Patient-Oriented Perspective on Chemokine Receptor Expression and Function in Glioma.

peer reviewedGliomas are severe brain malignancies, with glioblastoma (GBM) being the most aggressive one. Despite continuous efforts for improvement of existing therapies, overall survival remains poor. Over the last years, the implication of chemokines and their receptors in GBM development and progression has become more evident. Recently, large amounts of clinical data have been made available, prompting us to investigate chemokine receptors in GBM from a still-unexplored patient-oriented perspective. This study aims to highlight and discuss the involvement of chemokine receptors-CCR1, CCR5, CCR6, CCR10, CX3CR1, CXCR2, CXCR4, ACKR1, ACKR2, and ACKR3-most abundantly expressed in glioma patients based on the analysis of publicly available clinical datasets. Given the strong intratumoral heterogeneity characterizing gliomas and especially GBM, receptor expression was investigated by glioma molecular groups, by brain region distribution, emphasizing tissue-specific receptor functions, and by cell type enrichment. Our study constitutes a clinically relevant and patient-oriented guide that recapitulates the expression profile and the complex roles of chemokine receptors within the highly diversified glioma landscape. Additionally, it strengthens the importance of patient-derived material for development and precise amelioration of chemokine receptor-targeting therapies

GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity.

Immune responses highly depend on the effective trafficking of immune cells into and within secondary lymphoid organs (SLOs). Atypical chemokine receptors (ACKRs) scavenge chemokines to eliminate them from the extracellular space, thereby generating gradients that guide leukocytes. In contrast to canonical chemokine receptors, ACKRs do not induce classical intracellular signaling that results in cell migration. Recently, the closest relative of ACKR3, GPR182, has been partially deorphanized as a potential novel ACKR. We confirm and extend previous studies by identifying further ligands that classify GPR182 as a broadly scavenging chemokine receptor. We validate the "atypical" nature of the receptor, wherein canonical G-protein-dependent intracellular signaling is not activated following ligand stimulation. However, β-arrestins are required for ligand-independent internalization and chemokine scavenging whereas the C-terminus is in part dispensable. In the absence of GPR182 in vivo, we observed elevated chemokine levels in the serum but also in SLO interstitium. We also reveal that CXCL13 and CCL28, which do not bind any other ACKR, are bound and efficiently scavenged by GPR182. Moreover, we found a cooperative relationship between GPR182 and ACKR3 in regulating serum CXCL12 levels, and between GPR182 and ACKR4 in controlling CCL20 levels. Furthermore, we unveil a new phenotype in GPR182-KO mice, in which we observed a reduced marginal zone (MZ), both in size and in cellularity, and thus in the T-independent antibody response. Taken together, we and others have unveiled a novel, broadly scavenging chemokine receptor, which we propose should be named ACKR5

CXCL14 preferentially synergizes with homeostatic chemokine receptor systems

Reflecting their importance in immunity, the activity of chemokines is regulated on several levels, including tissue and context-specific expression and availability of their cognate receptor on target cells. Chemokine synergism, affecting both chemokine and chemokine receptor function, has emerged as an additional control mechanism. We previously demonstrated that CXCL14 is a positive allosteric modulator of CXCR4 in its ability to synergize with CXCL12 in diverse cellular responses. Here, we have extended our study to additional homeostatic, as well as a selection of inflammatory chemokine systems. We report that CXCL14 strongly synergizes with low (sub-active) concentrations of CXCL13 and CCL19/CCL21 in in vitro chemotaxis with immune cells expressing the corresponding receptors CXCR5 and CCR7, respectively. CXCL14 by itself was inactive, not only on cells expressing CXCR5 or CCR7 but also on cells expressing any other known conventional or atypical chemokine receptor, as assessed by chemotaxis and/or β-arrestin recruitment assays. Furthermore, synergistic migration responses between CXCL14 and inflammatory chemokines CXCL10/CXCL11 and CCL5, targeting CXCR3 and CCR5, respectively, were marginal and occasional synergistic Ca2+ flux responses were observed. CXCL14 bound to 300-19 cells and interfered with CCL19 binding to CCR7-expressing cells, suggesting that these cellular interactions contributed to the reported CXCL14-mediated synergistic activities. We propose a model whereby tissue-expressed CXCL14 contributes to cell localization under steady-state conditions at sites with prominent expression of homeostatic chemokines.publishe

Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future

The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

peer reviewedThe chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, β-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving β-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the β-arrestin-bias and the atypical nature of CXCR3-B

New pairings and deorphanization among the atypical chemokine receptor family — physiological and clinical relevance

Atypical chemokine receptors (ACKRs) form a small subfamily of receptors (ACKR1–4) unable to trigger G protein-dependent signaling in response to their ligands. They do, however, play a crucial regulatory role in chemokine biology by capturing, scavenging or transporting chemokines, thereby regulating their availability and signaling through classical chemokine receptors. ACKRs add thus another layer of complexity to the intricate chemokine–receptor interaction network. Recently, targeted approaches and screening programs aiming at reassessing chemokine activity towards ACKRs identified several new pairings such as the dimeric CXCL12 with ACKR1, CXCL2, CXCL10 and CCL26 with ACKR2, the viral broad-spectrum chemokine vCCL2/vMIP-II, a range of opioid peptides and PAMP-12 with ACKR3 as well as CCL20 and CCL22 with ACKR4. Moreover, GPR182 (ACKR5) has been lately proposed as a new promiscuous atypical chemokine receptor with scavenging activity notably towards CXCL9, CXCL10, CXCL12 and CXCL13. Altogether, these findings reveal new degrees of complexity of the chemokine network and expand the panel of ACKR ligands and regulatory functions. In this minireview, we present and discuss these new pairings, their physiological and clinical relevance as well as the opportunities they open for targeting ACKRs in innovative therapeutic strategies

How Carvedilol activates β₂-adrenoceptors

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β(1)-adrenoceptors, arrestin-biased signalling via β(2)-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol’s cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β(2)ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets