Islet antigen-pulsed dendritic cells expressing ectopic IL-35Ig protect nonobese diabetic mice from autoimmune diabetes.

Dendritic cells (DCs) are professional antigen presenting cells capable of orchestrating either stimulatory or regulatory immune responses mediated by T cells. Interleukin 35 (IL-35) is an immunosuppressive, heterodimeric cytokine belonging to the IL-12 family and known to be produced by regulatory T cells but not DCs. In this study, we explored the possible immunosuppressive effect of IL-35 ectopically expressed by splenic DCs from nonobese diabetic (NOD) mice, a prototypical model of autoimmune diabetes. After pulsing with the IGRP peptide (a dominant, diabetogenic autoantigen in NOD mice) and transfer in vivo, IL-35Ig- but not Ig-transfected DCs suppressed antigen specific, T cell-mediated responses in a skin test assay. More importantly, transfer of IL-35Ig-transfected, IGRP-pulsed DCs into prediabetic NOD mice induced a delayed and less severe form of diabetes, an effect accompanied by the increase of CD4(+)CD39(+) suppressive T cells in pancreatic lymph nodes. Our data therefore suggest that DCs overexpressing ectopic IL-35Ig might represent a powerful tool in negative vaccination strategies.

LPS-conditioned dendritic cells confer endotoxin tolerance contingent on tryptophan catabolism.

Dendritic cells (DCs) are specialized antigen-presenting cells with a bipolar nature. Depending on environmental factors, DCs will promote either inflammatory or anti-inflammatory effects. Lipopolysaccharide (LPS), a ligand of Toll-like receptor (TLR)4 and a most potent proinflammatory stimulus, is responsible for complex signaling events in different cell types, including DCs. LPS effects range from protective inflammation-capable of counteracting growth and dissemination of gram-negative bacteria - to hyperacute detrimental responses, as it occurs in endotoxic shock. Consistent with the plasticity of TLR4 signaling, a low dosage of LPS will induce a regulatory response capable of protecting mice against a subsequent, otherwise lethal challenge ('endotoxin tolerance'). By examining CD11c(+) DCs ('conventional' DCs, or cDCs), we investigated whether DC flexibility in promoting either inflammation or tolerance can be differentially affected by single vs. repeated exposure to LPS in vitro. cDCs stimulated twice with LPS expressed high levels of indoleamine 2,3-dioxygenase 1 (IDO1) - one of the most effective mediator of anti-inflammatory activity by DCs - and of TGF-ß, an immunoregulatory cytokine capable of upregulating IDO1 expression and function. In contrast, a single exposure to LPS failed to upregulate IDO1, and it was instead associated with high-level production of IL-6, a cytokine that promotes inflammation and proteolysis of IDO1. When adoptively transferred in vivo, only cDCs on double endotoxin exposure greatly improved the outcome of an otherwise lethal LPS challenge. The protective effect required that the transferred cDCs be fully competent for IDO1 and the host for TGF-ß production. Thus cDCs, conditioned by LPS in vitro to mimic an endotoxin-tolerant state, can protect recipients from endotoxic shock, pointing to adoptive transfer of tolerance as a new option for controlling potentially harmful responses to TLR4 signaling.

Forced IDO1 expression in dendritic cells restores immunoregulatory signalling in autoimmune diabetes.

Indoleamine 2,3-dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. We have recently demonstrated that IDO1 does not merely degrade tryptophan and produce immunoregulatory kynurenines, but it also acts as a signal-transducing molecule, independently of its enzymic function. IDO1 signalling activity is triggered in plasmacytoid dendritic cells (pDCs) by transforming growth factor-ß (TGF-ß), an event that requires the non-canonical NF-κB pathway and induces long-lasting IDO1 expression and autocrine TGF-ß production in a positive feedback loop, thus sustaining a stably regulatory phenotype in pDCs. IDO1 expression and catalytic function are defective in pDCs from non-obese diabetic (NOD) mice, a prototypic model of autoimmune diabetes. In the present study, we found that TGF-ß failed to activate IDO1 signalling function as well as up-regulate IDO1 expression in NOD pDCs. Moreover, TGF-ß-treated pDCs failed to exert immunosuppressive properties in vivo. Nevertheless, transfection of NOD pDCs with Ido1 prior to TGF-ß treatment resulted in activation of the Ido1 promoter and induction of non-canonical NF-κB and TGF-ß, as well as decreased production of the pro-inflammatory cytokines, interleukin 6 (IL-6) and tumour necrosis factor-α (TNF-α). Overexpression of IDO1 in TGF-ß-treated NOD pDCs also resulted in pDC ability to suppress the in vivo presentation of a pancreatic ß-cell auto-antigen. Thus, our data suggest that a correction of IDO1 expression may restore its dual function and thus represent a proper therapeutic manoeuvre in this autoimmune setting.

Cinnabarinic acid, an endogenous agonist of type-4 metabotropic glutamate receptor, suppresses experimental autoimmune encephalomyelitis in mice.

Cinnabarinic acid (CA) is an endogenous metabolite of the kynurenine pathway which acts as an orthosteric agonist of type-4 metabotropic glutamate receptor (mGlu4). We now report that systemic administration of CA (0.1-10 mg/kg, i.p.) was highly protective against experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG35-55) peptide, which models multiple sclerosis in mice. Full protection against EAE required daily injections of CA since the time of immunization, similarly to what reported for the mGlu4 enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1acarboxamide (PHCCC). CA treatment boosted an immune response dominated by regulatory T (Treg) cells at the expenses of Th17 cells. In addition, exogenous CA enhanced endogenous CA formation in lymphocytes, suggesting the occurrence of a positive feedback loop sustaining immune tolerance. To examine whether activation of mGlu4 could account for the protective activity of CA against EAE, we used mGlu4 knockout mice. As expected, these mice displayed a more severe form of EAE in response to immunization. CA was still protective against EAE in mGlu4-deficient mice, although its action was significantly reduced both at high and low CA doses. This suggests that the action of CA against neuroinflammation involves multiple mechanisms including the activation of mGlu4. These data further suggest that CA is one possible bridge between activation of the kynurenine pathway and immune tolerance aimed at restraining neuroinflammation.

Molecular typing of fluoroquinolone-resistant and fluoroquinolone-susceptible Escherichia coli isolated from blood of neutropenic cancer patients in a single center.

OBJECTIVE: To investigate the molecular epidemiology of fluoroquinolone-resistant (FQ-R) and fluoroquinolone-susceptible (FQ-S) bacteremic Escherichia coli isolates from neutropenic patients by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. METHODS: Nineteen FQ-R and 27 FQ-S isolates of E. coli, obtained from patients on a hematologic ward over a 7-year period, were genotyped by PFGE and RAPD using two different random primers (1247 and 1283). RESULTS: PFGE analysis was able to type all FQ-S isolates and most (17/19, 89%) FQ-R isolates of E. coli. All isolates were genotypically unrelated, with the exception of two indistinguishable FQ-R isolates from different patients in the same period. RAPD analysis typed all isolates, including those FQ-R isolates untypable by PFGE, but was unable to distinguish between some isolates that were different by PFGE. Using primer 1247, RAPD analysis identified six pairs and one triad, while primer 1283 identified seven pairs and one triad of indistinguishable isolates. CONCLUSIONS: No spread of epidemic FQ-R or FQ-S E. coli isolates was documented among neutropenic patients. RAPD analysis is a powerful genotyping method, but appeared to be less reproducible and discriminatory than PFGE for investigating E. coli isolates.