regulatory T cells

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein‐A repetitions predominant), LGALS3 (lectin, galactoside‐binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T‐cell receptor stimulation. Retroviral overexpression of GARP in antigen‐specific Th cells leads to an efficient and stable re‐programming of an effector T cell towards a regulatory T cell, which involves up‐regulation of FOXP3, LGALS3, LGMN and other Treg‐associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down‐regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down‐regulation of FOXP3. Moreover, down‐regulation of FOXP3 resulted in similar phenotypic changes and down‐regulation of GARP. This provides compelling evidence for a GARP‐FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor‐β induced Treg cells as we show here that the latter do not up‐regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T‐cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization

The pore‐forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI‐anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLOs ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts’ aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts’ associated receptors, kinases and adaptors.

Lipid rafts clustering and signalling by listeriolysin O.

Listeriolysin O, the major virulent determinant of Listeria monocytogenes, is known for forming pores on cholesterol-rich membranes. In the present study, we reveal its other facet, rafts clustering. By immunofluorescence microscopy, we show that the glycosylphosphatidylinositol-anchored proteins CD14 and CD24, which normally exhibit uniform distribution on J774 cells, undergo clustering upon treatment with LLO. The non-raft marker transferrin receptor is unaffected by such treatment. Rafts clustering might explain the induction of tyrosine phosphorylation observed on LLO-treated cells.

Potentiation of Epithelial Innate Host Responses by Intercellular Communication

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection.

CXCR2-mediated tumor-associated neutrophil recruitment is regulated by IFN-β

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor‐associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon‐beta (IFN‐β) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor‐bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN‐β appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN‐β, they were expressed significantly higher in tumor‐infiltrating neutrophils. Treatment of such neutrophils from tumor‐bearing Ifnb1−/− mice with recombinant IFN‐β downregulated CXCR2 ligand expression to wild‐type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN‐β‐sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.