Background: G protein-coupled receptors (GPCR) can form heteromers and thereby alter their signaling properties. Results: GPR55 and cannabinoid 1 (CB1) receptor signaling is modulated if receptors are co-expressed. Conclusion: GPR55 signaling is inhibited in the presence of CB1 receptors; in contrast, CB1 receptor-mediated signaling is enhanced if GPR55 is co-expressed. Significance: Cross-regulation of CB1 receptor and GPR55 may affect cell function when endogenously co-expressed.

No abstract

BackgroundAccumulation of activated eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid 2‐arachidonoylglycerol (2‐AG) has been proposed to elicit eosinophil migration in a CB 2 receptor/Gi/o‐dependent manner. However, it has been claimed recently that this process may also involve other mechanisms such as cytokine priming and the metabolism of 2‐AG into eicosanoids. Here, we explored the direct contribution of specific CB 2 receptor activation to human and mouse eosinophil effector function in vitro and in vivo.Methods In vitro studies including CB 2 expression, adhesion and migratory responsiveness, respiratory burst, degranulation, and calcium mobilization were conducted in human peripheral blood eosinophils and mouse bone marrow‐derived eosinophils. Allergic airway inflammation was assessed in mouse models of acute OVA‐induced asthma and directed eosinophil migration.Results CB 2 expression was significantly higher in eosinophils from symptomatic allergic donors. The selective CB 2 receptor agonist JWH‐133 induced a moderate migratory response in eosinophils. However, short‐term exposure to JWH‐133 potently enhanced chemoattractant‐induced eosinophil shape change, chemotaxis, CD11b surface expression, and adhesion as well as production of reactive oxygen species. Receptor specificity of the observed effects was confirmed in eosinophils from CB 2 knockout mice and by using the selective CB 2 antagonist SR144528. Of note, systemic application of JWH‐133 clearly primed eosinophil‐directed migration in vivo and aggravated both AHR and eosinophil influx into the airways in a CB 2‐specific manner. This effect was completely absent in eosinophil‐deficient ∆dblGATA mice.ConclusionOur data indicate that CB 2 may directly contribute to the pathogenesis of eosinophil‐driven diseases. Moreover, we provide new insights into the molecular mechanisms underlying the CB 2‐mediated priming of eosinophils. Hence, antagonism of CB 2 receptors may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophilic disorders.

Pro-resolution functions were reported for Prostaglandin D2 (PGD2) in colitis, but the role of its two receptors, DP and in particular CRTH2 are less well defined. We investigated DP and CRTH2 expression and function during human and murine ulcerative colitis (UC). Expression of receptors was measured by flow cytometry on peripheral blood leukocytes, and by immunohistochemistry and immunoblotting in colon biopsies of patients with active UC and healthy individuals. Receptor involvement in UC was evaluated in a mouse model of DSS colitis. DP and CRTH2 expression changed in leukocytes of patients with active UC in a differential manner. In UC patients, DP showed higher expression in neutrophils but lower in monocytes as compared to control subjects. In contrast, CRTH2 was decreased in eosinophils, NK and CD3+ T cells but not in monocytes and CD3+/CD4+ T cells. The decrease of CRTH2 on blood eosinophils clearly correlated with disease activity. DP correlated positively with disease activity in eosinophils but inversely in neutrophils. CRTH2 internalized upon treatment with PGD2 and 11-dehydroTXB2 in eosinophils of controls. Biopsies of UC patients revealed an increase of CRTH2-positive cells in the colonic mucosa and high CRTH2 protein content. The CRTH2 antagonist CAY10595 improved while the DP antagonist MK0524 worsened inflammation in murine colitis. DP and CRTH2 play differential roles in UC. Although expression of CRTH2 on blood leukocytes is downregulated in UC, CRTH2 is present in colon tissue where it may contribute to inflammation whereas DP likely promotes anti-inflammatory actions.

† These authors contributed equally to this work.Human cytomegalovirus (HCMV) encodes the seven transmembrane (7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Gα q /PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-κB (NF-κB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Background Bacille Calmette–Guérin (BCG) vaccination can potentially reduce the rate of respiratory infections in vulnerable populations. This study evaluates the safety and efficacy of VPM1002 (a genetically modified BCG) as prophylaxis against severe respiratory tract infections including COVID-19 in an elderly population. Methods In this phase III, randomized, double-blind, placebo-controlled, multicenter clinical trial, healthy elderly volunteers (n = 2064) were enrolled, randomized (1:1) to receive either VPM1002 or placebo, and followed up remotely for 240 days. The primary outcome was the mean number of days with severe respiratory infections at hospital and/or at home. Secondary endpoints included the incidence of self-reported fever, number of hospital and ICU admissions, and number of adverse events. Results A total of 31 participants in the VPM1002 group reported at least 1 day with severe respiratory disease and a mean number of days with severe respiratory disease of 9.39 ± 9.28 days while in the placebo group, 38 participants reported a mean of 14.29 ± 16.25 days with severe respiratory disease. The incidence of self-reported fever was lower in the VPM1002 group (odds ratio: 0.46; 95% CI: 0.28 to 0.74; p-value: 0.001) and consistent trends to less hospitalization and ICU admissions due to COVID-19 were observed after VPM1002-vaccination. Local reactions typical for BCG were observed in the VPM1002-vaccinated group, which were mostly of mild intensity. Conclusions Vaccination with VPM1002 is well tolerated and seems to have a prophylactic effect against severe respiratory diseases in the elderly. (ClinicalTrials.gov: NCT04435379)

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

The adhesion and degranulation promoting adaptor protein (ADAP) is a multifunctional scaffold involved in many different signaling pathways that are important for the function of T cells, including the inside-out and outside-in signaling of integrins, the activation of NF-κB, and the subsequent production of proinflammatory cytokines (e.g., IFN-γ and IL-2). Strikingly, despite its well-established role in T cells, previous studies did not distinguish between CD4 and CD8 T cells, and thus, it is unknown whether ADAP fulfills equally important functions in both T cell subsets. We show here that despite comparable ADAP expression levels in CD4 and CD8 T cells, their function is differentially dependent on ADAP. Whereas in vitro TCR-stimulation experiments revealed that activation, proliferation, and adhesion are severely compromised in CD4 T cells lacking ADAP, their CD8 counterparts are hardly affected by ADAP deficiency. Accordingly, antigen-specific in vivo stimulation of adoptively transferred CD8 T cells during Listeria monocytogenes (Lm) and influenza A virus (IAV) infection revealed only moderate effects of ADAP deficiency in terms of CD8 T cell activation, proliferation, and differentiation, which, however, did not impair pathogen-specific immunity. Thus, we show for the first time that ADAP fulfills different functions in CD4 and CD8 T cells, with CD8 T cells being less dependent on ADAP. Our data identify ADAP as a potential molecular target for T cell subset-specific therapeutic interventions.