Linh P. Nguyen

Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance.

Central tolerance can be mediated by peripheral dendritic cells (DCs) that transport innocuous antigens (Ags) to the thymus for presentation to developing T cells, but the responsible DC subsets remained poorly defined. Immature plasmacytoid DCs (pDCs) express CCR9, a chemokine receptor involved in migration of T cell precursors to the thymus. We show here that CCR9 mediated efficient thymic entry of endogenous or i.v. transfused pDCs. pDCs activated by Toll-like receptor (TLR) ligands downregulated CCR9 and lost their ability to home to the thymus. Moreover, endogenous pDCs took up subcutaneously injected fluorescent Ag and, in the absence of TLR signals, transported Ag to the thymus in a CCR9-dependent fashion. Injected, Ag-loaded pDCs effectively deleted Ag-specific thymocytes, and this thymic clonal deletion required CCR9-mediated homing and was prevented by infectious signals. Thus, peripheral pDCs can contribute to immune tolerance through CCR9-dependent transport of peripheral Ags and subsequent deletion of Ag-reactive thymocytes.

Role and species-specific expression of colon T cell homing receptor GPR15 in colitis

Lymphocyte recruitment maintains intestinal immune homeostasis but also contributes to inflammation. The orphan chemoattractant receptor GPR15 mediates regulatory T cell homing and immunosuppression in the mouse colon. We show that GPR15 is also expressed by mouse TH17 and TH1 effector cells and is required for colitis in a model that depends on the trafficking of these cells to the colon. In humans GPR15 is expressed by effector cells, including pathogenic TH2 cells in ulcerative colitis, but is expressed poorly or not at all by colon regulatory T (Treg) cells. The TH2 transcriptional activator GATA-3 and the Treg-associated transcriptional repressor FOXP3 robustly bind human, but not mouse, GPR15 enhancer sequences, correlating with receptor expression. Our results highlight species differences in GPR15 regulation and suggest it as a potential therapeutic target for colitis.

Leukocyte Trafficking to the Small Intestine and Colon

Leukocyte trafficking to the small and large intestines is tightly controlled to maintain intestinal immune homeostasis, mediate immune responses, and regulate inflammation. A wide array of chemoattractants, chemoattractant receptors, and adhesion molecules expressed by leukocytes, mucosal endothelium, epithelium, and stromal cells controls leukocyte recruitment and microenvironmental localization in intestine and in the gut-associated lymphoid tissues (GALTs). Naive lymphocytes traffic to the gut-draining mesenteric lymph nodes where they undergo antigen-induced activation and priming; these processes determine their memory/effector phenotypes and imprint them with the capacity to migrate via the lymph and blood to the intestines. Mechanisms of T-cell recruitment to GALT and of T cells and plasmablasts to the small intestine are well described. Recent advances include the discovery of an unexpected role for lectin CD22 as a B-cell homing receptor GALT, and identification of the orphan G-protein-coupled receptor 15 (GPR15) as a T-cell chemoattractant/trafficking receptor for the colon. GPR15 decorates distinct subsets of T cells in mice and humans, a difference in species that could affect translation of the results of mouse colitis models to humans. Clinical studies with antibodies to integrin α4β7 and its vascular ligand mucosal vascular addressin cell adhesion molecule 1 are proving the value of lymphocyte trafficking mechanisms as therapeutic targets for inflammatory bowel diseases. In contrast to lymphocytes, cells of the innate immune system express adhesion and chemoattractant receptors that allow them to migrate directly to effector tissue sites during inflammation. We review the mechanisms for innate and adaptive leukocyte localization to the intestinal tract and GALT, and discuss their relevance to human intestinal homeostasis and inflammation.

A Hypomorphic Allele of Aryl Hydrocarbon Receptor-Associated Protein-9 Produces a Phenocopy of the Ahr-Null Mouse

The aryl hydrocarbon receptor-associated protein-9 (ARA9) is a chaperone of the aryl hydrocarbon receptor (AHR). The AHR has been shown to play a late developmental role in the normal closure of a fetal hepatovascular shunt known as the ductus venosus (DV). Given that Ara9-null mice display early embryonic lethality, we generated a hypomorphic Ara9 allele (designated Ara9fxneo) that displays reduced ARA9 protein expression. In an effort to demonstrate the role of ARA9 protein in AHR-mediated DV closure, we used combinations of Ara9 wild-type [Ara9(+/+)], null [Ara9(-/-)], and hypomorphic [Ara9(fxneo/fxneo)] alleles to produce mice with a graded expression of the ARA9 protein. Liver perfusion studies demonstrated that although none of the Ara9(+/+) mice displayed a patent DV, the shunt was observed in 10% of the Ara9(+/fxneo) mice, 55% of the Ara9(+/-) mice, and 83% of the Ara9(fxneo/fxneo) mice. That expression level of ARA9 correlates with the frequency of a phenocopy of the Ahr-null allele supports the conclusion that the ARA9 protein is essential for AHR signaling during development.

D-amino acid oxidase generates agonists of the aryl hydrocarbon receptor from D-tryptophan

The aryl hydrocarbon receptor (AHR) is well-known for its role in mediating the toxic and adaptive responses to xenobiotic compounds. Recent studies also indicate that AHR ligands are endogenously produced and may be essential for normal development. Previously, we showed that the endogenous enzyme, aspartate aminotransferase (AST), generates the AHR proagonist, indole-3-pyruvic acid (I3P), by deamination of its substrate L-tryptophan. We hypothesized that other enzymatic pathways capable of producing I3P may generate AHR agonists in vivo. We now demonstrate that the enzyme d-amino acid oxidase (DAAO) catalyzes the production of AHR agonists through the enzymatic conversion of D-tryptophan to I3P. Moreover, we provide evidence that the nonenzymatic oxidation and condensation of I3P is a critical step in the generation of receptor agonists by DAAO and AST. Products of this process include two novel agonists, 1,3-di(1H-indol-3-yl)propan-2-one and 1-(1H-indol-3-yl)-3-(3H-indol-3-ylidene) propan-2-one [characterized in the accompanying paper, Chowdhury et al. ( 2009 ) Chem. Res. Toxicol. , DOI: 10.1021/tx9000418 ], both of which can potently activate the AHR at concentrations in the nanomolar range. These results show that endogenous AHR activity can be modulated by I3P production from amino acid precursors through multiple enzymatic pathways, including those catalyzed by DAAO and AST.

SU5416, a VEGF Receptor Inhibitor and Ligand of the AHR, Represents a New Alternative for Immunomodulation

The experimental compound SU5416 went as far as Phase III clinical trials as an anticancer agent, putatively because of its activity as a VEGFR-2 inhibitor, but showed poor results. Here, we show that SU5416 is also an aryl hydrocarbon receptor (AHR) agonist with unique properties. Like TCDD, SU5416 favors induction of indoleamine 2,3 dioxygenase (IDO) in immunologically relevant populations such as dendritic cells in an AHR-dependent manner, leading to generation of regulatory T-cells in vitro. These characteristics lead us to suggest that SU5416 may be an ideal clinical agent for treatment of autoimmune diseases and prevention of transplant rejection, two areas where regulatory ligands of the AHR have shown promise. At the same time, AHR agonism might represent a poor characteristic for an anticancer drug, as regulatory T-cells can inhibit clearance of cancer cells, and activation of the AHR can lead to upregulation of xenobiotic metabolizing enzymes that might influence the half-lives of co-administered chemotherapeutic agents. Not only does SU5416 activate the human AHR with a potency approaching 2,3,7,8-tetrachlorodibenzo-p-dioxin, but it also activates polymorphic murine receptor isoforms (encoded by the Ahrd and Ahrb1 alleles) with similar potency, a finding that has rarely been described and may have implications in identifying true endogenous ligands of this receptor.

Structural identification of Diindole agonists of the aryl hydrocarbon receptor derived from degradation of indole-3-pyruvic acid.

Aerobic incubation of the tryptophan transamination/oxidation product indole-3-pyruvic acid (I3P) at pH 7.4 and 37 degrees C yielded products with activity as Ah receptor (AHR) agonists. The extracts were fractionated using HPLC and screened for AHR agonist activity. Two compounds were identified as agonists: 1,3-di(1H-indol-3-yl)propan-2-one (1) and 1-(1H-indol-3-yl)-3-(3H-indol-3-ylidene) propan-2-one (2), with the potency of 2 being 100-fold > 1 [ Nguyen et al. ( 2009 ) Chem. Res. Toxicol. , DOI: 10.1021/tx900043s . ]. Both 1 and 2 showed UV spectra indicative of indole. The molecular formulas were established by high-resolution mass spectrometry (HRMS), and the structures were determined by a combination of NMR methods, including (1)H, natural abundance (13)C, and two-dimensional methods. An intermediate in the oxidation of I3P to 1 is 3-hydroxy-2,4-di(1H-indol-3-yl)butanal (HRMS established the presence of a compound with the formula C(20)H(19)N(2)O(2)). Compound 1 was converted to 2 in air or (faster) with mild oxidants, and 2 could be further oxidized to 1,3-di(3H-indol-3-ylidene)propan-2-one. Determination of the structures allowed estimation of the molar Ah receptor agonist activity of these natural products, similar in potency to known classical AHR inducers.

Reactive Oxygen Species Imaging in a Mouse Model of Inflammatory Bowel Disease

PurposeReactive oxygen species (ROS) are important contributors to inflammatory bowel disease (IBD); however, there are insufficient tools for their in vivo evaluation.ProceduresTo determine if a chemiluminescent ROS reporter, coelenterazine, would be a useful tool for the detection of immune cell activation, the macrophage cell line (RAW 264.7) was treated with phorbol myristate acetate (PMA). Additionally, coelenterazine was used to monitor the changes in ROS production over time in a mouse model of IBD.ResultsIn vitro, coelenterazine enabled the dynamic monitoring of the RAW 264.7 cell oxidative burst. In vivo, there were early, preclinical, changes in the localization and magnitude of coelenterazine chemiluminescent foci.ConclusionsCoelenterazine offers a high-throughput method for assessing immune cell activation in culture and provides a means for the in vivo detection and localization of ROS during IBD disease progression.

A Thermo-Sensitive Delivery Platform for Topical Administration of Inflammatory Bowel Disease Therapies

Systemic therapies for inflammatory bowel disease are associated with an increased risk of infections and malignancies. Topical therapies reduce systemic exposure, but can be difficult to retain or have limited proximal distribution. To mitigate these issues, we developed a thermo-sensitive platform, using a polymer-based system that is liquid at room temperature but turns into a viscous gel on reaching body temperature. After rectal administration to mice with dextran sulfate sodium-induced colitis, the platform carrying budesonide or mesalamine becomes more viscoelastic near body temperature. Mice given the drug-containing platform gained more weight and had reduced histologic and biologic features of colitis than mice given the platform alone or liquid drugs via enema. Image analysis showed that enemas delivered with and without the platform reached similar distances in the colons of mice, but greater colonic retention was achieved by using the platform.

Age-dependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system

Objective The enteric nervous system (ENS) undergoes neuronal loss and degenerative changes with age. The cause of this neurodegeneration is poorly understood. Muscularis macrophages residing in close proximity to enteric ganglia maintain neuromuscular function via direct crosstalk with enteric neurons and have been implicated in the pathogenesis of GI motility disorders like gastroparesis and postoperative ileus. The aim of this study was to assess whether ageing causes alterations in macrophage phenotype that contributes to age-related degeneration of the ENS. Design Longitudinal muscle and myenteric plexus from small intestine of young, mid-aged and old mice were dissected and prepared for whole mount immunostaining, flow cytometry, Luminex immunoassays, western blot analysis, enteric neural stem cell (ENSC) isolation or conditioned media. Bone marrow derived macrophages were prepared and polarised to classic (M1) or alternative (M2) activation states. Markers for macrophage phenotype were measured using quantitative RT-PCR. Results Ageing causes a shift in macrophage polarisation from anti-inflammatory ‘M2’ to proinflammatory ‘M1’ that is associated with a rise in cytokines and immune cells in the ENS. This phenotypic shift is associated with a neural response to inflammatory signals, increase in apoptosis and loss of enteric neurons and ENSCs, and delayed intestinal transit. An age-dependent decrease in expression of the transcription factor FoxO3, a known longevity gene, contributes to the loss of anti-inflammatory behaviour in macrophages of old mice, and FoxO3-deficient mice demonstrate signs of premature ageing of the ENS. Conclusions A shift by macrophages towards a proinflammatory phenotype with ageing causes inflammation-mediated degeneration of the ENS.