Duffy Antigen Receptor For Chemokines Research Articles

Abstract 4975: Breast tumor expression of DARC/ACKR1 and impacts on disease progression and immune cell recruitment in the tumor microenvironment

Abstract The Duffy Antigen Receptor for Chemokines (DARC/ACKR1) is an atypical chemokine receptor which promiscuously binds chemokines of both CC and CXC chemokine families. DARC/ACKR1 maintains homeostatic levels of chemokines in circulation through its expression on erythrocytes, and in tissues participates in chemokine transport, where chemokines are transported across the cell layer to help establish chemokine gradients. Establishment of these chemokine gradients is essential for proper immune cell trafficking. In our recent study, we have shown that DARC/ACKR1 is expressed on the tumor epithelium, and through in silico analysis of data from the Cancer Genome Atlas (TCGA), that higher DARC/ACKR1 expression is positively and significantly correlated with tumor-associated leukocyte abundance when using CIBERSORT deconvolution methods. Specifically, higher DARC/ACKR1 expression was linked with increases in B cell, T cell, monocyte and macrophage populations. Higher DARC/ACKR1 expression is also significantly associated with better survival outcomes, across all breast cancer molecular subtypes. To further evaluate DARC/ACKR1 expression and influences on the breast TME, we have employed multiple approaches to characterize how DARC/ACKR1 impacts immune cell recruitment and subsequent infiltration into the tumor. In our patient cohort, we have recently completed Hyperion imaging mass cytometry staining of tumors that have been scored by IHC as DARC/ACKR1 high or low expressing. We used a panel of approximately 30 markers, including structural, immune, and other markers of interest. Following imaging, we have used cell segmentation software to quantify marker expression on a cell-by-cell basis. Using this method, we are able to conduct single-cell analyses of our markers of interest, while also retaining the spatial composition of the cells. We have used the tSNE algorithm to identify cell populations that cluster uniquely within DARC/ACKR1 high or low tumor expressing groups, specifically identifying different clusters of immune cell populations, which show different spatial expression patterns in our comparison groups. In addition to investigations of immune cell recruitment to the TME, we have also developed a novel DARC/ACKR1 breast cancer transgenic mouse model to study how disease progression may differ with differing DARC/ACKR1 phenotypes. Briefly, DARC -/- female mice were crossed with the male C3(1)Tag +/0 breast cancer transgenic mouse to generate DARC +/-, C3(1)Tag +/0 male mice. These males were subsequently backcrossed to DARC -/- females, to generate the target DARC/ACKR1 +/- or -/-, C3(1)Tag+ mice. These mice show evidence of early disease beginning at 3.5 months of age, and progress to advanced disease by 5 months of age. We have collected tumor tissue and blood specimens from mice ranging from 3.5 to 7 months of age, to characterize disease progression over time between our DARC/ACKR1 expressing and non-expressing groups. We have observed differences in tumors characteristics between our DARC/ACKR1 expressing groups, and upon histological and immunofluorescent evaluation, we have begun to observe differences in immune cell recruitment, following similar patterns to what we observed in our in silico and patient cohort analyses. Citation Format: Rachel Martini, Brittany Jenkins, Dorrah Deeb, Hiranmayi Ravichandran, Paula Ginter, Esther Cheng, Syed Hoda, Dhananjay Chitale, Haythem Ali, Eleanor Walker, Jessica Bensenhaver, Olivier Elemento, Nancy Manley, Lisa Newman, Melissa Davis. Breast tumor expression of DARC/ACKR1 and impacts on disease progression and immune cell recruitment in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4975.

Abstract MP154: Deletion of the Duffy Antigen Receptor for Chemokines (DARC) Confers Protection Against Abdominal Aortic Aneurysm (AAA) Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by inflammation and matrix metalloproteinase (MMP)-mediated degradation of extra cellular matrix proteins leading to aortic dilation and potentially rupture. Inflammatory chemokines that promote AAA are modulated by binding to the Duffy antigen receptor for chemokines (DARC), a non-signaling receptor expressed primarily on erythrocytes. Interestingly, African Americans exhibit reduced frequency of AAA, and the majority of African descendent individuals do not express DARC on their erythrocytes. Here, we tested the hypothesis that DARC gene deletion protects against the development of AAA. Methods: The induction of AAA was performed using both angiotensin II (AngII) infusion and calcium chloride (CaCl 2 ) application models. Eight week old LDLR knockout (KO, control) and LDLR KO/DARC KO mice were infused with AngII via osmotic mini-pumps for four weeks. For the CaCl 2 application model, twelve week old DARC KO and WT control mice underwent laparotomy and 0.5 mol/L CaCl 2 was applied to the infrarenal aorta. Aortic dilation and AAA formation was assessed using ultrasound, mice were then euthanized and tissues were harvested for analysis. Results: The aortic diameter of LDLR KO/DARC KO mice was significantly lower relative to control mice after AngII infusion (P=0.02). There was no difference in the pressor response to AngII between groups. Furthermore, IL-6 levels were significantly reduced in the aortas of LDLR KO/DARC KO mice compared to control mice (P=0.001). Expression of MCP-1, which has strong affinity for DARC, tended to be higher in aortas from LDLR KO/DARC KO mice compared to control (P=0.063). In contrast, plasma IL-6 levels were similar in both LDLR KO/DARC KO and control mice, while MCP-1 tended to be lower in the plasma of LDLR KO/DARC KO (P=0.07). DARC KO mice likewise exhibited a trend toward reduced aortic dilation in the CaCl 2 application model compared to controls (P=0.09). Conclusions: DARC KO mice are protected against AAA formation, perhaps through differential regulation of aortic chemokine trafficking. Understanding the mechanisms by which loss of DARC confers protection from AAA formation may be relevant to ethnic differences in susceptibility to AAA.

Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion.

Protein-protein interactions (IPP) play an essential role in practically all biological processes, including those related to microorganism invasion of their host cells. It has been found that a broad repertoire of receptor-ligand interactions takes place in the binding interphase with host cells in malaria, these being vital interactions for successful parasite invasion. Several trials have been conducted for elucidating the molecular interface of interactions between some Plasmodium falciparum and Plasmodium vivax antigens with receptors on erythrocytes and/or reticulocytes. Structural information concerning these complexes is available; however, deeper analysis is required for correlating structural, functional (binding, invasion, and inhibition), and polymorphism data for elucidating new interaction hotspots to which malaria control methods can be directed. This review describes and discusses recent structural and functional details regarding three relevant interactions during erythrocyte invasion: Duffy-binding protein 1 (DBP1)–Duffy antigen receptor for chemokines (DARC); reticulocyte-binding protein homolog 5 (PfRh5)-basigin, and erythrocyte binding antigen 175 (EBA175)-glycophorin A (GPA).

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.

Two Genetically Distinct Plasmodium knowlesi Duffy Binding Protein Alpha Region II (PkDBPαII) Haplotypes Demonstrate Higher Binding Level to Fy(a+b+) Erythrocytes than Fy(a+b--) Erythrocytes.

Invasion of human erythrocytes by merozoites of Plasmodium knowlesi involves interaction between the P. knowlesi Duffy binding protein alpha region II (PkDBPαII) and Duffy antigen receptor for chemokines (DARCs) on the erythrocytes. Information is scarce on the binding level of PkDBPαII to different Duffy antigens, Fya and Fyb. This study aims to measure the binding level of two genetically distinct PkDBPαII haplotypes to Fy(a+b-) and Fy(a+b+) human erythrocytes using erythrocyte-binding assay. The binding level of PkDBPαII of Peninsular Malaysian and Malaysian Borneon haplotypes to erythrocytes was determined by counting the number of rosettes formed in the assay. Overall, the Peninsular Malaysian haplotype displayed higher binding activity than the Malaysian Borneon haplotype. Both haplotypes exhibit the same preference to Fy(a+b+) compared with Fy(a+b-), hence justifying the vital role of Fyb in the binding to PkDBPαII. Further studies are needed to investigate the P. knowlesi susceptibility on individuals with different Duffy blood groups.

High fat diet facilitate duffy antigen receptor for chemokine (DARC) expression in mononuclear cells by IL-22 dependent manner

Abstract Though interleukin-22 (IL-22) is recently suggested to play a role in immune-inflammatory system, however, the specific role of IL-22 for accumulation of adipose tissue in the context of obesity and its metabolic consequences has not been clearly documented. The purpose of this study is determine the role of IL-22 on adipose tissue accumulation and inflammation through mouse and human model. 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, which 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.

Deletion of the Duffy Antigen Receptor for Chemokines (DARC) Confers Protection Against Abdominal Aortic Aneurysm (AAA) Formation

IntroductionAbdominal aortic aneurysms (AAA) are characterized by inflammation and matrix metalloproteinase (MMP)‐mediated degradation of extra cellular matrix proteins leading to aortic dilation and potentially rupture. Inflammatory chemokines that promote AAA are modulated by binding to the Duffy antigen receptor for chemokines (DARC), a non‐signaling receptor expressed primarily on erythrocytes. Interestingly, African Americans exhibit reduced frequency of AAA, and greater than 70% of individuals of African descent do not express DARC on their erythrocytes due to evolutionary pressure from malaria. Here, we tested the hypothesis that DARC gene deletion protects against the development of AAA.MethodsThe induction of AAA was performed using both angiotensin II (AngII) infusion and calcium chloride (CaCl2) application models. In brief, eight week old LDLR knockout (KO, control) and LDLR KO/DARC KO mice were infused with angiotensin II via osmotic minipumps for four weeks at a rate of 1,000 ng/kg/min. For the CaCl2 application model, 12 week old WT and DARC KO mice underwent laparotomy and 0.5 mol/L was applied to the infrarenal aorta. For both models, aortic dilation and AAA formation was assessed using ultrasound, mice were then euthanized and tissues were harvested for histology and biochemical analysis.ResultsLDLR KO/DARC KO mice were protected from the formation of AngII‐induced AAA, as only ~20% developed an aneurysm compared to ~80% in the control group. Concordantly, the aortic diameter of LDLR KO/DARC KO mice was significantly lower relative to control mice after AngII infusion (P=0.02). There was no difference in the pressor response to AngII between groups, but LDLR KO/DARC KO mice exhibited reduced macrophage infiltration, and aortic dilation compared to the control group. Furthermore, IL‐6 levels were significantly reduced in the aortas of LDLR KO/DARC KO mice compared to control mice (P=0.001). Interestingly, however, expression of MCP‐1, which has strong affinity for DARC, tended to be higher in aortas from LDLR KO/DARC KO mice compared to control (P=0.063). In contrast, plasma IL‐6 levels were similar in both LDLR KO/DARC KO and control mice, while MCP‐1 tended to be lower in the plasma of LDLR KO/DARC KO (P=0.07). Interestingly, DARC appears to be upregulated in the aortas of mice in response to AngII. Furthermore, DARC KO mice likewise exhibited reduced aortic dilation in the CaCl2 application model compared to controls.ConclusionsDARC KO mice are protected against AAA formation, perhaps through differential regulation of aortic chemokine trafficking. Understanding the mechanisms by which loss of DARC confers protection from AAA formation may be relevant to ethnic differences in susceptibility to AAA.Support or Funding InformationThis study was supported by the AHA [19PRE34400043 to T.W.B] and the National Institutes of Health [HL124097, HL126949, HL134354 and AR070029 to N.L.W].

The Red Blood Cell-Inflammation Vicious Circle in Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disease caused by a single mutation in the β-globin gene, leading to the production of an abnormal hemoglobin called hemoglobin S (HbS), which polymerizes under deoxygenation, and induces the sickling of red blood cells (RBCs). Sickled RBCs are very fragile and rigid, and patients consequently become anemic and develop frequent and recurrent vaso-occlusive crises. However, it is now evident that SCD is not only a RBC rheological disease. Accumulating evidence shows that SCD is also characterized by the presence of chronic inflammation and oxidative stress, participating in the development of chronic vasculopathy and several chronic complications. The accumulation of hemoglobin and heme in the plasma, as a consequence of enhanced intravascular hemolysis, decreases nitric oxide bioavailability and enhances the production of reactive oxygen species (ROS). Heme and hemoglobin also represent erythrocytic danger-associated molecular pattern molecules (eDAMPs), which may activate endothelial inflammation through TLR-4 signaling and promote the development of complications, such as acute chest syndrome. It is also suspected that heme may activate the innate immune complement system and stimulate neutrophils to release neutrophil extracellular traps. A large amount of microparticles (MPs) from various cellular origins (platelets, RBCs, white blood cells, endothelial cells) is also released into the plasma of SCD patients and participate in the inflammation and oxidative stress in SCD. In turn, this pro-inflammatory and oxidative stress environment further alters the RBC properties. Increased pro-inflammatory cytokine concentrations promote the activation of RBC NADPH oxidase and, thus, raise the production of intra-erythrocyte ROS. Such enhanced oxidative stress causes deleterious damage to the RBC membrane and further alters the deformability of the cells, modifying their aggregation properties. These RBC rheological alterations have been shown to be associated to specific SCD complications, such as leg ulcers, priapism, and glomerulopathy. Moreover, RBCs positive for the Duffy antigen receptor for chemokines may be very sensitive to various inflammatory molecules that promote RBC dehydration and increase RBC adhesiveness to the vascular wall. In summary, SCD is characterized by a vicious circle between abnormal RBC rheology and inflammation, which modulates the clinical severity of patients.

Abstract P1-03-08: In vivo investigation of the Duffy antigen receptor for chemokines (DARC/ACKR1) and tumor-infiltrating immune cells in the breast tumor microenvironment

Abstract Recruitment of immune cells and subsequent infiltration of these cell populations into the tumor microenvironment (TME) can influence disease prognosis, and potential treatments for patients. Through studying the TME of breast tumors, we have investigated factors that modify immune cell recruitment and infiltration into the tumor area. In our recently published work, we analyzed the TCGA breast cancer (BC) cohort, where we have identified the Duffy Antigen Receptor for Chemokines / Atypical Chemokine Receptor 1 (DARC/ACKR1) as a putative driver of immune cell recruitment and infiltration to the breast TME. DARC/ACKR1 is an atypical chemokine receptor that promiscuously binds both the CC and CXC classes of chemokines. On the surface of red blood cells in circulation, DARC/ACKR1 maintains homeostatic levels of chemokines in circulation by binding chemokines when levels are high, and releasing them back into circulation when levels lower. In tissues, DARC/ACKR1 participates in chemokine transcytosis, working to move chemokines across the cell layer to establish chemokine gradients necessary for recruitment of respective immune cell populations. Through in silico analysis of TCGA BC cohort data, we performed CIBERSORT TME deconvolution methods to approximated levels of 22 tumor-associated immune cell populations. We showed that as DARC/ACKR1 expression increased in breast tumors, we also saw a significant increase in number of tumor-associated immune cells (p<0.0001). When looking at the specific cell populations, we found that B cells, T cells, monocytes and macrophage populations were significantly different between high, mid and low DARC/ACKR1 expressing tumors. To continue investigation of these findings in vivo, we developed a novel DARC/ACKR1 breast cancer transgenic mouse model. Briefly DARC/ACKR1 -/- mice were crossed with the C3(1)Tag breast cancer transgenic mouse model. Breast cancer Tg+; DARC/ACKR1 +/- males were then backcrossed to DARC/ACKR1 -/- females, to generate target female mice that were BC Tg+, and either DARC/ACKR1 +/- or DARC/ACKR1 -/-. Disease progression in the C3(1)Tag line follows a similar pattern to ductal carcinoma in situ (DCIS), through progression to advanced invasive disease. All mice develop DCIS beginning at approximately 12 weeks of age, and 100% of mice are affected with invasive disease by 6 months of age. We have collected tissue from mice from DCIS stage of disease through invasive disease. Blood was collected from each mouse, and the plasma was used to quantify the circulating chemokine profile. Tumors and mammary tissue were collected from each mouse and weighed, alongside spleen, liver, and potential metastatic tumor sites. Tumors and mammary glands were processed for immunohistochemistry and immunofluorescent staining procedures, where DARC/ACKR1 expression, immune cells, structural markers and chemokines of interest were assayed. We also took representative sections from each genotype of interest for Hyperion imaging mass cytometry analysis, where we can simultaneously analysis 27 different immune, structural and other markers of interest. From our preliminary results from in silico TCGA analysis, we expect to see changes in immune cell recruitment and infiltration, specifically in quantity of immune cells recruited and infiltrating, and in the composition of the tumor associated immune cells between our DARC/ACKR1 expression or non-expressing BC Tg+ mice. Citation Format: Rachel Martini, Lisa Newman, Nancy Manley, Melissa Davis. In vivo investigation of the Duffy antigen receptor for chemokines (DARC/ACKR1) and tumor-infiltrating immune cells in the breast tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-08.

Plasmodium yoelii Erythrocyte-Binding-like Protein Modulates Host Cell Membrane Structure, Immunity, and Disease Severity.

Erythrocyte-binding-like (EBL) proteins are known to play an important role in malaria parasite invasion of red blood cells (RBCs); however, any roles of EBL proteins in regulating host immune responses remain unknown. Here, we show that Plasmodium yoelii EBL (PyEBL) can shape disease severity by modulating the surface structure of infected RBCs (iRBCs) and host immune responses. We identified an amino acid substitution (a change of C to Y at position 741 [C741Y]) in the protein trafficking domain of PyEBL between isogenic P. yoelliinigeriensis strain N67 and N67C parasites that produce different disease phenotypes in C57BL/6 mice. Exchanges of the C741Y alleles altered parasite growth and host survival accordingly. The C741Y substitution also changed protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduced PyEBL binding to band 3, increased phosphatidylserine (PS) surface exposure, and elevated the osmotic fragility of iRBCs, but it did not affect invasion of RBCs in vitro The modified iRBC surface triggered PS-CD36-mediated phagocytosis of iRBCs, host type I interferon (IFN-I) signaling, and T cell differentiation, leading to improved host survival. This study reveals a previously unknown role of PyEBL in regulating host-pathogen interaction and innate immune responses, which may be explored for developing disease control strategies.IMPORTANCE Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). Plasmodium yoelii EBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies.

The Crucial Role of CXCL8 and Its Receptors in Colorectal Liver Metastasis.

CXCL8 (also known as IL-8) can produce different biological effects by binding to its receptors: CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC). CXCL8 and its receptors are associated with the development of various tumor types, especially colorectal cancer and its liver metastases. In addition to promoting angiogenesis, proliferation, invasion, migration, and the survival of colorectal cancer (CRC) cells, CXCL8 and its receptors have also been known to induce the epithelial-mesenchymal transition (EMT) of CRC cells, to help them to escape host immunosurveillance as well as to enhance resistance to anoikis, which promotes the formation of circulating tumor cells (CTCs) and their colonization of distant organs. In this paper, we will review the established roles of CXCL8 signaling in CRC and discuss the possible strategies of targeting CXCL8 signaling for overcoming CRC drug resistance and cancer progression, including direct targeting of CXCL8/CXCR1/2 or indirect targeting through the inhibition of CXCL8-CXCR1/2 signaling.

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.

Duffy antigen receptor for chemokines gene polymorphisms and malaria in Mangaluru, India

BackgroundDuffy blood group antigens serve as receptors for Plasmodium vivax invasion into erythrocytes, and they are determined by polymorphisms of the Duffy antigen receptor for chemokines (DARC), also known as Fy glycoprotein (FY). Duffy negativity, i.e., absence of the antigens, protects against P. vivax infection and is rare among non-African populations. However, data on DARC polymorphisms and their impact on Plasmodium infection in India are scarce.MethodsIn a case–control study among 909 malaria patients and 909 healthy community controls in Mangaluru, southwestern India, DARC polymorphisms T-33C (rs2814778), G125A (rs12075), C265T (rs34599082), and G298A (rs13962) were genotyped. Associations of the polymorphisms with the odds of malaria, parasite species and manifestation were assessed.ResultsAmong patients, vivax malaria (70%) predominated over falciparum malaria (9%) and mixed species infections (21%). DARC T-33C was absent and C265T was rare (1%). FYB carriage (deduced from DARC G125A) was not associated with the risk of malaria per se but it protected against severe falciparum malaria (P = 0.03), and hospitalization (P = 0.006) due to falciparum malaria. Vice versa, carriage of DARC 298A was associated with increased odds of malaria (aOR, 1.46 (1.07–1.99), P = 0.015) and vivax malaria (aOR, 1.60 (1.14–2.22), P = 0.006) and with several reported symptoms and findings of the patients.ConclusionThis report from southern India is the first to show an independent effect of the DARC 298A polymorphism on the risk of malaria. Functional studies are required to understand the underlying mechanism. Moreover, FYB carriage appears to protect against severe falciparum malaria in southern India.

Cytokine Levels in Inner Ear Fluid of Young and Aged Mice as Molecular Biomarkers of Noise-Induced Hearing Loss.

Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, frequently caused by noise trauma and aging, with inflammation being implicated in both pathologies. Here, we provide the first direct measurements of proinflammatory cytokines in inner ear fluid, perilymph, of adolescent and 2-year-old mice. The perilymph of adolescent mice exposed to the noise intensity resulting in permanent auditory threshold elevations had significantly increased levels of IL-6, TNF-α, and CXCL1 6 h after exposure, with CXCL1 levels being most elevated (19.3 ± 6.2 fold). We next provide the first immunohistochemical localization of CXCL1 in specific cochlear supporting cells, and its presumed receptor, Duffy antigen receptor for chemokines (DARC), in hair cells and spiral ganglion neurons. Our results demonstrate the feasibility of molecular diagnostics of SNHL using only 0.5 μL of perilymph, and motivate future sub-μL based diagnostics of human SNHL based on liquid biopsy of the inner ear to guide therapy, promote hearing protection, and monitor response to treatment.

Abstract 4630: In vivo characterization of the Duffy antigen receptor for chemokines (DARC/ACKR1) in breast cancer tumor progression

Abstract In studies of the tumor microenvironment (TME), factors that influence immune cell infiltration are of great interest, as these populations can influence disease prognosis, and potential treatment for patients. Through in silico analysis of TCGA breast cancer (BC) cohort data, we have identified the Duffy antigen receptor for chemokine/atypical chemokine receptor 1 (DARC/ACKR1) as a potential driver of immune cell infiltration in the BC TME. DARC/ACKR1 is an atypical chemokine receptor that modulates levels of chemokine both in circulation through expression on red blood cells, and participates in chemokine transcytosis in tissues through expression on both post-capillary venules of endothelial cells and epithelial cells. As chemokines establish gradients to recruit specific immune cell types, we hypothesize that the function of DARC/ACKR1 in chemokine level modulation also influences tumor-associated immune cell levels. In the TCGA BC cohort, we observe a strongly positive and significant correlation between DARC/ACKR1 expression and total number of tumor-associated leukocytes, specifically populations of B cells, T cell, monocytes and macrophages. To further characterize the role of DARC/ACKR1 in vivo, we have established a novel DARC knock-out BC transgenic mouse model to study DARC/ACKR1 in the BC TME. To develop our target experimental mice, Ackr1-/- female mice were crossed with Ackr1 +/-; C3(1)Tag +/0 to generate the target Ackr1 -/-; C3(1)Tag +/0 experimental mice. Experimental mice and the littermate controls were aged to 3.5 months, at which point they were euthanized. Blood was collected from the mice to characterize the circulating chemokine profile. Mammary glands containing tumors were fixed and paraffin embedded, followed by IHC and immunofluorescent staining for expression of DARC/ACKR1, target chemokines, immune cells, epithelial cells and endothelial cells. The remaining glands were dissociated, followed by fluorescent-activating cell sorting analysis (FACS) to quantify abundance of immune cells in the gland. Using these methods, we have defined the circulating chemokine profile alongside patterns of immune cell infiltration in our target DARC/ACKR1 knockout BC mice compared to littermate controls in mouse mammary tumors. Citation Format: Rachel N. Martini, Brittany D. Jenkins, Lisa A. Newman, Nancy Manley, Melissa B. Davis. In vivo characterization of the Duffy antigen receptor for chemokines (DARC/ACKR1) in breast cancer tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4630.

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

SummaryThe most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells, through a process which requires interaction of the Plasmodium vivax Duffy binding protein, PvDBP with its human receptor, the Duffy antigen receptor for chemokines, DARC. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to Plasmodium vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunised in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to DARC, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of Plasmodium vivax. This identified a broadly neutralising human monoclonal antibody which inhibited invasion of all tested strains of Plasmodium vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Impact of DARC rs12075 Variants on Liver Fibrosis Progression in Patients with Chronic Hepatitis C: A Retrospective Study.

The Duffy antigen receptor for chemokines (DARC) rs12075 polymorphism regulates leukocyte trafficking and proinflammatory chemokine homeostasis. Hepatitis C virus (HCV)-mediated liver fibrosis is associated with an uncontrolled inflammatory response. In this study, we evaluate the association between the DARC rs12075 polymorphism and liver stiffness progression in HCV-infected patients. We carried out a retrospective cohort study (repeated measures design) in 208 noncirrhotic patients with chronic hepatitis C (CHC) who had at least two liver stiffness measurements (LSM) with a separation of at least 12 months. We used generalized linear models to analyze the association between DARC rs12075 polymorphism and outcome variables. During a follow-up of 46.6 months, the percentage of patients with stages of fibrosis F0/F1 decreased (p < 0.001), while LSM values and the percentage of patients with cirrhosis increased (p < 0.001). This pattern of changes was maintained in each of the groups of patients analyzed according to their rs12075 genotypes (AA or AG/GG). However, the variations in liver stiffness characteristics were lower in patients with the rs12075 AG/GG genotype (AG/GG versus AA). Thereby, in the adjusted analysis, patients with the rs12075 AG/GG genotype had a lower risk of an increased value of LSM2/LSM1 arithmetic mean ratio (AMR = 0.83; p = 0.001) and of an increase in LSM ≥ 5 kPa (odds ratio (OR) = 0.28; p = 0.009). Besides, patients with rs12075 AG/GG had a lower risk of cirrhosis progression (OR = 0.24; p = 0.009). No significant associations were found for an increase in LSM ≥ 10 kPa. We found an association between the DARC rs12075 single nucleotide polymorphism (SNP) and CHC progression. Specifically, patients with the DARC rs12075 AG/GG genotype had a lower risk of liver fibrosis progression and development of cirrhosis.

Neutrophil Effector Functions Are Not Impaired in Duffy Antigen Receptor for Chemokines (DARC)-Null Black South Africans.

Neutrophils are well-recognized for their pathogen killing mechanisms and disorders of neutrophil count and function are associated with recurrent infections. The Duffy Antigen Receptor for Chemokines (DARC)-null genotype is predominant in sub-Saharan African ancestry populations and is the major genetic determinant of benign ethnic neutropenia which has been associated with increased risk of Human Immunodeficiency Virus (HIV)-1 acquisition and mother-to-child transmission. However, the impact of DARC-null-linked neutropenia on HIV disease progression remains controversial. While the DARC-null genotype is associated with low numbers of circulating neutrophils, the effects of the polymorphism on neutrophil functions is unknown. We investigated the impact of the DARC-null trait and lower absolute neutrophil counts (ANCs) on key neutrophil effector functions [proteolytic activity within the phagosome following Fc receptor-mediated phagocytosis, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation] in 20 HIV negative and 22 HIV-1 chronically infected black South Africans. Phagosome maturation was measured by flow cytometry following Fc-mediated uptake of IgG opsonized beads; ROS production was measured by chemi-luminescence after activation of neutrophils with phorbol 12-myristate 13-acetate (PMA). Activated neutrophils were also visualized by fluorescent microscopy for NET quantification. Study subjects were genotyped for the DARC trait using TaqMan allelic discrimination assays and ANCs were measured by full blood count. As expected, the DARC-null polymorphism was highly prevalent in our participant cohort (69%) and was strongly associated with lower ANCs in uninfected (p = 0.0007) and HIV-1 infected (p = 0.03) subjects. We observed enhanced proteolytic activity within the phagosome in the absence of DARC at 10 min (p = 0.05 and p = 0.009) and 60 min (p = 0.05 and p = 0.07) in uninfected and HIV-1 infected subjects, respectively. ROS was unaffected by DARC trait irrespective of HIV status. Furthermore, formation of NETs was reduced in neutrophils from DARC-null subjects (p = 0.04) following prolonged in vitro stimulation, but only in HIV-1 infected subjects. The data indicate differential neutrophil function in the absence of DARC that may be moderately modulated by HIV-1 infection but overall, the data suggest that DARC-null trait is not deleterious to neutrophil effector functions in African populations.

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.