Chemokine Scavenging Research Articles

Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies.

Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3.

International Union of Basic and Clinical Pharmacology. CXVIII. Update on the Nomenclature for Atypical Chemokine Receptors including ACKR5.

Chemokines signal through classical G protein-coupled receptors (GPCRs) to induce cell migration during development, immune homeostasis and multiple diseases. Over the last decade a subfamily of atypical chemokine receptors (ACKRs) was delineated from GPCRs based on their inability to trigger conventional G protein signaling or mediate cell migration in response to chemokines. These receptors nevertheless play an important role within the chemokine system by sequestering, transporting or internalizing chemokines thereby regulating their availability and shaping their gradients. GPR182, the recently deorphanized chemokine receptor, shares about 30% of sequence similarity with its closest relative ACKR3. GPR182 is mainly expressed on endothelial cells and was proposed to act as a scavenger regulating the availability of a large set of chemokines from the CXC, CC and XC families and to act cooperatively with ACKR3 and ACKR4. Unlike other ACKRs, GPR182 was shown to have a strong constitutive interaction with β-arrestins that is required for intracellular receptor trafficking and chemokine scavenging. Chemokine ligation of GPR182 has no additional detectable impact on β-arrestin recruitment. Genetic ablation of GPR182 affects spleen size, myelopoiesis, and serum chemokine levels, indicating its role in chemokine homeostasis and immune regulation. GPR182 was also reported to regulate immune responses to bloodborne antigens and tumorigenesis. Taken together, compelling cumulative evidence indicates that GPR182 does not trigger G protein-mediated signaling but acts as a scavenger for chemokines in vitro and in vivo strongly supporting its inclusion as ACKR5 in the systematic nomenclature of chemokine receptors. Significance Statement The summarized presented findings strongly support the designation of GPR182 as ACKR5 and its formal inclusion in the family of atypical chemokine receptors.

The atypical chemokine receptor 2 reduces T cell expansion and tertiary lymphoid tissue but does not limit autoimmune organ injury in lupus-prone B6lpr mice.

The atypical chemokine receptor 2 (ACKR2) is a chemokine scavenger receptor, which limits inflammation and organ damage in several experimental disease models including kidney diseases. However, potential roles of ACKR2 in reducing inflammation and tissue injury in autoimmune disorders like systemic lupus erythematosus (SLE) and lupus nephritis are unknown, as well as its effects on systemic autoimmunity. To characterize functional roles of ACKR2 in SLE, genetic Ackr2 deficiency was introduced into lupus-prone C57BL/6lpr (Ackr2-/- B6lpr) mice. Upon inflammatory stimulation in vitro, secreted chemokine levels increased in Ackr2 deficient tubulointerstitial tissue but not glomeruli. Moreover, Ackr2 expression was induced in kidneys and lungs of female C57BL/6lpr mice developing SLE. However, female Ackr2-/- B6lpr mice at 28 weeks of age showed similar renal functional parameters as wildtype (WT)-B6lpr mice. Consistently, assessment of activity and chronicity indices for lupus nephritis revealed comparable renal injury. Interestingly, Ackr2-/- B6lpr mice showed significantly increased renal infiltrates of CD3+ T and B cells, but not neutrophils, macrophages or dendritic cells, with T cells predominantly accumulating in the tubulointerstitial compartment of Ackr2-/- B6lpr mice. In addition, histology demonstrated significantly increased peribronchial lung infiltrates of CD3+ T cells in Ackr2-/- B6lpr mice. Despite this, protein levels of pro-inflammatory chemokines and mRNA expression of inflammatory mediators were not different in kidneys and lungs of WT- and Ackr2-/- B6lpr mice. This data suggests compensatory mechanisms for sufficient chemokine clearance in Ackr2-deficient B6lpr mice in vivo. Analysis of systemic autoimmune responses revealed comparable levels of circulating lupus-associated autoantibodies and glomerular immunoglobulin deposition in the two genotypes. Interestingly, similar to kidney and lung CD4+ T cell numbers and activation were significantly increased in spleens of Ackr2-deficient B6lpr mice. In lymph nodes of Ackr2-/- B6lpr mice abundance of activated dendritic cells decreased, but CD4+ T cell numbers were comparable to WT. Moreover, increased plasma levels of CCL2 were present in Ackr2-/- B6lpr mice, which may facilitate T cell mobilization into spleens and peripheral organs. In summary, we show that ACKR2 prevents expansion of T cells and formation of tertiary lymphoid tissue, but is not essential to limit autoimmune tissue injury in lupus-prone B6lpr mice.

Comparative Genomic Analysis Unveils Potential Factors Contributing to the Endangerment of Silurus lanzhouensis

Silurus lanzhouensis is a large endemic fish exclusively found in the middle and upper reaches of China’s Yellow River, with a restricted distribution range and a small population size and is listed as an endangered species. To investigate the potential genetic factors contributing to the observed decrease in the population of S. lanzhouensis, we have successfully assembled the first chromosome-level genome of S. lanzhouensis. This genome comprises a total of 30 chromosomes, with a size of 794.43 Mb, and 23,093 protein-coding genes. Comparative genomics analysis reveals that the genetic relationship between S. lanzhouensis and S. meridionalis Chen is the closest, and their common ancestor experienced two rounds of whole genome duplication (WGD) before diverging. Notably, amino acid metabolism-related pathways are enriched in the unique S. lanzhouensis gene family. In addition, we observed that the chemokine receptor and scavenger receptor gene families contracted in S. lanzhouensis, which may be associated with its diminished immune response. Our research findings provide valuable insights into the endangered mechanism of S. lanzhouensis and enhance our understanding of the evolution of Siluriformes fish.

Large chemokine binding spectrum of human and mouse atypical chemokine receptor GPR182 (ACKR5).

Atypical chemokine receptors (ACKRs) play pivotal roles in immune regulation by binding chemokines and regulating their spatial distribution without inducing G-protein activation. Recently, GPR182, provisionally named ACKR5, was identified as a novel ACKR expressed in microvascular and lymphatic endothelial cells, with functions in hematopoietic stem cell homeostasis. Here, we comprehensively investigated the chemokine binding profile of human and mouse GPR182. Competitive binding assays using flow cytometry revealed that besides CXCL10, CXCL12 and CXCL13, also human and mouse CXCL11, CXCL14 and CCL25, as well as human CCL1, CCL11, CCL19, CCL26, XCL1 and mouse CCL22, CCL24, CCL27 and CCL28 bind with an affinity of less than 100nM to GPR182. In line with the binding affinity observed in vitro, elevated serum levels of CCL22, CCL24, CCL25, and CCL27 were observed in GPR182-deficient mice, underscoring the role of GPR182 in chemokine scavenging. These data show a broader chemokine binding repertoire of GPR182 than previously reported and they will be important for future work exploring the physiological and pathophysiological roles of GPR182, which we propose to be renamed atypical chemokine receptor 5 (ACKR5).

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.

Atypical Chemokine Receptor 3 "Senses" CXC Chemokine Receptor 4 Activation Through GPCR Kinase Phosphorylation.

Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts the availability of the chemokine agonist CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we determined that GRK5 phosphorylation of ACKR3 results in more efficient chemokine scavenging and β-arrestin recruitment than phosphorylation by GRK2 in HEK293 cells. However, co-activation of CXCR4-enhanced ACKR3 phosphorylation by GRK2 through the liberation of Gβγ, an accessory protein required for efficient GRK2 activity. The results suggest that ACKR3 "senses" CXCR4 activation through a GRK2-dependent crosstalk mechanism, which enables CXCR4 to influence the efficiency of CXCL12 scavenging and β-arrestin recruitment to ACKR3. Surprisingly, we also found that despite the requirement for phosphorylation and the fact that most ligands promote β-arrestin recruitment, β-arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet-to-be-determined function for these adapter proteins. Since ACKR3 is also a receptor for CXCL11 and opioid peptides, these data suggest that such crosstalk may also be operative in cells with CXCR3 and opioid receptor co-expression. Additionally, kinase-mediated receptor cross-regulation may be relevant to other atypical and G protein-coupled receptors that share common ligands. SIGNIFICANCE STATEMENT: The atypical receptor ACKR3 indirectly regulates CXCR4-mediated cell migration by scavenging their shared agonist CXCL12. Here, we show that scavenging and β-arrestin recruitment by ACKR3 are primarily dependent on phosphorylation by GRK5. However, we also show that CXCR4 co-activation enhances the contribution of GRK2 by liberating Gβγ. This phosphorylation crosstalk may represent a common feedback mechanism between atypical and G protein-coupled receptors with shared ligands for regulating the efficiency of scavenging or other atypical receptor functions.

Exosomal release of the virus-encoded chemokine receptor US28 contributes to chemokine scavenging

The human cytomegalovirus (HCMV)-encoded chemokine receptor US28 contributes to various aspects of the viral life cycle and promotes immune evasion by scavenging chemokines from the microenvironment of HCMV-infected cells.In contrast to the plasma membrane localization of most human chemokinereceptors, US28 has a predominant intracellular localization. In this study, weused immunofluorescence and electron microscopy to determine thelocalization of US28 upon exogenous expression, as well as in HCMV-infectedcells. We observed that US28 localizes to late endosomal compartmentscalled multivesicular bodies (MVBs), where it is sorted in intraluminal vesicles. Live-cell total internal reflection fluorescence (TIRF) microscopy revealed that US28-containing MVBs can fuse with the plasma membrane, resulting inthe secretion of US28 on exosomes. Exosomal US28 binds thechemokines CX3CL1 and CCL5, and US28-containing exosomes inhibited theCX3CL1-CX3CR1 signaling axis. These findings suggest that exosomal releaseof US28contributes to chemokine scavenging and immune evasion by HCMV.

#4574 ATYPICAL CHEMOKINE RECEPTOR 4 IS EXPRESSED IN GLOMERULAR PARIETAL EPITHELIAL CELLS AND MITIGATES THE SEVERITY OF EXPERIMENTAL GLOMERULONEPHRITIS

Abstract Background and Aims Cell migration contributes to the innumerable physiological and pathological processes driven and directed by chemotactic cytokines (chemokines), small structurally homologous proteins signaling via their cognate GPCRs on motile cells. In addition to GPCRs, chemokines importantly interact with atypical chemokine receptors (ACKRs) that are characterized by expression in various stromal cells and distinctive ligand specificities. ACKR4 binds CCL19, CCL20, CCL21, CCL22 and CCL25, chemokines involved primarily in adaptive immune responses. Accordingly, ACKR4 is prominently expressed in the primary and secondary lymphoid organs, thymus, bone marrow, lymph nodes and spleen where it regulates distinct cell migratory steps driven by its cognate ligands. Furthermore, ACKR4 is expressed by dissimilar cell types in multiple parenchymal organs, the heart, the liver and the gut. Currently it is not entirely clear how chemokine scavenging by ACKR4 in these organs might affect their pathophysiology. Here we investigated the expression of ACKR4 in murine kidney and assessed its contribution to nephrotoxic serum nephritis (NTSN), an experimental murine model of immune complex glomerulonephritis. The pathomechanisms in NTSN are known to rely on the contribution of multiple chemokines and their receptors and functions of Th1 cells, Th17 cells, neutrophils and macrophages exerting pathogenic effects in both lymphoid organs and the kidney. Method The expression of renal ACKR4 was evaluated in healthy ACKR4-eGFP reporter mice as well as after the induction of NTSN. To investigate the contribution of ACKR4 to NTSN, ACKR4-deficient mice and WT controls were subjected to an anti-basal membrane immunization protocol and the parameters of immunopathogenesis and the ensuing kidney disease were evaluated at 7 and 14 days after the immunization. Results Multicolour immunofluorescence confocal microscopy revealed that ACKR4 is expressed in the kidney exclusively in the glomeruli by a discrete subset of parietal epithelial cells localizing adjacently to the vascular glomerular pole. In mice with NTSN, the overall proportion of ACKR4 expressing cells in glomeruli at the vascular pole did not change as compared to healthy mice, however ACKR4+ cells were not detected in glomeruli corresponding with the increased abundance of alpha-SMA, a marker of renal fibrosis. The ACKR4-deficient mice showed delayed antibody response following immunization and reduced readouts of cellular immunity. However, despite this, disease parameters of NTSN, including albuminuria, PAS-score and crescent formation were significantly increased in ACKR4-deficient mice as compared to the WT controls. Renin, a hormone secreted by the cells adjacent to ACKR4+ cells in the kidney was significantly increased in serum of nephritic ACKR4-/- mice. Conclusion We report for the first time that ACKR4 is expressed in the kidney by a small subpopulation of parietal epithelial cells. Reduced parameters of humoral and cellular immunity in ACKR4-deficient mice contrast with a more severe manifestations of NTSN seen in these mice, suggesting a tissue-specific functional bias of ACKR4 in the kidney that limits the development of NTSN. Furthermore, the immediate proximity of ACKR4 expressing cells to the juxtaglomerular apparatus and the observed increased renin secretion in ACKR4-/- mice suggest a potential contribution of ACKR4 to the regulation of renin-driven pathways, by a yet unclear mechanism.

Atlas of the anatomical localization of atypical chemokine receptors in healthy mice.

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3GFP/+, ACKR4GFP/+, GPR182mCherry/+, and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.

Cutaneous lesions in psoriatic arthritis are enriched in chemokine transcriptomic pathways

ObjectivesSkin from people with psoriasis has been extensively studied and is assumed to be identical to skin from those with psoriatic arthritis (PsA). Chemokines and the CC chemokine scavenger receptor ACKR2 are upregulated in uninvolved psoriasis. ACKR2 has been proposed as a regulator of cutaneous inflammation in psoriasis. The aim of this study was to compare the transcriptome of PsA skin to healthy control (HC) skin and evaluate ACKR2 expression in PsA skin.MethodsFull-thickness skin biopsies from HC, lesional and uninvolved skin from participants with PsA were sequenced on NovaSeq 6000. Findings were validated using qPCR and RNAscope.ResultsNine HC and nine paired PsA skin samples were sequenced. PsA uninvolved skin was transcriptionally similar to HC skin, and lesional PsA skin was enriched in epidermal and inflammatory genes. Lesional PsA skin was enriched in chemokine-mediated signalling pathways, but uninvolved skin was not. ACKR2 was upregulated in lesional PsA skin but had unchanged expression in uninvolved compared with HC skin. The expression of ACKR2 was confirmed by qPCR, and RNAscope demonstrated strong expression of ACKR2 in the suprabasal layer of the epidermis in PsA lesions.ConclusionChemokines and their receptors are upregulated in lesional PsA skin but relatively unchanged in uninvolved PsA skin. In contrast to previous psoriasis studies, ACKR2 was not upregulated in uninvolved PsA skin. Further understanding of the chemokine system in PsA may help to explain why inflammation spreads from the skin to the joints in some people with psoriasis.

Chemokine receptors in GtoPdb v.2023.1

Chemokine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Chemokine Receptors [438, 437, 32]) comprise a large subfamily of 7TM proteins that bind one or more chemokines, a large family of small cytokines typically possessing chemotactic activity for leukocytes. Additional hematopoietic and non-hematopoietic roles have been identified for many chemokines in the areas of embryonic development, immune cell proliferation, activation and death, viral infection, and as antibacterials, among others. Chemokine receptors can be divided by function into two main groups: G protein-coupled chemokine receptors, which mediate leukocyte trafficking, and "Atypical chemokine receptors", which may signal through non-G protein-coupled mechanisms and act as chemokine scavengers to downregulate inflammation or shape chemokine gradients [32].Chemokines in turn can be divided by structure into four subclasses by the number and arrangement of conserved cysteines. CC (also known as β-chemokines; n= 28), CXC (also known as α-chemokines; n= 17) and CX3C (n= 1) chemokines all have four conserved cysteines, with zero, one and three amino acids separating the first two cysteines respectively. C chemokines (n= 2) have only the second and fourth cysteines found in other chemokines. Chemokines can also be classified by function into homeostatic and inflammatory subgroups. Most chemokine receptors are able to bind multiple high-affinity chemokine ligands, but the ligands for a given receptor are almost always restricted to the same structural subclass. Most chemokines bind to more than one receptor subtype. Receptors for inflammatory chemokines are typically highly promiscuous with regard to ligand specificity, and may lack a selective endogenous ligand. G protein-coupled chemokine receptors are named acccording to the class of chemokines bound, whereas ACKR is the root acronym for atypical chemokine receptors [33]. There can be substantial cross-species differences in the sequences of both chemokines and chemokine receptors, and in the pharmacology and biology of chemokine receptors. Endogenous and microbial non-chemokine ligands have also been identified for chemokine receptors. Many chemokine receptors function as HIV co-receptors, but CCR5 is the only one demonstrated to play an essential role in HIV/AIDS pathogenesis. The tables include both standard chemokine receptor names [693] and aliases.

Atypical regulation of an atypical chemokine receptor: ACKR3 'senses' CXCR4 activation through phosphorylation.

ACKR3 is an arrestin-biased 7-transmembrane receptor that is unable to activate G proteins. The receptor shares the chemokine agonist, CXCL12, with the canonical G protein-coupled receptor (GPCR), CXCR4. The receptor pair plays a critical role in mediating cell migration during cardiac and CNS development as well as contributing to cancer growth and metastasis. Whereas CXCR4 drives cellular migration through G protein signaling, ACKR3 primarily acts to scavenge chemokines, which indirectly regulates migration through shaping the extracellular chemokine gradient. Scavenging is driven by constitutive internalization and recycling of the receptor and concomitant shuttling of CXCL12 to lysosomes for degradation. Since internalization of GPCRs is often dependent on phosphorylation and β-arrestins, we set out to resolve how GPCR kinases (GRKs) and β-arrestins influence ACKR3 scavenging. Despite robust β-arrestin recruitment by ACKR3, knockouts of β-arrestins had little impact on CXCL12 scavenging by the receptor; nevertheless, GRK phosphorylation is essential. Employing GRK knockout cells, we discovered that GRK5 is the dominant kinase for ACKR3 phosphorylation and primary driver of chemokine scavenging in HEK293 cells. In comparison the responses elicited by GRK2 were much more limited. Because GRK2 requires free Gβγ released by heterotrimeric G protein activation, we hypothesized that the muted GRK2 impact may be due to the lack of G protein coupling by ACKR3. Indeed, addition of Gβγ significantly enhanced GRK2-mediated ACKR3 phosphorylation and scavenging of CXCL12. Additionally, co-activation of CXCR4 produced sufficient Gβγ to promote GRK2 phosphorylation of ACKR3. These results suggest a mechanism for ACKR3 to ‘sense’ CXCR4 activation through GRK2 activity. Likewise, this would allow CXCR4 to influence CXCL12 scavenging as well as its own signaling by promoting β-arrestin recruitment to ACKR3.

The dual-function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct mechanisms.

C-C chemokine receptor 2 (CCR2) is a dual-function receptor. Similar to other G protein-coupled chemokine receptors, it promotes monocyte infiltration into tissues in response to the chemokine CCL2, and, like atypical chemokine receptors (ACKRs), it scavenges chemokine from the extracellular environment. CCR2 therefore mediates CCL2-dependent signaling as a G protein-coupled receptor (GPCR) and also limits CCL2 signaling as a scavenger receptor. We investigated the mechanisms underlying CCR2 scavenging, including the involvement of intracellular proteins typically associated with GPCR signaling and internalization. Using CRISPR knockout cell lines, we showed that CCR2 scavenged by constitutively internalizing to remove CCL2 from the extracellular space and recycling back to the cell surface for further rounds of ligand sequestration. This process occurred independently of G proteins, GPCR kinases (GRKs), β-arrestins, and clathrin, which is distinct from other "professional" chemokine scavenger receptors that couple to GRKs, β-arrestins, or both. These findings set the stage for understanding the molecular regulators that determine CCR2 scavenging and may have implications for drug development targeting this therapeutically important receptor.

348 Metabolic syndrome in psoriasis is associated with upregulation of CXCL16 on monocytes and a dysbalance in innate lymphoid cells

Psoriasis is frequently associated with the metabolic syndrome and occurs more often in obese individuals. In order to understand innate immune mechanisms mediating this inflammatory pattern we investigated expression of the chemokine and lipid scavenger receptor CXCL16 in patients with psoriasis and associated comorbidities. CXCL16 expression was enhanced on all monocyte subsets in psoriatic patients compared with healthy controls and positively correlated with psoriasis activity and severity index, body mass index and the risk for cardiovascular disease indicated by PROCAM score.

Metabolic Syndrome in Psoriasis Is Associated With Upregulation of CXCL16 on Monocytes and a Dysbalance in Innate Lymphoid Cells.

Psoriasis is frequently associated with the metabolic syndrome and occurs more often in obese individuals. In order to understand innate immune mechanisms mediating this inflammatory pattern we investigated expression of the chemokine and lipid scavenger receptor CXCL16 in patients with psoriasis and associated comorbidities. CXCL16 expression was enhanced on all monocyte subsets in psoriatic patients compared with healthy controls and positively correlated with psoriasis activity and severity index, body mass index and the risk for cardiovascular disease indicated by PROCAM score. The intensity of CXCL16 expression on monocytes further correlated with their capability to phagocytose oxidized LDL indicating the possibility to transform into foam cells in atherosclerotic plaques. Patients with psoriasis and atherosclerosis or obesity displayed elevated numbers of innate lymphoid cells in blood with specific increase of the IFN-γ or IL-17 producing ILC1 and ILC3 subpopulations. The expression of the CXCL16 receptor, CXCR6, was increased in ILCs and co-expressed with CCR6 but not CCR7 indicating their migratory potential to psoriatic skin or adipose tissue that is characterized by strong CXCL16 and CCL20 expression. This hypothesis was supported by the finding that the percentage of CXCR6 expressing ILCs was alleviated in blood of psoriatic patients. Together these data link a strong expression of CXCL16 to metabolic syndrome in psoriasis and indicate a possible link to ILC activation and tissue distribution in obese psoriatic patients. These data contribute to the understanding of the complex interaction of innate immunity and metabolic state in psoriasis.

GPR182 limits antitumor immunity via chemokine scavenging in mouse melanoma models

For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.

Nanoluciferase-based methods to monitor activation, modulation and trafficking of atypical chemokine receptors.

Chemokines regulate directed cell migration, proliferation and survival and are key components in various physiological and pathological processes. They exert their functions by interacting with seven-transmembrane domain receptors that signal through G proteins (GPCRs). Atypical chemokine receptors (ACKRs) play important roles in the chemokine-receptor network by regulating chemokine bioavailability for the classical receptors through chemokine sequestration, scavenging or transport. Currently, this subfamily of receptors comprises four members: ACKR1, ACKR2, ACKR3 and ACKR4. They differ notably from the classical chemokine receptors by their inability to elicit G protein-mediated signaling, which precludes the use of classical assays relying on the activation of G proteins and related downstream secondary messengers to investigate ACKRs. There is therefore a need for alternative approaches to monitor ACKR activation, modulation and trafficking. This chapter details sensitive and versatile methods based on Nanoluciferase Binary Technology (NanoBiT) and Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) to monitor ACKR2 and ACKR3 activity through the measurement of β-arrestin and GRK recruitment, and receptor trafficking, including internalization and delivery to early endosomes.

Chemokine receptors (version 2020.5) in the IUPHAR/BPS Guide to Pharmacology Database

Chemokine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Chemokine Receptors [431, 430, 32]) comprise a large subfamily of 7TM proteins that bind one or more chemokines, a large family of small cytokines typically possessing chemotactic activity for leukocytes. Additional hematopoietic and non-hematopoietic roles have been identified for many chemokines in the areas of embryonic development, immune cell proliferation, activation and death, viral infection, and as antibiotics, among others. Chemokine receptors can be divided by function into two main groups: G protein-coupled chemokine receptors, which mediate leukocyte trafficking, and "Atypical chemokine receptors", which may signal through non-G protein-coupled mechanisms and act as chemokine scavengers to downregulate inflammation or shape chemokine gradients [32].Chemokines in turn can be divided by structure into four subclasses by the number and arrangement of conserved cysteines. CC (also known as β-chemokines; n= 28), CXC (also known as α-chemokines; n= 17) and CX3C (n= 1) chemokines all have four conserved cysteines, with zero, one and three amino acids separating the first two cysteines respectively. C chemokines (n= 2) have only the second and fourth cysteines found in other chemokines. Chemokines can also be classified by function into homeostatic and inflammatory subgroups. Most chemokine receptors are able to bind multiple high-affinity chemokine ligands, but the ligands for a given receptor are almost always restricted to the same structural subclass. Most chemokines bind to more than one receptor subtype. Receptors for inflammatory chemokines are typically highly promiscuous with regard to ligand specificity, and may lack a selective endogenous ligand. G protein-coupled chemokine receptors are named acccording to the class of chemokines bound, whereas ACKR is the root acronym for atypical chemokine receptors [33]. There can be substantial cross-species differences in the sequences of both chemokines and chemokine receptors, and in the pharmacology and biology of chemokine receptors. Endogenous and microbial non-chemokine ligands have also been identified for chemokine receptors. Many chemokine receptors function as HIV co-receptors, but CCR5 is the only one demonstrated to play an essential role in HIV/AIDS pathogenesis. The tables include both standard chemokine receptor names [684] and aliases.

ACKR4 Recruits GRK3 Prior to β-Arrestins but Can Scavenge Chemokines in the Absence of β-Arrestins.

Chemokines are essential for guiding cell migration. Atypical chemokine receptors (ACKRs) contribute to the cell migration process by binding, internalizing and degrading local chemokines, which enables the formation of confined gradients. ACKRs are heptahelical membrane spanning molecules structurally related to G-protein coupled receptors (GPCRs), but seem to be unable to signal through G-proteins upon ligand binding. ACKR4 internalizes the chemokines CCL19, CCL21, and CCL25 and is best known for shaping functional CCL21 gradients. Ligand binding to ACKR4 has been shown to recruit β-arrestins that has led to the assumption that chemokine scavenging relies on β-arrestin-mediated ACKR4 trafficking, a common internalization route taken by class A GPCRs. Here, we show that CCL19, CCL21, and CCL25 readily recruited β-arrestin1 and β-arrestin2 to human ACKR4, but found no evidence for β-arrestin-dependent or independent ACKR4-mediated activation of the kinases Erk1/2, Akt, or Src. However, we demonstrate that β-arrestins interacted with ACKR4 in the steady-state and contributed to the spontaneous trafficking of the receptor in the absence of chemokines. Deleting the C-terminus of ACKR4 not only interfered with the interaction of β-arrestins, but also with the uptake of fluorescently labeled cognate chemokines. We identify the GPCR kinase GRK3, and to a lesser extent GRK2, but not GRK4, GRK5, and GRK6, to be recruited to chemokine-stimulated ACKR4. We show that GRK3 recruitment proceded the recruitment of β-arrestins upon ACKR4 engagement and that GRK2/3 inhibition partially interfered with steady-state interaction and chemokine-driven recruitment of β-arrestins to ACKR4. Overexpressing β-arrestin2 accelerated the uptake of fluorescently labeled CCL19, indicating that β-arrestins contribute to the chemokine scavenging activity of ACKR4. By contrast, cells lacking β-arrestins were still capable to take up fluorescently labeled CCL19 demonstrating that β-arrestins are dispensable for chemokine scavenging by ACKR4.