Claudia Volpi

The Combined Effects of Tryptophan Starvation and Tryptophan Catabolites Down-Regulate T Cell Receptor ζ-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 ζ-chain in murine CD8+ T cells. TCR ζ 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-β 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.

Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells

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 is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. 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 found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-β-driven tolerance in noninflammatory contexts.

Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-κB–dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4+ T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.

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-γ. Production of interleukin 6 required B7-1 (CD80), B7-2 (CD86) and p38 mitogen-activated protein kinase and prevented interferon-γ-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.

Aryl hydrocarbon receptor control of a disease tolerance defence pathway

Disease tolerance is the ability of the host to reduce the effect 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 of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, 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 signalling 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.

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-γ 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-γ-rich environment.

Cutting Edge: Autocrine TGF-β Sustains Default Tolerogenesis by IDO-Competent Dendritic Cells

CD8− and CD8+ dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8+ DCs relies on autocrine TGF-β, which sustains the activation of IDO in response to environmental stimuli. CD8− DCs do not produce TGF-β, yet externally added TGF-β induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8− DCs correlates with activation of the PI3K/Akt and noncanonical NF-κB pathways. These data are the first to link TGF-β signaling with IDO in controlling spontaneous tolerogenesis by DCs.

SOCS3 drives proteasomal degradation of indoleamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis

Despite their common ability to activate intracellular signaling through CD80/CD86 molecules, cytotoxic T lymphocyte antigen 4 (CTLA-4)-Ig and CD28-Ig bias the downstream response in opposite directions, the latter promoting immunity, and CTLA-4-Ig tolerance, in dendritic cells (DCs) with opposite but flexible programs of antigen presentation. Nevertheless, in the absence of suppressor of cytokine signaling 3 (SOCS3), CD28-Ig—and the associated, dominant IL-6 response—become immunosuppressive and mimic the effect of CTLA-4-Ig, including a high functional expression of the tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO). Here we show that forced SOCS3 expression antagonized CTLA-4-Ig activity in a proteasome-dependent fashion. Unrecognized by previous studies, IDO appeared to possess two tyrosine residues within two distinct putative immunoreceptor tyrosine-based inhibitory motifs, VPY115CEL and LLY253EGV. We found that SOCS3—known to interact with phosphotyrosine-containing peptides and be selectively induced by CD28-Ig/IL-6—would bind IDO and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. This event accounted for the ability of CD28-Ig and IL-6 to convert otherwise tolerogenic, IDO-competent DCs into immunogenic cells. Thus onset of immunity in response to antigen within an early inflammatory context requires that IDO be degraded in tolerogenic DCs. In addition to identifying SOCS3 as a candidate signature for mouse DC subsets programmed to direct immunity, this study demonstrates that IDO undergoes regulatory proteolysis in response to immunogenic stimuli.

Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans.

We analyzed the contribution of intracellular signaling to the functional plasticity of dendritic cells (DCs) presenting Candida albicans, a human commensal associated with severe diseases. Distinct intracellular pathways were activated by recognition of different fungal morphotypes in distinct DC subsets and in Peyers patches DCs. Inflammatory DCs initiated Th17/Th2 responses to yeasts through the adaptor myeloid differentiation factor-88 (MyD88), whereas tolerogenic DCs activate Th1/T regulatory cell (Treg) differentiation programs to hyphae involving Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) as an intermediary of signaling. In addition, signal transducer and activator of transcription 3 (STAT3), affecting the balance between canonical and non-canonical activation of nuclear factor-kappaB (NF-κB) and 2,3 indoleamine dioxygenase (IDO), pivotally contributed to DC plasticity and functional specialization. As Candida-induced tolerogenic DCs ameliorated experimental colitis, our data qualify Candida as a commensal with immunoregulatory activity, resulting from the orchestrated usage of multiple, yet functionally distinct, receptor–signaling pathways in DCs. Ultimately, affecting the local Th17/Treg balance might likely be exploited by the fungus for either commensalism or pathogenicity.

Metabotropic glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation

High amounts of glutamate are found in the brains of people with multiple sclerosis, an inflammatory disease marked by progressive demyelination. Glutamate might affect neuroinflammation via effects on immune cells. Knockout mice lacking metabotropic glutamate receptor-4 (mGluR4) were markedly vulnerable to experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis) and developed responses dominated by interleukin-17–producing T helper (TH17) cells. In dendritic cells (DCs) from those mice, defective mGluR4 signaling—which would normally decrease intracellular cAMP formation—biased TH cell commitment to the TH17 phenotype. In wild-type mice, mGluR4 was constitutively expressed in all peripheral DCs, and this expression increased after cell activation. Treatment of wild-type mice with a selective mGluR4 enhancer increased EAE resistance via regulatory T (Treg) cells. The high amounts of glutamate in neuroinflammation might reflect a counterregulatory mechanism that is protective in nature and might be harnessed therapeutically for restricting immunopathology in multiple sclerosis.