Novel neutrophil biology insights underlying atypical chemokine receptor-1/Duffy antigen receptor of chemokines-associated neutropenia.

PURPOSE OF REVIEW: Atypical chemokine receptor-1 (ACKR1)/Duffy antigen receptor of chemokines (DARC)-associated neutropenia (ADAN; OMIM 611862), previously named benign ethnic neutropenia, and present in two-thirds of individuals identifying as Black in the USA, is associated with mild to moderate decreases in peripheral neutrophil counts that nevertheless do not lead to increased infections. Consequently, recent initiatives have sought to establish normal neutrophil count reference ranges for ADAN, considering it a normal variant rather than a clinical disorder requiring medical intervention. RECENT FINDINGS: A limited number of studies elucidating the mechanism of neutropenia in ADAN has suggested that neutrophils may redistribute from peripheral blood to the tissues including the spleen: this might explain why ADAN is not associated with increased risks of infection since the total number of neutrophils in the body remains normal. In this review, we critically examine the research underlying the molecular basis of ADAN. SUMMARY: Insights into the biology of neutrophils and their trafficking may inform the clinical interpretation of neutropenia in ADAN. The bulk of research suggests that ADAN does not lead to a diminished host defense as do other forms of neutropenia. However, ADAN may lead to increased proinflammatory signaling, with possible implications for senescence of the immune system and predisposition to autoimmunity and cancer.

A subset of macrophages and monocytes in the mouse bone marrow express atypical chemokine receptor 1.

Duffy antigen receptor for chemokines (DARC)/CD234, also known as atypical chemokine receptor 1 (ACKR1), is a seven-transmembrane domain protein expressed on erythrocytes, vascular endothelium, and a subset of epithelial cells (Peiper et al., 1995). Previously, we reported that ACKR1 was expressed in bone marrow macrophages. ACKR1 interacts with CD82 on long-term repopulating hematopoietic stem cells (LT-HSCs) to maintain the dormancy of LT-HSCs during homeostasis (Hur et al., 2016). We also demonstrated that ACKR1 interacts with CD82 in HSCs from human umbilical cord blood (hUCB). These findings demonstrated that CD82 is a functional surface marker of LT-HSCs and this molecule maintains LT-HSC quiescence by interactions with ACKR1-expressing macrophages in mice and humans.

Prognostic significance of BIRC7/Livin, Bcl-2, p53, Annexin V, PD-L1, DARC, MSH2 and PMS2 in colorectal cancer treated with FOLFOX chemotherapy with or without aspirin.

Evasion of apoptosis is associated with treatment resistance and metastasis in colorectal cancer (CRC). Various cellular processes are associated with evasion of apoptosis. These include overexpression of pro-apoptotic proteins (including p53 and PD-L1), anti-apoptotic proteins (BIRC7/Livin and Bcl-2), chemokine receptors (including DARC), and dysregulation of DNA mismatch repair proteins (including MSH2 and PMS2). The aim of this study was to determine the effect of folinic acid, 5-FU and oxaliplatin (FOLFOX) as a single agent and aspirin plus FOLFOX in various combinations on the aforementioned proteins in human CRC, SW480 cell line and rat models of N-Methyl-N-Nitrosourea (NMU)-induced CRC. In addition, effects of the NMU-induced CRC and chemotherapeutic regimens on haematological and biochemical parameters in the rat models were studied. Immunohistochemistry, immunofluorescence and immunoblot techniques were used to study the expression pattern of the related proteins in the human CRC cells pre- and post-treatment. Double contrast barium enema, post-mortem examination and histological analyses were used to confirm tumour growth and the effect of the treatment in vivo in rat models. Notably, we found in human mucinous CRC, a significant increase in expression of the BIRC7/Livin post-FOLFOX treatment compared with pre-treatment (p = 0.0001). This increase provides new insights into the prognostic role of BIRC7/Livin in evasion of apoptosis and facilitation of treatment resistance, local recurrence and metastasis particularly among mucinous CRCs post-FOLFOX chemotherapy. These poor prognostic features in the CRC may be further compounded by the significant suppression of DARC, PD-L1, PMS2 and overexpression of MSH2 and anti-apoptotic Bcl-2 and p53 proteins observed in our study (p < 0.05). Importantly, we found a significant reduction in expression of BIRC7/Livin and reactivation of DARC and PD-L1 with a surge in Annexin V expression in rat models of CRC cells post-treatment with a sequential dose of aspirin plus FOLFOX compared with other treatments in vivo (p <0.05). The mechanistic rational of these effects underscores the importance of expanded concept of possible aspirin combination therapy with FOLFOX sequentially in future CRC management. Validation of our findings through randomized clinical trials of aspirin plus FOLFOX sequentially in patients with CRC is therefore warranted.

Identification of ACKR1 variants associated with altered Duffy phenotype expression in blood donors from southern Brazil.

The atypical chemokine receptor 1 gene (ACKR1) is responsible for the clinically significant Duffy blood group. The main antigens of this system, Fya and Fyb, can be related to a null or weak expression of the DARC protein. In the present work, we aimed to identify ACKR1 gene variants in blood donors from southern Brazil based on discrepancies between their serological and molecular typing results. Then, we analyzed the association of these variants with the expression of the Duffy phenotype. The Fy antigen types were determined via hemagglutination and real-time PCR (c.125 G > A, c.265C > T and c.-67T > C SNPs) tests in a sample composed of 382 regular repetitive voluntary blood donors to the Blood Bank of Hospital de Clínicas de Porto Alegre. An inconclusive correlation between phenotype-genotype analyses was found in 11 (2.88 %) donors, and the entire ACKR1 gene was sequenced in these samples. Our investigation found 11 genetic variants, four of which (c.-541C > T, c.21 + 150C > T, c.22-58A > G, and c.298 G > A SNPs) seem to have putative functional effects on the structure and expression of DARC undertaken for in silico analysis (SIFT, PolyPhen-2 and RegulomeDB). Molecular events can result in apparent discrepancies between red cell genotypes and phenotypes. Our findings provided insight into the molecular background of FY antigens to improve technical approaches for red cell genotyping.

Co-expression network analysis identified atypical chemokine receptor 1 (ACKR1) association with lymph node metastasis and prognosis in cervical cancer.

Cervical cancer (CC) is one kind of female cancer. With the development of bioinformatics, targeted specific biomarkers therapy has become much more valuable. GSE26511 was obtained from gene expression omnibus (GEO). We utilized a package called "WGCNA" to build co-expression network and choose the hub module. Search Tool for the Retrieval of Interacting Genes Database (STRING) was used to analyze protein-protein interaction (PPI) information of those genes in the hub module. A Plug-in called MCODE was utilized to choose hub clusters of PPI network, which was visualized in Cytoscape. Clusterprofiler was used to do functional analysis. Univariate and multivariate cox proportional hazards regression analysis were both conducted to predict the risk score of CC patients. Kaplan-Meier curve analysis was done to show the overall survival. Receiver operating characteristic (ROC) curve analysis was utilized to evaluate the predictive value of the patient outcome. Validation of the hub gene in databases, Gene set enrichment analysis (GSEA) and GEPIA were completed. We built co-expression network based on GSE26511 and one CC-related module was identified. Functional analysis of this module showed that extracellular space and Signaling pathways regulating pluripotency of stem cells were most related pathways. PPI network screened GNG11 as the most valuable protein. Cox analysis showed that ACKR1 was negatively correlated with CC progression, which was validated in Gene Expression Profiling Interactive Analysis (GEPIA) and datasets. Survival analysis was performed and showed the consistent result. GSEA set enrichment analysis was also completed. This study showed hub functional terms and gene participated in CC and then speculated that ACKR1 might be tumor suppressor for CC.

Interleukin-22 Induces the Infiltration of Visceral Fat Tissue by a Discrete Subset of Duffy Antigen Receptor for Chemokine-Positive M2-Like Macrophages in Response to a High Fat Diet.

Interleukin-22 (IL-22) is a cytokine with important functions in host defense and inflammatory responses and has recently been suggested to play a role in immune-inflammatory system in the context of obesity and its metabolic consequences. The specific cellular targets and mechanisms of IL-22-mediated obesity are largely unknown however. We here identified a previously unknown subset of monocyte-derived Duffy antigen receptors for chemokines (DARC)+ macrophages in epididymal fat adipose tissue and found that they are preferentially recruited into the crown-like structures of adipose tissue in the mouse upon high fat diet-induced obesity. Importantly, DARC+ macrophages highly express the IL-22 receptor (IL-22Ra1). Exposure to recombinant IL-22 shifts macrophages to an alternative M2 polarization pathway and augments DARC expression via a STAT5b signaling axis. STAT5b directly binds to the DARC promoter and a STAT5 inhibitor abrogates the IL-22-mediated induction of DARC. These M2-like DARC+ subpopulations of monocytes/macrophages were elevated in obese db/db mice compared to WT lean mice. Furthermore, subsets of CD14+ and/or CD16+ monocytes/macrophages within human peripheral blood mononuclear cell populations express DARC and the prevalence of these subsets is enhanced by IL-22 stimuli. This suggested that IL-22 is a critical cytokine that promotes the infiltration of adipose tissue macrophages, that regulate inflammatory processes. Taken together, our present findings provide important insights into the molecular mechanism by which IL-22 signal modulates DARC expression in M2-like macrophages.

Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

Human inborn errors of immunity to infection affecting cells other than leukocytes: from the immune system to the whole organism.

Studies of vertebrate immunity have traditionally focused on professional cells, including circulating and tissue-resident leukocytes. Evidence that non-professional cells are also intrinsically essential (i.e. not via their effect on leukocytes) for protective immunity in natural conditions of infection has emerged from three lines of research in human genetics. First, studies of Mendelian resistance to infection have revealed an essential role of DARC-expressing erythrocytes in protection against Plasmodium vivax infection, and an essential role of FUT2-expressing intestinal epithelial cells for protection against norovirus and rotavirus infections. Second, studies of inborn errors of non-hematopoietic cell-extrinsic immunity have shown that APOL1 and complement cascade components secreted by hepatocytes are essential for protective immunity to trypanosome and pyogenic bacteria, respectively. Third, studies of inborn errors of non-hematopoietic cell-intrinsic immunity have suggested that keratinocytes, pulmonary epithelial cells, and cortical neurons are essential for tissue-specific protective immunity to human papillomaviruses, influenza virus, and herpes simplex virus, respectively. Various other types of genetic resistance or predisposition to infection in human populations are not readily explained by inborn variants of genes operating in leukocytes and may, therefore, involve defects in other cells. The probing of this unchartered territory by human genetics is reshaping immunology, by scaling immunity to infection up from the immune system to the whole organism.

Atypical Chemokine Receptor 1 (DARC/ACKR1) in Breast Tumors Is Associated with Survival, Circulating Chemokines, Tumor-Infiltrating Immune Cells, and African Ancestry.

BACKGROUND: Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry. METHODS: Using computational The Cancer Genome Atlas (TCGA) analysis, case-control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile-associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors. RESULTS: In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P <0.0001) and anticorrelated with CXCL8/IL8 (P <0.0001). Sub-Saharan African-specific DARC/ACKR1 alleles likely drive these correlations. Relapse-free survival (RFS) and overall survival (OS) were significantly longer in individuals with DARC/ACKR1-high tumors (P <1.0 × 10-16 and P <2.2 × 10-6, respectively) across all molecular tumor subtypes. CONCLUSIONS: DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors. IMPACT: This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Traditional Chinese Medicine Extract from Huaier Increases the Expression of Duffy Antigen Receptor for Chemokines and Reduces the Expression of Its Ligands.

AIMS: The aim of the present study is to investigate whether the aqueous extract from Huaier, a traditional Chinese medicine (TCM), can affect the expression of Duffy antigen receptor for chemokines (DARC) and its ligands. Moreover, we compare the status of DARC in primary and metastatic breast cancer tissues from the same patient. METHODS: Immunohistochemistry was used to detect the expression of DARC in primary and metastatic focuses in 30 patients with breast cancer. The effect of Huaier aqueous extract on the expression of DARC and its ligands was investigated by quantitative real-time polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. RESULTS: The expression score of DARC in primary focuses was significantly higher than that in metastatic focuses, while changes of ER, PR, and HER2 receptors were not significantly different between primary and metastatic focuses. Huaier aqueous extract promoted the expression of DARC and reduced the secretion of CC chemokine ligand 2 (CCL-2), CXC chemokine ligand 8 (CXCL-8, IL-8), matrix metalloproteinase 2 (MMP-2), and CXC chemokine ligand 1 (CXCL-1). CONCLUSION: The present study demonstrates that difference in expression level of DARC between primary and metastatic focuses of breast cancer was significant, while differences in expression of ER, PR, and HER2 between primary and metastatic focuses were not significant. DARC may play a negative role in the metastasis of breast cancer. Traditional Chinese medicine extract from Huaier can increase DARC expression and reduce the expression of its ligands such as CCL-2, IL-8, MMP-2, and CXCL-1.

Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues.

BACKGROUND: Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice. METHODS: We generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. RESULTS: DARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues. CONCLUSIONS: Immunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.

Atypical chemokine receptor 1 on nucleated erythroid cells regulates hematopoiesis.

Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.

Duffy antigen receptor for chemokines (DARC) expressing in cancer cells inhibits tumor progression by suppressing CXCR2 signaling in human pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Chemokines play important roles in the progression of many malignancies; however, the role of chemokine receptor expression in clinical cases of PDAC is unclear. Moreover, little is known about DARC, a decoy receptor of CXC chemokines, in the regulation of tumor progression. METHODS: Functions of chemokine receptors were evaluated using surgical specimens collected from PDAC patients, and PDAC cell lines. RESULTS: CXCR2 expression had no impacts on predicting prognosis, but low DARC expression in cancer cells was an independent risk factor for poor prognosis. In PDAC with low DARC expression, tumor sizes were larger and vascular invasion was increased. High CXCR2 expression was a significant predictor for poor prognosis, only in PDAC patients with low DARC expression. CXCR2 signaling induced STAT3 activation in PDAC, resulting in promoting cell cycle progression, inhibiting apoptosis, inducing angiogenesis, and enhancing invasiveness. DARC inhibited STAT3 activation by down-regulating CXCR2 signaling. These effects were confirmed by EMSA in vitro. DARC knockdown significantly increased cell proliferation in CFPAC-1 cells with high DARC expression, by activating STAT3. In contrast, CXCR2 knockdown inhibited the proliferative effects of IL-8 in MIA PaCa-2 cells with low DARC expression. Moreover, the inhibitory effect of CXCR2 antagonist on PDAC cell proliferation was more powerful in MIA PaCa-2 cells than CFPAC-1 cells. CONCLUSIONS: DARC expressing in cancer cells inhibits PDAC progression by suppressing STAT3 activation through the inhibition of CXCR2 signaling. Therefore, DARC is a novel prognostic predictor and a potential therapeutic target for PDAC.

CD82/KAI1 Maintains the Dormancy of Long-Term Hematopoietic Stem Cells through Interaction with DARC-Expressing Macrophages.

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 in niche-mediated LT-HSC maintenance. We found that CD82/KAI1 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82(-/-) mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-ß1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC(+) BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche.

Expression of Duffy antigen receptor for chemokines (DARC) is down-regulated in colorectal cancer.

Duffy antigen receptor for chemokines (DARC) is a silent chemokine receptor which selectively binds angiogenic chemokines without inducing conventional signaling responses. DARC has been reported to inhibit the development of multiple cancers through clearance of angiogenic chemokines. However, its role in colorectal cancer (CRC) remains unclear. We investigated the expression of DARC in CRC and explored correlation of DARC expression with clinical pathological features and microvessel density (MVD). The protein expression levels of DARC were detected by immunohistochemistry in 90 CRC and 64 paired unaffected tissues. The mRNA levels of DARC were detected by quantitative real-time PCR in 15 CRC and paired unaffected tissues. MVD in CRC was also assessed by immunohistochemistry of CD34. We found that the mRNA and protein expression levels of DARC were significantly lower in CRC than in the unaffected tissues (p < 0.05). The DARC protein expression levels were positively correlated with DARC mRNA expression levels in both CRC (p < 0.001) and unaffected tissues (p < 0.001). We also found that DARC expression was significantly correlated with tumor differentiation (p < 0.001), lymph node metastasis (p < 0.01) and TNM stage (p < 0.05). Moreover, we observed a strong negative relationship between DARC expression and MVD in CRC (p < 0.001). We showed that DARC expression is down-regulated in CRC and associated with clinical pathological features and MVD of CRC. DARC might be involved in tumorigenesis, progression, angiogenesis, and metastasis of CRC.

Distribution of Duffy Antigen Receptor for Chemokines (DARC) and Risk of Prostate Cancer in Barbados, West Indies.

Blood typing across different racial groups has revealed that Caucasians predominantly test positive for the Duffy antigen/receptor for chemokines (DARC), while 70-95% of African-origin populations lack expression of DARC on their erythrocytes. Since men of African descent are known to have higher rates of prostate cancer (PC) and some animal studies have indicated anti-angiogenic effects associated with Duffy-positive mice, DARC-negativity may help to explain some of the racial differences in prostate tumorigenesis. The Prostate Cancer in a Black Population (PCBP) Study, a large case-control investigation including 1,007 incident PC cases and 1,005 controls, performed DARC testing on a subset of 1,295 participants (641 cases, 654 controls). The relationship between DARC expressivity and PC risk was evaluated using logistic regression models and findings are presented as odds ratios and 95% confidence intervals. More than three-quarters (76.5%) of African-Barbadian men lacked DARC expression, whereas almost three-fifths (59.3%) of White participants tested positive for the Duffy a and b alleles. DARC-negativity was not found to be associated with PC risk in the present investigation [OR 1.04, 95% CI (0.78, 1.37)], regardless of tumor grade. Findings from the PCBP study indicate that the majority of African-Barbadian men do not express DARC on their erythrocytes, yet absence of expression does not appear to be associated with PC development in this population.

Absence of multiple atypical chemokine binders (ACBs) and the presence of VEGF and MMP-9 predict axillary lymph node metastasis in early breast carcinomas.

The aim of this study was to determine the frequency of axillary lymph node (ALN) metastasis of early breast cancers by evaluating the status of DARC, D6 and CCX-CKR and the levels of VEGF and MMP-9. The status of DARC, D6 and CCX-CKR and the levels VEGF and MMP-9 were evaluated in ALN- (n = 130) and ALN + (n = 88) patients with T1 breast cancer by immunohistochemical staining. For ALN, likelihood ratio χ (2)-tests were used for univariate analysis and logistic regression for multivariate analysis. Univariate analysis identified the nuclear grade, VEGF and MMP-9 expression and absence of DARC, D6 and CCX-CKR as predictors of ALN involvement. When combining the three receptors (DARC, D6 and CCX-CKR) together, tumors with multiple absence (multi-absence, any two or three loss) had a higher likelihood of being ALN positive than non-multi-absence (coexpression of any two or three) tumors (56.2 vs. 27.9 %, P < 0.001). The final multivariate logistic regression revealed nuclear grade, VEGF, MMP-9 and non-multi-absence versus multi-absence to be independent predictors of ALN involvement; the odds ratio (OR) and 95 % CI for non-multi-absence tumors versus multi-absence were 0.469 (0.233-0.943). Multi-absence was also associated with the involvement of four or more lymph nodes among ALN + tumors. Moreover, tumors with multi-absence had higher VEGF (78.1 vs. 50.0 %, P < 0.001) and MMP-9 (81.3 vs. 36.1 %, P < 0.001) expression than non-multi-absence tumors. Our data highlight that the absence of DARC, D6 and CCX-CKR in combination, which is associated with higher VEGF and MMP-9 expression, predicts the presence and extent of ALN metastasis in breast cancer.

Regulation of motor function and behavior by atypical chemokine receptor 1.

Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines, stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo is lacking. Here we demonstrate that Ackr1 (-/-) mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 (-/-) mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 (+/-) had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 (-/-) mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1.

CXCL1 inhibits airway smooth muscle cell migration through the decoy receptor Duffy antigen receptor for chemokines.

Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit α. Interestingly, DARC- or heat shock protein 90 subunit α-deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC's ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK-signaling pathway.