Ulrike Schleicher

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.

Key role of splenic myeloid DCs in the IFN-alphabeta response to adenoviruses in vivo.

The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.

The innate immune response against Leishmania parasites.

Parasites of the genus Leishmania are the causative agents of cutaneous, mucocutaneous or visceral leishmaniasis. The parasite species and host genetic factors determine the quality of the immune response and thereby the outcome of the infection. Here, we summarize previously published and present novel data on several aspects of the early innate immune reaction to Leishmania (L.) major, L. braziliensis and L. infantum, which cause cutaneous, mucocutaneous or visceral leishmaniasis, respectively. We will focus on (1) the effector molecules that contribute to the control of the parasite in the skin, lymph nodes and/or spleen; and (2) on the pattern recognition receptors (Toll-like receptors, TLRs), cell types (myeloid dendritic cells, plasmacytoid dendritic cells), cytokines (IL-12, IFN-alpha/beta), and signaling pathways (Tyk2 kinase) that are necessary for the initial sensing of the parasites and the subsequent development of an efficient NK cell response.

TLR9 signaling is essential for the innate NK cell response in murine cutaneous leishmaniasis

Mice deficient for the TLR adaptor molecule MyD88 succumb to a local infection with Leishmania (L.) major. However, the TLR(s) that contribute to the control of this intracellular parasite remain to be defined. Here, we show that TLR9 was required for the induction of IL‐12 in bone marrow‐derived DC by intact L. major parasites or L. major DNA and for the early IFN‐γ expression and cytotoxicity of NK cells following infection with L. major in vivo. During the acute phase of infection TLR9–/– mice exhibited more severe skin lesions and higher parasite burdens than C57BL/6 wild‐type controls. Although TLR9 deficiency led to a transient increase of IL‐4, IL‐13 and arginase 1 mRNA and a reduced expression of iNOS at the site of infection and in the draining lymph nodes, it did not prevent the development of Th1 cells and the ultimate resolution of the infection. We conclude that TLR9 signaling is essential for NK cell activation, but dispensable for a protective T cell response to L. major in vivo.

Nitric oxide–mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection

NOS2-derived nitric oxide drives ferroportin-1–mediated iron export in Salmonella-infected macrophages, thus limiting bacterial growth.

Organ-specific and stage-dependent control of Leishmania major infection by inducible nitric oxide synthase and phagocyte NADPH oxidase

In the Leishmania major mouse model of cutaneous leishmaniasis inducible nitric oxide synthase (iNOS) is crucial for the killing of the parasite in the skin and draining lymph node. However, the effector mechanism operating against L. major in the spleen is unknown. As reactive oxygen intermediates might play a role, we analyzed macrophages and mice lacking the gp91phoxsubunit of the phagocyte NADPH oxidase (phox) for their ability to combat an infection with L. major. Macrophages from wild‐type and gp91phox–/– mice had an equal capacity to killL. major after activation by cytokines. Unlike iNOS, the activity of phox was dispensable for the resolution of the acute skin lesions and exerted only a limited effect on the containment of the parasites in the draining lymph node, but was essential for the clearance of L. major in the spleen. During the chronic phase of infection, parasites persisted at high levels in gp91phox–/– mice, and cutaneous lesions re‐emerged in approximately 60% of these mice. gp91phox deficiency did not impair the expression of iNOS or the production of TNF and IFN‐γ. These results demonstrate that iNOS and phox are both required for the control of L. major in vivo and display unexpected organ‐ and stage‐specific anti‐leishmanial effects.

The Mincle-Activating Adjuvant TDB Induces MyD88-Dependent Th1 and Th17 Responses through IL-1R Signaling

Successful vaccination against intracellular pathogens requires the generation of cellular immune responses. Trehalose-6,6-dibehenate (TDB), the synthetic analog of the mycobacterial cord factor trehalose-6,6-dimycolate (TDM), is a potent adjuvant inducing strong Th1 and Th17 immune responses. We previously identified the C-type lectin Mincle as receptor for these glycolipids that triggers the FcRγ-Syk-Card9 pathway for APC activation and adjuvanticity. Interestingly, in vivo data revealed that the adjuvant effect was not solely Mincle-dependent but also required MyD88. Therefore, we dissected which MyD88-dependent pathways are essential for successful immunization with a tuberculosis subunit vaccine. We show here that antigen-specific Th1/Th17 immune responses required IL-1 receptor-mediated signals independent of IL-18 and IL-33-signaling. ASC-deficient mice had impaired IL-17 but intact IFNγ responses, indicating partial independence of TDB adjuvanticity from inflammasome activation. Our data suggest that the glycolipid adjuvant TDB triggers Mincle-dependent IL-1 production to induce MyD88-dependent Th1/Th17 responses in vivo.

Type I IFN Modulates Host Defense and Late Hyperinflammation in Septic Peritonitis

TLRs are considered important for the control of immune responses during endotoxic shock or polymicrobial sepsis. Signaling by TLRs may proceed through the adapter proteins MyD88 or TIR domain-containing adaptor inducinng IFN-β. Both pathways can lead to the production of type I IFNs (IFN-αβ). In the present study, the role of the type I IFN pathway for host defense and immune pathology in sepsis was investigated using a model of mixed bacterial peritonitis. Systemic levels of IFN-αβ protein were markedly elevated during septic peritonitis. More detailed analyses revealed production of IFN-β, but not IFN-α subtypes, and identified CD11b+CD11c− macrophage-like cells as major producers of IFN-β. The results further demonstrate that in IFN-αβ receptor I chain (IFNARI)-deficient mice, the early recruitment of neutrophils to the infected peritoneal cavity was augmented, most likely due to an increased local production of MCP-1 and leukotriene B4. In the absence of IFNARI, peritoneal neutrophils also exhibited enhanced production of reactive oxygen intermediates and elevated expression of Mac-1. Conversely, administration of recombinant IFN-β resulted in reduced leukotriene B4 levels and decreased peritoneal neutrophil recruitment and activation. Analysis of the cytokine response to septic peritonitis revealed that IFNARI deficiency strongly attenuated late, but not early, hyperinflammation. In accordance with these findings, bacterial clearance and overall survival of IFNARI−/− mice were improved. Therefore, the present study reveals critical functions of the type I IFN pathway during severe mixed bacterial infections leading to sepsis. The results suggest that type I IFN exerts predominantly adverse effects under these conditions.

Toll-like receptor activation and hypoxia use distinct signaling pathways to stabilize hypoxia-inducible factor 1α (HIF1A) and result in differential HIF1A-dependent gene expression.

HIF1A is a transcription factor that plays a central role for the adaptation to tissue hypoxia and for the inflammatory response of myeloid cells, including DCs. HIF1A is stabilized by hypoxia but also by TLR ligands under normoxic conditions. The underlying signaling events leading to the accumulation of HIF1A in the presence of oxygen are still poorly understood. Here, we show that in contrast to hypoxic stabilization of HIF1A, normoxic, TLR‐mediated HIF1A accumulation in DCs follows a different pathway that predominantly requires MYD88‐dependent NF‐κB activity. The TLR‐induced HIF1A controls a subset of proinflammatory genes that are insufficiently induced following hypoxia‐mediated HIF1A induction. Thus, TLR activation and hypoxia stabilize HIF1A via distinct signaling pathways, resulting in differential HIF1A‐dependent gene expression.

Cytokines, Signaling Pathways, and Effector Molecules Required for the Control of Leishmania (Viannia) braziliensis in Mice

ABSTRACT Cutaneous leishmaniasis is caused by protozoan parasites of the genus Leishmania. The mechanisms of pathogen control have been established primarily in the mouse model of Leishmania major infection, but they might not hold true for other Leishmania species associated with cutaneous disease. Here, we analyzed the role of cytokines, signaling components, and effector molecules in the control of New World cutaneous leishmaniasis due to L. braziliensis. Unlike L. major, L. braziliensis caused small, nonulcerative, and self-healing skin swelling in C57BL/6 mice, as well as BALB/c mice. In contrast to the results obtained for L. mexicana, mice deficient for interleukin-12 or its key signaling molecule, signal transducer and activator of transcription 4, rapidly succumbed to severe visceral leishmaniasis. Infection of tumor necrosis factor knockout mice with L. braziliensis led to progressive, nonhealing skin lesions with erosions and hemorrhagic ulcerations, but in contrast to the results with L. major, only 20 to 30% of the mice developed fatal visceral disease. As seen with L. major, mice with a deleted inducible nitric oxide synthase gene (iNOS−/−) were unable to contain L. braziliensis in the skin, whereas the control of the parasite in the spleen remained unimpaired. Unlike what happens in L. major infections, NADPH oxidase had no impact on the course of disease in L. braziliensis-infected mice. These results not only define essential components of a protective immune response to L. braziliensis but also illustrate that the requirements for the control of cutaneous leishmaniasis vary between different parasite species.