Susan J. Abbondanzo

CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue.

Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyers patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation

The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7alpha-hydroxylase pathway.

GPR119 is required for physiological regulation of glucagon-like peptide-1 secretion but not for metabolic homeostasis

G protein-coupled receptor 119 (GPR119) is expressed in pancreatic islets and intestine, and is involved in insulin and incretin hormone release. GPR119-knockout (Gpr119(-/-)) mice were reported to have normal islet morphology and normal size, body weight (BW), and fed/fasted glucose levels. However, the physiological function of GPR119 and its role in maintaining glucose homeostasis under metabolic stress remain unknown. Here, we report the phenotypes of an independently generated line of Gpr119(-/-) mice under basal and high-fat diet (HFD)-induced obesity. Under low-fat diet feeding, Gpr119(-/-) mice show normal plasma glucose and lipids, but have lower BWs and lower post-prandial levels of active glucagon-like peptide 1 (GLP-1). Nutrient-stimulated GLP-1 release is attenuated in Gpr119(-/-) mice, suggesting that GPR119 plays a role in physiological regulation of GLP-1 secretion. Under HFD-feeding, both Gpr119(+)(/)(+) and Gpr119(-/-) mice gain weight similarly, develop hyperinsulinemia and hyperleptinemia, but not hyperglycemia or dyslipidemia. Glucose and insulin tolerance tests did not reveal a genotypic difference. These data show that GPR119 is not essential for the maintenance of glucose homeostasis. Moreover, we found that oleoylethanolamide (OEA), reported as a ligand for GPR119, was able to suppress food intake in both Gpr119(+)(/)(+) and Gpr119(-/-) mice, indicating that GPR119 is not required for the hypophagic effect of OEA. Our results demonstrate that GPR119 is important for incretin and insulin secretion, but not for appetite suppression.

Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease.

OBJECTIVE—FFAR1/GPR40 is a G-protein–coupled receptor expressed predominantly in pancreatic islets mediating free fatty acid–induced insulin secretion. However, the physiological role of FFAR1 remains controversial. It was previously reported that FFAR1 knockout (Ffar1−/−) mice were resistant to high-fat diet–induced hyperinuslinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. A more recent report suggested that although FFAR1 was necessary for fatty acid–induced insulin secretion in vivo, deletion of FFAR1 did not protect pancreatic islets against fatty acid–induced islet dysfunction. This study is designed to investigate FFAR1 function in vivo using a third line of independently generated Ffar1−/− mice in the C57BL/6 background. RESEARCH DESIGN AND METHODS—We used CL-316,243, a β3 adrenergic receptor agonist, to acutely elevate blood free fatty acids and to study its effect on insulin secretion in vivo. Ffar1+/+ (wild-type) and Ffar1−/− (knockout) mice were placed on two distinct high-fat diets to study their response to diet-induced obesity. RESULTS—Insulin secretion was reduced by ∼50% in Ffar1−/− mice, confirming that FFAR1 contributes significantly to fatty acid stimulation of insulin secretion in vivo. However, Ffar1+/+ and Ffar1−/− mice had similar weight, adiposity, and hyperinsulinemia on high-fat diets, and Ffar1−/− mice showed no improvement in glucose or insulin tolerance tests. In addition, high-fat diet induced comparable levels of lipid accumulation in livers of Ffar1+/+ and Ffar1−/− mice. CONCLUSIONS—FFAR1 is required for normal insulin secretion in response to fatty acids; however, Ffar1−/− mice are not protected from high-fat diet–induced insulin resistance or hepatic steatosis.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.

Leukocytes Expressing Green Fluorescent Protein as Novel Reagents for Adoptive Cell Transfer and Bone Marrow Transplantation Studies

Transgenic mice expressing green fluorescent protein (GFP) were generated to provide a source of labeled leukocytes for cell transfer studies. The transgene comprises the GFP coding region under the transcriptional control of the chicken ss-actin promoter and human cytomegalovirus enhancer. Mice expressing this GFP transgene were generated in the B6D2 and in the 129SvEv backgrounds. Flow cytometric analysis of cells from the blood, spleen, and bone marrow of these transgenic mice revealed that most leukocytes, including dendritic cells and memory T cells, express GFP. In allogeneic cell transfers, donor GFP+ splenocytes were detected in the spleen and mesenteric lymph nodes of recipient mice within 2 hours after transfer and for at least 9 days thereafter. In syngeneic experiments using 129-derived GFP+ donor splenocytes, donor cells were detected in multiple tissues of 129 recipients from 2 hours to 3 weeks after transfer. In bone-marrow transplantation experiments using irradiated allogeneic recipients, the percent of GFP+ donor cells in recipients at 3 weeks was comparable to that seen in similar tissues of GFP+ donor mice. These data demonstrate that GFP+ transgenic mice provide a ready source of GFP-expressing primary cells that can be easily monitored after their transfer to recipient animals.

Expression of a Novel Murine Type I IFN in the Pancreatic Islets Induces Diabetes in Mice

IFN-κ belongs to a recently identified subclass of type I IFNs. In this study, we report the cloning and preliminary characterization of the murine homologue of IFN-κ. The gene encodes a 200-aa protein which is 38.5% homologous to human IFN-κ. Murine IFN-κ contains four cysteines in analogous positions to those observed in the IFN-α and an additional fifth unique cysteine, C174. The murine gene is located on chromosome 4, where other type I murine IFN genes, IFN-α and IFN-β, are clustered. This region is syntenic with human chromosome 9 where the gene encoding IFN-κ and the type I IFN gene cluster are found. Mouse IFN-κ is expressed at low levels in peritoneal macrophages and its expression is up-regulated by dsRNA and IFN-γ. Similar to previously reported transgenic mice carrying type I and type II IFNs, transgenic mice overexpressing murine IFN-κ in the β cells of the pancreas develop overt diabetes with hyperglycemia. Histological characterization of pancreatic islets from these transgenic mice showed inflammatory infiltrates with corresponding destruction of β cells.

Nmur1−/− mice are not protected from cutaneous inflammation

Neuromedin U (Nmu) is a neuropeptide expressed primarily in the gastrointestinal tract and central nervous system. Previous reports have identified two G protein-coupled receptors (designated Nmur1 and Nmur2) that bind Nmu. Recent reports suggest that Nmu mediates immune responses involving mast cells, and Nmur1 has been proposed to mediate these responses. In this study, we generated mice with an Nmur1 deletion and then profiled the responses of these mice in a cutaneous inflammation model utilizing complete Freunds adjuvant (CFA). We report here that mice lacking Nmur1 had normal inflammation responses with moderate changes in serum cytokines compared to Nmur1(+/+) littermates. Although differences in IL-6 were observed in mice lacking Nmu peptide, these mice exhibited a normal response to CFA. Our data argues against a major role for Nmur1 in mediating the reported inflammatory functions of NmU.

HIV Cell Fusion Assay Phenotypic Screening Tool for the Identification of HIV Entry Inhibitors via CXCR4

The health and disease-related biology of the CXCR4 chemokine receptor presents the challenge of finding a small molecule that can bind CXCR4 and block T-cell tropic human immunodeficiency virus type 1 (HIV-1) cell entry, while preserving the ability of CXCR4 to respond to its native ligand, CXCL12. HIV entry into the host cell involves the interaction of the viral envelope glycoprotein gp120 binding to CD4, followed by a rearrangement in gp120, and subsequent interaction with the chemokine receptor CXCR4 or CCR5. These initial events can be re-created in a cell fusion assay that represents a surrogate system, mimicking the early stages of viral entry via these host cell receptors. In the current study, a T-tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from the T-tropic HIV NL4-3 and HeLa cells expressing CD4 and CXCR4. Detection of the cell fusion event was based on a Gal4/VP16-activated β-lactamase signal and was measured by automated microscopy or laser scanning plate cytometry. Changes in morphology associated with cell fusion were combined with β-lactamase activity to generate results with robust assay statistics in both 384-well and 1536-well plates. Compounds were subsequently characterized by CXCR4 signaling assays to eliminate functional antagonists and allow the identification of a function-sparing HIV entry inhibitor.