Thomas Suply

Oxysterols direct immune cell migration via EBI2.

Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

Regulation of breathing by CO2 requires the proton-activated receptor GPR4 in retrotrapezoid nucleus neurons

Receptor in the brain controls breathing Control of breathing in mammals depends primarily not on sensing oxygen, but rather on detecting concentrations of carbon dioxide in the blood. Failure of this system can cause potentially deadly sleep apnias. Taking a hint from insects, which use a heterotrimeric guanine nucleotide–binding protein-coupled receptor (GPCR) to sense carbon dioxide, Kumar et al. demonstrate that the GPCR GPR4 is essential to control breathing in mice. GPR4 senses protons generated by the formation of carbonic acid in the blood and works with a pH-sensitive potassium channel called TASK-2 in a set of brain cells that control breathing. Science, this issue p. 1255 A G protein–coupled receptor in the brain controls respiration. Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H+ and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency, and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2 (K2P5), a pH-sensitive K+ channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel, and essential central mediators of respiratory chemosensitivity.

Administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by UTE-MRI and histology.

Pulmonary fibrosis can be experimentally induced in small rodents by bleomycin. The antibiotic is usually administered via the intratracheal or intranasal routes. In the present study, we investigated the oropharyngeal aspiration of bleomycin as an alternative route for the induction of lung fibrosis in rats and mice. The development of lung injury was followed in vivo by ultrashort echo time magnetic resonance imaging (UTE-MRI) and by post-mortem analyses (histology of collagen, hydroxyproline determination, and qRT-PCR). In C57BL/6 mice, oropharyngeal aspiration of bleomycin led to more prominent lung fibrosis as compared to intranasal administration. Consequently, the oropharyngeal aspiration route allowed a dose reduction of bleomycin and, therewith, a model refinement. Moreover, the distribution of collagen after oropharyngeal aspiration of bleomycin was more homogenous than after intranasal administration: for the oropharyngeal aspiration route, fibrotic areas appeared all over the lung lobes, while for the intranasal route fibrotic lesions appeared mainly around the largest superior airways. Thus, oropharyngeal aspiration of bleomycin induced morphological changes that were more comparable to the human disease than the intranasal administration route did. Oropharyngeal aspiration of bleomycin led to a homogeneous fibrotic injury also in rat lungs. The present data suggest oropharyngeal aspiration of bleomycin as a less invasive means to induce homogeneous and sustained fibrosis in the lungs of mice and rats.

Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells

Background: TLQP-21 is a bioactive peptide for which the receptor(s) are unknown. Results: We demonstrate that C3AR1 is a receptor for TLQP-21. Conclusion: Many of the effects of TLQP-21 can be explained by C3AR1 activation. Significance: These results provide a bridge linking the regulation of metabolism and the activation of complement in rodents. TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.

Reduced pathological angiogenesis and tumor growth in mice lacking GPR4, a proton sensing receptor

The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.

Neuropeptide S receptor deficiency modulates spontaneous locomotor activity and the acoustic startle response

The present study investigated the phenotype of heterozygous and homozygous neuropeptide S receptor (Npsr) deficient C57BL/6 mice in NPS- and cocaine induced hyperactivity, spontaneous and reactive locomotor activity, elevated plus maze, conditioned fear, and prepulse inhibition of the acoustic startle response. In Npsr-deficient mice, a strong reduction of spontaneous locomotor activity and of the startle magnitude was observed; heterozygous mice had an intermediate phenotype. In the other experiments, Npsr deficiency leads to no or only a very modest phenotype. These results support an important role of neuropeptide S in regulating locomotor activity.

G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation

Background:A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflammation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa. Mtehods:OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1−/− and Ogr1+/+ (WT) residential peritoneal macrophages. Results:Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-&kgr;B inhibitors. Kaplan–Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1−/−/Il-10−/− mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways. Conclusions:OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-&kgr;B inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation.

The Proton-Activated Receptor GPR4 Modulates Glucose Homeostasis by Increasing Insulin Sensitivity

Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues.

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia

The identification of a natural ligand of the orphan chemoattractant receptor GPR15 provides mechanistic insight into the migration of lymphocytes in the skin. Deorphanizing a chemoattractant receptor The orphan G protein–coupled receptor GPR15 mediates the trafficking of lymphocytes to the colon and the skin and the recruitment of effector T cells to inflamed intestinal tissue. Suply et al. purified a natural ligand of GPR15 (GPR15L) from porcine colonic extracts. In vitro assays showed that GPR15L specifically activated GPR15, but not other chemoattractant receptors. Although migration assays suggested that GPR15L inhibited chemokine-induced T cell migration, mouse skin allotransplantations showed that GPR15L recruited CD8+ T cells to the graft and that loss of the ligand was associated with increased graft protection. Given that GPR15L mRNA is abundant in psoriatic lesions, these data suggest that targeting the GPR15-GPR15L axis may help in the treatment of inflammatory skin conditions. GPR15 is an orphan G protein–coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.