Neutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase (Arg) 1 expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells/macrophages, some of which coexpressed Arg1. In TNF-deficient mice Arg1 was hyperexpressed causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2 which is critical for pathogen control.

Myeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected with Leishmania major parasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4+ T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4Rα signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-α or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.

During the course of an inflammatory response, nitric oxide (NOC; formed by inducible NO synthase, iNOS) and superoxide (O 2 C À ; formed by NADPH oxidase or NOX2) are both generated in quantities that surpass physiological levels. [1] Consecutively, NOC and O 2 C À react at a diffusion-controlled rate (k % 10 10 m À1 s À1 ) to form peroxynitrite (ONOO À ) and other reactive species (such as NO 2 C and OHC), which induce cytotoxic effects through DNA damage, low-density lipoprotein oxidation, protein nitration and oxidation, aconitase inactivation, and inhibition of respiration.[2] Several attempts have been made to either inhibit NOC production, by designing selective iNOS inhibitors, [3a] or to mimic the activity of superoxide dismutase (SOD), by developing low-molecularweight metal complexes, [3b] to treat diseases characterized by hyperinflammation. However, the pharmacological application of iNOS inhibitors [3c] or SOD mimics [3d] is restricted by a certain lack of selectivity, stability, and/or bioavailability of these compounds. Mn II pentaazamacrocyclic complexes feature the presently most potent synthetic SOD mimics. They were discovered just a decade ago [4a] and have since entered phase II clinical trials in the USA.[4b] Their main advantage is reported to be a strict selectivity towards superoxide.[ ).[5b] We proposed a new dismutation mechanism which involves the formation of labile metal-nitrosyl complexes and leads to the catalytic removal of large amounts of NOC from solution [5b] [Eqs. (1) and (2)]. Therefore, this class of complexes might also act towards NOC generation during an inflammatory response.To clarify the pharmacological effects related to the chemistry of Mn II pentaazamacrocyclic SOD mimics, we used [Mn II (pyane)Cl 2 ] (Scheme 1) [6] as a general representative of this class of complexes and studied its effect on the production of O 2 C À and NOC by living cells.Macrophages are the main actors in the production of reactive oxygen (ROS) and reactive nitrogen (RNS) species in inflammation, [7] so they were selected as cell models in this study. Moreover, some of us have shown that electrochemistry at ultramicroelectrodes offers the unique possibility of in situ, real-time, and direct measurements of ROS and RNS generated by cells, including species that are short-lived like ONOO À .[8] Thus, the ROS/RNS production by single immu-

Arginase (Arg) 1 is expressed by hematopoietic (e.g., macrophages) and nonhematopoietic cells (e.g., endothelial cells) and converts L-arginine into ornithine and urea. The enzyme is implicated in tissue repair but also antagonizes the production of NO by type 2 NO synthase in myeloid cells and thereby impedes the control of intracellular parasites such as Leishmania major. In this study, we tested whether Arg1 is required for spontaneous healing of acute cutaneous leishmaniasis in C57BL/6 mice and for lifelong parasite persistence in draining lymph nodes (dLNs) of healed mice. In vitro, bone marrow-derived macrophages and lymphoid endothelial cells readily expressed Arg1 in response to IL-4 and/or IL-13, whereas skin or dLN fibroblasts failed to do so, even during hypoxia. In vivo, Arg1 was found in skin lesions and, to a much lower extent, also in dLNs of acutely infected C57BL/6 mice but became undetectable at both sites after healing. Deletion of Arg1 in hematopoietic and endothelial cells using Tie2Cre +/2 Arg1 fl/fl C57BL/6 mice abolished the expression of Arg1 in skin lesions and dLNs but did not affect development and resolution of skin lesions, parasite burden, NO production, or host cell tropism of L. major during the acute or persistent phase of infection. Similar to wild-type controls, parasites persisting in Arg1-deficient mice favored NO synthase 22negative areas and mainly resided in myeloid cells and fibroblasts. We conclude that Arg1 expression by hematopoietic and endothelial cells is completely dispensable for clinical resolution of cutaneous leishmaniasis and for long-term persistence of L.