Expression Of C-C Chemokine Receptor Type 2 Research Articles

CCR2 is a potential therapeutic target in peri-implantitis.

CCR2 (C-C chemokine receptor type 2) plays a crucial role in inflammatory and bone metabolic diseases; however, its role in peri-implantitis remains unclear. This study aimed to explore whether CCR2 contributes to peri-implantitis and the treatment effects of cenicriviroc (CVC) on peri-implant inflammation and bone resorption. The expression of CCR2 was studied using clinical tissue analysis and an in vivo peri-implantitis model. The role of CCR2 in promoting inflammation and bone resorption in peri-implantitis was evaluated in Ccr2-/- mice and wild-type mice. The effect of CVC on peri-implantitis was evaluated using systemic and local dosage forms. Human peri-implantitis tissues showed increased CCR2 and CCL2 levels, which were positively correlated with bone loss around the implants. Knocking out Ccr2 in an experimental model of peri-implantitis resulted in decreased monocyte and macrophage infiltration, reduced pro-inflammatory cytokine generation and impaired osteoclast activity, leading to reduced inflammation and bone loss around the implants. Treatment with CVC ameliorated bone loss in experimental peri-implantitis. CCR2 may be a potential target for peri-implantitis treatment by harnessing the immune-inflammatory response to modulate the local inflammation and osteoclast activity.

Chemokine Receptor 2 Targeted PET/CT Imaging Distant Metastases in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and treatment-refractory malignancies. The lack of an effective screening tool results in the majority of patients being diagnosed at late stages, which underscores the urgent need to develop more sensitive and specific imaging modalities, particularly in detecting occult metastases, to aid clinical decision-making. The tumor microenvironment of PDAC is heavily infiltrated with myeloid-derived suppressor cells (MDSCs) that express C-C chemokine receptor type 2 (CCR2). These CCR2-expressing MDSCs accumulate at a very early stage of metastasis and greatly outnumber PDAC cells, making CCR2 a promising target for detecting early, small metastatic lesions that have scant PDAC cells. Herein, we evaluated a CCR2 targeting PET tracer (68Ga-DOTA-ECL1i) for PET imaging on PDAC metastasis in two mouse models. Positron emission tomography/computed tomography (PET/CT) imaging of 68Ga-DOTA-ECL1i was performed in a hemisplenic injection metastasis model (KI) and a genetically engineered orthotopic PDAC model (KPC), which were compared with 18F-FDG PET concurrently. Autoradiography, hematoxylin and eosin (H&E), and CCR2 immunohistochemical staining were performed to characterize the metastatic lesions. PET/CT images visualized the PDAC metastases in the liver/lung of KI mice and in the liver of KPC mice. Quantitative uptake analysis revealed increased metastasis uptake during disease progression in both models. In comparison, 18F-FDG PET failed to detect any metastases during the time course studies. H&E staining showed metastases in the liver and lung of KI mice, within which immunostaining clearly demonstrated the overexpression of CCR2 as well as CCR2+ cell infiltration into the normal liver. H&E staining, CCR2 staining, and autoradiography also confirmed the expression of CCR2 and the uptake of 68Ga-DOTA-ECL1i in the metastatic foci in KPC mice. Using our novel CCR2 targeted radiotracer 68Ga-DOTA-ECL1i and PET/CT, we demonstrated the sensitive and specific detection of CCR2 in the early PDAC metastases in two mouse models, indicating its potential in future clinical translation.

The role of the 47-kDa membrane lipoprotein of Treponema pallidum in promoting maturation of peripheral blood monocyte-derived dendritic cells without enhancing C-C chemokine receptor type 7-mediated dendritic cell migration.

The 47-kDa membrane lipoprotein (Tp47) is the most representative membrane protein of Treponema pallidum (T. pallidum). Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) that connect innate and acquired immunity. The regulatory role of Tp47 on DCs remains unclear. To evaluate the effects of Tp47 on DC maturation and migration, and research the changes of the main chemokine C-C chemokine receptor type 7 (CCR7) involved in DC migration. A transwell assay was applied to assess the migration of DCs. Cytokines (interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α)) in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA), and the expression of cell surface markers (CD80, CD86, CD40, and human leukocyte antigen (HLA)-DR) and CCR7 was assessed using flow cytometry. The expression of CCR7 in DCs was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The Tp47 promoted DC phenotypic maturation, such as increased CD40, CD80, CD86, and HLA-DR expression, as well as DC functional maturation, thus stimulating DCs to secrete inflammatory cytokines, including IL-6, IL-10, IL-12, and TNF-α. At the same time, Tp47 did not enhance DC migration and did not increase the expression of CCR7. The Tp47 promoted the maturation of DCs while not enhancing CCR7-mediated DC migration ability. This may be one of the mechanisms by which T. pallidum escapes host immune clearance.

LPS differentially affects expression of CD14 and CCR2 in monocyte subsets of Post-STEMI patients with hyperglycemia

AimsFollowing ST-segment elevation myocardial infarction (STEMI), recruitment and activation of monocytes [classical (CD14++CD16-CCR2++), intermediate (CD14++CD16+CCR2+), non-classical (CD14LowCD16++CCR2Low)] are needed for myocardial wound healing. Monocyte surface receptor CC chemokine receptor type 2 (CCR2) is responsible for monocyte chemotaxis to sites of inflammation and the lipopolysaccharide (LPS)-binding protein co-receptor, CD14, is involved in pro-inflammatory monocyte activation. The purpose of this investigation was to determine the effects of ex-vivo LPS activation on monocyte subset CD14 and CCR2 expression in post-STEMI individuals with normal and elevated random blood glucose. MethodsPost-STEMI subjects were identified as normal random glucose (NG, <98 mg/dL, n = 13) or impaired random glucose (IG, ≥98 mg/dL, n = 26) and monocytes were analyzed for non-activated and LPS-activated (1 µg/mL for 4 h) CCR2 and CD14 expression. ResultsNon-activated intermediate monocytes from IG showed decreased CD14 expression when compared to NG, which was maintained following LPS-activation. The NG group showed a larger absolute reduction in classical CCR2 expression, leading to a significant difference between NG and IG following LPS-activation. ConclusionResults suggest a heightened response to pro-inflammatory activation in IG following STEMI, which may impair or delay post-STEMI myocardial healing, and thus increase the incidence of chronic heart failure. NIH 1R34HL121402.

Screening and evaluation of quality markers from Shuangshen Pingfei formula for idiopathic pulmonary fibrosis using network pharmacology and pharmacodynamic, phytochemical, and pharmacokinetic analyses

BackgroundThe Shuangshen Pingfei formula (SSPF), a classic Chinese medicine derivative formula, has been shown to exert therapeutic effects on idiopathic pulmonary fibrosis (IPF). However, the quality control compounds of SSPF remain unclear. PurposeTo select and confirm Q-markers of SSPF based on network pharmacology, cytobiology, animal-based pharmacodynamics, and phytochemical and pharmacokinetic analyses. MethodsA compound–target network was constructed based on previous research. In addition, high-degree compounds of SSPF were chosen as potential Q-marker candidates. Animal and cytological experiments were performed to verify key targets of IPF. Haematoxylin-eosin and Masson's trichrome staining were used to observe lung tissue pathology. Cytokine levels in the bronchoalveolar lavage fluid (BALF) were measured using ELISA kits. Gene and protein expression levels were determined using PCR and western blotting, respectively. The contents of Q-marker candidates in different batches of SSPF were then determined for traceability research, and the quality consistency of SSPF was objectively evaluated using principal component analysis (PCA). Finally, pharmacokinetic research was performed, and candidates with desirable metabolite and bioavailability parameters were confirmed as Q-markers of SSPF. ResultsThe compound–target network included 56 compounds and 14 therapeutic targets. Animal experiments showed that SSPF attenuates lung fibrosis. SSPF decreased CC motif chemokine 2 (CCL2) and CC chemokine receptor type 2 (CCR2) levels in the BALF and downregulated the gene and protein expression of IPF therapy-related molecules, such as 5-hydroxytryptamine receptor 2A (HTR2A), CCL2, and CCR2, in the lungs. Cell experiments showed that nine Q-marker candidates in SSPF regulated the expression of CCL2 and CCR2, as predicted. Phytochemical analysis and PCA indicated that the qualities of SSPF in the nine batches were relatively stable. Pharmacokinetic studies demonstrated that mangiferin, salvianolic acid B, tanshinone IIA, naringin, and glycyrrhizic acid could be effectively absorbed into rat plasma, which ensured desirable bioavailability and confirmed their roles as Q-markers to represent anti-pulmonary fibrotic activity. ConclusionOur study is an integrated strategy, based on network pharmacology with experimental verification and phytochemical and pharmacokinetic analyses that provides a novel approach for Q-marker selection and validation of SSPF for IPF treatment.

Abstract 12532: Extracellular Vesicles Released by Cardiosphere-Derived Cells Dampen the Pro-Inflammatory Monocyte Response Through Repression of C-C Chemokine Receptor Type 2 (CCR2)

Background: Following a myocardial infarction (MI), CCR2+ monocytes migrate toward the injury site along a CCL2 concentration gradient and play an integral role in removing debris and facilitating repair. Although required to orchestrate the pro-inflammatory response, prolonged activation of CCR2+ monocytes can lead to adverse outcomes. Extracellular vesicles (EVs) derived from cardiosphere-derived cells (CDCs) have been suggested to mediate cardioprotection in part via immunomodulation. We hypothesized that CDC-EVs influence CCR2+ monocyte recruitment and function. Methods: Blood samples were obtained from healthy human donors under approved institutional IRBs. Human monocytes were isolated from peripheral blood using density gradient centrifugation (Ficoll) followed by magnetic activated cell sorting (MACS). EVs were isolated from CDCs, normal human dermal fibroblasts (NHDFs, an inert population in models of ischemia), and CDCs transfected with a miR-146a antagomir. EVs were characterized according to established guidelines for size, morphology and protein expression. Monocytes were incubated with PBS, NHDF-EVs, CDC-EVs, or miR-146a deficient CDC-EVs and analyzed for CCR2 protein expression by flow cytometry and western blot and migration along a CCL2/MCP-1 gradient by in vitro chemotaxis assays. Results: CDC-EVs significantly reduced CD14 hi CD16 - monocyte surface expression of CCR2 by 50% as quantified by flow cytometry when compared with PBS and NHDF-EVs (p&lt;0.05, one-way ANOVA, n=4). This appeared to be mediated by miR-146a as monocytes treated with CDC-EVs deficient in miR-146a had no significant difference in CCR2 levels compared with cells treated with PBS and NHDF-EV controls. Importantly, reduced levels of monocyte CCR2 expression following CDC-EV treatment led to a greater than 35% functional reduction in monocyte migration towards CCL2 compared to PBS control (42.8 ± 3.2% vs. 68.3 ± 4.9%, p&lt;0.05, t-test, n=4). CCR2 expression remained suppressed for 8-12 hours and could be maintained with repeated doses. Conclusions: CDC-EVs modulate classical monocyte expression of CCR2 and migration towards CCL2/MCP-1 via miR-146a, elucidating a novel mechanism of CDC-EV immunomodulation.

CCR7 Has Potential to Be a Prognosis Marker for Cervical Squamous Cell Carcinoma and an Index for Tumor Microenvironment Change.

The tumor microenvironment (TME) has an essential role in the development of cervical squamous cell carcinoma (CSCC); however, the dynamic role of the stromal and immune cells is still unclear in TME. We downloaded data from The Cancer Genome Atlas (TCGA) database and applied ESTIMATE and CIBERSORT algorithms to measure the quantity of stromal and immune cells and the composition of tumor-infiltrating immune cell (TIC) in 253 CSCC cases. The protein-protein interaction (PPI) network and Cox regression analysis presented the differentially expressed genes (DEGs). Then, C-C chemokine receptor type 7 (CCR7) was screened out as a prognostic marker by the univariate Cox and intersection analysis of PPI. Further analysis showed a positive correlation between the expression of CCR7 and the survival of CSCC patients. The result of the Gene Set Enrichment Analysis (GSEA) of genes in the high CCR7 expression group displayed a predominant enrichment in immune-related pathways. An enrichment in metabolic activities was observed in the low CCR7 expression group. CIBERSORT analysis showed a positive correlation between Plasma cells, CD8+ T cells, and regulatory T cells and the CCR7 expression, suggesting that CCR7 might play a crucial role in maintaining the immunological dominance status for TME. Therefore, the expression level of CCR7 might help predict the survival of CSCC cases and be an index that the status of TME transitioned from immunological dominance to metabolic activation, which presented a new insight into the treatment of CSCC.

PU.1 regulates Ccr7 gene expression by binding to its promoter in naïve CD4+ T cells.

C‐C chemokine receptor type 7 (CCR7) is expressed on naïve T cells, B cells, and activated dendritic cells (DCs). We previously demonstrated that the transcription factor PU.1/Spi1 positively regulates the expression of CCR7 in DCs. In the present study, we investigated the role of PU.1 in CCR7 expression in T cells. To confirm whether PU.1 is involved in the expression of CCR7, we conducted a ChIP assay in various T cells purified from splenocytes and thymocytes and found that PU.1 binds to the Ccr7 promoter‐proximal region in spleen naïve CD4+ T cells, but not in thymocytes. Small interfering RNA‐mediated PU.1 knockdown resulted in decreased CCR7 expression in spleen naïve CD4+ T cells. Compared to naïve CD4+ T cells, Spi1 and Ccr7 mRNA levels decreased in Th1 and Th2 cells, in which PU.1 did not bind to the Ccr7 promoter, suggesting that CCR7 expression decreases due to the dissociation of PU.1 from the Ccr7 promoter during the development of effector T cells from naïve T cells. Collectively, we concluded that CCR7 expression level correlates with the binding level of PU.1 to the Ccr7 promoter and PU.1 acts as a transcriptional activator of the Ccr7 gene in naïve CD4+ T cells.

An assessment of the relationship between the expression of CCR7/CCL19 axis and selected regulatory miRNAs in non-small cell lung cancer

CC chemokine receptor type 7 (CCR7) and its ligands has been implicated in the occurrence and progression of NSCLC. Previous studies have revealed that the diagnostic value of CCR7/CCL19 axis in lung tumorigenesis remains controversial. The present study evaluates the relationship between the mRNA expression of CCR7/CCL19 axis and selected regulatory miRNAs in NSCLC patients. It analyzes the expression level of CCR7 mRNA and its ligand in tumor tissue in relation to expression level of two miRNAs: miR let-7a and miR-335, as transcriptional regulators of study genes. Twenty-seven patients (n = 27) were enrolled. The expression of the studied genes and miRNAs was evaluated by qPCR. Tumour tissue fragments, adjacent macroscopically-unchanged lung tissue (control) and patient serum were used as biological material for study. Elevated expression of CCR7 and CCL19 mRNA was observed in patients with metastasis to lymph nodes. We noticed upregulated miR-335 expression and downregulated miR let-7a expression in patient serum with regard to AJCC tumor staging. Higher miR-335 expression and lower miR let-7a expression level was observed in patients with metastasis to lymph node. The presence of changes observed in the expression level of miR-335 and miR let-7a in the serum of NSCLC patients in relation to lymph node metastases and tumor stage may serve as a non-invasive molecular biomarker of tumor progression; however, this observation requires further investigation.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-β treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.

Meta-analysis of the prognostic value of C-C chemokine receptor type 7 in patients with solid tumors.

BackgroundExpression of C-C chemokine receptor type 7 (CCR7) is associated with the prognosis of several cancers. The aim of this study was to conduct the meta-analysis to determine the prognostic value of CCR7 expression in solid tumors.Materials and methodsWe searched for relevant literature in the PubMed, Embase, and Cochrane Library databases (last updated on January 15, 2018). The associations of CCR7 expression with overall survival (OS), disease-free survival (DFS), recurrence-free survival (RFS), progress-free survival (PFS), and disease-specific survival (DSS) were estimated.ResultsIn total, 30 qualified studies including 3,413 patients were enrolled. The results revealed that higher expression of CCR7 predicted poorer OS (pooled HR =1.79; 95% CI =1.49–2.16; P<0.001) and PFS (pooled HR =2.18; 95% CI =1.49–3.18; P<0.001), but was not associated with DFS (pooled HR =1.69; 95% CI =0.79–3.61; P=0.175), RFS (pooled HR =1.29; 95% CI =0.48–3.44; P=0.618), or DSS (pooled HR =3.06; 95% CI =0.38–24.83; P<0.294).ConclusionFrom this meta-analysis, we concluded that high expression of CCR7 in tumor tissue is associated with poor survival in patients with solid tumors, and may be a prognostic biomarker for tumor progression.

C–C chemokine receptor type 2 mediates glomerular injury and interstitial fibrosis in focal segmental glomerulosclerosis

Glomerulosclerosis and tubulointerstitial fibrosis are hallmarks of chronic kidney injury leading to end-stage renal disease. Inflammatory mechanisms contribute to glomerular and interstitial scarring, including chemokine-mediated recruitment of leucocytes. In particular, accumulation of C-C chemokine receptor type 2 (CCR2)-expressing macrophages promotes renal injury and fibrotic remodelling in diseases like glomerulonephritis and diabetic nephropathy. The functional role of CCR2 in the initiation and progression of primary glomerulosclerosis induced by podocyte injury remains to be characterized. We analysed glomerular expression of CCR2 and its chemokine ligand C-C motif chemokine ligand 2 (CCL2) in human focal segmental glomerulosclerosis (FSGS). Additionally, CCL2 expression was determined in stimulated murine glomeruli and glomerular cells in vitro. To explore pro-inflammatory and profibrotic functions of CCR2 we induced adriamycin nephropathy, a murine model of FSGS, in BALB/c wild-type and Ccr2-deficient mice. Glomerular expression of CCR2 and CCL2 significantly increased in human FSGS. In adriamycin-induced FSGS, progressive glomerular scarring and reduced glomerular nephrin expression was paralleled by induced glomerular expression of CCL2. Adriamycin exposure stimulated secretion of CCL2 and tumour necrosis factor-α (TNF) in isolated glomeruli and mesangial cells and CCL2 in parietal epithelial cells. In addition, TNF induced CCL2 expression in all glomerular cell populations, most prominently in podocytes. In vivo, Ccr2-deficient mice with adriamycin nephropathy showed reduced injury, macrophage and fibrocyte infiltration and inflammation in glomeruli and the tubulointerstitium. Importantly, glomerulosclerosis and tubulointerstitial fibrosis were significantly ameliorated. Our data indicate that CCR2 is an important mediator of glomerular injury and progression of FSGS. CCR2- targeting therapies may represent a novel approach for its treatment.

Deletion of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Accelerates Atherosclerosis Regression and Increases C-C Chemokine Receptor Type 7 (CCR7) Expression in Plaque Macrophages.

We previously showed that mice lacking MΦLRP1-/- (low-density lipoprotein receptor-related protein 1 in macrophages) undergo accelerated atherosclerotic plaque formation due to changes in macrophages including increased apoptosis, decreased efferocytosis, and exaggerated transition to the inflammatory M1 phenotype. Here we sought to explore the role of macrophage low-density lipoprotein receptor-related protein 1 during regression of atherosclerosis since regressing plaques are characterized by transitioning of macrophages to M2 status as inflammation resolves. Apolipoprotein E-/- mice on a high-fat diet for 12 weeks were reconstituted with bone marrow from apolipoprotein E-producing wild-type or MΦLRP1-/- mice, and then placed on a chow diet for 10 weeks (n=9 to 11 mice/group). A cohort of apolipoprotein E-/- mice reconstituted with apolipoprotein E-/- bone marrow served as baseline controls (n=9). Plaques of both wild-type and MΦLRP1-/- bone marrow recipients regressed compared with controls (11% and 22%, respectively; P<0.05), and plaques of MΦLRP1-/- recipients were 13% smaller than those of wild-type recipients ( P<0.05). Recipients of MΦLRP1-/- marrow had 36% fewer M1 macrophages ( P<0.01) and 2.5-fold more CCR7 (C-C chemokine receptor type 7)-positive macrophages in the plaque relative to wild-type mice ( P<0.01). Additionally, in vivo studies of cellular egress showed a 4.6-fold increase in 5-ethynyl-2´-deoxyuridine-labeled CCR7+ macrophages in mediastinal lymph nodes. Finally, in vivo studies of reverse cholesterol transport showed a 1.4-fold higher reverse cholesterol transport in MΦLRP1-/- recipient mice ( P<0.01). Absence of macrophage low-density lipoprotein receptor-related protein 1 unexpectedly accelerates atherosclerosis regression, enhances reverse cholesterol transport, and increases expression of the motility receptor CCR7, which drives macrophage egress from lesions.

The role of UNC5b in ox-LDL inhibiting migration of RAW264.7 macrophages and the involvement of CCR7

The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. The C-C chemokine receptor type 7 (CCR7) is positively correlated with the macrophage migration. But the mechanism of CCR7 regulation is not fully clearness. In the present study, we demonstrates that expression in UNC5b and netrin-1 was enhanced in respond to ox-LDL in Raw264.7 macrophage and associated with decreasing cell migration. Interestingly, it was found that ox-LDL significantly downregulate CCR7 gene expression. The expression of CCR7 in mRNA and protein levels were decreased in ox-LDL treated Raw264.7 macrophage when we over expression of UNC5b with pcDNA3.1-UNC5b plasmid. We got the inverse results after silence UNC5b gene with siUNC5b. Meanwhile, the data show that in ox-LDL inducement, UNC5b down-regulated CCR7, and then inhibited macrophage migration. This novel phenomenon is of a crucial highlights to understand deeply the pathogenesis of atherosclerosis. The molecular mechanism of CCR7 regulation deserves intensive study.

Overexpression of hsa-miR-125a-5p enhances proliferation, migration and invasion of head and neck squamous cell carcinoma cell lines by upregulating C-C chemokine receptor type 7.

Head and neck squamous cell carcinoma (HNSCC) is usually diagnosed accompanied by lymph node metastasis. C-C chemokine receptor type 7 (CCR7) is associated with the invasion and metastasis of tumors in HNSCC through various signaling pathways. The role of hsa-miR-125a-5p in HNSCC remains unclear. The present study was performed to investigate the association between hsa-miR-125a-5p and CCR7 in HNSCC. Reverse transcription-quantitative polymerase chain reaction was applied to analyze the expression of hsa-miR-125a-5p in clinical samples. Cell Counting Kit-8, Transwell and wound healing assays were used to detect cell proliferation, invasion, and metastasis, respectively, following overexpression of hsa-miR-125a-5p. Changes in protein expression of CCR7 were observed using western blotting. In the survival analysis, Student's t-tests and log rank tests were performed to analyze the association between the expression of hsa-miR-125a-5p, and HNSCC according to the Cancer Genome Atlas database. The expression of hsa-miR-125a-5p was identified to be significantly lower in cancer tissue compared with the corresponding adjacent normal tissues in clinical samples (P=0.038). The results of western blotting indicated that there was a positive regulatory association between hsa-miR-125a-5p and CCR7. Furthermore, overexpression of hsa-miR-125a-5p significantly enhanced the ability of cell proliferation, migration and invasion in HNSCC, with upregulation of CCR7. The results of survival analysis revealed that patients in the low expression group of hsa-miR-125a-5p tended to have longer survival times compared with the high expression group (P=0.045). Altogether, the data raised the possibility that hsa-miR-125a-5p has a significant role in promoting cancer in HNSCC, which may provide a basis for the treatment of HNSCC in molecular targeted therapy. Further studies are required to ascertain the role of hsa-miR-125a-5p in other HNSCC cell lines and in vivo.

Relationship between C-C chemokine receptor type 2 and P38 mitogen-activated protein kinase signaling pathway in the spinal cord of rats with bone cancer pain

Objective: To investigate the relationship between C-C chemokine receptor type 2(CCR2) and P38 mitogen-activated protein kinase (P38MAPK) signaling pathway in the spinal cord of rats and further clarify the mechanism of bone cancer pain (BCP). Methods: A total of 92 healthy female SD rats, of which 60 were subjected to behavioral tests using a ciliary mechanical stimulation needle. SD rats were randomly divided into six groups: sham operation group (group S), bone cancer pain group (group B), sham operation + DMSO solvent group (group SD), bone cancer pain + DMSO solvent group (group BD), sham operation + RS102895 CCR2 inhibitor group (group SR), bone cancer pain + RS102895 CCR2 inhibitor group (group BR), and Von Frey was used in the behavioral test. Another 32 SD rats were randomly divided into the following 8 groups (n=4): sham operation group (group S), bone cancer pain 5 d group (group B5), bone cancer pain 9 d group (group B9), bone cancer pain 14 d group (group B14), bone cancer pain + DMSO solvent group (group BD), bone cancer pain + RS102895 CCR2 inhibitor 0.5 h group (group BR0.5 h), bone cancer pain + RS102895 CCR2 inhibitor 4 h group (group BR4 h), bone cancer pain + RS102895 CCR2 inhibitor 12 h group (group BR12 h). Western blot was used to detect the expression of P38, p-P38 and CCR2 in spinal cord of rats. Results: At day 5, 7, 9, 14, 21 post-injection, mechanical withdrawal thresholds of group S were(30.9±1.5), (31.9±1.2), (32.0±1.1), (31.6±1.5), (32.2±1.4)g respectively, the mechanical withdrawal thresholds of group B were( 26.4±0.7), (24.4±0.8), (21.4±0.8), (13.5±0.4), (9.9±0.2)g respectively, the mechanical withdrawal thresholds in group B decreased obviously versus group S, and the differences were statistically significant(t=-13.177, -16.660, -23.778, -35.574, -48.401, all P<0.01). At day 9 post-injection, the mechanical withdrawal thresholds in SD, BD, SR and BR groups were (32.4±1.7), (19.4±1.1), (32.1±1.3), (26.3±1.0) g respectively, the difference was statistically significant (F=224.681, P<0.01), and the mechanical withdrawal thresholds in group BD decreased obviously versus group SD, while the mechanical withdrawal thresholds in group BR increased obviously versus group BD. The expression levels of p-P38 in spinal cord of group S, group B5, group B9 and group B14 were(0.08±0.03), (0.20±0.05), (0.40±0.17), (0.65±0.14)respectively, the expression levels of CCR2 were(0.08±0.04), (0.18±0.05), (0.30±0.09), (0.58±0.07)respectively, the difference was statistically significant(F=19.123, 40.746, all P<0.01), and the expression of p-P38 and CCR2 in group B9 were showed a significant up-regulation versus group S. The expression levels of p-P38 in spinal cord of group BD, group BR0.5 h, group BR4 h and group BR12 h were (0.57±0.06), (0.17±0.11), (0.03±0.01), (0.25±0.11)respectively, and the difference was statistically significant(F=29.582, P<0.01). The expression of p-P38 in group BR0.5 h, BR4 h, BR12 h showed a significant down-regulation versus group BD. Conclusion: CCR2 in the spinal cord may be involved in the development of bone cancer pain by activating P38MAPK signaling pathway in rats.

C-C Chemokine Receptor Type 2-Dependent Migration of Myeloid-Derived Suppressor Cells in Protection of Islet Transplants.

Islet transplantation is a promising therapeutic approach to restore the physical response to blood glucose in type 1 diabetes. Current chronic use of immunosuppressive reagents for preventing islet allograft rejection is associated with severe complications. In addition, many of the immunosuppressive drugs are diabetogenic. The induction of transplant tolerance to eliminate the dependency on immunosuppression is ideal, but remains challenging. Addition of hepatic stellate cells allowed generation of myeloid-derived suppressor cells (MDSC) from precursors in mouse bone marrow. Migration of MDSC was examined in an islet allograft transplant model by tracking the systemic administered MDSC from CD45.1 congenic mice. The generated MDSC were expressed C-C chemokine receptor type 2 (CCR2), which was enhanced by exposure to interferon-γ. A single systemic administration of MDSC markedly prolonged survival of islet allografts without requirement of immunosuppression. Tracking the administered MDSC showed that they promptly migrated to the islet graft sites, at which point they exerted potent immune suppressive activity by inhibiting CD8 T cells, enhancing regulatory T cell activity. MDSC generated from CCR2 mice failed to be mobilized and lost tolerogenic activity in vivo, but sustained suppressive activity in vitro. MDSC migration was dependent on expression of CCR2, whereas CCR2 does not directly participate in immune suppression. Expression of CCR2 needs to be closely monitored for quality control purpose when MDSC are generated in vitro for immune therapy.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6-/- mice contain less inflammatory (CCR2hiCX3CR1lo) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2hiCX3CR1lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2hiCX3CR1lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis.

Abstract 2545: Upregulated lncRNA-HNGA1, a target of miR-378a, contributes to aerobic glycolysis of head and neck squamous cell carcinoma through increasing levels of the C-C chemokine receptor type 7 (CCR7)

Abstract Introduction: Long non-coding RNAs (lncRNAs) have been regarded as key regulators in aerobic glycolysis of human cancer. However, the role and function of lncRNAs in Head and Neck Squamous Cell Carcinoma (HNSCC) aerobic glycolysis remain unclear. Here, we report a novel lncRNA, HNGA1, which could promote HNSCC aerobic glycolysis and malignancy by competing for miR-378a binding to regulate CCR7. Materials and methods: Microarrays were performed to explore the lncRNA/miRNA profiles in tissues samples. qRT-PCR and functional analysis were used to confirm the expression and role of lncRNA/miRNA. Bioinformatics approach and luciferase assay were used to verify the miRNA target gene and the interation between lncRNA and miRNA. Nude mouse model was utilized to observe the effect of lncRNA/miRNA in vivo. Tissue array was performed to explore the association between lncRNA and postoperative survival. Results: 1. LncRNA HNSCC glycolysis-associated 1 (HNGA1) was up-regulated in tumor tissues, while miR-378a was down-regulated significantly. These observations were confirmed in 60 pairs of HNSCC tissues/non-tumor tissues samples and 7 cohorts of HNSCC cell lines. 2. Silencing of HNGA1 inhibited HNSCC cells proliferation and glycolysis, while overexpression of HNGA1 had the opposite effect. 3. Ectopic expression of miR-378a repressed HNSCC cells proliferation and glycolysis, whereas miR-378a inhibition resulted in the opposite effect. MiR-378a could repress the CCR7 expression by binding to the 3’-UTR region of CCR7 directly. 4. There was an inverse correlation between HNGA1 and miR-378a in HNSCC specimens. Moreover, miR-378a suppressed HNGA1’s expression and function by directly binding to HNGA1. In addition, HNGA1 could reverse the inhibitory effect of miR-378a on HNSCC cells, which might act as an endogenous ‘sponge’ by competing for miR-378a binding to regulate CCR7. 5. The xenograft mouse model unveiled the suppressive effects of miR-378a on HNSCC tumor growth and glycolysis, while HNGA1 could accelerate this process. 6. The clinicopathological findings suggested that the up-regulation of HNGA1 in HNSCC patients was associated with the poorly differentiated degree and more metastasis. Moreover, the results of tissue array showed that HNGA1 was correlated with postoperative survival. Conclusion: Taken together, our data highlights the pivotal role of HNGA1 in HNSCC aerobic glycolysis. More importantly, we elucidate a novel lncRNA-miRNA-mRNA regulatory network that is HNGA1-miR-378a-CCR7 axis in HNSCC malignancy and progression. Citation Format: Yun Wang. Upregulated lncRNA-HNGA1, a target of miR-378a, contributes to aerobic glycolysis of head and neck squamous cell carcinoma through increasing levels of the C-C chemokine receptor type 7 (CCR7) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2545. doi:10.1158/1538-7445.AM2017-2545