C-C Motif) Receptor 2 Research Articles

Single-Cell Analysis Reveals the Cellular and Molecular Changes of Liver Injury and Fibrosis in Mice During the Progression of Schistosoma japonicum Infection

Schistosomiasis is a parasitic disease that poses a serious threat to human health. However, the pathogenic mechanism during the progression of Schistosoma japonicum infection remains unclear. In order to elucidate this mechanism, we used single-cell RNA sequencing (scRNA-seq) to investigate the transcriptome characteristics of the cellular (single-cell) landscape in the livers of mice infected with Schistosoma japonicum, which were divided into three groups: uninfected mice (0 week (w)), infected mice at 6 w post-infection (the acute phase), and infected mice at 10 w post-infection (the chronic phase). A total of 31,847 liver cells were included and clustered into 21 groups. The cells and T-cells had high heterogeneity in the liver during the progression of schistosome infection. The number and intensity of the intercellular interactions significantly increased at 6 w after infection but decreased at 10 w. The inflammatory signaling pathways chemoattractant cytokine ligand (CCL)5-chemokine C-C-motif receptor (CCR)5 between macrophages and T-cells were predominant at 6 w post-infection; the CCL6-CCR2 signaling pathway between macrophages was predominant at 10 w. The CD80 signaling pathway related to T-cell activation was increased at 6 w after infection, and increased expression of its receptor CD28 on the surfaces of CD4+ and CD8+ T-cells was confirmed by flow cytometry, suggesting an increase in their activation. In addition, scRNA-seq and quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed that the intercellular communication between secretory phosphoprotein 1 (SPP1)-cluster of differentiation (CD44), insulin-like growth factor (IGF)-1-IGF1r and visfatin-insulin receptor (Insr) associated with bone metabolism and insulin metabolism was increased and enhanced in the liver at 6 w post-infection. Overall, we provide the comprehensive single-cell transcriptome landscape of the liver in mice during the progression of schistosome infection and delineate the key cellular and molecular events involved in schistosome infection-induced liver injury and fibrosis. The elevated CCL5-CCR5 and CCL6-CCR2 signaling pathways in the liver may be a drug target for liver injury and fibrosis caused by schistosome infection, respectively.

RNA-binding protein SYNCRIP contributes to neuropathic pain through stabilising CCR2 expression in primary sensory neurones

BackgroundNerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to the genesis of neuropathic pain. SYNCRIP, an RNA-binding protein, is critical for the stabilisation of gene expression. Whether SYNCRIP participates in nerve injury-induced alterations in DRG gene expression and nociceptive hypersensitivity is unknown. MethodsThe expression and distribution of SYNCRIP in mouse DRG after chronic constriction injury (CCI) of the unilateral sciatic nerve were assessed. Effect of microinjection of Syncrip small interfering RNA into the ipsilateral L3 and L4 DRGs on the CCI-induced upregulation of chemokine (C-C motif) receptor 2 (CCR2) and nociceptive hypersensitivity were examined. Additionally, effects of microinjection of adeno-associated virus 5 expressing full length Syncrip mRNA (AAV5-Syncrip) on basal DRG CCR2 expression and nociceptive thresholds were observed. ResultsSYNCRIP is expressed predominantly in DRG neurones, where it co-exists with CCR2. Levels of Syncrip mRNA and SYNCRIP protein in injured DRG increased time-dependently on days 3–14 after CCI. Blocking this increase through microinjection of Syncrip small interfering RNA into injured DRG attenuated CCI-induced upregulation of DRG CCR2 and development and maintenance of nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of AAV5-Syncrip elevated CCR2 expression in microinjected DRGs, enhanced the responses to mechanical, heat, and cold stimuli, and induced ongoing pain in naive mice. Mechanistically, SYNCRIP bound to 3-UTR of Ccr2 mRNA and stabilised its expression in DRG neurones. ConclusionsSYNCRIP contributes to the induction and maintenance of neuropathic pain likely through stabilising expression of CCR2 in injured DRG. SYNCRIP may be a potential target for treating this disorder.

M2-like tumor-associated macrophage-secreted CCL2 facilitates gallbladder cancer stemness and metastasis

BackgroundThe predominant immune cells in solid tumors are M2-like tumor-associated macrophages (M2-like TAMs), which significantly impact the promotion of epithelial-mesenchymal transition (EMT) in tumors, enhancing stemness and facilitating tumor invasion and metastasis. However, the contribution of M2-like TAMs to tumor progression in gallbladder cancer (GBC) is partially known.MethodsImmunohistochemistry was used to evaluate the expression of M2-like TAMs and cancer stem cell (CSC) markers in 24 pairs of GBC and adjacent noncancerous tissues from patients with GBC. Subsequently, GBC cells and M2-like TAMs were co-cultured to examine the expression of CSC markers, EMT markers, and migratory behavior. Proteomics was performed on the culture supernatant of M2-like TAMs. The mechanisms underlying the induction of EMT, stemness, and metastasis in GBC by M2-like TAMs were elucidated using proteomics and transcriptomics. GBC cells were co-cultured with undifferentiated macrophages (M0) and analyzed. The therapeutic effect of gemcitabine combined with a chemokine (C-C motif) receptor 2 (CCR2) antagonist on GBC was observed in vivo.ResultsThe expression levels of CD68 and CD163 in M2-like TAMs and CD44 and CD133 in gallbladder cancer stem cells (GBCSCs) were increased and positively correlated in GBC tissues compared with those in neighboring noncancerous tissues. M2-like TAMs secreted a significant amount of chemotactic cytokine ligand 2 (CCL2), which activated the MEK/extracellular regulated protein kinase (ERK) pathway and enhanced SNAIL expression after binding to the receptor CCR2 on GBC cells. Activation of the ERK pathway caused nuclear translocation of ELK1, which subsequently led to increased SNAIL expression. GBCSCs mediated the recruitment and polarization of M0 into M2-like TAMs within the GBC microenvironment via CCL2 secretion. In the murine models, the combination of a CCR2 antagonist and gemcitabine efficiently inhibited the growth of subcutaneous tumors in GBC.ConclusionsThe interaction between M2-like TAMs and GBC cells is mediated by the chemokine CCL2, which activates the MEK/ERK/ELK1/SNAIL pathway in GBC cells, promoting EMT, stemness, and metastasis. A combination of a CCR2 inhibitor and gemcitabine effectively suppressed the growth of subcutaneous tumors. Consequently, our study identified promising therapeutic targets and strategies for treating GBC.

Chemokine receptor hetero-oligomers regulate monocyte chemotaxis.

It is known that stress influences immune cell function. The underlying molecular mechanisms are unclear. We recently reported that many chemokine receptors (CRs) heteromerize with α1-adrenoceptors (α1-ARs) through which CRs are regulated. Here, we show that arginine vasopressin receptor 1A (AVPR1A) heteromerizes with all human CRs, except chemokine (C-X-C motif) receptor (CXCR)1, in recombinant systems and that such heteromers are detectable in THP-1 cells and human monocytes. We demonstrate that ligand-free AVPR1A differentially regulates the efficacy of CR partners to mediate chemotaxis and that AVPR1A ligands disrupt AVPR1A:CR heteromers, which enhances chemokine (C-C motif) receptor (CCR)1-mediated chemotaxis and inhibits CCR2-, CCR8-, and CXCR4-mediated chemotaxis. Using bioluminescence resonance energy transfer to monitor G protein activation and CRISPR/Cas9 gene-edited THP-1 cells lacking AVPR1A or α1B-AR, we show that CRs that share the propensity to heteromerize with α1B/D-ARs and AVPR1A exist and function within interdependent hetero-oligomeric complexes through which the efficacy of CRs to mediate chemotaxis is controlled. Our findings suggest that hetero-oligomers composed of CRs, α1B/D-ARs, and AVPR1A may enable stress hormones to regulate immune cell trafficking.

Hybrid Membrane-Coated Nanoparticles for Precise Targeting and Synergistic Therapy in Alzheimer's Disease.

The blood brain barrier (BBB) limits the application of most therapeutic drugs for neurological diseases (NDs). Hybrid cell membrane-coated nanoparticles derived from different cell types can mimic the surface properties and functionalities of the source cells, further enhancing their targeting precision and therapeutic efficacy. Neuroinflammation has been increasingly recognized as a critical factor in the pathogenesis of various NDs, especially Alzheimer's disease (AD). In this study, a novel cell membrane coating is designed by hybridizing the membrane from platelets and chemokine (C-C motif) receptor 2 (CCR2) cells are overexpressed to cross the BBB and target neuroinflammatory lesions. Past unsuccessful endeavors in AD drug development underscore the challenge of achieving favorable outcomes when utilizing single-mechanism drugs.Two drugs with different mechanisms of actions into liposomes are successfully loaded to realize multitargeting treatment. In a transgenic mouse model for familial AD (5xFAD), the administration of these drug-loaded hybrid cell membrane liposomes results in a significant reduction in amyloid plaque deposition, neuroinflammation, and cognitive impairments. Collectively, the hybrid cell membrane-coated nanomaterials offer new opportunities for precise drug delivery and disease-specific targeting, which represent a versatile platform for targeted therapy in AD.

The myokine CCL5 recruits subcutaneous preadipocytes and promotes intramuscular fat deposition in obese mice.

Intramuscular fat (IMF) refers to the lipid stored in skeletal muscle tissue. The number and size of intramuscular adipocytes are the primary factors that regulate IMF content. Intramuscular adipocytes can be derived from either in situ or ectopic migration. In this study, it was discovered that the regulation of IMF levels is achieved through the chemokine (C-C motif) ligand 5 (CCL5)/chemokine (C-C motif) receptor 5 (CCR5) pathway by modulating adipocyte migration. In coculture experiments, C2C12 myotubes were more effective in promoting the migration of 3T3-L1 preadipocytes than C2C12 myoblasts, along with increasing CCL5. Correspondingly, overexpressing the CCR5, one of the receptors of CCL5, in 3T3-L1 preadipocytes facilitated their migration. Conversely, the application of the CCL5/CCR5 inhibitor, MARAVIROC (MVC), reduced this migration. In vivo, transplanted experiments of subcutaneous adipose tissue (SCAT) from transgenic mice expressing green fluorescent protein (GFP) provided evidence that injecting recombinant CCL5 (rCCL5) into skeletal muscle promotes the migration of subcutaneous adipocytes to the skeletal muscle. The level of CCL5 in skeletal muscle increased with obesity. Blocking the CCL5/CCR5 axis by MVC inhibited IMF deposition, whereas elevated skeletal muscle CCL5 promoted IMF deposition in obese mice. These results establish a link between the IMF and the CCL5/CCR5 pathway, which could have a potential application for modulating IMF through adipocyte migration.NEW & NOTEWORTHY C2C12 myotubes attract 3T3-L1 preadipocyte migration regulated by the chemokine (C-C motif) ligand 5 (CCL5)/ chemokine (C-C motif) receptor 5 (CCR5) axis. High levels of skeletal muscle-specific CCL5 promote the migration of subcutaneous adipocytes to skeletal muscle and induce the intramuscular fat (IMF) content.

Chemokine receptor CCR1 regulates macrophage activation through mTORC1 signaling in nonalcoholic steatohepatitis

Background and aimsChemokine (CC motif) receptor 1 (CCR1) promotes liver fibrosis in mice. However, its effects on nonalcoholic steatohepatitis (NASH) remain unclear. Therefore, the present study aimed to investigate the role of CCR1 in the progression of NASH. MethodsHuman serum and liver tissues were obtained from patients with NASH and controls. Systemic (Ccr1−/−) and liver macrophage-knockout Ccr1 (Ccr1LKD) mice were fed a high-cholesterol and high-fat (CL) diet for 12 weeks or a methionine/choline-deficient (MCD) diet for 4 weeks. BX471 was used to pharmacologically inhibit CCR1 in CL-fed mice. ResultsCCR1 was significantly upregulated in liver samples from patients with NASH and in animal models of dietary-induced NASH. In the livers of mice fed a CL diet for 12 weeks, the CCR1 protein colocalized with F4/80+ macrophages rather than with hepatic stellate cells. Compared to their wild-type littermates, Ccr1−/− mice fed with the CL or MCD diet showed inhibition of NASH-associated hepatic steatosis, inflammation, and fibrosis. Mechanistically, Ccr1 deficiency suppressed macrophage infiltration and activation by attenuating the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Similar results were observed in Ccr1LKD mice administered the CL diet. Moreover, CCR1 inhibition by BX471 effectively suppressed NASH progression in CL-fed mice. ConclusionsCcr1 deficiency mitigated macrophage activity by inhibiting mTORC1 signaling, thereby preventing the development of NASH. Notably, the CCR1 inhibitor BX471 protected against NASH. These findings would help in developing novel strategies for the treatment of NASH.

Ginsenoside Rh1, a novel casein kinase II subunit alpha (CK2α) inhibitor, retards metastasis via disrupting HHEX/CCL20 signaling cascade involved in tumor cell extravasation across endothelial barrier

Tumor cell extravasation across endothelial barrier has been recognized as a pivotal event in orchestrating metastasis formation. This event is initiated by the interactions of extravasating tumor cells with endothelial cells (ECs). Therefore, targeting the crosstalk between tumor cells and ECs might be a promising therapeutic strategy to prevent metastasis. In this study, we demonstrated that Rh1, one of the main ingredients of ginseng, hindered the invasion of breast cancer (BC) cells as well as diminished the permeability of ECs both in vitro and in vivo, which was responsible for the attenuated tumor cell extravasation across endothelium. Noteworthily, we showed that ECs were capable of inducing the epithelial-mesenchymal transition (EMT) and invadopodia of BC cells that are essential for tumor cell migration and invasion through limiting the nuclear translocation of hematopoietically expressed homeobox (HHEX). The decreased nuclear HHEX paved the way for initiating the CCL20/CCR6 signaling axis, which in turn contributed to damaged endothelial junctions, uncovering a new crosstalk mode between tumor cells and ECs. Intriguingly, Rh1 inhibited the kinase activity of casein kinase II subunit alpha (CK2α) and further promoted the nuclear translocation of HHEX in the BC cells, which resulted in the disrupted crosstalk between chemokine (C-C motif) ligand 20 (CCL20) in the BC cells and chemokine (C-C motif) receptor 6 (CCR6) in the ECs. The prohibited CCL20-CCR6 axis by Rh1 enhanced vascular integrity and diminished tumor cell motility. Taken together, our data suggest that Rh1 serves as an effective natural CK2α inhibitor that can be further optimized to be a therapeutic agent for reducing tumor cell extravasation.

Tfh and Tfr Cells in Autoimmune Diseases

An immunological condition known as autoimmunity causes the excessive generation of autoantibodies against self-antigen and is characterized by enhanced T-cell activation and extra-stimulated B-cells. The development of lymphatic follicle germinal centers (GCs), the maturation of B cells, and differentiation into plasma cells are all significantly aided by follicular helper T cells (Tfh). Tfh cells express the transcriptional regulator B cell lymphoma 6 (BCL-6), C-X-C chemokine receptor 5 (CXCR5), inducible T cell co-stimulator (ICOS), and programmed cell death protein 1 (PD-1). The production of interleukin (IL)-21 and low expression of the chemokine (C-C motif) receptor 7 (CCR7) define Tfh cells. Additionally, Tfh cells are a diverse population of cells with the potential to co-express minute quantities of transcription factors, such as T-box expressed in T cells (T-bet), GATA-binding protein 3 (GATA-3), and retinoic acid receptor-related orphan receptor (ROR-t). Tfh cells that also produce IL-21, IL-4, IL-17, and IFN-γ are referred to as Tfh1, Tfh2, and Tfh17 cells, respectively. The control of humoral immunity is carried out by follicular regulatory (Tfr) cells that express Forkhead box protein 3 (Foxp3). Tfr cells can, however, decrease T-B cell interactions through the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) while promoting B cell maturation through IL-10. In the context of autoimmunity, the role of Tfh and Tfr cells is still not fully understood. We intend to present the most recent data on the characteristics and function of Tfh and Tfr cells under conditions of autoimmunity in this review.

Myeloid-specific blockade of notch signaling alleviates dopaminergic neurodegeneration in Parkinson's disease by dominantly regulating resident microglia activation through NF-κB signaling.

Yolk sac-derived microglia and peripheral monocyte-derived macrophages play a key role during Parkinson's disease (PD) progression. However, the regulatory mechanism of microglia/macrophage activation and function in PD pathogenesis remains unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and activation. In this study, with an 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride-induced acute murine PD model, we found that Notch signaling was activated in amoeboid microglia accompanied by a decrease in tyrosine hydroxylase (TH)-positive neurons. Furthermore, using myeloid-specific RBP-J knockout (RBP-JcKO) mice combined with a PD model, our results showed that myeloid-specific disruption of RBP-J alleviated dopaminergic neurodegeneration and improved locomotor activity. Fluorescence-activated cell sorting (FACS) analysis showed that the number of infiltrated inflammatory macrophages and activated major histocompatibility complex (MHC) II+ microglia decreased in RBP-JcKO mice compared with control mice. Moreover, to block monocyte recruitment by using chemokine (C-C motif) receptor 2 (CCR2) knockout mice, the effect of RBP-J deficiency on dopaminergic neurodegeneration was not affected, indicating that Notch signaling might regulate neuroinflammation independent of CCR2+ monocyte infiltration. Notably, when microglia were depleted with the PLX5622 formulated diet, we found that myeloid-specific RBP-J knockout resulted in more TH+ neurons and fewer activated microglia. Ex vitro experiments demonstrated that RBP-J deficiency in microglia might reduce inflammatory factor secretion, TH+ neuron apoptosis, and p65 nuclear translocation. Collectively, our study first revealed that RBP-J-mediated Notch signaling might participate in PD progression by mainly regulating microglia activation through nuclear factor kappa-B (NF-κB) signaling.

Increased Interleukin-17-Producing γδT Cells in the Brain Exacerbate the Pathogenesis of Sepsis-Associated Encephalopathy and Sepsis-Induced Anxiety in Mice.

Overactivated microglia play a key role in sepsis-associated encephalopathy (SAE), although the involvement of T cells is unclear. γδT cells in the brain and meninges regulate normal fear responses via interleukin (IL)-17 in healthy mice. In our sepsis model, the mice showed exacerbated anxious behavior at 10 days post-induction (dpi). At 8 dpi, IL-17 mRNA was significantly upregulated in the brains of septic mice compared with those of control mice. Simultaneously, the number of γδT cells increased in the brains of septic mice in a severity-dependent manner. Additionally, IL-17-producing γδT cells, expressing both the C-X-C motif receptor (CXCR) 6 and the C-C motif receptor (CCR) 6, increased in mice brains, dependent on the severity of sepsis. The frequency of γδT cells in the meninges fluctuated similarly to that in the brain, peaking at 8 dpi of sepsis. Behavioral tests were performed on septic mice after the continuous administration of anti-γδTCR (α-γδTCR) or anti-IL-17A (α-IL-17A) antibodies to deplete the γδT cells and IL-17A, respectively. Compared with IgG-treated septic mice, α-γδTCR- and α-IL-17A-treated septic mice showed suppressed microglial activation and improvements in anxious behavior. These results suggested that CCR6+CXCR6+ IL-17-producing γδT cells in the brain and meninges promote the exacerbation of SAE and sepsis-induced psychological disorders in mice.

Silver nanoparticles combat Salmonella Typhimurium: Suppressing intracellular infection and activating dendritic cells

Salmonella Typhimurium (ST) can hide inside cells, avoid antibiotic therapy and being killed by host’s immune system to cause persistent infection in humans and animals. Metal nanoparticles are regarded as an alternative to overcome the above limitations, silver nanoparticles especially have been applied in combating drug-resistant bacteria. However, the therapeutic effects of silver nanoparticles against intracellular infection and their impacts on host immunity remain an area of further investigation. In this work, we synthesized Ganoderma extract-capped silver nanoparticles (Ag@Ge) and explored the therapeutic potential and immune adjuvant effects of Ag@Ge against intracellular ST. Firstly, Ag@Ge had a small particle size of 35.52±7.46 nm, good stability, and biocompatibility. Then, Ag@Ge effectively entered RAW 264.7 cells, suppressed intracellular ST infection. Furthermore, Ag@Ge activated mouse dendritic cells (DCs) in vitro, evidenced by increased phenotypic markers (CD80/CD86/CD40/major compatibility complex II (MHCII)) expression and cytokine and chemokine (interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL-2), and chemokine (C-C motif) receptor-7 (CCR-7)) transcription. More notably, the combination of Ag@Ge with inactivated ST recruited intestinal DCs to mitigate ST infection in mice, evidenced by decreased body weight loss and bacterial loads in the tissues (liver, jejunum, and colon), and improved platelets count. The above findings indicate that Ag@Ge has the potential as an alternative nano-antibiotic against intracellular ST infection.

Abstract B013: Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas

Abstract The tumor microenvironment (TME), at both the primary tumor and sites of metastases, is a key determinant in the success of metastatic colonization. Cancer-associated fibroblasts (CAFs) are a predominant cell type in the TME that, through deposition and remodeling of extracellular matrix (ECM) components, can mediate the TME and promote cancer progression. In addition to cancer progression and metastasis, CAFs have also been shown to mediate resistance to chemotherapy and radiotherapy. Therefore, there is a need to identify the factors which activate and recruit CAFs to solid tumors. Though cytokine arrays, we identified CC motif chemokine ligand 5 (CCL5) as a secreted factor which is increased upon co-culture of esophageal cancer cell lines and CAFs. Using CRISPR/Cas9 technology, we determined that CCL5 is predominantly tumor-cell derived and loss of tumor-cell derived CCL5 reduced esophageal carcinoma cell proliferation in vitro and in vivo. We propose that this reduction in cell proliferation is mediated by ERK1/2 signaling as loss of tumor-cell derived CCL5 reduces expression of p-ERK1/2 and cyclin D1, an ERK1/2 target gene which is essential for cell cycle progression. Interestingly, the loss of tumor-derived CCL5 also reduced the percentage of CAFs, identified by αSMA positivity, recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists called Maraviroc. We treated xenografts generated from esophageal carcinoma cell lines or patient derived tumor fragments with Maraviroc. Maraviroc treated xenografts had reduced tumor volume, CAF recruitment and ERK1/2 signaling. Thus, mimicking the effects of the genetic loss of CCL5. Analysis of the TCGA database, identified that high CCL5 or CCR5 expression is associated with poorer prognosis in low grade esophageal carcinomas. Together, we propose that this data highlights the importance of CCL5 in esophageal carcinomas with regards to CAF recruitment and tumor cell proliferation, and that the maraviroc in vivo experiments suggest that targeting the CCL5-CCR5 axis may have therapeutic potential in esophageal carcinomas. Citation Format: Karen J. Dunbar, Qiaosi Tang, Tatiana A. Karakasheva, Anil K. Rustgi. Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B013.

EXPRESSION OF CHEMOKINE (C-C MOTIF) RECEPTOR7 IN PROSTATE CANCER TISSUE OF YOUNG PATIENTS AND IN METASTATIC CANCER CELLS.

Chemokine (C-C motif) receptor 7 (CCR7) is a chemokine receptor involved in the carcinogenesis of several types of tumors due to its promoting action in epithelial-mesenchymal transition events, invasion, angiogenesis and metastasis. However, its role in prostate cancer (PCa) remains unclear. To evaluate CCR7expression by immunohistochemistry in prostate tumors from young patients and to determine the possible relationship with the clinicopathological characteristics. We analyzed retrospectively paraffin-embedded tissue sections from 23young PCa (≤ 55years old) patients and evaluated the transcriptomic expression in the TCGA database. Expression of CCR7was observed in 15cases (65%). The tissue samples from younger patients (≤ 50years) were mostly positive in 72.7% (8/11) of cases. High grade GS (≥ 3) tumors were CCR7-positive in 71% cases. The malignant cells present in lymph nodes were CCR7positive in 100% cases. The bioinformatic analysis showed a high CCR7expression associated with the presence of metastasis (FC = 2.6, p = 0.03) in the Cancer Genome Atlas (TCGA) PCa cohort (PRAD). We showed that CCR7expression in tumors from young patients is associated with the early onset of the disease and could also be related to lymph node metastasis.

Gene Expression Profiling of Peripheral Blood Mononuclear Cells in Type 2 Diabetes: An Exploratory Study.

Background and Objectives: Visceral obesity is associated with chronic low-grade inflammation that predisposes to metabolic syndrome. Indeed, infiltration of adipose tissue with immune-inflammatory cells, including 'classical' inflammatory M1 and anti-inflammatory 'alternative' M2 macrophages, causes the release of a variety of bioactive molecules, resulting in the metabolic complications of obesity. This study examined the relative expression of macrophage phenotypic surface markers, cholesterol efflux proteins, scavenger receptors, and adenosine receptors in human circulating peripheral blood mononuclear cells (PBMCs), isolated from patients with type 2 diabetes mellitus (T2DM), with the aim to phenotypically characterize and identify biomarkers for these ill-defined cells. Materials and Methodology: PBMCs were isolated from four groups of adults: Normal-weight non-diabetic, obese non-diabetic, newly diagnosed with T2DM, and T2DM on metformin. The mRNA expression levels of macrophage phenotypic surface markers (interleukin-12 (IL-12), C-X-C motif chemokine ligand 10 (CXCL10), C-C motif chemokine ligand 17 (CCL17), and C-C motif receptor 7 (CCR7)), cholesterol efflux proteins (ATP-binding cassette transporter-1 (ABCA1), ATP binding cassette subfamily G member 1 (ABCG1), and sterol 27-hydroxylase (CYP27A)), scavenger receptors (scavenger receptor-A (SR-A), C-X-C motif ligand 16 (CXCL16), and lectin-like oxidized LDL receptor-1 (LOX-1)), and adenosine receptors (adenosine A2A receptor (A2AR) and adenosine A3 receptor (A3R)) were measured using qRT-PCR. Results: In PBMCs from T2DM patients, the expression of IL-12, CCR7, ABCA1, and SR-A1 was increased, whereas the expression of CXCL10, CCL17, ABCG1,27-hydroxylase, LOX-1, A2AR and A3R was decreased. On the other hand, treatment with the antidiabetic drug, metformin, reduced the expression of IL-12 and increased the expression of 27-hydroxylase, LOX-1, CXCL16 and A2AR. Conclusions: PBMCs in the circulation of patients with T2DM express phenotypic markers that are different from those typically present in adipose tissue M1 and M2 macrophages and could be representative of metabolically activated macrophages (MMe)-like cells. Our findings suggest that metformin alters phenotypic markers of MMe-like cells in circulation.

Heteromerization between α1B -adrenoceptor and chemokine (C-C motif) receptor 2 biases α1B -adrenoceptor signaling: Implications for vascular function.

Previously, we reported that chemokine (C-C motif) receptor 2 (CCR2) heteromerizes with α1B -adrenoceptor (α1B -AR) in leukocytes, through which α1B -AR controls CCR2. Whether such heteromers are expressed in human vascular smooth muscle cells (hVSMCs) is unknown. Bioluminescence resonance energy transfer confirmed formation of recombinant CCR2:α1b -AR heteromers. Proximity ligation assays detected CCR2:α1B -AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α1B -AR heteromerization per se enhanced α1B -AR-mediated Gαq -coupling. Chemokine (C-C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gαq activation via α1B -AR, cross-recruited β-arrestin to and induced internalization of α1B -AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α1B -AR heteromers biases α1B -AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function.

Mechanism of Panlongqi Tablets intervening in vertebral artery type of cervical spondylosis in rats through PI3K/AKT signaling pathway based on network pharmacology and experimental verification

This study aimed to further explore the relevant mechanism of action by network pharmacology integrated with animal experimental verification based on previous proven effective treatment of vertebral artery type of cervical spondylosis(CSA) by Panlongqi Tablets. Bionetwork analysis was performed to establish drug-disease interaction network, and it was found that the key candidate targets of Panlongqi Tablets were enriched in multiple signaling pathways related to CSA pathological links, among which phosphatidylinositol 3-kinase(PI3 K)/serine-threonine kinase(AKT/PKB) signaling pathway was the most significant. Further, mixed modeling method was used to build the CSA rat model, and the rats were divided into normal, model, Panlongqi Tablets low-, medium-and high-dose(0.16, 0.32, 0.64 g·kg~(-1)) and Jingfukang Granules(positive drug, 1.35 g·kg~(-1)) groups. After successful modeling, the rats were administered for 8 consecutive weeks. Pathological changes of rat cervical muscle tissues were detected by hematoxylin-eosin(HE) staining, and the content of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), vascular endothelial cell growth factor(VEGF) and chemokine(C-C motif) ligand 2(CCL2) in rat serum and/or cervical tissues was determined by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to detect the protein expression levels of chemokine(C-C motif) receptor 2(CCR2), PI3 K, AKT, phosphorylated AKT(p-AKT), I-kappa-B-kinase beta(IKK-beta/IKKβ), nuclear factor kappa B(NF-κB P65) and phosphorylated nuclear factor kappa B(NF-κB p-P65) in rat cervical tissues, and positive expression of p-NF-κB P65 in rat cervical muscle tissues was detected by immunofluorescence. The results showed that Panlongqi Tablets at different doses improved the degree of muscle fibrosis and inflammation in cervical muscle tissues of CSA rats, and reduced the content of inflammatory factors IL-1β, TNF-α, VEGF, CCL2 and CCR2 in serum and/or cervical tissues. The protein expression levels of PI3 K, p-AKT, IKKβ and p-NF-κB P65 as well as the nuclear entry of p-NF-κB P65 in cervical tissues were down-regulated. These findings suggest that Panlongqi Tablets can significantly inhibit the inflammatory response of CSA rats, and the mechanism of action may be related to the down-regulation of the activation of PI3 K/AKT signaling pathway.

The Chemokine (C-C Motif) Receptor 2 Antagonist INCB3284 Reduces Fluid Requirements and Protects From Hemodynamic Decompensation During Resuscitation From Hemorrhagic Shock.

OBJECTIVES:Clinical correlations suggest that systemic chemokine (C-C motif) ligand (CCL) 2 release may contribute to blood pressure regulation and the development of hemodynamic instability during the early inflammatory response to traumatic-hemorrhagic shock. Thus, we investigated whether blockade of the principal CCL2 receptor chemokine (C-C motif) receptor (CCR) 2 affects blood pressure in normal animals, and hemodynamics and resuscitation fluid requirements in hemorrhagic shock models.DESIGN:Randomized prospective treatment study.SETTING:University laboratory.SUBJECTS:Male Sprague-Dawley rats.INTERVENTIONS:First, treatment of healthy anesthetized rats with increasing doses of INCB3284 or vehicle. Second, rats were hemorrhaged for 30 minutes, followed by treatment with the CCR2 antagonist INCB3284 (1.1 and 5.5 μmol/kg), the CCR5 antagonist Maraviroc (=control, 5.5 μmol/kg) or vehicle, and subsequent fluid resuscitation to maintain blood pressure until t = 90 minutes. Third, treatment of rats with 5 μmol/kg INCB3284 or vehicle after hemorrhage and fluid resuscitation until t = 300 minutes.MEASUREMENTS AND MAIN RESULTS:INCB3284 did not affect intrinsic function of isolated rat resistance arteries in pressure myography experiments. Blood pressure in anesthetized vehicle-treated animals continuously decreased by 0.09 ± 0.01 mm Hg/min (p < 0.001) but remained constant after INCB3284 injections. Systemic concentrations of the CCR2 agonists CCL2, CCL5, and CCL11 increased during hemorrhage and fluid resuscitation. INCB3284 dose-dependently reduced fluid requirements by 58% ± 11% in short-term experiments, whereas Maraviroc and vehicle-treated animals were indistinguishable. When resuscitation was performed until t = 300 minutes, INCB3284 reduced fluid requirements by 62% ± 6%, prevented from hemodynamic decompensation, reduced mortality from 50% with vehicle treatment to zero, and reduced overall tissue wet-weight/dry-weight ratios.CONCLUSIONS:Our findings suggest that CCR2 is involved in the regulation of normal cardiovascular function and during the cardiovascular stress response to hemorrhagic shock and fluid resuscitation. The present study identifies CCR2 as a drug target to reduce fluid requirements and to prevent death from hemodynamic decompensation during resuscitation from hemorrhagic shock.

CCR4 and CCR5 Involvement in Monocyte-Derived Macrophage Migration in Neuroinflammation.

Microglia, resident macrophages in the brain, play major roles in neuroinflammation after an acute many neurological diseases, including stroke. Our recent animal stroke model showed that interleukin (IL)-4 and IL-13 released by microglia are converted into monocyte-derived macrophages. However, the correlation with the migration mechanism of these cells is still unclear. This study aimed to clarify the effect of these cells on their migration and to identify potential targets that influence neuroinflammatory conditions. Inflammatory conditions were induced by lipopolysaccharide (LPS) treatment in in vitro and in vivo models. Cell migration was observed using transwell assay, and target chemokines were screened using the proteome profiler array in the in vitro model. Intravital, IVIS, and CLARITY imaging were used in the in vivo model. After LPS (1 ng/ml) treatment in BV2 (microglia cell line) and J774 (monocyte/macrophage cell line) cells, BV2 migration was approximately two-fold more enhanced compared to J774 migration. Overall, six types of chemokine C-C motif ligands (CCLs) were detected from the BV2 conditioned medium with LPS. These CCLs were related to C-C motif receptor (CCR)4 and CCR5. In the in vivo model, CCR4 and CCR5 antagonist significantly inhibited the migration of monocyte-derived macrophages to brain tissue following LPS (5 µg) treatment. In conclusion, the chemokines released by microglia may influence migration of monocyte-derived macrophages in necroinflammation conditions inducted by microglial activation. CCR4 and CCR5 expressed on monocyte-derived macrophages interacted with these chemokines and induced migration. Therefore, CCR4 and CCR5 may be explored as new therapeutic targets for neuroinflammation.

CCR2 as a Marker of Carotid Plaque Disease Severity

It continues to be debated whether patients with asymptomatic carotid artery stenosis (ACAS) benefit from carotid endarterectomy (CEA). It is unknown which patients with ACAS have more vulnerable carotid plaques and can have symptomatic progression leading to stroke. Previous studies have demonstrated that a hallmark of plaque vulnerability is increased populations of inflammatory cells such as monocytes and macrophages expressing chemokine (C-C motif) receptor 2 (CCR2). Although the presence of CCR2 has been reported in human plaque specimens, it is currently unknown whether CCR2 content correlates with inflammation and disease severity in carotid artery plaques.