Biomedical subjects

Roberta Bianchi

Publications and source records attributed to Roberta Bianchi.

At least 19 recordsLinked to original sources

PubMed2006

Tryptophan catabolism generates autoimmune-preventive regulatory T cells.

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that its effects include the emergence of a regulatory phenotype in naive CD4(+)CD25(-) cells via the general control non-depressing 2 (GCN2) protein kinase mediated induction of the forkhead transcription factor Foxp3. These cells are capable of effective control of diabetogenic T cells in vivo.

Animals↗

PubMed2006

S100B binding to RAGE in microglia stimulates COX-2 expression.

Besides exerting regulatory roles within astrocytes, the Ca2+-modulated protein of the EF-hand type S100B is released into the brain extracellular space, thereby affecting astrocytes, neurons, and microglia. However, extracellular effects of S100B vary, depending on the concentration attained and the protein being trophic to neurons up to nanomolar concentrations and causing neuronal apoptosis at micromolar concentrations. Effects of S100B on neurons are transduced by receptor for advanced glycation end products (RAGE). At high concentrations, S100B also up-regulates inducible NO synthase in and stimulates NO release by microglia by synergizing with bacterial endotoxin and IFN-gamma, thereby participating in microglia activation. We show here that S100B up-regulates cyclo-oxygenase-2 expression in microglia in a RAGE-dependent manner in the absence of cofactors through independent stimulation of a Cdc42-Rac1-JNK pathway and a Ras-Rac1-NF-kappaB pathway. Thus, S100B can be viewed as an astrocytic endokine, which might participate in the inflammatory response in the course of brain insults, once liberated into the brain extracellular space.

Animals↗

PubMed2006

Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO.

Dendritic cell (DC) tryptophan catabolism has emerged in recent years as a major mechanism of peripheral tolerance. However, there are features of this mechanism, initiated by IDO, that are still unclear, including the role of enzymes that are downstream of IDO in the kynurenine pathway and the role of the associated production of kynurenines. In this study, we provide evidence that 1) murine DCs express all enzymes necessary for synthesis of the downstream product of tryptophan breakdown, quinolinate; 2) IFN-gamma enhances transcriptional expression of all of these enzymes, although posttranslational inactivation of IDO may prevent metabolic steps that are subsequent and consequent to IDO; 3) overcoming the IDO-dependent blockade by provision of a downstream quinolinate precursor activates the pathway and leads to the onset of suppressive properties; and 4) tolerogenic DCs can confer suppressive ability on otherwise immunogenic DCs across a Transwell in an IDO-dependent fashion. Altogether, these data indicate that kynurenine pathway enzymes downstream of IDO can initiate tolerogenesis by DCs independently of tryptophan deprivation. The paracrine production of kynurenines might be one mechanism used by IDO-competent cells to convert DCs lacking functional IDO to a tolerogenic phenotype within an IFN-gamma-rich environment.

Animals↗

PubMed2006

The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor zeta-chain and induce a regulatory phenotype in naive T cells.

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that the short-term, combined effects of tryptophan deprivation and tryptophan catabolites result in GCN2 kinase-dependent down-regulation of the TCR zeta-chain in murine CD8+ T cells. TCR zeta down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25- T cells via TGF-beta induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69-, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells.

Animals↗

PubMed2005

Toward the identification of a tolerogenic signature in IDO-competent dendritic cells.

Although much is known about the transcriptional profiles of dendritic cells (DCs) during maturation, the molecular switches critical for the induction of a tolerogenic program in DC subsets are still obscure. We examined the gene-expression profiles of murine splenic CD8+ DCs rendered highly tolerogenic by interferon-gamma (IFN-gamma), which activates the enzyme indoleamine 2,3-dioxygenase (IDO, encoded by Indo) and thus initiates the immunosuppressive pathway of tryptophan catabolism. By examining the expression of a series of relevant genes in IDO+ compared with IDO- DCs, we found consistent and selective association of the IDO-competent phenotype with down-modulation of the Tyrobp gene, encoding the signaling adapter DAP12, which typically associates with activating receptors. Down-modulation of Tyrobp involved IFN consensus sequence binding protein (ICSBP), a transcription factor also known as IRF-8. In murine and human monocyte-derived DCs, silencing DAP12 expression imparted IDO functional competence to IDO- cells, whereas silencing IRF-8 in IDO+ counterparts abolished IDO expression and function. Thus, IRF-8 is required in tolerogenic DCs for the positive regulation of Indo and the negative regulation of Tyrobp. Overall, these studies reveal the occurrence of a simple and evolutionarily conserved code in the control of tolerance by an ancestral metabolic enzyme.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells.

Murine plasmacytoid dendritic cells (pDCs) have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) function and mediate immunosuppression in specific settings; yet, the conditions of spontaneous versus induced activity have remained unclear. We have used maneuvers known to up-regulate IDO in different cell types and have examined the relative efficacy and mechanisms of the induced activity in splenic pDCs, namely, after specific receptor engagement by CTLA-4-Ig, CD200-Ig or CD28-Ig, the latter in combination with silenced expression of the suppressor of cytokine signaling 3 (SOCS3) gene. We found that pDCs (CD11c+ mPDCA-1+ 120G8+) do not express IDO and are not tolerogenic under basal conditions. B7-1 engagement by CTLA-4-Ig, CD200R1 engagement by CD200-Ig and B7-1/B7-2 engagement by CD28-Ig in SOCS3-deficient pDCs were each capable of initiating IDO-dependent tolerance via different mechanisms. IFN-gamma was the major cytokine responsible for CTLA-4-Ig effects, and type I IFNs for those of CD200-Ig. Immunosuppression by CD28-Ig in the absence of SOCS3 required IFN-gamma induction and IFN-like actions of IL-6. Therefore, although pDCs do not mediate IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by pDCs in the mouse.

Animals↗

PubMed2005

Cutting edge: silencing suppressor of cytokine signaling 3 expression in dendritic cells turns CD28-Ig from immune adjuvant to suppressant.

CTLA-4-Ig and CD28-Ig are both agonist ligands of B7 coreceptor molecules on mouse dendritic cells (DCs), yet they bias the downstream response in opposite directions, and CTLA-4-Ig promotes tolerance, whereas CD28-Ig favors the onset of immunity. Although B7 engagement by either ligand leads to a mixed cytokine response, a dominant IL-6 production in response to CD28-Ig prevents the IFN-gamma-driven induction of immunosuppressive tryptophan catabolism mediated by IDO. In the present study, we show that silencing the expression of suppressor of cytokine signaling 3 (SOCS3) in DCs by RNA interference renders CD28-Ig capable of activating IDO, likely as a result of unrestrained IFN-gamma signaling and IFN-gamma-like actions of IL-6. Thus, in the absence of SOCS3, CD28-Ig becomes immunosuppressive and mimics the action of CTLA-4-Ig on tryptophan catabolism.

Abatacept↗

PubMed2005

Enhanced tryptophan catabolism in the absence of the molecular adapter DAP12.

DAP12 is an immunoreceptor tyrosine-based activation motif-bearing membrane adapter molecule expressed by different cell types. Although several receptors associate with DAP12 in murine dendritic cells (DC), the function of these receptors is as yet unknown. Here we report that splenic mature DC with DAP12 overexpression are characterized by an impaired tolerogenic potential. In contrast, inhibition of DAP12 function results in enhanced tolerogenesis and constitutive expression of immunosuppressive tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO). Increased resistance to experimental encephalomyelitis is observed in DAP12 knockin mice, which is dependent on IDO expression. Therefore, DAP12-related receptors act as negative regulators of IDO-mediated tolerance in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Efficacy and tolerability of multiple drug therapy in HIV-infected children.

OBJECTIVES: To characterize the efficacy and tolerability of multiple drug therapy (MDT) among heavily pre-treated HIV-infected children. METHODS: An observational study of seven children treated with 4-7 antiretroviral agents. MDT regimens were chosen with regard to past antiretroviral exposure and genotypic resistance data. Five children received MDT once, one child twice and one child four times. All patients had AIDS and severe CD4+ depletion and failed >2 PI-based HAART regimens. RESULTS: Virologic response, defined as a > or =log10 decrease in plasma HIV-1 RNA at week 24, was achieved in 7/11 MDT. Successful MDT kept a sustained viral suppression (<50 copies/ml) at longest follow-up (72-96 weeks). Successful MDT obtained a great immune recovery: the median rise in absolute and percentage of CD4+ cells was 261 and 4 at week 24 and it reached 480 and 16 at 72-96 weeks. Adverse events were common but generally manageable. Mild/moderate gastrointestinal complaints and laboratory abnormalities were detected in 5/11 and 8/11 MDT. Grade 2 severity pancreatitis occurred in one case with chronic active hepatitis C. Pancreatitis resolved within 30 days of MDT interruption. CONCLUSIONS: MDT may be a therapeutic option in children who failed to respond to most standard HAART regimens.

Acquired Immunodeficiency Syndrome↗

PubMed2005

CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig.

The synthetic immunomodulator cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) initiates effects in human monocyte-derived dendritic cells (DC) that rely on immunosuppressive tryptophan catabolism. However, it is unable to induce suppressive properties in DC matured by CD40 engagement. Thus, CD40-driven events may physiologically set human DC free from restraint by regulatory cells expressing surface CTLA-4.

Abatacept↗

PubMed2004

S100B-stimulated NO production by BV-2 microglia is independent of RAGE transducing activity but dependent on RAGE extracellular domain.

The Ca(2+)-modulated protein, S100B, is expressed in high abundance in and released by astrocytes. At the low levels normally found in the brain, extracellular S100B acts as a trophic factor, protecting neurons against oxidative stress and stimulating neurite outgrowth through its binding to the receptor for advanced glycation end products (RAGE). However, upon accumulation in the brain extracellular space, S100B might be detrimental to neurons. At relatively high concentrations, S100B stimulates NO release by microglia in the presence of lipid A or interferon-gamma (IFN-gamma). We analyzed further the S100B-microglia interaction to elucidate the molecular mechanism by which the protein brings about this effect. We found that S100B increased NO release by BV-2 microglia by stimulating reactive oxygen species (ROS) production and activating the stress-activated kinases, p38 and JNK. However, S100B stimulated NO production to the same extent in microglia overexpressing a transduction-incompetent mutant of RAGE and in microglia overexpressing full-length RAGE, with a significantly smaller effect in mock-transfected microglia. This suggests that the RAGE transducing activity has little or no role in S100B-stimulated NO production by microglia, whereas RAGE extracellular domain is important, probably serving to concentrate S100B on the BV-2 cell surface. On the other hand, S100B stimulated NF-kappaB transcriptional activity in BV-2 microglia in a manner that was strictly dependent on RAGE transducing activity, pointing to additional, RAGE-mediated effects of the protein on microglia that remain to be investigated.

Cell Line↗

PubMed2004

S100B increases proliferation in PC12 neuronal cells and reduces their responsiveness to nerve growth factor via Akt activation.

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner.

Animals↗

PubMed2004

CTLA-4-Ig activates forkhead transcription factors and protects dendritic cells from oxidative stress in nonobese diabetic mice.

Prediabetes and diabetes in nonobese diabetic (NOD) mice have been targeted by a variety of immunotherapies, including the use of a soluble form of cytotoxic T lymphocyte antigen 4 (CTLA-4) and interferon (IFN)-gamma. The cytokine, however, fails to activate tolerogenic properties in dendritic cells (DCs) from highly susceptible female mice early in prediabetes. The defect is characterized by impaired induction of immunosuppressive tryptophan catabolism, is related to transient blockade of the signal transducer and activator of transcription (STAT)1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. Here, we show that soluble CTLA-4 imparts suppressive properties to DCs from early prediabetic NOD female mice through mechanisms that rely on autocrine signaling by IFN-gamma. Although phosphorylation of STAT1 in response to IFN-gamma is compromised in those mice, CTLA-4 obviates the defect. IFN-gamma-driven expression of tryptophan catabolism by CTLA-4-immunoglobulin is made possible through the concomitant activation of the Forkhead Box class O (FOXO) transcription factor FOXO3a, induction of the superoxide dismutase gene, and prevention of peroxynitrite formation.

Abatacept↗

PubMed2004

CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86.

Bidirectional signaling along the B7-CTLA-4 coreceptor pathway enables reciprocal conditioning of T cells and dendritic cells. Although T cells can instruct dendritic cells to manifest tolerogenic properties after CTLA-4 engagement of B7, such a B7-mediated signaling is not known to occur in response to CD28. Here we show that mouse dendritic cells were induced by soluble CD28 to express interleukin 6 and interferon-gamma. Production of interleukin 6 required B7-1 (CD80), B7-2 (CD86) and p38 mitogen-activated protein kinase and prevented interferon-gamma-driven expression of immunosuppressive tryptophan catabolism. In vivo, an adjuvant activity of soluble CD28 was demonstrated as enhanced T cell-mediated immunity to tumor and self peptides and protection against microbial and tumor challenge. Thus, different ligands of B7 can signal dendritic cells to express functionally distinct effector responses.

Animals↗

PubMed2004

Murine plasmacytoid dendritic cells initiate the immunosuppressive pathway of tryptophan catabolism in response to CD200 receptor engagement.

In this study, using a soluble CD200-Ig fusion protein, we provide evidence that murine dendritic cells (DCs) possess a functional CD200R, whose engagement results in the reinforcement or appearance of immunosuppressive properties in these cells. In particular, the plasmacytoid subset (CD11c+B220+120G8+) of splenic DCs (pDCs) is induced by CD200-Ig to express the enzyme IDO, which initiates the tolerogenic pathway of tryptophan catabolism. As a result, pDCs are capable of suppressing Ag-specific responses in vivo when transferred into recipient hosts after treatment with CD200-Ig. IDO induction in pDCs through CD200R engagement requires type I IFNR signaling. Although the release of IFN-alpha may contribute to the full expression of CD200-Ig activity, autocrine IFN-alpha is unlikely to mediate alone the effects of CD200R engagement. These data prospect novel functions for both pDCs and the CD200-CD200R pair in the mouse. At the same time, these data underscore the possible unifying role of the IDO mechanism in immune tolerance.

Animals↗

PubMed2004

Thyroid dysfunction in antiretroviral treated children.

BACKGROUND: A high rate of thyroid disorders has been described in HIV-infected adults treated with highly active antiretroviral therapy (HAART), but data on children are lacking. We aimed to assess thyroid function in pediatric patients. METHODS: Fifty-two HIV-infected children receiving HAART were assessed for signs of thyroid dysfunction and serum concentrations of thyrotropin (TSH), free thyroxin (FT4), free triiodothyronine (FT3), thyroglobulin (TG), reverse triiodothyronine (rT3), anti-TG and antimicrosomal (anti-TSM) antibodies. RESULTS: Eighteen (35%) children showed thyroid abnormalities: isolated low FT4 value in 16; subclinical hypothyroidism in 1; and symptomatic hypothyroidism in 1. Children with low FT4 values as compared with the 34 children without thyroid dysfunction were similar for stage of disease, number of patients with undetectable HIV-RNA, FT3, TSH, TG, rT3, anti-TSM and anti-TG values, whereas they had shorter duration of HAART exposure (P = 0.019) and lower CD4 cell percentage (P = 0.035). The thyrotropin-releasing hormone (TRH) test was normal in all children with low FT4 values. Among children with low FT4, FT4 concentrations correlated positively with CD4 cell percentage (P < 0.05) and duration of HAART exposure (P < 0.05). The case with subclinical hypothyroidism had high basal TSH (7.3 microunits/ml), normal TSH response to TRH test and normal FT4, FT3, TG, rT3, anti-TG and anti-TSM antibodies. The case with symptomatic hypothyroidism had low FT4 (6.6 pg/ml) and high TSH (44 microunits/ml), TG (55 ng/ml), anti-TG (666 IU/ml) and anti-TSM (123 IU/ml). CONCLUSION: Thyroid abnormalities occur frequently in HAART-treated children even in the absence of clinical symptoms. These data suggest a need of regular thyroid function monitoring.

Adolescent↗

PubMed2004

Longitudinal changes of bone mineral density and metabolism in antiretroviral-treated human immunodeficiency virus-infected children.

Highly active antiretroviral therapy (HAART) may be a contributory factor for a decreased bone mass and altered bone metabolism in HIV-infected children. However, the evolution of bone mineral density (BMD) and bone metabolism during HAART has not been studied yet. In the current longitudinal study we monitored the changes of BMD and bone metabolism over a period of 12 months. Thirty-two HIV-infected children (15 girls and 17 boys), aged from 6.3 to 17.7 yr, with a long duration of HAART exposure (40.0 months at baseline) were enrolled in the study. As a control group, 381 healthy volunteers of comparable age were assessed. BMD was measured at the lumbar spine and whole skeleton by dual-energy x-ray absorptiometry. Bone-specific alkaline phosphatase (BALP, as bone formation index) and N-terminal telopeptide of type I collagen (as bone resorption index) were measured in serum and urine, respectively. BMD values at baseline were significantly lower at all skeletal sites than those of control subjects. The annual increment of spine BMD was comparable to normal, whereas that of the whole skeleton was significantly lower (P < 0.04). BALP and N-terminal telopeptide of type I collagen concentrations were significantly higher compared with controls at baseline and at follow-up. BALP annual changes of HIV patients were significantly different from normal. Our data confirm the presence of low BMD and bone metabolism derangement in HIV-infected children treated with HAART. The role of possible therapeutic approach to restore bone mass and metabolism should be assessed in pediatrics.

Absorptiometry, Photon↗

PubMed2003

Modulation of tryptophan catabolism by regulatory T cells.

Regulatory T (T(R)) cells manifest constitutive expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), but the function of CTLA-4 in mediating the regulatory function of T(R) cells is unclear. We show here that mouse CD4+CD25+ cells, either resting or induced to overexpress CTLA-4 by treatment with antibody to CD3, initiated tryptophan catabolism in dendritic cells through a CTLA-4-dependent mechanism. This process required B7 expression and cytokine production by the dendritic cells. In contrast, T(R) cells cultured in the presence of bacterial lipopolysaccharide induced tryptophan catabolism by dendritic cells in a CTLA-4-independent but cytokine-dependent way. Thus, regulation of immunosuppressive tryptophan catabolism in dendritic cells might represent a major mechanism of action of T(R) cells.

Animals↗