Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon γ (IFN-γ) is the primary IDO inducer to preventhyperinflammatory responses, yet the enzyme is also responsible for longer-term self-tolerance effects. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor β (TGF-β) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We reveal that IDO is involved in intracellular signaling events responsible for selfamplification and maintenance of a stably regulatory phenotype in pDCs. Thus IDO has a tonic, non-enzymic function that contributes to TGF-β-driven tolerance in non-inflammatory contexts. _____________________________________________________________________Immune regulation is a highly evolved biologic response capable of fine-tuning inflammation and innate immunity, but also of modulating adaptive immunity and establishing tolerance to self 1,2 . Amino acid catabolism is an ancestral survival strategy that can additionally control immune responses in mammals 3 . Several metabolic enzymes are known to possess a second function, which allows them to meet additional functional challenges and needs inside the cell 4 . Three distinct enzymes, namely tryptophan 2,3-dioxygenase (TDO; confined to the liver), IDO (also referred to as IDO1), and the IDO paralogue indoleamine 2,3-dioxygenase-2 (IDO2) catalyze the same rate-limiting step of tryptophan (Trp) catabolism along a common pathway, which leads to Trp starvation and the production of Trp metabolites collectively known as kynurenines [5][6][7] . However, IDO alone is recognized as an authentic regulator of immunity in a variety of physiopathologic conditions, including pregnancy, infection, allergy, autoimmunity, chronic inflammation, transplantation, and immuno-escaping tumoral mechanisms 8,9 .Normally expressed at low basal levels, IDO is rapidly induced by IFN-γ in DCs [8][9][10] . The IFN-γ-IDO axis is considered to be a phylogenetically conserved mechanism of restricting microbial growth and avoiding potentially harmful (hyper) inflammatory responses in the host 11 . However, its regulatory function in pregnancy and long-term prevention of immunopathology have been unclear [8][9][10] . Autocrine or paracrine signaling through transforming growth factor β (TGF-β) can also induce long-term, IDO-dependent effects 12 . The TGF-β-IDO axis was shown to mediate 3 durable regulatory functions, with a primary role in the generation and maintenance of regulatory T (T reg ) cells 13 .Functional flexibility and fostering of T reg responses are features typical of CD11c low B220 high plasmacytoid DCs (pDCs), which are capable of activating but also suppressing both inflammatory or innate responses and adaptive immunity [14][15][16][17] .Although different forms of immunosuppressive mechanisms are exploited by pDCs in distinct environmental conditions 15 , IDO is one of the...

Summary Disease tolerance is the ability of the host to reduce the impact of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (“endotoxin tolerance”). We found that a first exposure to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase 2, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR complex-associated Src kinase activity promoted IDO1 phosphorylation and signaling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in gram-negative and gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Apoptotic signalling by p53 occurs at both transcriptional and nontranscriptional levels, as p53 may act as a direct apoptogenic stimulus via activation of the intrinsic mitochondrial pathway. HOPS is a highly conserved, ubiquitously expressed shuttling protein with an ubiquitin-like domain. We generated Hops À/À mice and observed that they are viable with no apparent phenotypic defects. However, when treated with chemotherapeutic agents, Hops À/À mice display a significant reduction in apoptosis, suggesting an impaired ability to respond to genotoxic stressors. We show that HOPS acts as a regulator of cytoplasmic p53 levels and function. By binding p53, HOPS inhibits p53 proteasomal degradation and favours p53 recruitment to mitochondria and apoptosis induction. By interfering with importin a, HOPS further increases p53 cytoplasmic levels. Thus, HOPS promotes the p53-dependent mitochondrial apoptosis pathway by preserving cytoplasmic p53 from both degradation and nuclear uptake.

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, gamma-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.