Atypical Chemokine Receptor Research Articles

Decoy Receptors Regulation by Resveratrol in Lipopolysaccharide-Activated Microglia.

Resveratrol is a polyphenol that acts as antioxidants do, protecting the body against diseases, such as diabetes, cancer, heart disease, and neurodegenerative disorders, such as Alzheimer's (AD) and Parkinson's diseases (PD). In the present study, we report that the treatment of activated microglia with resveratrol after prolonged exposure to lipopolysaccharide is not only able to modulate pro-inflammatory responses, but it also up-regulates the expression of decoy receptors, IL-1R2 and ACKR2 (atypical chemokine receptors), also known as negative regulatory receptors, which are able to reduce the functional responses promoting the resolution of inflammation. This result might constitute a hitherto unknown anti-inflammatory mechanism exerted by resveratrol on activated microglia.

CXCR7 as a novel therapeutic target for advanced prostate cancer.

Chemokines and their cognate receptors comprise an intricate signaling network that becomes high-jacked by cancer cells for uncontrollable tumor growth and dissemination. ACKR3 (Atypical Chemokine Receptor 3), traditionally called CXCR7, is up-regulated in many cancers, including advanced prostate cancer, and represents promising targets for therapeutic intervention. Unlike typical G protein-coupled receptors such as CXCR4, CXCR7, once bound by its cognate ligand CXCL12, initiates the recruitment of β-arrestin instead of G proteins, and results in rapid internalization and degradation of CXCL12, functioning as a scavenger receptor. However, recent evidence suggests that CXCR7 may be more than a scavenger or auxiliary receptor of CXCR4 and that it may play essential roles in regulating cancer progression, some of which are independent of CXCR4 and its ligands, such as CXCL12. Constitutively active CXCR7 binds to β-arrestin. This protein complex internalizes to form a scaffold for assembling and activating various cytoplasmic kinases necessary for cell survival and tumor growth. Here we review and discuss the up-to-date knowledge on CXCR7 regulation and function and how this new understanding guides the development of CXCR7 inhibitors, focusing on prostate cancer.

Chemokine Receptors—Structure-Based Virtual Screening Assisted by Machine Learning

Chemokines modulate the immune response by regulating the migration of immune cells. They are also known to participate in such processes as cell-cell adhesion, allograft rejection, and angiogenesis. Chemokines interact with two different subfamilies of G protein-coupled receptors: conventional chemokine receptors and atypical chemokine receptors. Here, we focused on the former one which has been linked to many inflammatory diseases, including: multiple sclerosis, asthma, nephritis, and rheumatoid arthritis. Available crystal and cryo-EM structures and homology models of six chemokine receptors (CCR1 to CCR6) were described and tested in terms of their usefulness in structure-based drug design. As a result of structure-based virtual screening for CCR2 and CCR3, several new active compounds were proposed. Known inhibitors of CCR1 to CCR6, acquired from ChEMBL, were used as training sets for two machine learning algorithms in ligand-based drug design. Performance of LightGBM was compared with a sequential Keras/TensorFlow model of neural network for these diverse datasets. A combination of structure-based virtual screening with machine learning allowed to propose several active ligands for CCR2 and CCR3 with two distinct compounds predicted as CCR3 actives by all three tested methods: Glide, Keras/TensorFlow NN, and LightGBM. In addition, the performance of these three methods in the prediction of the CCR2/CCR3 receptor subtype selectivity was assessed.

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.

ACKR4a induces autophagy to block NF-κB signaling and apoptosis to facilitate Vibrio harveyi infection

Autophagy and apoptosis are two recognized mechanisms of resistance to bacterial invasion. However, bacteria have likewise evolved the ability to evade immunity. In this study, we identify ACKR4a, a member of an atypical chemokine receptor family, as a suppressor of the NF-κB pathway, which cooperates with Beclin-1 to induce autophagy to inhibit NF-κB signaling and block apoptosis, facilitating Vibrio harveyi infection. Mechanistically, V. harveyi-induced Ap-1 activates ACKR4a transcription and expression. ACKR4a forms a complex with Beclin-1 and MyD88, respectively, inducing autophagy and transporting MyD88 into the lysosome for degradation to suppress inflammatory cytokine production. Meanwhile, ACKR4a-induced autophagy blocks apoptosis by inhibiting caspase8. This study proves for the first time that V. harveyi uses both autophagy and apoptosis to evade innate immunity, suggesting that V. harveyi has evolved the ability to against fish immunity.

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.

Abstract C035: ACKR1 expression in stromal cells regulates immune infiltration in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC), a highly aggressive and metastatic subtype of breast cancer, lacks expression of the estrogen and progesterone receptors and amplification of HER2 and comprises 15-20% of all breast cancer. Incidence and mortality of TNBC are higher in Black Americans than in other racial/ethnic groups. Conversely, Black patients are underrepresented in clinical breast cancer drug trials, limiting our understanding of differential treatment responses. Leukocyte infiltration is a key modulator of TNBC outcome, and Black patients show immunological differences that may contribute to differential outcomes in TNBC. About 60-80% of Black Americans are homozygous for a polymorphism in the Atypical Chemokine Receptor 1 (ACKR1) promoter region that causes loss of ACKR1 expression on red blood cells, also known as the “Duffy-null” serotype. ACKR1 is expressed on tumor cells, red blood cells, and endothelial cells, and can act as a chemokine sink or chemokine concentrator, depending on the context. Whole-tumor RNA analysis shows that overall loss of ACKR1 expression in TNBC tumors correlates strongly with decreased survival, but it is unclear which ACKR1-expressing cell type(s) contribute to this effect. Under inflammatory conditions, ACKR1 expression in endothelial cells localizes chemotactic cytokines at the endothelial junction, and loss of endothelial ACKR1 blocks the ability of leukocytes to extravasate through the endothelial barrier into surrounding tissues. We hypothesized that loss of endothelial/stromal ACKR1 reduces immune infiltration and activation in TNBC and racial/ethnic differences in ACKR1 expression may contribute to health disparities. To determine the role of ACKR1 in tumor immune infiltration, we chose a syngeneic immunocompetent mouse model to incorporate species-matched stromal-tumor interactions. We orthotopically inoculated E0771.LMB cells, a C57BL6/J syngeneic TNBC cell line, into ACKR1null and ACKR1het female mice. Tumor growth was measured triweekly, and tumor mass was measured at the 28-day endpoint. To measure immune infiltration, we fluorescently stained representative tumor cryosections with CD45 to detect all classes of infiltrating hematopoietic cells involved in immune response. There was no significant difference in tumor volume and mass among the ACKR1null and ACKR1het controls. Tumors implanted in ACKR1het controls showed broadly infiltrated leukocytes clusters, while tumors in ACKR1nullhosts showed sparse and infrequent leukocyte infiltration. Conclusion: Stromal ACKR1 does not have a strong effect on primary tumor growth. Loss of stromal ACKR1 reduces infiltration of leukocytes into the tumor microenvironment even when ACKR1 is not manipulated in the tumor cells, suggesting that ACKR1 in endothelial or other stromal cells regulates immune response in TNBC. Impact: Understanding the immunogenic functions of ACKR1 allelic variation will elucidate mechanisms of racially distinct therapeutic response in TNBC, enhance precision oncology, and promote cancer health equity. Citation Format: Chinwe Obianuju Ewenighi-Amankwah, Tanner Roach, Joseph Dufraine, Naiche Adler, Jan Kitajewski. ACKR1 expression in stromal cells regulates immune infiltration in triple negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C035.

Relationship between astrocyte damage and different levels of cerebrospinal fluid markers and prognosis in patients with subarachnoid hemorrhage.

The aim of the study was to explore the relationship between astrocyte damage and different levels of cerebrospinal fluid markers and prognosis in patients with subarachnoid hemorrhage (SAH). A total of 168 SAH patients diagnosed and treated in the emergency department of our hospital during the period October 2019 to February 2022 were randomly selected as the study subjects. The severity of these patients' condition was evaluated by Hunt-Hess grading and these subjects were graded as the low-level group (78 patients) and high-level group (90 patients) according to the evaluation results. The Extended Disability Status Scale (EDSS) score was employed to evaluate the astrocyte damage. The content of atypical chemokine receptor 3 (ACKR3), Connexin 43 (Cx43), oxygenated hemoglobin (HbO 2 ), and endothelin (ET) in cerebrospinal fluid was measured. The relationship between the content of ACKR3, Cx43, HbO 2 , and ET in cerebrospinal fluid with EDSS score was analyzed through Pearson correlation analysis. Multivariate logistic regression analysis was adopted to analyze the risk factors. ACKR3 was mainly expressed in the cytoplasm of cerebrospinal fluid monocytes, and Cx43 was mainly expressed in the cell membrane and cytoplasm. Patients in the high-level group had markedly higher expression rates of ACKR3 and Cx43 positive cells in cerebrospinal fluid than those in the low-level group ( p < 0.05). Patients in the high-level group had higher content of HbO 2 and ET in cerebrospinal fluid and EDSS score than patients in the low-level group ( p < 0.05). The content of ACKR3, Cx43, HbO 2 , and ET in cerebrospinal fluid of SAH patients was positively correlated with EDSS scores ( p < 0.05). Systolic blood pressure, Hunt-Hess grade, rebleeding, emotional control, EDSS score, ACKR3, Cx43 positive cell rate, and HbO 2 and ET expression levels were independent risk factors for the prognosis of SAH patients ( p < 0.05). Astrocyte damage in SAH patients was positively correlated with the content of ACKR3, Cx43, HbO 2 , and ET in cerebrospinal fluid. These indicators increased significantly with the increasing severity of the disease, and had certain value in reflecting the patient's condition. Astrocyte damage combined with cerebrospinal fluid markers had potential value in evaluating the severity and prognosis of patients.

Abstract C050: The role of PD-L1, ACKR/DARC, Livin and Annexin A5 in malignant tumours of the bone, prostate, breast, eye/orbit, nasopharynx, and metastatic carcinomas to the brain, neck and lymph node among West Africans in Zaria, Nigeria

Abstract Cancer ranks as a leading cause of death and an important barrier to increasing life expectancy worldwide. Globally, there are estimated 19.3 million new cancer cases and about 10 million cancer mortality in 2020. Should current cancer trends continue, Africa’s cancer burden is projected to reach an alarming 1.4 million new cases and 1 million deaths by 2030. Thus, racial/ethnic disparities in cancer morbidity and mortality continue to widen globally but molecular biology and genomic studies rarely interrogate cancer in diverse disadvantaged populations. Effective control of tumour growth in cancer microenvironment is facilitated by cellular interaction between innate and adaptive immune cells. The programmed death ligand-1 (PD-L1), an immune checkpoint expressed on tumour cells, facilitates the escape of immunosurveillance in cancer by interacting with programmed death-1 (PD-1) to initiate apoptosis of the immune cells. The interaction between these immune cells may be mediated by atypical chemokine receptor 1 (ACKR1)/Duffy antigen receptor for chemokines (DARC) towards activation of tumour-specific immune response by chemoattraction of leukocytes to the inflammatory sites. Livin/BIRC7 protein overexpression is a key component of evasion of apoptosis in cancer cells. Interestingly, Annexin A5, an important member of Annexin family of proteins with high affinity to phosphatidylserine, has been reported to serve as an important component of apoptosis by stimulating immunogenicity of tumor cells. In this pilot study, we report using immunohistochemistry, RNA-seq and whole exome sequencing the expression pattern and functional significance of PD-L1, ACKR/DARC, Livin/BIRC7 and Annexin A5 in malignant tumours of the bone, prostate, breast, eye/orbit, nasopharynx, and metastatic carcinomas to the brain, neck and lymph node among West Africans in Zaria, Nigeria. In osteosarcoma and osteogenic sarcoma of the bone, we observed negative expression of ACKR/DARC and PD-L1, and overexpression of Livin and Annexin A5 proteins. However, a mucoepidermoid carcinoma of the index finger shows strong expression of ACKR/DARC, PD-L1 and Annexin A5 with poor expression of Livin protein. The results also show negative expression of ACKR/DARC, PD-L1 and Livin proteins from retinoblastoma, nasopharyngeal carcinoma, metastatic adenosquamous carcinoma to the parietal lobe of the brain, Metastatic squamous neck cancer, metastatic carcinoma of the lymph nodes and invasive breast carcinoma no special type (NST) grade 3. Prostate adenocarcinomas with Gleason grade 9 show negative expression of ACKR/DARC and PD-L1 proteins. All the tumours studied are strongly positive for Annexin A5 expression. The diverse expression pattern of ACKR/DARC, PD-L1, Livin and Annexin A5 in malignant tumours of the bone, retina, nasopharynx, breast, prostate and metastatic carcinomas to the brain, neck and lymph may be of significant importance in tumour behaviour and prognosis. Citation Format: Faruk Mohammed, Melissa B. Davis, Clayton C. Yates, Halimatu Sadiya Musa. The role of PD-L1, ACKR/DARC, Livin and Annexin A5 in malignant tumours of the bone, prostate, breast, eye/orbit, nasopharynx, and metastatic carcinomas to the brain, neck and lymph node among West Africans in Zaria, Nigeria [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C050.

CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

Background: Lung cancer is a malignant tumor with metastatic potential. Chemokine ligand 14 (CXCL14) has been reported to be associated with different cancer cell migration and invasion. However, few studies have explored the function of CXCL14 and its specific receptor in lung cancer metastasis. This study aims to determine the mechanism of CXCL14-promoted cancer metastasis. Methods: The expression of CXCL14, atypical chemokine receptor 2 (ACKR2), and epithelial mesenchymal transition (EMT) markers was evaluated by the public database of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), Western blot, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and immunofluorescence (IF). Migration and wound healing assays were used to observe the motility of cancer cells. A luciferase reporter assay was performed to analyze transcription factor activity. The metastasis of lung cancer cells was evaluated in an orthotopic model. Results: We have presented that overexpression of CXCL14 and ACKR2 was observed in lung cancer datasets, human lung tumor sections, and lung cancer cells. Furthermore, the migration of CXCL14-promoted lung cancer cells was determined in vitro and in vivo. In particular, ACKR2 knockdown abolished CXCL14-induced cancer cell motility. Additionally, ACKR2 was involved in CXCL14-triggered phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src signaling pathway and subsequently upregulated nuclear factor κB (NF-κB) transcription activity leading to EMT and migration of lung cancer cells. These results indicated that the CXCL14/ACKR2 axis played an important role in lung cancer metastasis. Conclusion: This study is the first to reveal the function of CXCL14 in promoting EMT and metastasis in lung cancer. As a specific receptor for CXCL14 in lung cancer, ACKR2 mediates CXCL14-induced signaling that leads to cell motility. Our findings can be used as a prognostic biomarker of lung cancer metastasis.

Evaluation of Atypical Chemokine Receptor Expression in T Cell Subsets.

Chemokines are molecules that pertain to a family of small cytokines and can generate cell chemotaxis through the interaction with their receptors. Chemokines can trigger signaling via conventional G-protein-coupled receptors or through atypical chemokine receptors. Currently, four atypical chemokine receptors have been are described (ACKR1, ACKR2, ACKR3 and ACKR4). ACKRs are expressed in various cells and tissues, including T lymphocytes. These receptors' main function is related to the internalization and degradation of chemokines, as well as to the inflammation control. However, the expression of these receptors in human T lymphocytes is unclear in the literature. The objective of this study was to evaluate the expression of ACKRs in different subpopulations of T lymphocytes. For this, peripheral blood from healthy donors was used to analyze the expression of ACKR2, ACKR3 and ACKR4 by immunophenotyping CD4, CD8 T lymphocytes and, in their subsets, naive, transition and memory. Results obtained in this study demonstrated that ACKR2, ACKR3 and ACKR4 receptors were expressed by T lymphocytes subsets in different proportions. These receptors are highly expressed in the cytoplasmic milieu of all subsets of T lymphocytes, therefore suggesting that their expression in plasma membrane is regulated after transcription, and it must be dependent on a stimulus, which was not identified in our study. Thus, regarding ACKRs function as scavenger receptors, at least for the ACKR3, this function does not impair the chemotaxis exert for their ligand compared to the typical counterpart receptor.

CCRL2 affects the sensitivity of myelodysplastic syndrome and secondary acute myeloid leukemia cells to azacitidine.

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

Single-cell transcriptomic analysis reveals differential cell subpopulations and distinct phenotype transition in normal and dissected ascending aorta

BackgroundAcute thoracic aortic dissection (ATAD) is a fatal condition characterized by tear of intima, formation of false lumen and rupture of aorta. However, the subpopulations of normal and dissected aorta remain less studied.MethodsSingle-cell RNA sequencing was performed including 5 patients with ATAD and 4 healthy controls. Immunohistochemistry and immunofluorescence were used to verify the findings.ResultsWe got 8 cell types from human ascending aorta and identified 50 subpopulations including vascular smooth muscle cells (VSMCs), endothelial cells, fibroblasts, neutrophils, monocytes and macrophages. Six transmembrane epithelial antigen of prostate 4 metalloreductase (STEAP4) was identified as a new marker of synthetic VSMCs. CytoTRACE identified subpopulations with higher differentiation potential in specified cell types including synthetic VSMCs, enolase 1+ fibroblasts and myeloid-derived neutrophils. Synthetic VSMCs-derived C-X-C motif chemokine ligand 12 (CXCL12) might interact with neutrophils and fibroblasts via C-X-C motif chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), respectively, which might recruit neutrophils and induce transdifferentitation of fibroblasts into synthetic VSMCs.ConclusionWe characterized signatures of different cell types in normal and dissected human ascending aorta and identified a new marker for isolation of synthetic VSMCs. Moreover, we proposed a potential mechanism that synthetic VSMCs might interact with neutrophils and fibroblasts via CXCL12-CXCR4/ACKR3 axis whereby deteriorating the progression of ATAD, which might provide new insights to better understand the development and progression of ATAD.

Phosphoproteomic mapping of CCR5 and ACKR2 signaling properties.

ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3min) and long (30min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.

Atypical CXCL12 signaling enhances neutrophil migration by modulating nuclear deformability.

To reach inflamed tissues from the circulation, neutrophils must overcome physical constraints imposed by the tissue architecture, such as the endothelial barrier or the three-dimensional (3D) interstitial space. In these microenvironments, neutrophils are forced to migrate through spaces smaller than their own diameter. One of the main challenges for cell passage through narrow gaps is the deformation of the nucleus, the largest and stiffest organelle in cells. Here, we showed that chemokines, the extracellular signals that guide cell migration in vivo, modulated nuclear plasticity to support neutrophil migration in restricted microenvironments. Exploiting microfabricated devices, we found that the CXC chemokine CXCL12 enhanced the nuclear pliability of mouse bone marrow-derived neutrophils to sustain their migration in 3D landscapes. This previously uncharacterized function of CXCL12 was mediated by the atypical chemokine receptor ACKR3 (also known as CXCR7), required protein kinase A (PKA) activity, and induced chromatin compaction, which resulted in enhanced cell migration in 3D. Thus, we propose that chemical cues regulate the nuclear plasticity of migrating leukocytes to optimize their motility in restricted microenvironments.

Incidence of Typical Neutrophil Count with Fy(a-b-) Status Among Hematology Referrals for Neutropenia at an Urban Safety-Net Hospital

Typical neutrophil count with Fy(a-b-) status, known variously as Duffy null phenotype or Benign Ethnic Neutropenia, causes asymptomatic neutropenia without clinical sequelae.[i][ii] Fy(a-b-) phenotype is correlated to homozygosity of a single nucleotide polymorphism in the promoter region of atypical chemokine receptor 1 (ACKR1; formerly known as DARC), a decoy chemokine receptor, and may protect against malaria infection.[iii][iv] This genotype is enriched in malaria-endemic regions.[v] 25% to 50% of people of African ancestry in the United States are thought to have Fy(a-b-) status and are often erroneously identified as having pathologically low neutrophil counts. We performed a retrospective chart review of new neutropenia referrals to Hematology Clinic at Boston Medical Center (BMC) to evaluate diagnostic patterns for typical neutrophil count with Fy(a-b-) status. Overall, 103 new referrals for evaluation of neutropenia were made to BMC Hematology Clinic from 1/2020 to 2/2022, of which 78 were included for further analysis. Demographic and laboratory data are summarized in the accompanying Table. Typical neutrophil count with Fy(a-b-) status was identified as the primary etiology for neutropenia in 64.1% (50 of 78 referrals), of which 41 (82%) identified as African American or Black or were born in an African or Caribbean country. Of patients suspected of having Duffy null phenotype, 66% (33 of 50 patients) underwent confirmatory blood bank testing, and 97% (32 of 33) of these patients were found to have Fy(a-b-) status. Average cost of laboratory visit was calculated for each new referral using the Outpatient Price Transparency Tool from BMC Billing Department, which is based on chargemaster prices for the hospital. Patients with typical neutrophil count with Fy(a-b-) status on average had lower, but not negligible, cost of workup ($363.82 vs $689.46; p<.005 by unpaired student's T test assuming unequal variance) and were statistically less likely to have a follow up appointment (p=0.039 by unpaired student's T test assuming unequal variance) compared to those without. Though this phenotype reflects normal variation of white cell count in non-White populations, it is a "...label that illustrates how common phenotypes seen in non-White individuals continue to be considered nonstandard in the medical community." Workup of referrals that have typical neutrophil count with Fy(a-b-) status is not currently recommended, unless they have a change in their baseline white cell count or new, atypical features concerning for a secondary etiology.[vi] In our study, asymptomatic referrals were the majority and represent an unnecessary cost burden on an overtaxed healthcare system. Fy(a,b) antigen testing provides a low-cost alternative to hematology referral for confirmation of Fy(a-b-) status for patients or providers who require reassurance. Expanded use of serological Fy(a,b) antigen testing by primary care providers for patients with ancestry from malaria-endemic regions and chronic, asymptomatic neutropenia could reduce cost of care and referrals to Hematology clinic. [i] Ortiz MV, Meier ER, Hsieh MM. Identification and Clinical Characterization of Children With Benign Ethnic Neutropenia. J Pediatr Hematol Oncol. 2016 Apr;38(3):e140-3. doi: 10.1097/MPH.0000000000000528. PMID: 26925714; PMCID: PMC5102334. [ii] Merz LE, Achebe M. When non-Whiteness becomes a condition. Blood. 2021 Jan 7;137(1):13-15. doi: 10.1182/blood.2020008600. PMID: 33181819. [iii] Reich D et al. Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene. PLoS Genet. 2009 Jan;5(1):e1000360. doi: 10.1371/journal.pgen.1000360. Epub 2009 Jan 30. PMID: 19180233; PMCID: PMC2628742. [iv] Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med. 1976 Aug 5;295(6):302-4. doi: 10.1056/NEJM197608052950602. PMID: 778616. [v] Haddy TB, Rana SR, Castro O. Benign ethnic neutropenia: what is a normal absolute neutrophil count? J Lab Clin Med. 1999 Jan;133(1):15-22. doi: 10.1053/lc.1999.v133.a94931. PMID: 10385477. [vi] Gibson C, Berliner N. How we evaluate and treat neutropenia in adults. Blood (2014) 124 (8): 1251-1258. https://doi.org/10.1182/blood-2014-02-482612 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Atypical Chemokine Receptor 4 Is Expressed in Kidney Glomeruli and Mitigates the Severity of Experimental Glomerulonephritis

Atypical Chemokine Receptor 4 Is Expressed in Kidney Glomeruli and Mitigates the Severity of Experimental Glomerulonephritis

Histone deacetylase 2 regulates STAT1-dependent upregulation of atypical chemokine receptor 3 to induce M2 macrophage migration and immune escape in hepatocellular carcinoma

Atypical chemokine receptor 3 (ACKR3) has been linked to the tumor microenvironment. This work investigates the effects of ACKR3 and its regulatory molecules on the chemotactic migration of tumor-associated macrophages (TAMs) in hepatocellular carcinoma (HCC). RT-qPCR and western blot assays identified elevated ACKR3 and HDAC2 levels in HCC tissues and cells. Knockdown or overexpression of ACKR3 was induced in HCC cells through vectors of lentivirus plasmids, and then the conditioned medium of the HCC cells was collected to stimulate TAMs. The stimulated TAMs were co-cultured with CD3+ T cells. ACKR3 knockdown in HCC cells inhibited migration of TAMs and promoted M1 polarization, which restored the activity of T cells. Histone deacetylase 2 (HDAC2) recruited signal transducer and activator of transcription 1 (STAT1) to the ACKR3 promoter to activate ACKR3 transcription. HDAC2 silencing suppressed nuclear translocation of STAT1 and decreased ACKR3 expression. HDAC2 knockdown in HCC cells similarly suppressed TAM migration, promoted M1 polarization, and restored T cell function, but these changes were inversed by ACKR3 upregulation. HDAC2 or ACKR3 silencing weakened tumor growth and immune escape in mice. In conclusion, this study demonstrates that HDAC2 upregulates ACKR3 via STAT1 to induce migration of M2 macrophages and immune escape in HCC.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a β-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 μM) and C11 (EC50 = 11.4 μM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 μM) and 27 (LN6023, EC50 = 3.5 μM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Structural snapshot of a β-arrestin-biased receptor

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to β-arrestins (βarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor.