Showing papers on "Chemokine receptor CCR5 published in 2019"

CCR5 antagonism by maraviroc inhibits Hodgkin lymphoma microenvironment interactions and xenograft growth.

Abstract: Classic Hodgkin lymphoma tumor cells express a functional CCR5 receptor, and tumor tissues express high CCL5 levels, suggesting that CCL5-CCR5 signaling is involved in tumor-microenvironment formation and tumor growth. Using the CCR5 antagonist, maraviroc, and a neutralizing anti-CCL5 antibody, we found that CCL5 secreted by classic Hodgkin lymphoma cells recruited mesenchymal stromal cells and monocytes. The “education” of mesenchymal stromal cells by tumor cell-conditioned medium enhanced mesenchymal stromal cells’ proliferation and CCL5 secretion. In turn, educated mesenchymal stromal cell-conditioned medium increased the clonogenic growth of tumor cells and monocyte migration, but these effects were reduced by maraviroc. Monocyte education by tumor cell-conditioned medium induced their growth and reprogrammed them towards immunosuppressive tumor-associated macrophages that expressed IDO and PD-L1 and secreted IL-10, CCL17 and TGF-β. Educated monocyte-conditioned medium slowed the growth of phytohemagglutinin-activated lymphocytes. Maraviroc decreased tumor cell growth and synergized with doxorubicin and brentuximab vedotin. A three-dimensional heterospheroid assay showed that maraviroc counteracted both the formation and viability of heterospheroids generated by co-cultivation of tumor cells with mesenchymal stromal cells and monocytes. In mice bearing tumor cell xenografts, maraviroc reduced tumor growth by more than 50% and inhibited monocyte accumulation, without weight loss. Finally, in classic Hodgkin lymphoma human tumor tissues, CCL5 and CD68 expression correlated positively, and patients with high CCL5 levels had poor prognosis. In conclusion, since the present challenges are to find molecules counteracting the formation of the immunosuppressive tumor microenvironment or new, less toxic drug combinations, the repurposed drug maraviroc may represent a new opportunity for classic Hodgkin lym phoma treatment.

CCR5 blockage by maraviroc: a potential therapeutic option for metastatic breast cancer.

Abstract: Bone metastasis is observed in up to 70% of breast cancer patients. The currently available treatment options are palliative in nature. Chemokine receptor 5 (CCR5) has gained attention as therapeutic target in various malignancies. Here, we investigated the effects of targeting CCR5 by its antagonist maraviroc in metastatic breast cancer cells. In response to maraviroc exposure, cytotoxicity was assessed using an MTT proliferation assay, whereas the effects on colony formation and migration were assessed using colony formation, transwell chamber migration and scratch wound healing assays, respectively. Apoptosis-related activities were investigated using nuclear staining, annexin-V FITC staining and Western blotting. Cell cycle changes were analysed using flow cytometry and qRT-PCR for cell cycle relevant genes. A nude rat model for breast cancer bone metastasis was used to evaluate the in vivo efficacy of CCR5 targeting by maraviroc. Circulatory levels of the three cognate ligands for CCR5 (CCL3, CCL4, CCL5) were analysed in sera of breast cancer patients using ELISA. We found that blockade of CCR5 attenuated the proliferation, colony formation and migration of metastatic breast cancer cells, and induced apoptosis and arrest in the G1 phase of the cell cycle. Expression profiling highlighted the involvement of cell cycle related signalling cascades. We also found that treatment with maraviroc significantly inhibited bone metastasis in nude rats implanted with MDA-MB-231 breast cancer cells. Finally, we found that the circulatory levels of three cognate ligands for the CCR5 receptor varied between breast cancer patients and healthy controls. Our findings indicate that targeting CCR5 may be an effective strategy to combat breast cancer bone metastasis.

Modeling interaction between gp120 HIV protein and CCR5 receptor

Abstract: The study of the process of HIV entry into the host cell and the creation of biomimetic nanosystems that are able to selectively bind viral particles and proteins is a high priority research area for the development of novel diagnostic tools and treatment of HIV infection. Recently, we described multilayer nanoparticles (nanotraps) with heparin surface and cationic peptides comprising the N-terminal tail (Nt) and the second extracellular loop (ECL2) of CCR5 receptor, which could bind with high affinity some inflammatory chemokines, in particular, Rantes. Because of the similarity of the binding determinants in CCR5 structure, both for chemokines and gp120 HIV protein, here we expand this approach to the study of the interactions of these biomimetic nanosystems and their components with the peptide representing the V3 loop of the activated form of gp120. According to surface plasmon resonance results, a conformational rearrangement is involved in the process of V3 and CCR5 fragments binding. As in the case of Rantes, ECL2 peptide showed much higher affinity to V3 peptide than Nt (KD = 3.72 × 10-8 and 1.10 × 10-6 M, respectively). Heparin-covered nanoparticles bearing CCR5 peptides effectively bound V3 as well. The presence of both heparin and the peptides in the structure of the nanotraps was shown to be crucial for the interaction with the V3 loop. Thus, short cationic peptides ECL2 and Nt proved to be excellent candidates for the design of CCR5 receptor mimetics.

CCR5 receptor antagonism inhibits hepatitis C virus (HCV) replication in vitro.

Abstract: Background and aim The hepatitis C virus (HCV) is a single-strand RNA virus that infects millions of people worldwide. Recent advances in therapy have led to viral cure using two- and three- drug combinations of direct acting inhibitors of viral replication. CCR5 is a chemokine receptor that is expressed on hepatocytes and represents a key co-receptor for HIV. We evaluated the effect of CCR5 blockade or knockdown on HCV replication in Huh7.5JFH1 cells. Methods Cells were exposed to varying concentrations of maraviroc (CCR5 inhibitor), cenicriviroc (CCR2/CCR5 inhibitor), sofosbuvir (nucleotide polymerase inhibitor), or raltegravir (HIV integrase inhibitor). Results HCV RNA was detected utilizing two qualitative strand-specific RT-PCR assays. HCV core antigen and NS3 protein was quantified in the supernatant and cell lysate, respectively. siRNA was utilized to knockdown CCR5 gene expression in hepatocytes. Alternatively, anti-CCR5 antibodies were employed to block the receptor. Supernatant levels of HCV RNA (expressed as fold change) were not reduced in the presence of raltegravir but were reduced 8.55-fold and 12.42-fold with cenicriviroc and maraviroc, respectively. Sofosbuvir resulted in a 16.20-fold change in HCV RNA levels. HCV core and NS3 protein production was also reduced in a dose-dependent manner. Two distinct anti-CCR5 antibodies also resulted in a significant reduction in HCV protein expression, as did siRNA knockdown of CCR5 gene expression. Conclusions These data provide evidence that CCR5 modulation could have a significant effect on HCV replication in an in vitro system. Further evaluation of the role of CCR5 inhibition in clinical settings may be warranted.

A single nucleotide variant in the promoter region of the CCR5 gene increases susceptibility to arthritis encephalitis virus in goats

Abstract: The small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses that includes caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus (MVV). SRLVs affect the production and welfare of sheep and goats worldwide. There is currently no effective treatment. Their high mutation rate precludes vaccine development, making innovative control measures necessary. A variant of the chemokine (C-C motif) receptor 5 (CCR5) gene is reportedly involved in resistance to human immunodeficiency (HIV) infection in humans and to SRLV in sheep. The aim of this study was to analyse the genetic structure and variability of the CCR5 gene in goats and to carry out a cross-sectional study to investigate the role of CCR5 genetic variants in controlling susceptibility/resistance to CAEV. The variant g.1059 T located in the promoter region revealed an interesting association with high proviral loads (a 2.8-fold increased risk). A possible explanation could be an alteration of the transcriptional level. Overexpression of the CCR5 receptor on the cell surface may increase virus internalization and proviral load as a consequence. Our findings could be advantageously used to reduce the susceptibility of goat herds to CAEV by negatively selecting animals carrying the g.1059 T mutation. Eliminating animals predisposed to high proviral loads could also limit the development of clinical signs and the spread of the virus, since these animals are also highly efficient in shedding the virus.

Chemokine receptor CCR5 correlates with functional CD8+ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

Abstract: C-C chemokine receptor 5 (CCR5) plays an essential role in HIV pathogenesis as the major coreceptor on CD4+ T cells used by HIV, yet the function of CCR5 on CD8 T cells is not well understood. Furthermore, the immunologic effects of the CCR5 inhibitor maraviroc (MVC), despite approval for clinical use, have not yet been well evaluated for their potential effects on cytotoxic T-cell responses. In this study, we characterized the development and function of CCR5+CD8+ T cells in rhesus macaques with or without Simian immunodeficiency virus (SIV) infection. We also investigated the effects of the CCR5 antagonist MVC on functional CCR5+CD8+ T-cell responses in vitro. The data show that CCR5+CD8+ T cells have an effector memory phenotype and increase with age in systemic and mucosal lymphoid tissues as a heterogeneous population of polyfunctional CD8 T cells. In addition, CCR5 is highly expressed on SIV gag-specific (CM9+) CD8+ T cells in SIV-infected macaques, yet CCR5+CD8+ T cells are significantly reduced in mucosal lymphoid tissues with disease progression. Furthermore, in vitro MVC treatment reduced activation and cytokine secretion of CD8+ T cells via a CCR5-independent pathway. These findings suggest that surface CCR5 protein plays an important role in differentiation and activation of CD8+ T cells. Although MVC may be helpful in reducing chronic inflammation and activation, it may also inhibit virus-specific CD8+ T-cell responses. Thus optimal use of CCR5 antagonists either alone or in combination with other drugs should be defined by further investigation.-Wang, X., Russell-Lodrigue, K. E., Ratterree, M. S., Veazey, R. S., Xu, H. Chemokine receptor CCR5 correlates with functional CD8+ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

Predominance of the heterozygous CCR5 delta-24 deletion in African individuals resistant to HIV infection might be related to a defect in CCR5 addressing at the cell surface.

Abstract: Introduction The chemokine receptor CCR5 is the main co-receptor for R5-tropic HIV-1 variants. We have previously described a novel 24-base pair deletion in the coding region of CCR5 among individuals from Rwanda. Here, we investigated the prevalence of hCCR5 Delta 24 in different cohorts and its impact on CCR5 expression and HIV-1 infection in vitro. Methods We screened hCCR5 Delta 24 in a total of 3232 individuals which were either HIV-1 uninfected, high-risk HIV-1 seronegative and seropositive partners from serodiscordant couples, Long-Term Survivors, or HIV-1 infected volunteers from Africa (Rwanda, Kenya, Guinea-Conakry) and Luxembourg, using a real-time PCR assay. The role of the 24-base pair deletion on CCR5 expression and HIV infection was assessed in cell lines and PBMC using mRNA quantification, confocal analysis, flow and imaging cytometry. Results and Discussion Among the 1661 patients from Rwanda, 12 individuals were heterozygous for hCCR5 Delta 24 but none were homozygous. Although heterozygosity for this allele may not confer complete resistance to HIV-1 infection, the prevalence of the mutation was 2.41% (95%CI: 0.43; 8.37) in 83 Long-Term Survivors (LTS) and 0.99% (95%CI: 0.45; 2.14) in 613 HIV-1 exposed seronegative members as compared with 0.35% (95% Cl: 0.06; 1.25) in 579 HIV-1 seropositive members. The prevalence of hCCR5 Delta 24 was 0.55% (95%CI: 0.15; 1.69) in 547 infants from Kenya but the mutation was not detected in 224 infants from Guinea-Conakry nor in 800 Caucasian individuals from Luxembourg. Expression of hCCR5 Delta 24 in cell lines and PBMC showed that the hCCR5 Delta 24 protein is stably expressed but is not transported to the plasma membrane due to a conformational change. Instead, the mutant receptor was retained intracellularly, colocalized with an endoplasmic reticulum marker and did not mediate HIV-1 infection. Co-transfection of hCCR5 Delta 24 and wtCCR5 did not indicate a transdominant negative effect of CCR5 Delta 24 on wtCCR5. Conclusions Our findings indicate that hCCR5 Delta 24 is not expressed at the cell surface. This could explain the higher prevalence of the heterozygous hCCR5 Delta 24 in LTS and HIV-1 exposed seronegative members from serodiscordant couples. Our data suggest an East-African localization of this deletion, which needs to be confirmed in larger cohorts from African and non-African countries.

CCR5 deficiency enhances hepatic innate immune cell recruitment and inflammation in a murine model of acute hepatitis B infection

Abstract: Human genetic studies demonstrate a link between the 32-bp deletion that produces a nonfunctional CCR5 receptor and enhanced recovery from acute hepatitis B virus (HBV) infection. To investigate the role of CCR5 in immune responses to acute HBV, we intravenously infected Ccr5+/+ (WT) and Ccr5-/- (KO) mice with a replication-incompetent adenovirus containing the overlapping HBV1.3 construct (AdHBV), or vector control. At day 3 following AdHBV infection, analysis of intrahepatic leukocytes (IHL) showed KO mice had increased CD11b+ NK cells compared to WT (18.2% versus 7.6% of live IHL, P < 0.01). These CD11b+ NK cells were nonresident (CD49a- ) and had capacity to degranulate and produce IFN-γ following stimulation. At day 3, plasma CXCL10 was significantly increased in KO, but not WT, mice receiving AdHBV as compared to vector control, while CXCR3 expression on hepatic CD11b+ NK cells in AdHBV-treated KO mice was significantly lower than that in uninfected mice, suggesting these NK cells are recruited along the CXCL10-CXCR3 axis. At days 7 and 14, no differences between genotypes were observed in number, or HBV-specific function, of intrahepatic CD8+ T cells. Instead, at day 14, KO mice had increased intrahepatic proinflammatory monocytes compared to WT mice (17.56% versus 6.57% of live IHL, P = 0.014), corresponding with an increase in plasma alanine aminotransferase and intrahepatic IL-1β observed in KO mice. Taken together, these findings demonstrate that loss of CCR5 signaling drives a more robust inflammatory liver microenvironment early in acute HBV infection via enrichment of hepatic innate immune cells.

Association analysis and allelic distribution of deletion in CC chemokine receptor 5 gene (CCR5Δ32) among breast cancer patients of Pakistan.

Abstract: Chemokine CC receptor type 5 (CCR5) is a cell surface receptor that has high affinity for chemotropic cytokines called chemokines. The CCR5 gene contains a 32 base pairs (bp) deletion (CCR5Δ32). This deletion may result in a malformed and nonfunctional receptor, reported to be responsible for the development and dissemination of different cancers. CCR5Δ32 exists in two allelic forms i.e. deletion (D) and wild type (WT). This study aims to detect the role of CCR5Δ32 in breast cancer development. Blood samples were collected from breast cancer patients (330) and controls of same gender (306). Along with this histopathologically diagnosed malignant tissue samples were also excised from breast lesions of 100 patients. Genetic variations within the blood and tissue samples were examined by PCR then observed through gel electrophoresis and confirmed by direct DNA sequencing. Obtained DNA sequences were aligned and analyzed by MEGA6 software. Genotypic and association analyses were done by SPSS software version 17.0. Deletion of 32 bp in CCR5 gene has been analyzed. Genotypic variations of CCR5Δ32 are; homozygous wild type (WT/WT), heterozygous deletion (WT/D) and homozygous deletion (D/D). Statistical analyses of CCR5Δ32 data revealed that WT/D was significantly higher in blood samples of breast cancer patients (7.27% (24/330)) as compare to controls (1.30% (4/306)). In tumor tissue samples WT/WT being the most frequent genotype (99.00% (99/100)) with 1.00 (1/100) of D/D which suggested that it may be acquired. Hence, association analysis showed that CCR5Δ32 is positively associated with breast cancer in Pakistan (p < 0.001). The risk ratio of CCR5Δ32 was 5.6610 (95% confidence interval: 2.0377 to 15.7267) and odds ratio was calculated to be 6.0335 (95% confidence interval: 2.1288 to 17.0999) which signifies that deletion also increases the risk of breast cancer development. Moreover, association analyses also revealed that clinicopathological features do not have any impact on the CCR5Δ32 genotype of breast cancer. This suggests that deletion of 32 bp in CCR5 gene may be associated with breast cancer. CCR5 signals the activation and migration of immune cells at the site of tumor formation. Because of deletion; deformed CCR5 receptor might be unable to express and function properly which may subdue the immunity against cancer hence, leading to its progression.

Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models.

Abstract: Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggest that CCR5+ CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 h of SP exposure (P = 0.03). Meanwhile, NK cells retain the capacity to produce limited amounts of MIP-1α/MIP-1β in response to anti-HIV-1 antibody-dependent stimulation (median, 9.5% MIP-1α+ and/or MIP-1β+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three of the animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design.IMPORTANCE The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility.

Molecular binding mode of PF-232798, a clinical anti-HIV candidate, at chemokine receptor CCR5

Abstract: The chemokine receptor CCR5 is an important anti-HIV (human immunodeficiency virus) drug target owning to its pivotal role in HIV-1 viral entry as a co-receptor. Here, we present a 2.9 A resolution crystal structure of CCR5 bound to PF-232798, a second-generation oral CCR5 antagonist currently in phase II clinical trials. PF-232798 and the marketed HIV drug maraviroc share a similar tropane scaffold with different amino (N)- and carboxyl (C)- substituents. Comparison of the CCR5–PF-232798 structure with the previously determined structure of CCR5 in complex with maraviroc reveals different binding modes of the two allosteric antagonists and subsequent conformational changes of the receptor. Our results not only offer insights into the phenomenon that PF-232798 has higher affinity and alternative resistance profile to maraviroc, but also will facilitate the design of new anti-HIV drugs.

Transplant success reignites interest in reprogramming cells against HIV.

Abstract: Editing cells to lack the CCR5 receptor may prove more feasible for more people. Editing cells to lack the CCR5 receptor may prove more feasible for more people.

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Co-receptor CCR5

Abstract: The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine RANTES (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders-of-magnitude higher than that of RANTES itself. It has been hypothesized that the ultra-high potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy (FCCS) assay for saturation and competition binding experiments. The FCCS assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES, and about twenty-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site; whereas, the native chemokines (RANTES and MIP1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.