A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.

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-ß (TGF-ß) 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-ß-driven tolerance in noninflammatory contexts.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Class IA PI3Ks regulate subcellular and functional dynamics of IDO1.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

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.

IL-35Ig-expressing dendritic cells induce tolerance via Arginase 1.

The cytokine interleukin IL-35 is known to exert strong immunosuppressive functions. Indoleamine 2,3-dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL-35 and the activity of those enzymes. We transfected a single chain IL-35Ig gene construct in murine splenic DCs (DC35 ) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL-35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC35 , and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed-type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC35 was associated with the detection of CD25+ CD39+ , rather than Foxp3+ , regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC35 appears to be an early event in IL-35Ig-mediated immunosuppression.

Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

Distinct roles of immunoreceptor tyrosine-based motifs in immunosuppressive indoleamine 2,3-dioxygenase 1.

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the initial, rate-limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1-dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine-based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP-1 and SHP-2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine-phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half-life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM-associated tyrosines with phospho-mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 - but not ITIM2 mutant - did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen-specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1-dependent events will occur in a local microenvironment.

A Multischool Outbreak Due to Salmonella enterica serovar Napoli Associated with Elevated Rates of Hospitalizations and Bacteremia, Milan, Italy, 2014.

A multischool outbreak of salmonellosis caused by Salmonella enterica serovar Napoli was investigated in the province of Milan from October to November 2014, following an increase in school absenteeism coinciding with two positive cases. Epidemiological studies detected 47 cases in four primary schools: 46 children and 1 adult woman (51.4% males and 48.6% females, median age 8.9). From these, 14 cases (29.8%) were severe and resulted in hospitalization, including 6 children (12.8%) who developed an invasive salmonellosis. The epidemic curve revealed an abnormally long incubation period, peaking 1 week after the first confirmed case. Twenty-five available isolates were typed by pulsed-field gel electrophoresis showing an identical pattern. The isolate belongs to ST474, an ST composed exclusively of Salmonella Napoli human strains isolated in France and Italy. Antibiotic resistance analysis showed resistance to aminoglycosides, correlating with the presence of the aminoglycoside resistance gene aadA25 in its genome. Trace-back investigations strongly suggested contaminated ham as the most likely food vehicle, which was delivered by a common food center on 21 October. Nevertheless, this ingredient could not be retrospectively investigated since it was no longer available at the repository. This represents the largest Salmonella Napoli outbreak ever reported in Italy and provides a unique scenario for studying the outcome of salmonellosis caused by this emerging and potentially invasive nontyphoidal serotype.

Mutation of threonine 34 in mouse podoplanin-Fc reduces CLEC-2 binding and toxicity in vivo while retaining antilymphangiogenic activity.

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.

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.

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.

A nonenzymatic biosensor based on gold electrodes modified with peptide self-assemblies for detecting ammonia and urea oxidation.

We have developed a nonenzymatic biosensor for the detection of ammonia and urea oxidation based on the deposition of peptide microstructures onto thiolated gold electrodes. FF-MNSs/MCP/Au assemblies were obtained by modifying gold substrates with 4-mercaptopyridine (MCP), followed by coating with l,l-diphenylalanine micro/nanostructures (FF-MNSs) grown in the solid-vapor phase. Benzene rings and amide groups with peptide micro/nanostructures interact with synthetic NH4(+) receptors through cation-π and hydrogen bonding. AuOH clusters on the Au surface provided the catalytic sites. The application of a predetermined concentration of analytes at the peptide interfaces activated the catalytic sites. We observed a relationship between the stability of films and the crystal structure of peptides, and we organized the FF-MNSs into an orthorhombic symmetry that was the most suitable assembly for creation of our biosensors. At 0.1 mol L(-1) NaOH, these FF-MNSs/MCP/Au electrodes have electrocatalytic properties regarding ammonia and urea oxidation that are comparable to those of enzyme-based architectures. Under optimal conditions, the electrocatalytic response is proportional to the ammonia and urea concentration in the range 0.1-1.0 mmol L(-1). The sensitivity was calculated as 2.83 and 81.3 µA mmol L(-1) cm(-2) for ammonia and urea, respectively, at +0.40 V (vs SCE). Our detection method is easy to follow, does not require a mediator or enzyme, and has strong potential for detecting urea via nonenzymatic routes.

A GpC-rich oligonucleotide acts on plasmacytoid dendritic cells to promote immune suppression.

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-ß-dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3+ T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.

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-kappaB-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.

Long pentraxin 3/tumor necrosis factor-stimulated gene-6 interaction: a biological rheostat for fibroblast growth factor 2-mediated angiogenesis.

OBJECTIVE: Angiogenesis is regulated by the balance between pro- and antiangiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor 2 (FGF2) is a major proangiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally coexpressed with its ligand tumor necrosis factor-stimulated gene-6 (TSG-6), a secreted glycoprotein that cooperates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis. METHODS AND RESULTS: Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine subcutaneous Matrigel plug assay, the inhibition being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals. CONCLUSION: TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the coexpression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2.

Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies.

PURPOSE: Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. EXPERIMENTAL DESIGN: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single-cell RNA-sequencing of whole blood treated with CD20-TCB together with bulk RNA-sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity. RESULTS: Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1ß, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1ß, MCP-1, MIP-1α, MIP-1ß, and IP-10 release. Endothelial cells contribute to IL6 and IL1ß release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1ß). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB-mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity. CONCLUSIONS: Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs. See related commentary by Luri-Rey et al., p. 4320.

S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release.

The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia.

Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin.

Podoplanin is a small transmembrane protein required for development and function of the lymphatic vascular system. To investigate the effects of interfering with its function, we produced an Fc fusion protein of its ectodomain. We found that podoplanin-Fc inhibited several functions of cultured lymphatic endothelial cells and also specifically suppressed lymphatic vessel growth, but not blood vessel growth, in mouse embryoid bodies in vitro and in mouse corneas in vivo. Using a keratin 14 expression cassette, we created transgenic mice that overexpressed podoplanin-Fc in the skin. No obvious outward phenotype was identified in these mice, but surprisingly, podoplanin-Fc-although produced specifically in the skin-entered the blood circulation and induced disseminated intravascular coagulation, characterized by microthrombi in most organs and by thrombocytopenia, occasionally leading to fatal hemorrhage. These findings reveal an important role of podoplanin in lymphatic vessel formation and indicate the potential of podoplanin-Fc as an inhibitor of lymphangiogenesis. These results also demonstrate the ability of podoplanin to induce platelet aggregation in vivo, which likely represents a major function of lymphatic endothelium. Finally, keratin 14 podoplanin-Fc mice represent a novel genetic animal model of disseminated intravascular coagulation.

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, VPY(115)CEL and LLY(253)EGV. 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.