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BACKGROUND: ACKR1 blood group genes exhibit a high degree of polymorphisms with varying allele distribution seen among different populations and ethnic groups. The study aimed to genotype ACKR1 antigens and to establish FY allele frequency among the individuals with the Bombay (Oh) blood group phenotype. MATERIALS AND METHODS: ACKR1 phenotype and genotype frequencies were estimated on 160 individuals typed as Oh and were compared with 100 non-oh blood donors from Mumbai, India by molecularly genotyping via PCR-RFLP. RESULTS: The allelic and genotypic frequency of T(-67)C polymorphism showed the dominance of T allele and TT genotype [OR= 3.26 (0.59-17.99)] in both the study groups. The ACKR1 null (Fya-b-) phenotype was not found in the tested group. While the genotypic combination among the Oh group individuals was FYA/FYB (45.3 %), FYA/FYA (42.7 %), and FYB/FYB (12 %), in the non-Oh group donors, it was observed as FYA/FYB (53.3 %), FYA/FYA (39.1 %), and FYB/FYB (7.6 %). The haplotype TGGGC occurred in 38.4 % of the Oh group, but in non-Oh donors, it was found to be 50.9 % [OR = 1.820 (1.196-2.771)], and the difference was statistically significant (p = 0.005). Similarly, the TGGGT haplotype was found at a frequency of 12.7 % in non-Oh donors and 27.1 % in Oh group [OR= 0.411 (0.234-0.722)] (p = 0.001). CONCLUSIONS: This study shows the prevalence of ACKR1 gene polymorphisms, including weak ACKR1 antigens in Oh individuals with a high frequency of haplotype TGGGC. The present study demonstrated for the first time the genotypes FyBweak, FyAweak and Fy Aweak/FyBESon RBC membranes in Indian subjects with Oh phenotype.

Asunto(s)

Polimorfismo Genético , Humanos , India , Femenino , Masculino , Genotipo , Frecuencia de los Genes , Adulto , Antígenos de Grupos Sanguíneos/genética , Sistema del Grupo Sanguíneo Duffy/genética , Receptores de Superficie Celular/genética

RESUMEN

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.

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Microscopía por Crioelectrón , Sistema del Grupo Sanguíneo Duffy , Receptores de Superficie Celular , Humanos , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/química , Sistema del Grupo Sanguíneo Duffy/metabolismo , Sistema del Grupo Sanguíneo Duffy/química , Transducción de Señal , Sitios de Unión , Quimiocinas/metabolismo , Quimiocinas/química , Unión Proteica

RESUMEN

Sarcoma is a malignant tumor originating from mesenchymal tissue with a poor prognosis. Atypical chemokine receptor 1 (ACKR1) is found closely related to cancer progression. However, the effects of ACKR1 in soft tissue sarcoma have not been well investigated. Therefore, our present study is devoted to analyze the functions of ACKR1 in sarcoma progression and its potential mechanism. We detected the expression of ACKR1 in the Cancer Genome Atlas (TCGA)-pan-cancer database, TCGA-Sarcoma from TCGA databases, and GSE21122 from Gene Expression Omnibus (GEO) database. The relationships between ACKR1 expression, clinicopathological data, and survival status were evaluated in the TCGA-Sarcoma database. Moreover, overexpression negative control (OE-NC) and overexpression ACKR1 (OE-ACKR1) were used to further verify the effects of ACKR1 overexpression in the progression of sarcoma cells by using Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), cell counting kit-8 (CCK-8), 5-Ethyny-2'-Deoxyuridine (EdU), wound healing, transwell assay, and flow cytometry assays. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) analyses were carried out to explore the potential enriched biological process of ACKR1 expression in sarcoma. Furthermore, tumor-immune system interactions databases (TISIDB) were applied to further confirm the relations between ACKR1 and tumor immune microenvironment in sarcoma. Our study found that ACKR1 is downregulated in multiple cancers (including sarcoma), and low expression of ACKR1 is related to poor survival status in sarcoma. The biological experiments found that promoting expression of ACKR1 can suppress sarcoma cell proliferation, migration, invasion, promote cell apoptosis, and arrest cell cycle. The GO-KEGG, GSEA, and TISIDB analysis showed that ACKR1 is related to the tumor immune microenvironment. In conclusion, low expression of ACKR1 presented as an independent prognostic biomarker in sarcoma. Overexpression of ACKR1 can significantly suppress cell progression ability in sarcoma by regulating the immune microenvironment.

Asunto(s)

Sarcoma , Humanos , Sarcoma/genética , Apoptosis , Proliferación Celular/genética , Citometría de Flujo , Pronóstico , Microambiente Tumoral/genética

RESUMEN

INTRODUCTION: Angiogenesis involves the development of new blood vessels. Biochemical signals start this process in the body, which is followed by migration, growth, and differentiation of endothelial cells that line the inside wall of blood vessels. This process is vital for the growth of cancer cells and tumors. MATERIALS AND METHODS: We started our analysis by composing a list of genes that have a validated impact in humans with respect to angiogenesis-related phenotypes. Here, we have investigated the expression patterns of angiogenesis-related genes in the context of previously published single-cell RNA-Seq data from prostate and breast cancer samples. RESULTS: Using a protein-protein interaction network, we showed how different modules of angiogenesis-related genes are overexpressed in different cell types. In our results, genes, such as ACKR1, AQP1, and EGR1, showed a strong cell type-dependent overexpression pattern in the two investigated cancer types, which can potentially be helpful in the diagnosis and follow-up of patients with prostate and breast cancer. CONCLUSION: Our work demonstrates how different biological processes in distinct cell types contribute to the angiogenesis process, which can provide clues regarding the potential application of targeted inhibition of the angiogenesis process.

Asunto(s)

Neoplasias de la Mama , Neoplasias de la Próstata , Masculino , Humanos , Células Endoteliales/metabolismo , Angiogénesis , Neoplasias de la Próstata/genética , Neoplasias de la Mama/patología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo

RESUMEN

BACKGROUND/AIM: Tyrosine kinase inhibitor (TKI) therapy, a principal treatment for advanced non-small cell lung cancer (NSCLC), frequently encounters the development of drug resistance. The tumor microenvironment (TME) plays a critical role in the progression of NSCLC, yet the relationship between endothelial cells (ECs) and cancer-associated fibroblasts (CAFs) subpopulations in TKI treatment resistance remains largely unexplored. MATERIALS AND METHODS: The BioProject database PRJNA591860 project was used to analyze scRNA-seq data including 49 advanced-stage NSCLC samples across three different time points: pre-targeted therapy (naïve), post-partial response (PR) to targeted therapy, and post-progressive disease (PD) stage. The data involved clustering stromal cells into multiple CAFs and ECs subpopulations. The abundance changes and functions of each cluster during TKI treatment were investigated by KEGG and GO analysis. Additionally, we identified specific transcription factors and metabolic pathways via DoRothEA and scMetabolism. Moreover, cell-cell communications between PD and PR stages were compared by CellChat. RESULTS: ECs and CAFs were clustered and annotated using 49 scRNA-seq samples. We identified seven ECs subpopulations, with OIT3 ECs showing enrichment in the PR phase with a drug-resistance phenotype, and ACKR1 ECs being prevalent in the PD phase with enhanced cell adhesion. Similarly, CAFs were clustered into 7 subpopulations. PLA2G2A CAFs were predominant in PR, whereas POSTN CAFs were prevalent in PD, characterized by an immunomodulatory phenotype and increased collagen secretion. CellChat analysis showed that ACKR1 ECs strongly interacted with macrophage through the CD39 pathway and POSTN CAFs secreted Tenascin-C (TNC) to promote the progression of epithelial cells, primarily malignant ones, in PD. CONCLUSION: This study reveals that POSTN CAFs and ACKR1 ECs are associated with resistance to TKI treatment, based on single-cell sequencing.

Asunto(s)

Fibroblastos Asociados al Cáncer , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Perfilación de la Expresión Génica , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Microambiente Tumoral/genética

RESUMEN

INTRODUCTION: Clozapine is the most effective antipsychotic for treatment-resistant schizophrenia, but it is markedly underutilized, particularly in the US Black population, partly because of concern over clozapine-associated low absolute neutrophil count (ANC). People of African descent have a lower normative ANC range than the White population, which is associated with a specific "ACKR1-null" ("Duffy null") CC genotype (SNP rs2814778) on the ACKR1 gene, termed benign ethnic neutropenia (BEN). The range of ANC variability and safety of clozapine have not been established in people with BEN or examined prospectively in people of African descent. METHODS: We completed a multisite, 6-month, prospective, open-label clinical trial of clozapine treatment in people of African descent with schizophrenia spectrum disorders for whom clozapine was clinically indicated, with or without the ACKR1-null genotype. We examined clozapine safety and weekly ANC during clozapine treatment and evaluated ANC variability by ACKR1-null genotype, sex, study site, and clozapine dosing using repeated measures analysis of covariance. Genotype was assayed using TaqMan® technology. RESULTS: We enrolled 274 participants, of whom 227 (82.8 %) completed 6 months of clozapine treatment. There was one case of severe neutropenia (<500 cells/mm3) (0.36 %) over 1467.6 person-months of clozapine exposure. This participant recovered without sequelae after discontinuation of clozapine. Of the 249 participants with known genotypes, 199 (79.9 %) had the ACKR1-null genotype. Neutropenia (<1500 cells/mm3) occurred significantly more often in the ACKR1-null group (33 % [65/199]) than in those with the T allele (6 % (3/50); p < 0.001). Fourteen (5 %) patients discontinued due to adverse events. Rates of infection and fever were low and sialorrhea was the commonest side effect (N = 187, 68 %). CONCLUSION: To our knowledge, this is the largest prospective clozapine trial in people of African descent. Severe neutropenia was rare, despite the high prevalence (80 %) of the ACKR1-null genotype. Our findings suggest that clozapine can be used safely in Black patients including those with BEN.

RESUMEN

Atypical chemokine receptors (ACKRs) form a small subfamily of receptors (ACKR1-4) unable to trigger G protein-dependent signaling in response to their ligands. They do, however, play a crucial regulatory role in chemokine biology by capturing, scavenging or transporting chemokines, thereby regulating their availability and signaling through classical chemokine receptors. ACKRs add thus another layer of complexity to the intricate chemokine-receptor interaction network. Recently, targeted approaches and screening programs aiming at reassessing chemokine activity towards ACKRs identified several new pairings such as the dimeric CXCL12 with ACKR1, CXCL2, CXCL10 and CCL26 with ACKR2, the viral broad-spectrum chemokine vCCL2/vMIP-II, a range of opioid peptides and PAMP-12 with ACKR3 as well as CCL20 and CCL22 with ACKR4. Moreover, GPR182 (ACKR5) has been lately proposed as a new promiscuous atypical chemokine receptor with scavenging activity notably towards CXCL9, CXCL10, CXCL12 and CXCL13. Altogether, these findings reveal new degrees of complexity of the chemokine network and expand the panel of ACKR ligands and regulatory functions. In this minireview, we present and discuss these new pairings, their physiological and clinical relevance as well as the opportunities they open for targeting ACKRs in innovative therapeutic strategies.

Asunto(s)

Relevancia Clínica , Transducción de Señal , Ligandos , Quimiotaxis , Unión Proteica

RESUMEN

Background: At present, research on immunogenic cell death (ICD) is mainly associated with cancer therapy. Little is known about the role of ICD in cardiovascular disease, especially in ascending thoracic aortic aneurysms (ATAA). Method: ATAA single-cell RNA (scRNA) sequencing data were analyzed to identify the involved cell types and determine their transcriptomic characteristics. The chi-square test, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, Gene Set Enrichment Analysis (GSEA), and CellChat for cell-to-cell communication analysis from the Gene Expression Omnibus (GEO) database were used. Result: A total of 10 cell types were identified, namely, monocytes, macrophages, CD4 T/NK (CD4+ T cells and natural killer T cells), mast cells, B/Plasma B cells, fibroblasts, endothelial cells, cytotoxic T cells (CD8+ T cells, CTLs), vascular smooth muscle cells (vSMCs), and mature dendritic cells (mDCs). A large number of inflammation-related pathways were present in the GSEA results. A large number of ICD-related pathways were found in the KEGG enrichment analysis of differentially expressed genes in endothelial cells. The number of mDCs and CTLs in the ATAA group was significantly different from that in the control group. A total of 44 pathway networks were obtained, of which 9 were associated with ICD in endothelial cells (CCL, CXCL, ANNEXIN, CD40, IL1, IL6, TNF, IFN-II, GALECTIN). The most important ligand-receptor pair by which endothelial cells act on CD4 T/NK cells, CTLs and mDCs is CXCL12-CXCR4. The most important ligand-receptor pair by which endothelial cells act on monocytes and macrophages is ANXA1-FPR1. The most important ligand-receptor pair by which CD4 T/NK cells and CTLs act on endothelial cells is CCL5-ACKR1. The most important ligand-receptor pair that myeloid cells (macrophages, monocytes and mDCs) act on endothelial cells is CXCL8-ACKR1. Moreover, vSMCs and fibroblasts mainly promote inflammatory responses through the MIF signaling pathway. Conclusion: ICD is present in ATAA and plays an important role in the development of ATAA. The target cells of ICD may be mainly endothelial cells, in which the aortic endothelial cell ACKR1 receptor can not only promote T-cell infiltration through the CCL5 ligand but also promote myeloid cell infiltration through the CXCL8 ligand. ACKR1 and CXCL12 may become target genes for ATAA drug therapy in the future.

Asunto(s)

Aneurisma de la Aorta Torácica , Células Endoteliales , Humanos , Ligandos , Células Endoteliales/metabolismo , Muerte Celular Inmunogénica , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/metabolismo , Linfocitos T CD8-positivos/metabolismo

RESUMEN

The chemokine network is comprised of a family of signal proteins that encode messages for cells displaying chemokine G-protein coupled receptors (GPCRs). The diversity of effects on cellular functions, particularly directed migration of different cell types to sites of inflammation, is enabled by different combinations of chemokines activating signal transduction cascades on cells displaying a combination of receptors. These signals can contribute to autoimmune disease or be hijacked in cancer to stimulate cancer progression and metastatic migration. Thus far, three chemokine receptor-targeting drugs have been approved for clinical use: Maraviroc for HIV, Plerixafor for hematopoietic stem cell mobilization, and Mogalizumab for cutaneous T-cell lymphoma. Numerous compounds have been developed to inhibit specific chemokine GPCRs, but the complexity of the chemokine network has precluded more widespread clinical implementation, particularly as anti-neoplastic and anti-metastatic agents. Drugs that block a single signaling axis may be rendered ineffective or cause adverse reactions because each chemokine and receptor often have multiple context-specific functions. The chemokine network is tightly regulated at multiple levels, including by atypical chemokine receptors (ACKRs) that control chemokine gradients independently of G-proteins. ACKRs have numerous functions linked to chemokine immobilization, movement through and within cells, and recruitment of alternate effectors like ß-arrestins. Atypical chemokine receptor 1 (ACKR1), previously known as the Duffy antigen receptor for chemokines (DARC), is a key regulator that binds chemokines involved in inflammatory responses and cancer proliferation, angiogenesis, and metastasis. Understanding more about ACKR1 in different diseases and populations may contribute to the development of therapeutic strategies targeting the chemokine network.

Asunto(s)

Compuestos Heterocíclicos , Neoplasias , Humanos , Movilización de Célula Madre Hematopoyética , Neoplasias/metabolismo , Receptores de Quimiocina/metabolismo , Quimiocinas/metabolismo

RESUMEN

【Objective】 To determine the effect of storage time on the chemokine storage and chemokine scavenging function of erythrocyte atypical chemokine receptor 1(ACKR1). 【Methods】 Samples from six bags of red blood cells product, split into two gruoups(10 mL each), were stored in a refrigerator and sampled on day 5 and day 25 during storage, respectively, to measure the concentrations of CCL5, CXCL8 and CCL11 and the ACKR1 chemokine scavenging function of erythrocytes. In addition, 42 erythrocyte products(1 mL each), stored for 5 to 25 days, were sampled to measure the expression of ACKR1 and the concentrations of chemokines CCL5, CXCL8 and CCL11 on the erythrocyte membrane. 【Results】 Compared with RBCs stored for 5 days, no difference in the concentration of CCL5(467.7±250.2 pg/mL vs 586.9±209.5pg/mL, P>0.05) and CCL11(122.2±30.3pg/mL vs 125.5 ±32.7pg/mL, P>0.05)were noticed in erythrocyte lysates stored for 25 days, but the concentrations of CXCL8 decreased significantly(42.4±5.3pg/mL vs 24.3± 5.9pg/mL, P0.05). 【Conclusion】 Erythrocytes are the main places for storing ACKR1 binding chemokines. During the storage, the chemokine scavenging of erythrocyte ACKR1 and some intracellular the ACKR1 binding chemokines are reduced.

RESUMEN

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Asunto(s)

Envejecimiento/inmunología , Transporte Biológico/inmunología , Inflamación/inmunología , Neutrófilos/inmunología , Animales , Quimiocina CXCL1/inmunología , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Femenino , Uniones Intercelulares/inmunología , Pulmón/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-8B/inmunología , Vénulas/inmunología

RESUMEN

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton-Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE-LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE's rim domain contribute to hemolysis, namely residues 57-75 (DR1) and residues 182-196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE-DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.

Asunto(s)

Proteínas Bacterianas , Sistema del Grupo Sanguíneo Duffy , Eritrocitos , Exotoxinas , Proteínas Hemolisinas , Receptores de Superficie Celular , Staphylococcus aureus , Animales , Humanos , Ratones , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistema del Grupo Sanguíneo Duffy/química , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/metabolismo , Eritrocitos/química , Eritrocitos/metabolismo , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/metabolismo , Dominios Proteicos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo

RESUMEN

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

Asunto(s)

Sistema del Grupo Sanguíneo Duffy/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Bases , Brasil , Humanos , Fenotipo

RESUMEN

BACKGROUND: Blood-retinal barrier cells are known to exhibit a massive phenotypic change during experimental autoimmune uveitis (EAU) development. In an attempt to investigate the mechanisms of blood-retinal barrier (BRB) breakdown at a global level, we studied the gene regulation of total retinal cells and retinal endothelial cells during non-infectious uveitis. METHODS: Retinal endothelial cells were isolated by flow cytometry either in Tie2-GFP mice (CD31+ CD45- GFP+ cells), or in wild type C57BL/6 mice (CD31+ CD45- endoglin+ cells). EAU was induced in C57BL/6 mice by adoptive transfer of IRBP1-20-specific T cells. Total retinal cells and retinal endothelial cells from naïve and EAU mice were sorted and their gene expression compared by RNA-Seq. Protein expression of selected genes was validated by immunofluorescence on retinal wholemounts and cryosections and by flow cytometry. RESULTS: Retinal endothelial cell sorting in wild type C57BL/6 mice was validated by comparative transcriptome analysis with retinal endothelial cells sorted from Tie2-GFP mice, which express GFP under the control of the endothelial-specific receptor tyrosine kinase promoter Tie2. RNA-Seq analysis of total retinal cells mainly brought to light upregulation of genes involved in antigen presentation and T cell activation during EAU. Specific transcriptome analysis of retinal endothelial cells allowed us to identify 82 genes modulated in retinal endothelial cells during EAU development. Protein expression of 5 of those genes (serpina3n, lcn2, ackr1, lrg1 and lamc3) was validated at the level of inner BRB cells. CONCLUSION: Those data not only confirm the involvement of known pathogenic molecules but further provide a list of new candidate genes and pathways possibly implicated in inner BRB breakdown during non-infectious posterior uveitis.

Asunto(s)

Enfermedades Autoinmunes/diagnóstico , Células Endoteliales/patología , Inmunidad Celular , Retina/patología , Linfocitos T/inmunología , Uveítis/diagnóstico , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Barrera Hematorretinal , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Uveítis/inmunología , Uveítis/metabolismo

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BACKGROUND: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. MATERIALS AND METHODS: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. RESULTS: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. DISCUSSION: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a "longitudinal" overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

RESUMEN

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

Asunto(s)

Sistema del Grupo Sanguíneo Duffy/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Mapas de Interacción de Proteínas , Receptores de Superficie Celular/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Biomarcadores de Tumor/genética , Biología Computacional/métodos , Sistema del Grupo Sanguíneo Duffy/biosíntesis , Sistema del Grupo Sanguíneo Duffy/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Metástasis Linfática , Pronóstico , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/metabolismo , Tasa de Supervivencia , Neoplasias del Cuello Uterino/metabolismo

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Chemokines regulate directed cell migration, proliferation and survival and are key components in cancer biology. They exert their functions by interacting with seven-transmembrane domain receptors that signal through G proteins (GPCRs). A subgroup of four chemokine receptors known as the atypical chemokine receptors (ACKRs) has emerged as essential regulators of the chemokine functions. ACKRs play diverse and complex roles in tumor biology from tumor initiation to metastasis, including cancer cell proliferation, adherence to endothelium, epithelial-mesenchymal transition (EMT), extravasation from blood vessels, tumor-associated angiogenesis or protection from immunological responses. This chapter gives an overview on the established and emerging roles that the atypical chemokine receptors ACKR1, ACKR2, ACKR3 and ACKR4 play in the different phases of cancer development and dissemination, their clinical relevance, as well as on the hurdles to overcome in ACKRs targeting as cancer therapy.

Asunto(s)

Antineoplásicos/uso terapéutico , Quimiocinas/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Receptores de Quimiocina/antagonistas & inhibidores , Animales , Proliferación Celular , Humanos , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Receptores de Quimiocina/metabolismo , Transducción de Señal

RESUMEN

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Asunto(s)

Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Sistema del Grupo Sanguíneo Duffy/metabolismo , Células Endoteliales/efectos de los fármacos , Exotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Receptores de Superficie Celular/metabolismo , Infecciones Estafilocócicas/patología , Staphylococcus aureus/patogenicidad , Animales , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Exotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Staphylococcus aureus/metabolismo , Análisis de Supervivencia

RESUMEN

Benign neutropenia, observed in different ethnic groups, is the most common form of neutropenia worldwide. A specific single nucleotide polymorphism, rs2814778, located at the promoter of the ACKR1 (previously termed DARC) gene, which disrupts a binding site for the GATA1 erythroid transcription factor, resulting in a ACKR1-null phenotype, was found to serve as a predictor of low white blood cell and neutrophil counts in African-Americans and Yemenite Jews. Individuals with benign neutropenia due to the ACKR1-null allele have been found to have an increased susceptibility to human immunodeficiency virus infection and, on the other hand, a protective effect against malaria. The associated protective effect may explain the spread of the ACKR1-null allele by natural selection. The reviewed relationships between ACKR1 polymorphism and various pathological states may have important clinical implications to individuals with and without benign neutropenia. Potential mechanisms for ACKR1 (previously termed DARC) modulation during neutrophil recruitment to inflammation, and chemokine bioavailability in the circulation and in local tissue are reviewed and discussed.

Asunto(s)

Sistema del Grupo Sanguíneo Duffy , Factor de Transcripción GATA1 , Neutropenia , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular , Elementos de Respuesta/inmunología , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/inmunología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/inmunología , Predisposición Genética a la Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1/inmunología , Humanos , Malaria/genética , Malaria/inmunología , Malaria/patología , Neutropenia/genética , Neutropenia/inmunología , Neutropenia/patología , Neutrófilos/inmunología , Neutrófilos/patología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología

RESUMEN

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

Asunto(s)

Quimiocina CXCL1 , Quimiocina CXCL2 , Sistema del Grupo Sanguíneo Duffy , Neutrófilos , Receptores de Superficie Celular , Migración Transendotelial y Transepitelial , Animales , Músculos Abdominales/efectos de los fármacos , Músculos Abdominales/inmunología , Músculos Abdominales/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/genética , Quimiocina CXCL2/inmunología , Quimiocina CXCL2/metabolismo , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/inmunología , Sistema del Grupo Sanguíneo Duffy/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/inmunología , Uniones Intercelulares/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/genética , Migración Transendotelial y Transepitelial/inmunología , Factor de Necrosis Tumoral alfa/farmacología