Dissecting the autocrine and paracrine roles of the CCR2-CCL2 axis in tumor survival and angiogenesis

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX₃CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX₃CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX₃CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx₃cr1 ^gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX₃CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.     

Influence of the CCR2-V64I Polymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) from CCR2-V64I heterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by either CCR2b- or CCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by both CCR2 allele receptor types provides a mechanistic link that might help explain the in vivo effects of CCR2 gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.     

Structural analysis of human CCR2b and primate CCR2b by molecular modeling and molecular dynamics simulation

CCR2b, a chemokine receptor for MCP-1, -2, -3, -4, plays an important role in a variety of diseases involving infection, inflammation, and/or injury, as well as being a coreceptor for HIV-1 infection. Two models of human CCR2b (hCCR2b) were generated by homology modeling and 1 ns restrained molecular dynamics (MD) simulation. In one only C113-C190 forms a disulfide bond (SS model); in another the potential C32-C277 disulfide bond was formed (2SS model). Analysis of the structures and averaged displacements of Calpha atoms of the N-terminal residues shows that the main differences between the SS and 2SS models lie in a region D25YDYGAPCHKFD36; in the extracellular part of the 2SS model the accessible surfaces of N12, F23, Y26, Y28 and F35 are obviously raised and a more stable H-bond net is formed. The potential energy of the 2SS-water assembly finally fluctuated around -43,020 kJ x mol(-1), which is about 302 kJ x mol(-1) lower than that of the SS-water assembly. All these results suggest that the 2SS model is more favorable. The CCR2b genes of 17 primates were sequenced and four CCR2b models for primates Ateles paniscus (A. pan), Hylobates leucogyneus(H. leu), Papio cynocephalus (P. cyn) and Trachypithecus francoist ( T. fra) were generated based on the 2SS model. A comparison of hCCR2b with primate CCR2b also supports the importance of the region D25YDYGAPCHKFD36. Electrostatic potential maps of human and primate CCR2b all display the dipolar characteristics of CCR2b with the negative pole located in the extracellular part and a strong positive pole in the cytoplasmic part. Based on the CCR2b model, we suggest that the main functional residues fall in the D25YDYGAPCHKFD36 region, and the negative electrostatic feature is a non-specific, but necessary, factor for ligands or gp120/CD4 binding.     

Small molecule inhibitors of the CCR2b receptor. Part 1: Discovery and optimization of homopiperazine derivatives

N,N'-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the sigma factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.     

Hematopoietic CC-chemokine receptor 2 (CCR2) competent cells are protective for the cognitive impairments and amyloid pathology in a transgenic mouse model of Alzheimer's disease

Monocytes emigrate from bone marrow, can infiltrate into brain, differentiate into microglia and clear amyloid β (Aβ) from the brain of mouse models of Alzheimer’s disease (AD). Here we show that these mechanisms specifically require CC-chemokine receptor 2 (CCR2) expression in bone marrow cells (BMCs). Disease progression was exacerbated in APP_Swe/PS1 mice (transgenic mice expressing a chimeric amyloid precursor protein [APPSwe] and human presenilin 1 [PS1]) harboring CCR2-deficient BMCs. Indeed, transplantation of CCR2-deficient BMCs enhanced the mnesic deficit and increased the amount of soluble Aβ and expression of transforming growth factor (TGF)-β1 and TGF-β receptors. By contrast, transplantation of wild-type bone marrow stem cells restored memory capacities and diminished soluble Aβ accumulation in APP_Swe/PS1 and APP_Swe/PS1/CCR2^−/− mice. Finally, gene therapy using a lentivirus-expressing CCR2 transgene in BMCs prevented cognitive decline in this mouse model of AD. Injection of CCR2 lentiviruses restored CCR2 expression and functions in monocytes. The presence of these cells in the brain of non-irradiated APP_Swe/PS1/CCR2^−/− mice supports the concept that they can be used as gene vehicles for AD. Decreased CCR2 expression in bone marrow–derived microglia may therefore play a major role in the etiology of this neurodegenerative disease.     

N-Benzylindole-2-carboxylic acids: potent functional antagonists of the CCR2b chemokine receptor

Screening of the corporate database led to the discovery of a novel series of N-benzylindole-2-carboxylic acid CCR2b chemokine receptor antagonists. These compounds demonstrate high affinity and functional inhibition of the CCR2b receptor. A discussion of the structure-activity relationships is presented, together with evidence for a highly selective receptor binding profile.     

The CC chemokine eotaxin (CCL11) is a partial agonist of CC chemokine receptor 2b

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.     

CCR2 Regulates the Uptake of Bone Marrow-Derived Fibroblasts in Renal Fibrosis

Recent studies have shown that bone marrow-derived fibroblasts contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment of bone marrow-derived fibroblasts into the kidney are incompletely understood. Bone marrow-derived fibroblasts express the chemokine receptor - CCR2. In this study, we tested the hypothesis that CCR2 participates in the recruitment of fibroblasts into the kidney during the development of renal fibrosis. Bone marrow-derived collagen-expressing GFP⁺ fibroblasts were detected in the obstructed kidneys of chimeric mice transplanted with donor bone marrow from collagen α1(I)-GFP reporter mice. These bone marrow-derived fibroblasts expressed PDGFR-β and CCR2. CCR2 knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors expressing the hematopoietic marker-CD45 and the mesenchymal markers-PDGFR-β or procollagen I in the obstructed kidneys compared with wild-type mice. Furthermore, CCR2 knockout mice displayed fewer bone marrow-derived myofibroblasts and expressed less α-SMA or FSP-1 in the obstructed kidneys compared with wild-type mice. Consistent with these findings, genetic deletion of CCR2 inhibited total collagen deposition and suppressed expression of collagen I and fibronectin. Moreover, genetic deletion of CCR2 inhibits MCP-1 and CXCL16 gene expression associated with a reduction of inflammatory cytokine expression and macrophage infiltration, suggesting a linear interaction between two chemokines/ligand receptors in tubular epithelial cells. Taken together, our results demonstrate that CCR2 signaling plays an important role in the pathogenesis of renal fibrosis through regulation of bone marrow-derived fibroblasts. These data suggest that inhibition of CCR2 signaling could constitute a novel therapeutic approach for fibrotic kidney disease.     

Ly-6G+CCR2- myeloid cells rather than Ly-6ChighCCR2+ monocytes are required for the control of bacterial infection in the central nervous system

Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b(+)Ly-6G(+)CCR2(-) granulocytes, also CD11b(+)Ly-6C(high)CCR2(+) but not Ly-6C(low)CCR2(-) monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b(+)Ly-6C(high)CCR2(+) inflammatory monocytes modulated local CXCL2 and IL-1beta production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6C(high)CCR2(+) inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b(+)Ly-6G(+)CCR2(-) myeloid cells are involved in the restriction of the extracellular bacteria.     

Specific Binding and Chemotactic Activity of mBD4 and Its Functional Orthologue hBD2 to CCR6-expressing Cells

β-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the β-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG₁ to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these β-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the β-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the β-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and β-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the β-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific β-defensin receptor interactions.     

CCR2 expression correlates with prostate cancer progression

Although the primary role of chemokines and their receptors is controlling the trafficking of leukocytes during inflammatory responses, they also play pleoitropic roles in cancer development. There is emerging evidence that cancer cells produce chemokines that induce tumor cell proliferation or chemotaxis in various cancer types. We have previously reported that MCP-1 acts as a paracrine and autocrine factor for prostate cancer (PCa) growth and invasion. As the cellular effects of MCP-1 are mediated by CC chemokine receptor 2 (CCR2), we hypothesized that CCR2 may contribute PCa progression. Accordingly, we first determined CCR2 mRNA and protein expression in various cancer cell lines, including PCa and other cancer types. All cells expressed CCR2 mRNA and protein, but in PCa, more aggressive cancer cells such as C4-2B, DU145, and PC3 expressed a higher amount of CCR2 compared with the less aggressive cancer cells such as LNCaP or non-neoplastic PrEC and RWPE-1 cells. Further, we found a positive correlation between CCR2 expression and PCa progression by analyzing an ONCOMINE gene array database. We confirmed that CCR2 mRNA was highly expressed in PCa metastatic tissues compared with the localized PCa or benign prostate tissues by real-time RT-PCR. Finally, CCR2 protein expression was examined by immunohistochemical staining on tissue microarray specimens from 96 PCa patients and 31 benign tissue controls. We found that CCR2 expression correlated with Gleason score and clinical pathologic stages, whereas lower levels of CCR2 were expressed in normal prostate tissues. These results suggest that CCR2 may contribute to PCa development.     

Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198

The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC₅₀ of 22.8 nm but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV–VI of CCR5 with an IC₅₀ of 55 nm. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC₅₀ of 122 nm) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC₅₀ of 1023 nm) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile¹⁹⁸.     

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.     

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates, Like HIV-1 Isolates, Frequently Use CCR5 but Show Promiscuity in Coreceptor Usage

Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors.     

Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2

The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca²⁺ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor β-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.     

Rs7853346 Polymorphism in lncRNA-PTENP1 and rs1799864 Polymorphism in CCR2 are Associated with Radiotherapy-Induced Cognitive Impairment in Subjects with Glioma Via Regulating PTENP1/miR-19b/CCR2 Signaling Pathway

LncRNA-PTENP1 was reported to promote multiple myeloma cancer stem cell proliferation, and the G allele of rs7853346 polymorphism in lncRNA-PTENP1 was demonstrated to enhance the effect of lncRNA-PTENP1. In this study, we aimed to study the potential effect of lncRNA-PTENP1 and CCR2 mRNA polymorphisms on cognitive impairment in glioma patients. In this study, 279 glioma patients were recruited and grouped according to their genotypes of rs7853346 in PTENP1 and rs1799864 in CCR1. Pathogenic parameters were collected from patients before radiotherapy (month 0) or at month 1 and month 3 after radiotherapy to study the effect of rs7853346 and rs1799864 on cognitive impairment. Sequence analysis, luciferase assay, real-time PCR, and Western blot were performed to study the regulatory relationships between lncRNA-PTENP1, miR-18b, and CCR2. The glioma patient groups exhibited no significant differences concerning basic characteristics. However, the CG&GG/GG genotype alleviated radiotherapy-induced cognitive impairment by exhibiting the highest MMSE among the four groups. On the contrary, parameters including the severity of depression, bladder control, global health status, itchy skin, and weakness of legs all showed no difference among different patient groups at month 0, month 1, and month 3. Also, a long-term positive effect of CG&GG/GG genotype on role functioning and social functioning was also observed after radiotherapy. Compared with patients carrying the CC genotype of rs7853346, the expression of lncRNA-PTENP1 was reduced while the miR-19b level was elevated in patients carrying the CG&GG genotypes of rs7853346. Moreover, the expression of CCR2 mRNA was the highest in the CC/GA&AA group and the lowest in the CG&GG/GG group. Subsequent sequence analysis and luciferase assay indicated that miR-19b could bind to lncRNA-PTENP1 and 3’UTR of CCR2 mRNA, and the knockdown of lncRNA-PTENP1 led to evident up-regulation of miR-19b and down-regulation of CCR2 mRNA/protein in a cellular model, thus verifying the presence of the lncRNA-PTENP1/miR-19b/CCR2 mRNA signaling pathway. In conclusion, by studying the changes in the key parameters of glioma patients who were subjected to radiotherapy, we concluded that the rs7853346 polymorphism in lncRNA-PTENP1 and the rs1799864 polymorphism in CCR2 could independently affect cognitive impairment, while a more significant combined effect on cognitive impairment was exerted in glioma patients via the signaling pathway of PTENP1/miR-19b/CCR2.

     

C-C Chemokine Receptor 2 Inhibitor Ameliorates Hepatic Steatosis by Improving ER Stress and Inflammation in a Type 2 Diabetic Mouse Model

Hepatic steatosis is the accumulation of excess fat in the liver. Recently, hepatic steatosis has become more important because it occurs in the patients with obesity, type 2 diabetes, and hyperlipidemia and is associated with endoplasmic reticulum (ER) stress and insulin resistance. C-C chemokine receptor 2 (CCR2) inhibitor has been reported to improve inflammation and glucose intolerance in diabetes, but its mechanisms remained unknown in hepatic steatosis. We examined whether CCR2 inhibitor improves ER stress-induced hepatic steatosis in type 2 diabetic mice. In this study, db/db and db/m (n = 9) mice were fed CCR2 inhibitor (2 mg/kg/day) for 9 weeks. In diabetic mice, CCR2 inhibitor decreased plasma and hepatic triglycerides levels and improved insulin sensitivity. Moreover, CCR2 inhibitor treatment decreased ER stress markers (e.g., BiP, ATF4, CHOP, and XBP-1) and inflammatory cytokines (e.g., TNFα, IL-6, and MCP-1) while increasing markers of mitochondrial biogenesis (e.g., PGC-1α, Tfam, and COX1) in the liver. We suggest that CCR2 inhibitor may ameliorate hepatic steatosis by reducing ER stress and inflammation in type 2 diabetes mellitus.     

Role of the chemokine receptors CCR1, CCR2 and CCR4 in the pathogenesis of experimental dengue infection in mice

Dengue virus (DENV), a mosquito-borne flavivirus, is a public health problem in many tropical countries. Recent clinical data have shown an association between levels of different chemokines in plasma and severity of dengue. We evaluated the role of CC chemokine receptors CCR1, CCR2 and CCR4 in an experimental model of DENV-2 infection in mice. Infection of mice induced evident clinical disease and tissue damage, including thrombocytopenia, hemoconcentration, lymphopenia, increased levels of transaminases and pro-inflammatory cytokines, and lethality in WT mice. Importantly, infected WT mice presented increased levels of chemokines CCL2/JE, CCL3/MIP-1α and CCL5/RANTES in spleen and liver. CCR1^-/- mice had a mild phenotype with disease presentation and lethality similar to those of WT mice. In CCR2^-/- mice, lethality, liver damage, levels of IL-6 and IFN-γ, and leukocyte activation were attenuated. However, thrombocytopenia, hemoconcentration and systemic TNF-α levels were similar to infected WT mice. Infection enhanced levels of CCL17/TARC, a CCR4 ligand. In CCR4^-/- mice, lethality, tissue injury and systemic inflammation were markedly decreased. Despite differences in disease presentation in CCR-deficient mice, there was no significant difference in viral load. In conclusion, activation of chemokine receptors has discrete roles in the pathogenesis of dengue infection. These studies suggest that the chemokine storm that follows severe primary dengue infection associates mostly to development of disease rather than protection.     

Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC(50) 3.2 nM; MCP-1-Induced Chemotaxis IC(50) 0.83 nM; Ca(2+) Flux IC(50) 7.5 nM].