Duffy Antigen Receptor for Chemokines (DARC) Nanodisc-Based Biosensor for Detection of Staphylococcal Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus (S. aureus) is an opportunistic infectious pathogen, which causes a high mortality rate during bloodstream infections. The early detection of virulent strains in patients' blood samples is of medical interest for rapid diagnosis. The main virulent factors identified in patient isolates include leukocidins that bind to specific membrane receptors and lyse immune cells and erythrocytes. Duffy antigen receptor for chemokines (DARC) on the surface of specific cells is a main target of leukocidins such as gamma-hemolysin AB (HlgAB) and leukocidin ED (LukED). Among them, HlgAB is a conserved and critical leukocidin that binds to DARC and forms pores on the cell membranes, leading to cell lysis. Current methods are based on ELISA or bacterial culture, which takes hours to days. For detecting HlgAB with faster response and higher sensitivity, we developed a biosensor that combines single-walled carbon nanotube field effect transistors (swCNT-FETs) with immobilized DARC receptors as biosensing elements. DARC was purified from a bacterial expression system and successfully reconstituted into nanodiscs that preserve binding capability for HlgAB. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed an increase of the DARC-containing nanodisc size in the presence of HlgAB, indicating the formation of HlgAB prepore or pore complexes. We demonstrate that this sensor can specifically detect the leukocidins HlgA and HlgAB in a quantitative manner within the dynamic range of 1 fM to 100 pM with an LOD of 0.122 fM and an LOQ of 0.441 fM. The sensor was challenged with human serum spiked with HlgAB as simulated clinical samples. After dilution for decreasing nonspecific binding, it selectively detected the toxin with a similar detection range and apparent dissociation constant as in the buffer. This biosensor was demonstrated with remarkable sensitivity to detect HlgAB rapidly and has the potential as a tool for fundamental research and clinical applications, although this sensor cannot differentiate between HlgAB and LukED as both have the same receptor.

Unveiling P. vivax invasion pathways in Duffy-negative individuals.

Vivax malaria has long been thought to be absent from sub-Saharan Africa owing to the high proportion of individuals lacking the Duffy antigen receptor for chemokines (DARC) in their erythrocytes. The interaction between P. vivax Duffy-binding protein (PvDBP) and DARC is assumed to be the main pathway used by merozoites to invade reticulocytes. However, the increasing number of reports of vivax malaria cases in genotypically Duffy-negative (DN) individuals has raised questions regarding the P. vivax invasion pathway(s). Here, we show that a subset of DN erythroblasts transiently express DARC during terminal erythroid differentiation and that P. vivax merozoites, irrespective of their origin, can invade DARC+ DN erythroblasts. These findings reveal that a large number of DN individuals may represent a silent reservoir of deep P. vivax infections at the sites of active erythropoiesis with low or no parasitemia, and it may represent an underestimated biological problem with potential clinical consequences in sub-Saharan Africa.

Designing an effective malaria vaccine targeting Plasmodium vivax Duffy-binding protein.

Malaria caused by the Plasmodium vivax parasite is a major global health burden. Immunity against blood-stage infection reduces parasitemia and disease severity. Duffy-binding protein (DBP) is the primary parasite protein responsible for the invasion of red blood cells and it is a leading subunit vaccine candidate. An effective vaccine, however, is still lacking despite decades of interest in DBP as a vaccine candidate. This review discusses the reasons for targeting DBP, the challenges associated with developing a vaccine, and modern structural vaccinology methods that could be used to create an effective DBP vaccine. Next-generation DBP vaccines have the potential to elicit a broadly protective immune response and provide durable and potent protection from P. vivax malaria.

The DARC Side of Inflamm-Aging: Duffy Antigen Receptor for Chemokines (DARC/ACKR1) as a Potential Biomarker of Aging, Immunosenescence, and Breast Oncogenesis among High-Risk Subpopulations.

The proclivity of certain pre-malignant and pre-invasive breast lesions to progress while others do not continues to perplex clinicians. Clinicians remain at a crossroads with effectively managing the high-risk patient subpopulation owing to the paucity of biomarkers that can adequately risk-stratify and inform clinical decisions that circumvent unnecessary administration of cytotoxic and invasive treatments. The immune system mounts the most important line of defense against tumorigenesis and progression. Unfortunately, this defense declines or "ages" over time-a phenomenon known as immunosenescence. This results in "inflamm-aging" or the excessive infiltration of pro-inflammatory chemokines, which alters the leukocyte composition of the tissue microenvironment, and concomitant immunoediting of these leukocytes to diminish their antitumor immune functions. Collectively, these effects can foster the sequelae of neoplastic transformation and progression. The erythrocyte cell antigen, Duffy antigen receptor for chemokines(DARC/ACKR1), binds and internalizes chemokines to maintain homeostatic levels and modulate leukocyte trafficking. A negative DARC status is highly prevalent among subpopulations of West African genetic ancestry, who are at higher risk of developing breast cancer and disease progression at a younger age. However, the role of DARC in accelerated inflamm-aging and malignant transformation remains underexplored. Herein, we review compelling evidence suggesting that DARC may be protective against inflamm-aging and, therefore, reduce the risk of a high-risk lesion progressing to malignancy. We also discuss evidence supporting that immunotherapeutic intervention-based on DARC status-among high-risk subpopulations may evade malignant transformation and progression. A closer look into this unique role of DARC could glean deeper insight into the immune response profile of individual high-risk patients and their predisposition to progress as well as guide the administration of more "cyto-friendly" immunotherapeutic intervention to potentially "turn back the clock" on inflamm-aging-mediated oncogenesis and progression.

COVID-19: Mechanistic Model of the African Paradox Supports the Central Role of the NF-κB Pathway.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has expanded into a global pandemic, with more than 220 million affected persons and almost 4.6 million deaths by 8 September 2021. In particular, Europe and the Americas have been heavily affected by high infection and death rates. In contrast, much lower infection rates and mortality have been reported generally in Africa, particularly in the sub-Saharan region (with the exception of the Southern Africa region). There are different hypotheses for this African paradox, including less testing, the young age of the population, genetic disposition, and behavioral and epidemiological factors. In the present review, we address different immunological factors and their correlation with genetic factors, pre-existing immune status, and differences in cytokine induction patterns. We also focus on epidemiological factors, such as specific medication coverage, helminth distribution, and malaria endemics in the sub-Saharan region. An analysis combining different factors is presented that highlights the central role of the NF-κB signaling pathway in the African paradox. Importantly, insights into the interplay of different factors with the underlying immune pathological mechanisms for COVID-19 can provide a better understanding of the disease and the development of new targets for more efficient treatment strategies.

Hematologic safety of palbociclib in combination with endocrine therapy in patients with benign ethnic neutropenia and advanced breast cancer.

BACKGROUND: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, including palbociclib, are approved to treat hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer (ABC) and are associated with hematologic toxicity. African American women, who are underrepresented in CDK4/6 inhibitor clinical trials, may experience worse neutropenia because of benign ethnic neutropenia. The authors specifically investigated the hematologic safety of palbociclib in African American women with HR-positive/HER2-negative ABC. METHODS: PALINA was a single-arm, open-label, investigator-initiated study of palbociclib (125 mg daily; 21 days on and 7 days off) plus endocrine therapy (ET) in African American women who had HR-positive/HER2-negative ABC and a baseline absolute neutrophil count ≥1000/mm3 (ClinicalTrials.gov identifier NCT02692755). The primary outcome was the proportion of patients who completed 12 months of therapy without experiencing febrile neutropenia or treatment discontinuation because of neutropenia. Single nucleotide polymorphism analysis was used to assess Duffy polymorphism status. RESULTS: Thirty-five patients received ≥1 dose of palbociclib plus ET; 19 had a Duffy null polymorphism (cytosine/cytosine). There were no reports of febrile neutropenia or permanent study discontinuation because of neutropenia. Significantly more patients with the Duffy null versus the wild-type variant had grade 3 and 4 neutropenia (72.2% vs 23.1%; P = .029) and required a palbociclib dose reduction (55.6% vs 7.7%; P = .008). Patients with the Duffy null versus the wild-type variant had lower overall relative dose intensity (mean ± SD, 81.89% ± 15.87 and 95.67% ± 5.89, respectively; P = .0026) and a lower clinical benefit rate (66.7% and 84.6%, respectively). CONCLUSIONS: These findings suggest that palbociclib is well tolerated in African American women with HR-positive/HER2-negative ABC. Duffy null status may affect the incidence of grade 3 neutropenia, dose intensity, and possibly clinical benefit.

Renal allograft DARCness in subclinical acute and chronic active ABMR.

Gene 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.

Presence of additional Plasmodium vivax malaria in Duffy negative individuals from Southwestern Nigeria.

BACKGROUND: Malaria in sub-Saharan Africa (sSA) is thought to be mostly caused by Plasmodium falciparum. Recently, growing reports of cases due to Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax have been increasingly observed to play a role in malaria epidemiology in sSA. This in fact is due to the usage of very sensitive diagnostic tools (e.g. PCR), which have highlighted the underestimation of non-falciparum malaria in this sub-region. Plasmodium vivax was historically thought to be absent in sSA due to the high prevalence of the Duffy negativity in individuals residing in this sub-continent. Recent studies reporting detection of vivax malaria in Duffy-negative individuals from Mali, Mauritania, Cameroon challenge this notion. METHODS: Following previous report of P. vivax in Duffy-negative individuals in Nigeria, samples were further collected and assessed RDT and/or microscopy. Thereafter, malaria positive samples were subjected to conventional PCR method and DNA sequencing to confirm both single/mixed infections as well as the Duffy status of the individuals. RESULTS: Amplification of Plasmodium gDNA was successful in 59.9% (145/242) of the evaluated isolates and as expected P. falciparum was the most predominant (91.7%) species identified. Interestingly, four P. vivax isolates were identified either as single (3) or mixed (one P. falciparum/P. vivax) infection. Sequencing results confirmed all vivax isolates as truly vivax malaria and the patient were of Duffy-negative genotype. CONCLUSION: Identification of additional vivax isolates among Duffy-negative individuals from Nigeria, substantiate the expanding body of evidence on the ability of P. vivax to infect RBCs that do not express the DARC gene. Hence, such genetic-epidemiological study should be conducted at the country level in order to evaluate the true burden of P. vivax in Nigeria.

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.

Engagement Rules That Underpin DBL-DARC Interactions for Ingress of Plasmodium knowlesi and Plasmodium vivax Into Human Erythrocytes.

Malaria parasite erythrocytic stages comprise of repeated bursts of parasites via cyclical invasion of host erythrocytes using dedicated receptor-ligand interactions. A family of erythrocyte-binding proteins from Plasmodium knowlesi (Pk) and Plasmodium vivax (Pv) attach to human Duffy antigen receptor for chemokines (DARC) via their Duffy binding-like domains (DBLs) for invasion. Here we provide a novel, testable and overarching interaction model that rationalizes even contradictory pieces of evidence that have so far existed in the literature on Pk/Pv-DBL/DARC binding determinants. We further address the conundrum of how parasite-encoded Pk/Pv-DBLs recognize human DARC and collate evidence for two distinct DARC integration sites on Pk/Pv-DBLs.

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.

The Duffy antigen receptor for chemokines regulates asthma pathophysiology.

BACKGROUND: The Duffy antigen receptor for chemokines (DARC) is an atypical receptor that regulates pro-inflammatory cytokines. However, the role of DARC in asthma pathophysiology is unknown. OBJECTIVE: To determine the role of DARC in allergic airways disease in mice, and the association between DARC single nucleotide polymorphisms (SNPs) and clinical outcomes in patients with asthma. METHODS: Mice with targeted disruption of the Darc gene (Darc∆E2 ) or WT mice were challenged over 3 weeks with house dust mite (HDM) antigen. Allergic airways disease was assessed 24 hours and 7 days following the final challenge. Additionally, associations between DARC SNPs and clinical outcomes were analysed in a cohort of poorly controlled asthmatics. RESULTS: Total airway inflammation following HDM did not differ between Darc∆E2 and WT mice. At 24 hours, Darc∆E2 mice had increased airway hyperresponsiveness; however, at 7 days airway hyperresponsiveness had completely resolved in Darc∆E2 but persisted in WT mice. In poorly controlled asthmatics, DARC SNPs were associated with worse asthma control at randomization and subsequent increased risk of healthcare utilization (odds ratio 3.13(1.37-7.27), P=.0062). CONCLUSIONS AND CLINICAL RELEVANCE: Our animal model and human patient data suggest a novel role for DARC in the temporal regulation in asthma pathophysiology and symptoms.

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.

Naturally Acquired Binding-Inhibitory Antibodies to Plasmodium vivax Duffy Binding Protein and Clinical Immunity to Malaria in Rural Amazonians.

BACKGROUND: Antibodies to the cysteine-rich domain II of Plasmodium vivax Duffy binding protein (PvDBP) can inhibit binding of this parasite ligand to its receptor on red blood cells, the Duffy antigen/receptor for chemokines. These binding-inhibitory antibodies (BIAbs) also inhibit P. vivax invasion of reticulocytes in vitro. METHODS: To investigate whether naturally acquired anti-PvDBP antibodies are associated with reduced risk of clinical malaria in a population exposed to low levels of P. vivax transmission, we measured total levels of immunoglobulin G antibodies to 5 PvDBP variants and used a functional in vitro assay to quantify their binding-inhibitory activity in a cohort of 466 rural Amazonians followed up for up to 37 months. RESULTS: No association between total immunoglobulin G antibody responses to any PvDBP variant and risk of symptomatic, laboratory-confirmed vivax malaria was observed in this cohort. However, a Cox proportional hazards model, adjusted for age, sex, and genotype for the Duffy antigen/receptor for chemokines, showed a >40% decrease in the prospective risk of clinical vivax malaria in subjects with the strongest BIAb responses (upper and middle terciles). High BIAb responses were mostly PvDBP variant transcending and stable over time. CONCLUSIONS: Strong naturally acquired BIAb responses are associated with a reduced risk of clinical P. vivax malaria in rural Amazonians.

Potential Role of DARC-Chemokine Interaction in the Recruitment of Osteoclast Precursors in Response to Bacterial Lipopolysaccharide Challenge.

Duffy antigen receptor for chemokines (DARC) binds to a number of pro-inflammatory chemokines, and since chemokines are known to regulate trafficking of osteoclast (OC) precursors, we predicted that DARC would regulate OC recruitment to sites of inflammation by modulating chemokine activity. To test this hypothesis, we evaluated the mRNA expression of Darc and the chemokines known to bind to DARC, in endothelial cells treated with bacterial lipopolysaccharide (LPS). The mRNA expression of Mcp-1, Rantes, Darc and Ccr5 was significantly increased in endothelial cells in response to LPS treatment. Blocking the function of DARC with neutralizing antibody partially abrogated the effect of LPS on the mRNA expression of Mcp-1 and Rantes. In vivo, mice with targeted disruption of Darc gene (Darc-KO) and control wild-type (WT) mice were used to assess the role of DARC in response to single LPS application on the top of parietal bones. Five hours post-LPS injection, local expression of Cd14 mRNA (a marker of inflammatory monocytes) was significantly increased in both lines of mice. However, the magnitude of increase was greater in WT mice compared with Darc-KO mice suggesting a role for DARC in mediating the recruitment of monocytes in response to LPS. Histological staining for tartrate-resistant acid phosphatase (TRAP) in calvaria sections taken from the injection sites revealed a significant reduction in TRAP-labeled surface per bone surface in response to LPS in Darc-KO mice compared with WT mice. Based on these findings, we concluded that DARC regulates recruitment of OC precursors at the inflammation site, probably through regulation of chemokines transcytosis across endothelial cell barrier.

The role of Duffy antigen receptor for chemokines in keloids.

This study aims to determine the relationship between Duffy antigen receptor for chemokines (DARC) and keloid pathogenesis. DARC expression was determined by immunohistochemistry, real-time PCR, and Western blot analysis. Cell proliferation was assessed by CCK-8 assay. Cell migration and invasion abilities were measured by the shift assay. Levels of CC chemokine ligand 2 (CCL2), CXC chemokine ligand 8 (CXCL8), and matrix metalloproteinase 2 (MMP2) were detected by real-time PCR and ELISA. Our results showed that DARC levels were elevated in human keloid fibroblasts. After knocking down DARC, cell proliferation was not altered, whereas the migration and invasion abilities of keloid fibroblasts were significantly elevated. Additionally, the mRNA expression levels of CCL2, CXCL8, and MMP2 were not influenced by DARC knockdown. However, the secretion of CCL2, but not CXCL8 or MMP2, was significantly increased after DARC knockdown. Our results suggest that DARC might inhibit the secretion of CCL2. Moreover, DARC knockdown increases the migration and invasion abilities of keloid fibroblasts.

Immunogenicity of single versus mixed allele vaccines of Plasmodium vivax Duffy binding protein region II.

The Duffy binding protein (DBP) of Plasmodium vivax is vital for host erythrocyte invasion. DBP region II (DBPII) contains critical residues for receptor recognition and anti-DBPII antibodies have been shown to inhibit erythrocyte binding and invasion, thereby making the molecule an attractive vaccine candidate against P. vivax blood stages. Similar to other blood-stage antigens, allelic variation within the DBPII and associated strain-specific immunity is a major challenge for development of a broadly effective vaccine against P. vivax malaria. We hypothesized that immunization with a vaccine composed of multiple DBP alleles or a modified epitope DBP (DEKnull) will be more effective in producing a broadly reactive and inhibitory antibody response to diverse DBPII alleles than a single allele vaccine. In this study, we compared single, naturally occurring DBPII allele immunizations (Sal1, 7.18, P) and DEKnull with a combination of (Sal1, 7.18, P) alleles. Quantitative analysis by ELISA demonstrated that the multiple allele vaccine tend to be more immunogenic than any of the single allele vaccines when tested for reactivity against a panel of DBPII allelic variants whereas DEKnull was less immunogenic than the mixed-allele vaccine but similar in reactivity to the single allele vaccines. Further analysis for functional efficacy by in vitro erythrocyte-binding inhibition assays demonstrated that the multiple allele immunization produced a stronger strain-neutralizing response than the other vaccination strategies even though inhibition remained biased toward some alleles. Overall, there was no correlation between antibody titer and functional inhibition. These data suggest that a multiple allele vaccine may enhance immunogenicity of a DBPII vaccine but further investigation is required to optimize this vaccine strategy to achieve broader coverage against global P. vivax strains.