Duffy Antigen Receptor For Chemokines Expression Research Articles

The Darc Side of Vivax Malaria in Africa: Unveiling Invasion Pathways into Duffy-Negative Erythroblasts

Background & Objectives: Plasmodium vivax malaria was long thought to be absent from sub-Saharan Africa owing to the high prevalence of people lacking the Duffy antigen receptor for chemokines (DARC) on their erythrocytes. The interaction between P. vivax Duffy binding protein (PvDBP) and DARC is assumed to be the main mechanism used by P. vivax merozoites to invade human erythrocytes. However, the increasing numbers of vivax malaria cases in Duffy-negative African individuals has raised questions about alternative P. vivax invasion pathway(s) other than PvDBP-DARC interaction. Since P. vivax has a tropism for CD71+ immature reticulocytes and the hematopoietic niches of the bone marrow may be a hidden reservoir of P. vivax parasites, we hypothesized that P. vivax merozoites may be able to invade erythroblasts derived from Duffy-positive (DP) and Duffy-negative (DN) individuals. Therefore, our objectives were to (i) investigate the expression of DARC during DP and DN erythropoiesis and (ii) examine the infection of DP and DN erythroblasts with P. vivax merozoites in vitro to study and identify host receptors used by P. vivax to invade erythroblasts. Methods: We performed in vitro erythropoiesis assays (two-phase culture) of genotypically DN and DP erythroid progenitors. Erythroid differentiation, CD71 and DARC expression profiles were monitored using flow cytometry, microscopy, and western blot over time during phase 2. In vitro invasion assays were conducted using DP and DN erythroblasts to investigate whether genotypically DN erythroblasts could be invaded by P. vivax. At D7 of phase 2, we thawed and cultured parasite isolates obtained from two Malagasy and seven Ethiopian patients infected with P. vivax. After approximately 24-30 hours of in vitro maturation (at D8 of phase 2), each P. vivax parasite culture was placed in co-culture with either DP or DN erythroblasts for 24-48 hours. We examined the infection of DP and DN erythroblasts by P. vivax parasites by light and confocal microscopy. Results: Using in vitro erythropoiesis, we show that both genotypically DP and DN erythroblasts have similar kinetics of terminal erythroid differentiation. Moreover, we found similar CD71 expression profiles in both DP and DN erythroblasts. Interestingly, we observed that a subset of DN erythroblasts transiently expressed DARC during erythropoiesis. This was confirmed using both flow cytometry and western blot analysis. In vitro invasion assays conducted on both DP and DN erythroblasts using parasite isolates, showed that P. vivax merozoites were able to invade both DP and DN erythroblasts (Figure 1). We found that all the DP and DN erythroblasts that were infected with P. vivax were CD71+ and DARC+. These results confirm that P. vivax can invade DP and DN erythroblasts expressing both DARC and CD71, irrespective of the origin of the isolates. Conclusion: This study provides the first biological evidence for the ability of P. vivax to invade genotypically DN erythroblasts and highlights that African populations may represent a silent reservoir of P. vivax infections, with major implications for the control and elimination of vivax malaria in sub-Saharan Africa.

Abstract 18831: The Protective Effect of Duffy Antigen Receptor for Chemokines Gene Deletion on Abdominal Aortic Aneurysm Formation: Role of Lysyl Oxidase

Introduction: Epidemiologic studies indicate that Blacks are protected against abdominal aortic aneurysm (AAA), despite similar risk factors as Caucasians. Approximately 70% of Blacks harbor a variant in the Duffy Antigen Receptor for Chemokines (DARC), a non-signaling receptor expressed primarily on erythrocytes which binds and regulates inflammatory chemokines. Since chemokines play a critical role in AAA, we tested the hypothesis that lack of DARC expression on erythrocytes protects against AAA, and we investigated the mechanisms. Methods: Mice lacking DARC on hematopoietic cells were created by mating DARC flox/flox mice with Vav1-Cre mice. At 10-14 weeks of age, both Vav1-Cre positive (DARC HKO ) and Vav1-Cre - mice [wild type (WT) littermate controls] received PCSK9 adeno-associated virus injection and were started on high fat diet. After 1 week, mice were infused with angiotensin II (AngII, 1,000 ng/kg/min) via osmotic minipump for 26 days to induce AAA. Results: Compared to WT, there was no difference in blood pressure or cholesterol levels in AngII-infused DARC HKO mice, which were confirmed to lack DARC on erythrocytes by flow cytometry. Aortic diameter and AAA incidence were significantly decreased in DARC HKO compared to WT mice (n=16-20, p=0.0257), in conjunction with reduced elastin degradation and macrophage infiltration. We did not find differences in levels of DARC-bound chemokines that could account for the protection against AAA. Interestingly, we found that the level and activity of lysyl oxidase (LOX), a cytokine-like enzyme that crosslinks extracellular matrix proteins and protects against AAA, were higher in DARC HKO mice compared to WT mice. Conclusions: Loss of DARC on hematopoietic cells protects against AAA formation, likely by upregulating aortic LOX. These findings provide novel insight into ethnic differences in susceptibility to AAA.

Association of the Duffy-null allele and endometrial cancer characteristics: An effort to explore racial disparity in Black women with endometrial cancer.

5603 Background: Efforts are needed to identify factors that account for the disparately high mortality in Black women with endometrial cancer. The Duffy antigen receptor for chemokines (DARC) is expressed on erythrocytes and endothelial cells and modulates levels of inflammatory chemokines in circulation and in tissues. The African ancestry-specific Duffy-null allele, in which erythrocyte expression and function of DARC is lost, is associated with worse breast cancer phenotypes and survival. Here, we characterize Duffy-null status and associated prognostic and clinical outcomes in patients with endometrial cancer. Methods: Patients with endometrial cancer were prospectively consented, and germline DNA was extracted from saliva or blood samples. Duffy-null genotype was determined using an allelic discrimination assay. Associations between Duffy-null genotype status and clinicopathologic characteristics were performed using Fisher’s Exact analysis. Results: 33 patients were enrolled (Table); 17 (51.5%) were Duffy-null, 5 (15.2%) were Duffy-null allele carriers, and 11 (33.3%) were non-carriers. Compared with non-carriers, Duffy-null patients were more likely to be Black (100% v 9.1%, p<0.0001) and have non-endometrioid histology (64.7% v 10%, p=0.0173). There was a trend towards higher rates of grade 3 histology (70.6% v 36.6%), intact tumor mismatch repair (MMR) (86.7% v 54.6%), estrogen receptor (ER) negative tumor status (25% v 10%), advanced stage (37.5% v 18.2%), and positive lymph nodes (25% v 0%) (Table). Over a minimum of 12 months of follow up, only Duffy-null individuals died from disease (4 v 0, p=0.0006). Conclusions: Duffy-null status was associated Black race, aggressive histologic subtypes, intact MMR mechanisms, and worse survival. Further investigation is needed to explore the underlying mechanism of the Duffy-null phenotype on endometrial cancer outcomes to better elucidate risk factors and underlying causes that may contribute to endometrial cancer disparities.[Table: see text]

Abstract 6165: DARC/ACKR1 expression is associated with immune landscape changes among triple negative breast tumors

Abstract Recent work defining the immune landscape across breast cancer (BC) subtypes have revealed that TNBC tumors have increased immunogenicity, and these increases in immune cell infiltration are associated with better prognosis and outcomes. Our recent work has proposed the Duffy Antigen Receptor for Chemokines (DARC), as a potential driver of immune regulation in breast tumors. DARC/ACKR1 is an atypical chemokine receptor that promiscuously binds both pro-inflammatory CC and pro-angiogenic CXC class chemokines and plays a role in chemokine gradient establishment both in circulation, and at sites of inflammation via chemokine transcytosis. In the BC context, we and others have reported DARC/ACKR1 protein expression on breast tumor epithelial cells by IHC and have also shown a range of gene expression in breast tumors from bulk RNAseq data in the TCGA cohort. Specifically, alongside CIBERSORT deconvolution of tumor associated immune cell populations, we have shown a strong positive correlation between DARC/ACKR1 gene expression and total tumor-associated leukocyte (TAL) abundance across all BC subtypes. To investigate the role of DARC/ACKR1 specifically in the TNBC tumor microenvironment (TME), we have analyzed (1) in silico bulk RNAseq data from two independent TNBC cohorts, (2) imaging mass cytometry data from TNBC patients and (3) murine model of DARC/ACKR1 BC progression in a basal-like BC transgenic mouse model. Our in silico data shows strong positive correlation of DARC/ACKR1 expression and TAL abundance, when DARC/ACKR1 expression is both dichotomized and analyzed as a continuous variable. Immune cell populations increasing with DARC/ACKR1 expression include B cell, T cell, monocyte and macrophage populations, which was consistent with our previous findings across all BC subtypes. IMC analysis of TNBC FFPE sections to determine protein expression of various immune and structural markers on the single-cell level between DARC/ACKR1 high or low expressing tumors show cells from DARC/ACKR1 high expressing tumors clustered distinctly from DARC/ACKR1 low expressing tumors. Image analysis also revealed increased infiltration among our DARC/ACKR1 high tumors. Finally, our murine model of DARC/ACKR1 in TNBC, shows that tumor development followed similar disease progression as human BC, following early through late-stage disease. DARC/ACKR1 deficient mice were found to have larger tumor volume and more palpable tumors, where DARC/ACKR1 expressing mice show co-localization of DARC and immune cell expression. This is the first work to describe the role of DARC/ACKR1 in the TNBC TME in bulk tumor transcriptomic data, spatial single-cell protein level data, and murine BC tumor model. Further investigation of factors that drive immune response in TNBC tumors, like DARC/ACKR1, may lead the way to novel therapeutic options or biomarkers for better outcomes for women with TNBC. Citation Format: Rachel Martini, Kiel Telesford, Brittany Lord, Hiranmayi Ravichandran, Olivier Elemento, Nancy Manley, Michele Monteil, Lisa Newman, Upender Manne, Clayton Yates, Melissa B. Davis. DARC/ACKR1 expression is associated with immune landscape changes among triple negative breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6165.

Renal allograft DARCness in subclinical acute and chronic active ABMR.

SummaryGene expression profiling of renal allograft biopsies revealed the Duffy antigen receptor for chemokines (DARC) as being strikingly upregulated in antibody‐mediated rejection (ABMR). DARC has previously been shown to be associated with endothelial injury. This study aimed at assessing the value of DARC immunohistochemistry as diagnostic marker in ABMR. The study was performed on 82 prospectively collected biopsies of a clinically well‐defined population (BORTEJECT trial, NCT01873157) of DSA‐positive patients with gene expression data available for all biopsies. Diagnostic histologic assessment of biopsies was performed according to the Banff diagnostic scheme. DARC expression was focally accentuated, on peritubular capillaries (PTC) mostly in areas of interstitial fibrosis and/or inflammation. DARC positivity was associated with diagnosis of ABMR and correlated with DARC gene expression levels detected by microarray analysis. Still, as previously described, a substantial number of biopsies without signs of rejection showed DARC‐positive PTC. We did not observe significantly reduced graft survival in cases showing histologic signs of ABMR and being DARC‐positive, as compared to DARC‐negative ABMR. In summary, the upregulation of DARC, detected by immunohistochemistry, is associated with but not specific for ABMR. We did not observe reduced graft survival in DARC‐positive patients.

The effect of erythrocyte transfusion on macrophage pyroptosis and inflammation in a sepsis model.

Sepsis is one of most common causes of death in the intensive care unit (ICU) due to infection and inflammation. The Duffy antigen receptor for chemokines (DARC) regulates pro-inflammatory cytokines, thus playing an important role in inflammation. This study aimed to elucidate the correlation among erythrocyte transfusion, macrophage pyroptosis and inflammation in the progression of sepsis. Alanine aminotransferase (ALT/GPT) activity was measured with the ALT/GPT activity measurement kit (Jiancheng Bio, Nanjing, China) according to the kit manual. The ET-1 concentration was measured with enzyme-linked immunosorbent assay (ELISA) using the endothelin-1 (ET-1) measurement kit (Jiancheng Bio) according to the kit manual. Apoptosis was evaluated using flow cytometry-based Annexin V staining assay. The cells were collected using centrifugation and resuspended in binding buffer. Ultrastructural analysis of pyroptotic body, the levels of interleukin (IL)-1β, IL-18, IL-33, MIP-2, CXCL8, reactive oxygen species (ROS), and LTB4 were measured with ELISA. Our results showed that septic rats had impaired hepatic function and ET-1 levels. Erythrocyte transfusion upregulated DARC expression in the sepsis model. Erythrocyte transfusion also affected pyroptosis in macrophages, reduced the production of inflammatory cytokines, such as IL-1β, IL-18 and IL-33, and alleviated cytotoxicity in the sepsis model. Erythrocyte transfusion may function as a therapeutic tool against sepsis by regulating pyroptosis, inflammation and cytotoxicity.

Abstract B058: In silico and in vivo analysis of the Duffy Antigen Receptor for Chemokines (DARC) in the breast tumor microenvironment

Abstract In clinic, we observe that women of African descent have higher incidence rates of breast cancer (BC). These women are disproportionately diagnosed with the most aggressive subtype of BC, triple-negative BC, compared to women belonging to any other ancestry groups. To investigate factors driving this disparity, our work focuses on the tumor microenvironment (TME), specifically through the lens of an atypical chemokine receptor. The Duffy Antigen Receptor for Chemokines (DARC) is a nonsignaling, atypical chemokine receptor that binds two structural classes of chemokines. DARC modulates chemokine availability in circulation and participates in chemokine transport in tissues, to recruit immune cells back to sites of inflammation. DARC additionally serves as a portal of entry for the malaria-causing parasite Plasmodium vivax. In Sub-Saharan Africa, where malaria is endemic, a regulatory variant arose removing DARC expression from red blood cells (RBCs). This mutation, known as the Duffy-Null allele, was highly beneficial in this population, and quickly rose to fixation. Despite being called Duffy-Null, these individuals retain DARC expression in other cell types, only lacking expression on RBCs. Knowing that the Duffy-Null allele is prevalent in this population of women, we aim to investigate how DARC tumor expression and Duffy-Null status impacts the TME in silico through analysis of The Cancer Genome Atlas (TCGA) data, and in vivo through establishment of mouse models that depict Duffy-Null status in the BC TME. Using TCGA data, we see that DARC tumor gene expression is significantly lower among African-Americans compared to Whites. Using the CIBERSORT online platform, we quantified tumor-associated leukocyte (TAL) abundance in the TME. Overall, we found that tumors with high DARC expression have significantly higher total TAL abundance (p < 0.0001), and that DARC expression and TAL abundance are positively and significantly correlated (R = 0.545, p < 0.0001). Specifically, DARC expression influenced levels of B cell, T cell, monocyte and macrophage population in the TME. In our mouse models, we have utilized the C3(1)Tag BC transgenic mouse model, alongside a DARC knock-out mouse. To better recapitulate the Duffy-Null phenotype in our mouse model, our approach is to generate a BC/Duffy-Null bone marrow derived (BMD) chimera, where a DARC+/-; C3(1)Tag+/0 recipient will receive bone marrow from a DARC -/- donor. The resulting chimera mice will retain DARC expression in all cells, aside from bone marrow-derived cells from the null donor. In the C3(1)Tag line, disease onset is at approximately 12 weeks, at which time we will assess changes in disease progression in our chimeras compared to controls by collecting total number of tumors, tumor location, tumor size and weight, and hematoxylin and eosin staining of tumor sections, and immune cell infiltration quantification. Citation Format: Rachel Martini, Brittany Jenkins, Clayton Yates, Lisa Newman, Nancy Manley, Melissa Davis. In silico and in vivo analysis of the Duffy Antigen Receptor for Chemokines (DARC) in the breast tumor microenvironment [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B058.

Differential interaction between DARC and SDF-1 on erythrocytes and their precursors

The Duffy Antigen Receptor for Chemokines (DARC) is expressed on erythrocytes and on endothelium of postcapillary venules and splenic sinusoids. Absence of DARC on erythrocytes, but not on endothelium, is referred to as the Duffy negative phenotype and is associated with neutropenia. Here we provide evidence that stromal cell-derived factor 1 (SDF-1), the chemokine that restricts neutrophil precursors to the bone marrow, binds to erythrocyte progenitors in a DARC-dependent manner. Furthermore, we show that SDF-1 binding to DARC is dependent on the conformation of DARC, which gradually changes during erythroid development, resulting in the absence of SDF-1 binding to mature erythrocytes. However, SDF-1 binding to erythrocytes was found to be inducible by pre-treating erythrocytes with IL-8 or with antibodies recognizing specific epitopes on DARC. Taken together, these novel findings identify DARC on erythrocyte precursors as a receptor for SDF-1, which may be of interest in beginning to understand the development of neutropenia in situations where DARC expression is limited.

Abstract A090: The Duffy Antigen Receptor for Chemokines (DARC) influences levels of tumor-associated leukocytes in the breast tumor microenvironment

Abstract The regulation of immune cell infiltration into the tumor microenvironment can influence disease prognosis. The specific populations that are present can inform potential treatment options. This work investigates immune cell regulation in the breast cancer tumor microenvironment, specifically how an atypical chemokine receptor, known as the Duffy Antigen Receptor for Chemokines (DARC/ACKR1) can influence levels of leukocyte populations in the breast tumor environment. DARC is a nonsignaling receptor able to bind both the CC and CXC classes of chemokines, and mainly functions to modulate levels of chemokines in circulation, and aid in chemokine transport in tissues. In this regard, DARC expression may determine the profile of immune response.To investigate DARC expression and its effects on tumor-associated leukocyte (TAL) populations, we obtained RNA-seq data from The Cancer Genome Atlas (TCGA) Breast Cancer cohort. We proceeded through our analysis with those samples denoted as primary tumor samples (n=400). To estimate the relative abundance of TAL populations, we used the CIBERSORT online platform, which estimates fractions of 22 different TAL populations based on gene expression data. After completing the CIBERSORT analysis, we proceeded with only those samples that had a significant CIBERSORT output (n=167). In our analysis, we found that the total abundance of all TALs was significantly higher in tumors that have high DARC expression (p < 0.0001), and that DARC expression and the TAL abundance are positively and significantly correlated (R = 0.545, p < 0.0001). When we investigated specific TAL populations, we saw that B cell, T-cell, monocyte and macrophage populations were significantly increased in tumors with high DARC expression. Citation Format: Rachel Martini, Brittany D. Jenkins, Clayton Yates, Lisa Newman, Melissa Davis. The Duffy Antigen Receptor for Chemokines (DARC) influences levels of tumor-associated leukocytes in the breast tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A090.

Abstract A63: Characterizing the role of ancestry-specific variants of the Duffy Antigen Receptor for Chemokines (DARC) gene in the breast tumor microenvironment

Abstract Chemokines play an important role in cancer, as they direct infiltrating immune cells and mediate angiogenesis, tumor progression, and direct metastasis to distal sites. This work focuses on an atypical chemokine receptor, named the Duffy Antigen Receptor for Chemokines (DARC). DARC is a nonsignaling receptor that can bind two structural classes of inflammatory chemokines, and mainly acts as a regulator of chemokine homeostasis by removing chemokines from circulation when expressed on red blood cells, or moving them from sites of inflammation into circulation to recruit appropriate immune cell types. The purpose of this study is to investigate how polymorphic variants of the DARC gene found across global populations influence ancestry-specific immune responses, and how these changes in the immune response influence the breast tumor microenvironment. A well-studied example of this is a variant restricted to recent decedents of sub-Saharan Africa, known as the Duffy-Null allele. This variant removes expression of DARC from red blood cells, causing those individuals carrying the Duffy-Null allele to lose the ability to sequester chemokines in circulation. In terms of immune response, Duffy-Null status is shown to be predictive of chronic low white blood cell counts observed in individuals of African descent. By using 1000 Genomes Project variant calling data, we have been able to identify additional gene variants restricted to varying global populations. Our initial study is focused on variants found in the regulatory region of the gene, as these variants will likely change how the DARC gene will be expressed. Preliminarily, using data from the Genotype-Tissue Expression project (GTEx), we have been able to show that our DARC gene variants are also tissue-specific expression quantitative trait loci, showing significant changes in tissue-specific gene expression in association with our variants of interest. By studying these variants with panels of human breast cell lines, ancestry-specific cell lines, and human tissue, we will be able to identify these ancestry-specific changes in immune response, and resulting breast tumor microenvironment. Citation Format: Rachel N. Martini, Brittany D. Jenkins, Melissa B. Davis. Characterizing the role of ancestry-specific variants of the Duffy Antigen Receptor for Chemokines (DARC) gene in the breast tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A63.

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.

Abstract 5119: In vivo characterization of the Duffy Antigen Receptor for Chemokines (DARC) in the breast cancer microenvironment

Abstract Chemokines are small inflammatory molecules that, in combination with their receptors, recruit lymphocytes to influence immune and inflammatory responses. This work specifically follows an atypical chemokine receptor, known as the Duffy Antigen Receptor for Chemokines (DARC). DARC is a non-signaling receptor able to bind both the CC and CXC chemokine classes, and mainly functions to regulate homeostatic levels of chemokines in circulation, and aid in chemokine transcytosis from sites of inflammation to mediate immune response. DARC serves as a portal of entry for the malaria-causing parasite Plasmodium vivax. In Sub-Saharan Africa, where malaria is endemic, a regulatory variant arose removing DARC expression from red blood cells (RBCs). This mutation, known as the Duffy-Null allele, was highly beneficial in this population, as removal of DARC from RBCs conferred immunity to Duffy-Null individuals. The Duffy-Null allele quickly rose to fixation in this population, and today recent decedents of Sub-Saharan Africans carry this allele. In the context of breast cancer (BCa) we are particularly interested in how Duffy-Null status impacts the tumor microenvironment. In clinic, we observe that pre-menopausal women of African descent have higher incidence rates of BCa, and that regardless of age, these women are disproportionately diagnosed with the most aggressive molecular subtype of BCa, triple-negative BCa. Knowing the important role that DARC plays in immune response, and that the Duffy-Null allele is prevalent in this population of women, we aim to investigate how Duffy-Null status impacts the tumor microenvironment in vivo through establishment of mouse models that depict Duffy-Null status in the BCa environment. To do this, we are using the C3(1)-TAg BCa transgenic mouse, alongside a DARC-KO mouse to characterize cancer progression and aggressiveness in BCa transgenic mice with varying global DARC expression. At 6 and 12 week time points, mice were sacrificed and the following information was obtained: total number of tumors, tumor location, tumor size and weight, and hematoxylin and eosin staining of tumor sections. Removal of DARC could lead to an excess of chemokines in the tumor microenvironment, promoting processes of tumor progression and metastasis, leading to the more aggressing tumor phenotypes we observe in women of African descent in clinic. Citation Format: Rachel Martini, Nancy Manley, Melissa Davis. In vivo characterization of the Duffy Antigen Receptor for Chemokines (DARC) in the breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5119.

DARC extracellular domain remodeling in maturating reticulocytes explains Plasmodium vivax tropism

AbstractPlasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71high/RNAhigh (∼0.016%), CD71low/RNAhigh (∼0.059%), CD71neg/RNAhigh (∼0.37%), CD71neg/RNAlow (∼0.55%), and erythrocytes CD71neg/RNAneg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71high/RNAhigh reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.

The Duffy null genotype is associated with a lower level of CCL2, leukocytes and neutrophil count but not with the clinical outcome of HTLV-1 infection.

Purpose. Chemokines are important in the immune response against viral infections, and may play a role in human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) pathogenesis. Polymorphisms in the Duffy antigen receptor for chemokines (DARC), such as rs12075 (A>G; FY*B>FY*A) and rs281477 (-46T>C; GATA-1 box) may influence circulating concentrations of proinflammatory chemokines. We investigate whether Duffy genotypes influence the HTLV-1 proviral load (PVL) level, HTLV-1 infection outcome and chemokine concentrations in HTLV-1 asymptomatic carriers (AC=162), HAM/TSP patients (HAM=135) and seronegative individuals (SN=71).Methodology. Quantification of plasmatic IL8, CCL2 and CCL5 were performed by flow cytometry and Duffy genotypes were investigated by real-time PCR. HTLV-1 PVL was quantified in peripheral blood. To control for spurious association, individual ancestry profiles in AC and HAM groups were investigated.Results/Key findings. PVL and IL8 level were significantly higher in the HAM group than in the AC group, but were not associated with Duffy genotypes. The highest CCL2 and CCL5 levels were seen in the SN group, and there was no difference when comparing the infected groups. The level of CCL5 was not associated with Duffy genotypes. The polymorphism -46 C/C that abrogates the DARC expression on the erythrocytes was significantly associated with lower levels of CCL2, neutrophil and white blood cell (WBC) counts in HTLV-1-infected individuals.Conclusion. We conclude that although the Duffy null genotype was associated with leukopenia, neutropenia and lower levels of CCL2, the data do not suggest the influence of Duffy genotypes on the neurologic outcome of HTLV-1 infection, but may be a confounding factor in comparison HTLV-1-infected populations with different ancestries, especially when defining inflammatory biomarkers.

The expression of Duffy antigen receptor for chemokines by epithelial ovarian cancer decreases growth potential.

Epithelial ovarian cancer (EOC) is one of the main causes of cancer-associated mortality in females with gynecological malignancies. Duffy antigen receptor for chemokines (DARC) has previously been reported to be involved in tumor growth and the inhibition of tumor metastasis. However, the association between DARC and EOC remains unknown. The aim of the present study was to investigate the expression of DARC in the SKOV3 human epithelial ovarian cancer cell line with the establishment of a subcutaneous model in nude mice. To investigate the effects of DARC on the tumorigenesis of human epithelial ovarian cancer cells, GV287-DARC-L.V lentiviral vectors containing a DARC overexpression construct were transfected into SKOV3 cells. The present study revealed that transfection with DARC reduced the viability of SKOV3 cells in vitro by performing an MTT assay. SKOV3-DARC and SKOV3-negative control (NC) cells cultured in vitro were injected into nude mice to establish a subcutaneous model. The ovarian tumor volumes and the tumor weights were observed. Immunohistochemistry to detect CD31 expression was used to determine the microvessel density (MVD) in SKOV3-DARC and SKOV3-NC tumors. The results of the present study revealed that DARC-induced inhibition of tumor growth was associated with MVD in xenograft tumors. This suggested that DARC was a negative regulator of tumor growth in EOC, primarily via the inhibition of tumor angiogenesis.

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

BackgroundPancreatic 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. MethodsFunctions of chemokine receptors were evaluated using surgical specimens collected from PDAC patients, and PDAC cell lines. ResultsCXCR2 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. ConclusionsDARC 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.

Staphylococcus Aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes

Introduction: Staphylococcus aureus is a major human pathogen. Infections by this bacterium range from minor skin and soft tissue infections, to more invasive and life threating infections like sepsis, osteomyelitis, and pneumonia. In order to successfully infect the mammalian host, S. aureus has to overcome iron scarcity within the host. As such, S. aureus is thought to produce toxins that lyse erythrocytes, releasing hemoglobin, a critical iron source for S. aureus in mammals. The bi-component β-barrel pore-forming leukocidins kill human neutrophils and were mostly considered as virulence factors fighting the host innate immune system. We show here thatHlgAB and LukED are two potent hemolytic leukocidins against human erythrocytes. Furthermore, we describe the identification of the Duffy antigen receptor for chemokines (DARC) as the red blood cell receptor for both LukED and HlgAB leukocidins.Results: We took advantage of erythrocyte samples from different Duffy (Fy) genotyped individuals from the French National Blood Transfusion Institute. We evaluated the receptor expression on the surface of erythrocytes (Figure 1). DARC-negative erythrocytes (phenotype Fya-/b-) were fully resistant to HlgAB and LukED. Compared to individuals expressing normal levels of DARC (phenotypes Fya+/b+, Fya+/b+weak, Fya+/a+, and Fyb+/b+), individuals expressing intermediate (phenotype Fyb+weak/b+weak) or very low levels of DARC (phenotype Fyb-/b+weak) showed intermediate susceptibility to both HlgAB and LukED (Figure 1). These data demonstrate that the hemolytic activity of HlgAB and LukED is dependent on the expression of DARC at the red blood cell surface. To better understand the interaction of HlgAB and LukED with DARC, we screened HEK293T cells transfected with plasmids encoding DARC mutants (Tournamille et al, 2003). We demonstrate that while both HlgAB and LukED bind to DARC with nanomolar affinities, they do so by recognizing different domains of the receptor. To directly evaluate whether LukED and HlgAB can promote bacterial replication as a result of erythrocyte lysis, S. aureus was grown in iron-starved medium supplemented with cell-free extracts of erythrocytes treated with LukED or HlgAB. We observed that HlgAB and LukED were each capable of promoting S. aureus growth in a DARC and hemoglobin scavenging system (IsdBH)-dependent manner. These in vitro studies were supported by a murine bacteremia model.Discussion: By combining human studies of DARC polymorphisms with gain and loss of function experiments and biochemical analyses, we demonstrate that DARC is necessary and sufficient to render host cells susceptible to LukED and HlgAB. By targeting DARC, HlgAB and LukED support S. aureus growth in a hemoglobin-acquisition dependent-manner. Thus, these findings provide the missing link of how S. aureus targets and lyses erythrocytes to release one of the scarcest nutrient within the mammalian host. Human epidemiological studies comparing the severity of S. aureus infection in patients with DARC positive or DARC negative erythrocytes are now required to evaluate the contribution of DARC-mediated hemolysis in human staphylococcal diseases. Given the resistance of DARC negative erythrocytes to the parasites Plasmodium vivax and P. knowlesi, and now to the hemolytic activity of the bacterium S. aureus, our findings suggest the possibility of a positive selection event in response to these important human pathogens.(A) Susceptibility of human erythrocytes to S. aureus β-barrel pore forming toxins. The dashed line indicates 50% hemolysis. n = 6.(B) Levels of DARC and CD55 on erythrocytes of donors with different Fy phenotypes. The dashed line indicates the detection threshold. n = 2-7 ± SEM.(C,E) Susceptibility of human erythrocytes with different Fy phenotypes to HlgAB (C) and LukED (E). The dashed line indicates 50% hemolysis. n = 2-7 ± SEM.(D, F) Correlation of half-maximal effective concentrations (EC50) of HlgAB (D) and LukED (F) with the total number of receptors expressed on the erythrocyte surface.For Fyb+weak/Fyb- donors, LukED EC50 could not be calculated. n = 2-7 ± SEM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1)/Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele.

The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups.

Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes.

In order for Staphylococcus aureus to thrive inside the mammalian host, the bacterium has to overcome iron scarcity. S.aureus is thought to produce toxins that lyse erythrocytes, releasing hemoglobin, the most abundant iron source in mammals. Here we identify the Duffy antigen receptor for chemokines (DARC) as the receptor for the S.aureus hemolytic leukocidins LukED and HlgAB. By assessing human erythrocytes with DARC polymorphisms, we determined that HlgAB- and LukED-mediated lysis directly relates to DARC expression. DARC is required for S.aureus-mediated lysis of human erythrocytes, and DARC overexpression is sufficient to render cells susceptible to toxin-mediated lysis. HlgA and LukE bind directly to DARC through different regions, and by targeting DARC, HlgAB and LukED support S.aureus growth in a hemoglobin-acquisition-dependent manner. These findings elucidate how S.aureus targets and lyses erythrocytes to release one of the scarcest nutrients within the mammalian host.

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.