Heike Schindler

Repetitive Injections of Dendritic Cells Matured with Tumor Necrosis Factor α Induce Antigen-specific Protection of Mice from Autoimmunity

Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-α (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-α induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10–producing CD4+ T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-α results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10–producing T cells in vivo and prevent EAE.

IFN-γ production by antigen-presenting cells: mechanisms emerge

Abstract The suggestion that antigen-presenting cells (APCs) produce interferon γ (IFN-γ) is controversial because it conflicts with the initial paradigm in which the production of IFN-γ was restricted to lymphoid cells. However, some answers to this skepticism have been provided by recent findings of high-level production and intracellular expression of IFN-γ by interleukin-12 (IL-12)-stimulated macrophages and dendritic cells. New data are now emerging to explain the mechanism of production of IFN-γ vby APCs. As in lymphoid cells, IL-12-induced IFN-γ production in APCs requires signal transducer and activator of transcription 4 (STAT4), although the precise molecular events that govern the transcription of the gene encoding IFN-γ are enigmatic still. Understanding these processes in lymphoid, and now nonlymphoid, cells remains an important challenge.

Partial impairment of cytokine responses in Tyk2-deficient mice.

To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2-/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2-/- mice display reduced responses to IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma signaling is also impaired in Tyk2-/- mice. Tyk2-/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2-/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors.

Type 1 Interferon (IFNα/β) and Type 2 Nitric Oxide Synthase Regulate the Innate Immune Response to a Protozoan Parasite

Abstract Type 2 nitric oxide synthase (NOS2) is required for the Th1-dependent healing of infections with intracellular microbes, including Leishmania major . Here, we demonstrate the expression and define the function of NOS2 during the innate response to L. major . At day 1 of infection, genetic deletion or functional inactivation of NOS2 abolished the IFNγ and natural killer cell response, increased the expression of TGFβ, and caused parasite spreading from the skin and lymph node to the spleen, liver, bone marrow, and lung. Induction of NOS2 was dependent on IFNα/β. Neutralization of IFNα/β mimicked the phenotype of NOS2 −/− mice. Thus, IFNα/β and NOS2 are critical regulators of the innate response to L. major.

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4.

Macrophages release IFN-γ on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-γ by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-γ production in NK cells, was not required for IFN-γ production by these macrophages. IL-12 alone already induced the expression of IFN-γ mRNA, but nuclear translocation of STAT4, the release of IFN-γ protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-κB, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-β) also suppressed macrophage IFN-γ production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-γ by macrophages and point to a diversity in the signals required for IFN-γ production by various cell types.

Regulation of type 2 nitric oxide synthase by type 1 interferons in macrophages infected with Leishmania major.

We recently reported that the infection of macrophages with Leishmania major led to the release of type 1 interferons (IFN‐α /β ). Moreover, at day 1 of infection of mice with L. major, IFN‐α /β  was required for the expression of type 2 (inducible) NO synthase (NOS2 or iNOS) which, however, was restricted to a few macrophages in the dermis. Here, we further characterized the regulation of NOS2 by IFN‐α /β . Macrophages that were either simultaneously or sequentially exposed to L. major promastigotes and IFN‐α /β  expressed NOS2 and anti‐leishmanial activity. In contrast, when high amounts of IFN‐α /β  were used or when IFN‐α /β  was added to the macrophages 2 h prior to the parasites, almost no induction of NOS2 was observed. After pretreatment with IFN‐α /β , tyrosine phosphorylation and nuclear DNA binding of Stat1α, the degradation of the NF‐κB inhibitor (IκBα and β), and the nuclear translocation of NF‐κB were strongly impaired compared with macrophages exposed to IFN‐α /β  and L. major simultaneously. Thus, IFN‐α /β  exerts agonistic or antagonistic effects on the expression of NOS2 in macrophages infected with a microbial pathogen, depending on the sequence of the stimuli and the amount of IFN‐α /β  added. The limited number of NOS2‐positive macrophages at day 1 of infection in vivo might result from a blockage of non‐infected macrophages by IFN‐α /β  that is released by neighboring infected cells.

Control of Leishmania major in the absence of Tyk2 kinase

IL‐12 is indispensable for the control of many intracellular pathogens, but the components of the signaling pathway that are essential for its function in vivo are incompletely understood. Here, we investigated in the Leishmania major mouse model whether Tyk2 kinase is required for the generation of a protective immune response. Unlike C57BL/6 controls, Tyk2–/–mice developed severe skin lesions after infection that frequently ulcerated, but ultimately healed. NK cell cytotoxicity was absent in infected Tyk2–/– mice, even after IL‐12 pretreatment, which correlated with a STAT4 activation defect. IFN‐α / β, which was still able to activate STAT1 in Tyk2–/– NK cells, reconstituted their cytotoxic activity, but not their IL‐12responsiveness. The IL‐12‐induced production of IFN‐γ by NK cells and CD8+ T cells was strongly suppressed in Tyk2–/– mice at day 1 of infection, but partly regained during the late phase of infection. Tyk2–/– CD4+ T cells developed into Th1 cells (although in a delayed fashion) and infected Tyk2–/– mice expressed normals levels of inducible NO synthase. Thus, Tyk2 is required for the IL‐12 response of NK cells and CD8+ T cells in L. major‐infected mice, but not for the generation of Th1 cells and the ultimate control of the disease.

IFN‐γ inhibits the production of latent transforming growth factor‐β1 by mouse inflammatory macrophages

Transforming growth factor (TGF)‐β is a multifunctional cytokine, which in mammals exists in three isoforms (TGF‐β1, 2 and 3). It is synthesized by a variety of cells including macrophages, and exerts potent immunoregulatory effects such as the inhibition of Th1 development and the suppression or reversal of IFN‐γ‐induced macrophage activation. In this study we analyzed the effect of IFN‐γ on the production of TGF‐β1 by thioglycolate‐elicited mouse peritoneal macrophages under serum‐free conditions. Untreated macrophages released TGF‐β1 in its latent form, which became detectable in a capture ELISA specific for active TGF‐β1 after acid activation of the culture supernatants. Treatment with IFN‐γ reduced the amount of latent TGF‐β1 in the culture supernatants in a dose‐dependent fashion. The effect of IFN‐γ was confirmed by a newly developed Western blot system for the detection of mouse TGF‐β1 protein. IFN‐γ only weakly (16 – 24 %) reduced the levels TGF‐β1 mRNA at early and late time points of stimulation, and no evidence was obtained that IFN‐γ suppresses the secretion of latent TGF‐β1. Thus, inhibition of TGF‐β1 production by IFN‐γ is most likely due to decreased synthesis and/or stability of the TGF‐β1 protein, and might be important for the generation of fully activated macrophages and a Th1 response.