C-X-C Motif) Receptor 4 Research Articles

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.

Reg4 deficiency aggravates pancreatitis by increasing mitochondrial cell death and fibrosis

Regenerating gene family member 4 (Reg4) has been implicated in acute pancreatitis, but its precise functions and involved mechanisms have remained unclear. Herein, we sought to investigate the contribution of Reg4 to the pathogenesis of pancreatitis and evaluate its therapeutic effects in experimental pancreatitis. In acute pancreatitis, Reg4 deletion increases inflammatory infiltrates and mitochondrial cell death and decreases autophagy recovery, which are rescued by the administration of recombinant Reg4 (rReg4) protein. In chronic pancreatitis, Reg4 deficiency aggravates inflammation and fibrosis and inhibits compensatory cell proliferation. Moreover, C-X-C motif ligand 12 (CXCL12)/C-X-C motif receptor 4 (CXCR4) axis is sustained and activated in Reg4-deficient pancreas. The detrimental effects of Reg4 deletion are attenuated by the administration of the approved CXCR4 antagonist plerixafor (AMD3100). Mechanistically, Reg4 mediates its function in pancreatitis potentially via binding its receptor exostosin-like glycosyltransferase 3 (Extl3). In conclusion, our findings suggest that Reg4 exerts a therapeutic effect during pancreatitis by limiting inflammation and fibrosis and improving cellular regeneration.

Synthesis, Screening, and Evaluation of Theranostic Molecular CPCR4-Based Probe Targeting CXCR4.

Chemokines and chemokine receptors are indispensable to play a key role in the development of malignant tumors. As one of the most widely expressed chemokine receptors, chemokine (C-X-C motif) receptor 4 (CXCR4) has been a popular research focus. In most tumors, CXCR4 expression is significantly upregulated. Moreover, integrated nuclide diagnosis and therapy targeting CXCR4 show great potential. [68Ga]Ga-pentixafor, a radioligand targeting CXCR4, exhibits a strong affinity for CXCR4 both in vivo and in vitro. However, [177Lu]Lu-pentixather, the therapeutic companion of [68Ga]Ga-pentixafor, requires significant refinement to mitigate its pronounced hepatic biodistribution. The objective of this study was to synthesize theranostic molecular tracers with superior CXCR4 targeting functions. The Daudi cell line, which highly expressed CXCR4, and the MM.1S cell line, which weakly expressed CXCR4, were used in this study. Based on the pharmacophore cyclo (-d-Tyr-n-me-d-Orn-l-Arg-L-2-NAL-Gly-) (CPCR4) of pentixafor, six tracers were synthesized: [124I]I-1 ([124I]I-CPCR4), [99mTc]Tc-2 ([99mTc]Tc-HYNIC-CPCR4), [124I]I-3 ([124I]I-pentixafor), [18F]AlF-4 ([18F]AlF-NETA-CPCR4), [99mTc]Tc-5 ([99mTc]Tc-MAG3-CPCR4) and [124I]I-6 ([124I]I-pentixafor-Ga) and their radiochemical purities were all higher than 95%. After positron emission tomography (PET)/single-photon emission computed tomography (SPECT) imaging, the [124I]I-6 group exhibited the best target-nontarget ratio. At the same time, comparing the [68Ga]Ga-pentixafor group with the [124I]I-6 group, we found that the [124I]I-6 group had a better target-nontarget ratio and lower uptake in nontarget organs. Therefore, compound 6 was selected for therapeutic radionuclide (131I) labeling, and the tumor-bearing animal models were treated with [131I]I-6. The volume of the tumor site was significantly reduced in the treatment group compared with the control group, and no significant side effects were found. [124I]I-6 and [131I]I-6 showed excellent affinity for targeting CXCR4, and they showed great potential for the integrated diagnosis and treatment of tumors with high CXCR4 expression.

A novel phthalein component ameliorates neuroinflammation and cognitive dysfunction by suppressing the CXCL12/CXCR4 axis in rats with vascular dementia

Ethnopharmacological relevanceChuanxiong, a plant of the Umbelliferae family, is a genuine medicinal herb from Sichuan Province. Phthalides are one of its main active components and exhibit good protective effect against cerebrovascular diseases. However, the mechanism by which phthalides exert neuroprotective effects is still largely unclear. Aim of the studyIn this study, we extracted a phthalein component (named as QBT) from Ligusticum Chuanxiong, and investigated its neuroprotective effects against vascular dementia (VaD) rats and the underlying mechanism, focusing on the chemokine 12 (CXCL12)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis. MethodsA rat model of VaD was established, and treated with QBT. Cognitive dysfunction in VaD rats was assessed using the Y-maze, new object recognition, and Morris water maze tests. Neuronal damage and inflammatory response in VaD rats were examined through Nissl staining, immunofluorescence, enzyme-linked immunospecific assay, and western blotting analysis. Furthermore, the effects of QBT on CXCL12/CXCR4 axis and its downstream signaling pathways, Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor-κB (NF-κB), were investigated in VaD rats and BV2 microglial cells exposed to oxygen glucose deprivation. ResultsQBT significantly alleviated cognitive dysfunction and neuronal damage in VaD rats, along with inhibition of VaD-induced over-activation of microglia and astrocytes and inflammatory response. Moreover, QBT exhibited anti-inflammatory effects by inhibiting the CXCL12/CXCR4 axis and its downstream JAK2/STAT3 and PI3K/AKT/NF-κB pathways, thereby attenuating the neuroinflammatory response both in vivo and in vitro. ConclusionQBT effectively mitigated neuronal damage and cognitive dysfunction in VaD rats, exerting neuroprotective effects by suppressing neuroinflammatory response through inhibition of the CXCL12/CXCR4 axis.

PU.1 alleviates the inhibitory effects of cigarette smoke on endothelial progenitor cell function and lung-homing through Wnt/β-catenin and CXCL12/CXCR4 pathways.

Endothelial progenitor cells (EPCs) dysfunction is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The transcription factor PU.1 is essential for the maintenance of stem/progenitor cell homeostasis. However, the role of PU.1 in COPD and its effects on EPC function and lung-homing, remain unclear. This study aimed to explore the protective activity of PU.1 and the underlying mechanisms in a cigarette smoke extract (CSE)-induced emphysema mouse model. C57BL/6 mice were treated with CSE to establish a murine emphysema model and injected with overexpressed PU.1 or negative control adeno-associated virus. Morphometry of lung slides, lung function, and apoptosis of lung tissues were evaluated. Immunofluorescence co-localization was used to analyze EPCs homing into the lung. Flow cytometry was performed to detect EPC count in lung tissues and bone marrow (BM). The angiogenic ability of BM-derived EPCs cultured in vitro was examined by tube formation assay. We determined the expression levels of PU.1, β-catenin, C-X-C motif ligand 12 (CXCL12), C-X-C motif receptor 4 (CXCR4), stem cell antigen-1 (Sca-1), and stemness genes. CSE exposure significantly reduced the expression of PU.1 in mouse lung tissues, BM, and BM-derived EPCs. PU.1 overexpression attenuated CSE-induced emphysematous changes, lung function decline, and apoptosis. In emphysematous mice, PU.1 overexpression markedly reversed the decreased proportion of EPCs in BM and promoted the lung-homing of EPCs. The impaired angiogenic ability of BM-derived EPCs induced by CSE could be restored by the overexpression of PU.1. In addition, PU.1 upregulation evidently reversed the decreased expression of β-catenin, CXCL12, CXCR4, Scal-1, and stemness genes in mouse lung tissues, BM, and BM-derived EPCs after CSE exposure. PU.1 alleviates the inhibitory effects of CSE on EPC function and lung-homing via activating the canonical Wnt/β-catenin pathway and CXCL12/CXCR4 axis. While further research is needed, our research may indicate a potential therapeutic target for COPD patients.

Exercise training inhibits macrophage-derived IL-17A-CXCL5-CXCR2 inflammatory axis to attenuate pulmonary fibrosis in mice exposed to silica

Exposure to crystalline silica leads to health effects beyond occupational silicosis. Exercise training's potential benefits on pulmonary diseases yield inconsistent outcomes. In this study, we utilized experimental silicotic mice subjected to exercise training and pharmacological interventions, including interleukin-17A (IL-17A) neutralizing antibody or clodronate liposome for macrophage depletion. Findings reveal exercise training's ability to mitigate silicosis progression in mice by suppressing scavenger receptor B (SRB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and Toll-like receptor 4 (TLR4) pathways. Macrophage-derived IL-17A emerges as primary source and trigger for silica-induced pulmonary inflammation and fibrosis. Exercise training effectively inhibits IL-17A-CXC motif chemokine ligand 5 (CXCL5)-Chemokine (C-X-C motif) Receptor 2 (CXCR2) axis in silicotic mice. Our study evidences exercise training's potential to reduce collagen deposition, preserve elastic fibers, slow pulmonary fibrosis advancement, and enhance pulmonary function post silica exposure by impeding macrophage-derived IL-17A-CXCL5-CXCR2 axis.

Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

BackgroundExcess polymorphonuclear neutrophil (PMN) recruitment or excessive neutrophil extracellular trap (NET) formation can lead to the development of multiple organ dysfunction during sepsis. M2 macrophage-derived exosomes (M2-Exos) have exhibited anti-inflammatory activities in some inflammatory diseases to mediate organ functional protection, but their role in treating sepsis-related acute lung injury (ALI) remains unclear. In this study, we sought to investigate whether M2-Exos could prevent potentially deleterious inflammatory effects during sepsis-related ALI by modulating abnormal PMN behaviours.MethodsC57BL/6 wild-type mice were subjected to a caecal ligation and puncture (CLP) mouse model to mimic sepsis in vivo, and M2-Exos were administered intraperitoneally 1 h after CLP. H&E staining, immunofluorescence and immunohistochemistry were conducted to investigate lung tissue injury, PMN infiltration and NET formation in the lung. We further demonstrated the role of M2-Exos on PMN function and explored the potential mechanisms through an in vitro coculture experiment using PMNs isolated from both healthy volunteers and septic patients.ResultsHere, we report that M2-Exos inhibited PMN migration and NET formation, alleviated lung injury and reduced mortality in a sepsis mouse model. In vitro, M2-Exos significantly decreased PMN migration and NET formation capacity, leading to lipid mediator class switching from proinflammatory leukotriene B4 (LTB4) to anti-inflammatory lipoxin A4 (LXA4) by upregulating 15-lipoxygenase (15-LO) expression in PMNs. Treatment with LXA4 receptor antagonist attenuated the effect of M2-Exos on PMNs and lung injury. Mechanistically, prostaglandin E2 (PGE2) enriched in M2-Exos was necessary to increase 15-LO expression in PMNs by functioning on the EP4 receptor, upregulate LXA4 production to downregulate chemokine (C-X-C motif) receptor 2 (CXCR2) and reactive oxygen species (ROS) expressions, and finally inhibit PMN function.ConclusionsOur findings reveal a previously unknown role of M2-Exos in regulating PMN migration and NET formation through lipid mediator class switching, thus highlighting the potential application of M2-Exos in controlling PMN-mediated tissue injury in patients with sepsis.

G protein activation via chemokine (C-X-C motif) receptor 4 and α1b -adrenoceptor is ligand and heteromer-dependent.

It is unknown whether heteromerization between chemokine (C-X-C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3) and α1b -adrenoceptor (α1b -AR) influences effects of the CXCR4/ACKR3 agonist chemokine (C-X-C motif) ligand 12 (CXCL12) and the noncognate CXCR4 agonist ubiquitin on agonist-promoted G protein activation. We provide biophysical evidence that both ligands stimulate CXCR4-mediated Gαi activation. Unlike CXCL12, ubiquitin fails to recruit β-arrestin. Both ligands differentially modulate the conformation of CXCR4:ACKR3 heterodimers and its propensity to hetero-trimerize with α1b -AR. CXCR4:ACKR3 heterodimerization reduces the potency of CXCL12, but not of ubiquitin, to activate Gαi. Ubiquitin enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from hetero-oligomers comprising CXCR4. CXCL12 enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from CXCR4:α1b -AR heterodimers and reduces phenylephrine-stimulated α1b -AR-promoted Gαq activation from ACKR3 comprising heterodimers and trimers. Our findings suggest heteromer and ligand-dependent functions of the receptor partners.

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.

Multi-omics reveals Dengzhan Shengmai formulation ameliorates cognitive impairments in D-galactose-induced aging mouse model by regulating CXCL12/CXCR4 and gut microbiota.

Dengzhan Shengmai (DZSM), a traditional Chinese medicine formulation, has been administered extensively to elderly individuals with cognitive impairment (CI). However, the underlying mechanisms by which Dengzhan Shengmai improves cognitive impairment remains unknown. This study aimed to elucidate the underlying mechanism of the effect of Dengzhan Shengmai on aging-associated cognitive impairment via a comprehensive combination of transcriptomics and microbiota assessment. Dengzhan Shengmai was orally administered to a D-galactose-induced aging mouse model, and evaluation with an open field task (OFT), Morris water maze (MWM), and histopathological staining was performed. Transcriptomics and 16S rDNA sequencing were applied to elucidate the mechanism of Dengzhan Shengmai in alleviating cognitive deficits, and enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (PCR), and immunofluorescence were employed to verify the results. The results first confirmed the therapeutic effects of Dengzhan Shengmai against cognitive defects; specifically, Dengzhan Shengmai improved learning and impairment, suppressed neuro loss, and increased Nissl body morphology repair. Comprehensive integrated transcriptomics and microbiota analysis indicated that chemokine CXC motif receptor 4 (CXCR4) and its ligand CXC chemokine ligand 12 (CXCL12) were targets for improving cognitive impairments with Dengzhan Shengmai and also indirectly suppressed the intestinal flora composition. Furthermore, in vivo results confirmed that Dengzhan Shengmai suppressed the expression of CXC motif receptor 4, CXC chemokine ligand 12, and inflammatory cytokines. This suggested that Dengzhan Shengmai inhibited CXC chemokine ligand 12/CXC motif receptor 4 expression and modulated intestinal microbiome composition by influencing inflammatory factors. Thus, Dengzhan Shengmai improves aging-related cognitive impairment effects via decreased CXC chemokine ligand 12/CXC motif receptor 4 and inflammatory factor modulation to improve gut microbiota composition.

Sustained induction of IP-10 by MRP8/14 via the IFNβ-IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice.

As a damage-associated molecular pattern, the myeloid-related protein 8/14 (MRP8/14) heterodimer mediates various inflammatory diseases, such as sepsis. However, how MRP8/14 promotes lung injury by regulating the inflammatory response during endotoxemia remains largely unknown. This study aims at illuminating the pathological functions of MRP8/14 in endotoxemia. An endotoxemic model was prepared with wild-type and myeloid cell-specific Mrp8 deletion (Mrp8ΔMC) mice for evaluating plasma cytokine levels. Lung injury was evaluated by hematoxylin and eosin (H&E) staining, injury scoring and wet-to-dry weight (W/D) ratio. The dynamic profile of interferon γ (IFNγ)-inducible protein 10 (IP-10) mRNA expression induced by macrophage MRP8/14 was determined by quantitative real-time polymerase chain reaction (qPCR). Immunoblotting was used to evaluate the increase in IP-10 level induced by activation of the JAK-STAT signaling pathway. Luciferase reporter assay was performed to detect the involvement of IRF7 in Ip-10 gene transcription. In vivo air pouch experiments were performed to determine the biological function of IP-10 induced by MRP8/14. Experiments with Mrp8ΔMC mice showed that MRP8/14 promoted the production of cytokines, including IP-10, in the bronchoalveolar lavage fluid (BALF) and lung injury in endotoxic mice. The result of qPCR showed sustained expression of Ip-10 mRNA in macrophages after treatment with MRP8/14 for 12h. Neutralization experiments showed that the MRP8/14-induced Ip-10 expression in RAW264.7 cells was mediated by extracellular IFNβ. Western blotting with phosphorylation-specific antibodies showed that the JAK1/TYK2-STAT1 signaling pathway was activated in MRP8/14-treated RAW264.7 cells, leading to the upregulation of Ip-10 gene expression. IRF7 was further identified as a downstream regulator of the JAK-STAT pathway that mediated Ip-10 gene expression in macrophages treated with MRP8/14. In vivo air pouch experiments confirmed that the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway was required for chemokine (C-X-C motif) receptor 3 (CXCR3)+ T lymphocyte migration, which promoted lung injury in the context of endotoxemia. In summary, our study demonstrates that MRP8/14 induces sustained production of IP-10 via the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway to attract CXCR3+ T lymphocytes into lung tissues and ultimately results in lung injury by an excessive inflammatory response in the context of endotoxemia.

Transcriptomic analysis reveals the promotion of lymph node metastasis by Helicobacter pylori infection via upregulating chemokine (C-X-C motif) receptor 2 expression in gastric carcinoma

Gastric carcinoma (GC) progression is mainly caused by local aggression and lymph node metastasis. However, some patients with early T-stage disease have lymph node metastasis, whereas some patients with late T-stage disease do not have lymph node metastasis, which indicates that invasion and metastasis are not always sequential in some GC patients. In the present study, the data of 101 GC cases were acquired from TCGA and divided into T-late-N-negative and T-early-N-positive groups according to pathological stages. A total of 338 genes were identified as differential genes between the T-late-N-negative and T-early-N-positive groups. GSEA showed that epithelial cell signaling in the Helicobacter pylori (HP) infection pathway was enriched in the T-early-N-positive group. MB staining indicated that the HP infection rate was 63% (39/62) in N-positive patients compared to 42% (16/38) in N-negative patients. To investigate the potential mechanism, we focused on the gene chemokine (C-X-C motif) receptor 2 (CXCR2), which was not only clustered in the gene set of epithelial cells signaling in the HP infection pathway but also significantly upregulated in T-early-N-positive GC by the analysis of the different genes based on the TCGA dataset. A meta-analysis showed that CXCR2 expression was positively correlated with N-stage but not with T-stage in GC. This study indicated that invasion and metastasis could be independent processes driven by different molecular mechanisms in some GC patients. HP infection was a potential factor that promoted lymph node metastasis by upregulating CXCR2 expression.

Reduced CXCR4 expression in associated with extramedullary and predicts poor survival in newly diagnosed multiple myeloma

ABSTRACT Background Multiple myeloma (MM), a bone marrow-resident hematological malignancy of plasma cells, has remained largely incurable despite the recent advancement in novel therapies. The heterogeneity of myeloma cells makes risk stratification of MM important for therapeutic regimen planning. Research design and methods No immunohistochemical (IHC) predictive and prognostic marker of MM has been constructed yet. Herein, the prognostic value of chemokine (C-X-C motif) receptor 4 (CXCR4) expression in 48 newly diagnosed MM patients was explored using IHC. Correlations between CXCR4 expression and clinical features of MM were analyzed. Results CXCR4-positive patients significantly outperformed CXCR4-negative patients in both 3-year estimated overall survival (93.8% vs 45.8%, P = 0.0392) and progression-free survival (57.1% vs 40.9%, P = 0.0436). Conclusions The incidence of extramedullary lesions in CXCR4-negative patients increased significantly compared with CXCR4-positive patients. Plasma cells that reduce CXCR4 expression have poor prognosis and increase the incidence of extramedullary lesions.

CXCL12/CXCR7/β-arrestin1 biased signal promotes epithelial-to-mesenchymal transition of colorectal cancer by repressing miRNAs through YAP1 nuclear translocation

BackgroundChemokine CXC motif receptor 7 (CXCR7) is an atypical G protein-coupled receptor (GPCR) that signals in a biased fashion. CXCL12/CXCR7 biased signal has been reported to play crucial roles in multiple stages of colorectal cancer (CRC). However, the mechanism of CXCL12/CXCR7 biased signal in promoting CRC progression and metastasis remains obscure.ResultsWe demonstrate that CXCR7 activation promotes EMT and upregulates the expression of Vimentin and doublecortin-like kinase 1 (DCLK1) in CRC cells with concurrent repression of miR-124-3p and miR-188-5p through YAP1 nuclear translocation. Cell transfection and luciferase assay prove that these miRNAs regulate EMT by targeting Vimentin and DCLK1. More importantly, CXCL12/CXCR7/β-arrestin1-mediated biased signal induces YAP1 nuclear translocation, which functions as a transcriptional repressor by interacting with Yin Yang 1 (YY1) and recruiting YY1 to the promoters of miR-124-3p and miR-188-5p. Pharmacological inhibitor of YAP1 suppresses EMT and tumor metastasis upon CXCR7 activation in vivo in tumor xenografts of nude mice and inflammatory colonic adenocarcinoma models. Clinically, the expression of CXCR7 is positively correlated with nuclear YAP1 levels and EMT markers.ConclusionsOur studies reveal a novel mechanism and clinical significance of CXCL12/CXCR7 biased signal in promoting EMT and invasion in CRC progression. These findings highlight the potential of targeting YAP1 nuclear translocation in hampering CXCL12/CXCR7 biased signal-induced metastasis of colorectal cancer.

SDF‑1α/CXCR4 signaling promotes capillary tube formation of human retinal vascular endothelial cells by activating ERK1/2 and PI3K pathways invitro.

The purpose of this study is to address the effect and mechanism of stromal cell-derived factor-1 (SDF-1)α/chemokine (C-X-C motif) receptor 4 (CXCR4) signaling on capillary tube formation of human retinal vascular endothelial cells (HRECs). The expression of CXCR4 in HRECs was quantified by reverse transcription (RT-PCR) and western blotting. The effects of SDF-1α/CXCR4 signaling in capillary tube formation and migration of HRECs was examined using three-dimensional Matrigel assay and wound scratching assay respectively in vitro. Cell proliferation of HRECs was examined using cell counting kit (CCK)-8 assay in the presence of different concentrations of SDF-1α protein. The effect of SDF-1α/CXCR4 signaling in HREC expression of VEGF, basic fibroblast growth factor (bFGF), IL-8 and intercellular cell adhesion molecule (ICAM)-1 was examined using RT-PCR and western blotting. RT-PCR and western blot analysis revealed CXCR4 was expressed in HRECs. The number of intact capillary tubes formed by HRECs in the presence of SDF-1α was markedly more compared with a PBS treated control group. However, it was reduced with treatment with an CXCR4 antagonist. Wound scratching assay showed a significant increase in the number of migrated HRECs under SDF-1α stimulation and the number was reduced with treatment with an CXCR4 antagonist. RT-PCR and western blotting showed that SDF-1α significantly promoted VEGF, bFGF, IL-8 and ICAM-1 expression in HRECs. The proliferation of HRECs in the presence of SDF-1α was promoted in a dosage-dependent manner. SDF-1α/CXCR4 signaling can increase HREC capillary tube formation through promoting HREC migration, proliferation and expression of VEGF, bFGF, IL-8 and ICAM-1.

Aging Promotes Chronic Stress-Induced Depressive-Like Behavior by Activating NLRP1 Inflammasome-Driven Inflammatory Signaling in Mice.

NLRP1 inflammasome has been reported to participate in many neurological disorders. Our previous study has demonstrated that NLRP1 inflammasome is implicated in chronic stress-induced depressive-like behaviors in mice. Age has been reported to be related to depression. Here we examine whether NLRP1 inflammasome is involved in the effect of age on depressive disorder. Two chronic stress stimuli, chronic social defeat stress (CSDS) and repeat social defeat stress (RSDS), were used to establish a depression model in mice of different ages. We found that aged mice exhibited worse depressive-like behaviors and locomotor activity compared to young mice. Interestingly, the expression of hippocampal NLRP1 inflammasome complexes and the levels of the inflammatory cytokines were increased in an age-dependent manner. Also, chronic stress-induced increase in the expression of the hippocampal chemokine C-X-C motif ligand 1 (CXCL1), and its cognate receptor, CXC-motif receptor 2 (CXCR2), was more remarkable in aged mice than that in young mice. Moreover, aged mice exhibited lower hippocampal BDNF levels compared to young mice. Hippocampal Nlrp1a knockdown reduced the levels of pro-inflammatory cytokines and the expression of CXCL1/CXCR2, restored BDNF levels, and alleviated chronic stress-induced depressive-like behaviors in aged mice. Our results suggest that NLRP1 inflammasome-CXCL1/CXCR2-BDNF signaling contributes to the effect of age on chronic stress-induced depressive-like behavior in mice.

α1B‐Adrenoceptor is Required for Chemokine (C‐X‐C Motif) Receptor 4 Mediated Chemotaxis in THP1 Cells

IntroductionChemokine (C‐X‐C motif) receptor 4 (CXCR4) is a key regulator of leukocyte trafficking under physiological conditions and in numerous inflammatory disease processes. Previously, we demonstrated that CXCR4 hetero‐oligomerizes with α1B‐adrenoceptors (ARs), through which the receptors cross‐talk. The existence and function of these hetero‐oligomers in leukocytes, however, is unknown. Thus, we tested whether such heteromeric complexes are detectable in the human monocytic cell line THP1 and in freshly isolated human monocytes, and evaluated how α1B‐AR knockdown by CRISPR/Cas9 gene editing in THP1 cells affects CXCR4‐mediated chemotaxis.MethodsPeripheral blood mononuclear cells were isolated by density gradient centrifugation from human whole blood; monocytes were then isolated via negative selection using MACS LS, an indirect magnetic labeling system. Proximity ligation assays (PLA) were used to visualize individual receptors and receptor‐receptor interactions in THP1 cells and freshly isolated monocytes. CRISPR/Cas9 gene editing was utilized to create a α1B‐AR knockout strain (ADRA1BKO); wild type THP1 cells were used as a negative control (ADRA1Bwt). Cell migration toward the cognate CXCR4 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) was tested employing a 96 well Boyden chamber. Transmigrated cells were counted automatically utilizing high contrast bright field and post‐imaging particle analyses. The chemotactic index (ChI) was calculated as the ratio of cells that transmigrated in the presence versus the absence of CXCL12. Data were analyzed with ANOVA/Tukey’s post‐hoc. Data are mean ± SE from n=4 experiments performed on different days. A 2‐tailed p<0.05 was considered significant.ResultsPLA confirmed the presence of endogenously and constitutively expressed CXCR4:α1B‐AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α1B‐AR and CXCR4:α1B‐AR heteromers were not detectable in ADRA1BKO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1Bwtcells showed a bell‐shaped chemotactic dose response to CXCL12 with a maximal ChI of 9.1±1.1 at 100 nM. CRISPR/Cas9 knockout of α1B‐AR reduced the chemotactic response to CXCL12 by 89±52% (p<0.05). ADRA1BKO results were replicated in an additional α1B‐AR knockout strain to rule out clonal effects.ConclusionOur observations suggest that CXCR4 heteromerizes with α1B‐AR in human monocytes and that CXCL12‐mediated chemotaxis depends on the formation of CXCR4:α1B‐AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4‐mediated chemotaxis in monocytes/leukocytes is controlled.

CXCR4 antagonist AMD3100 enhances therapeutic efficacy of transcatheter arterial chemoembolization in rats with hepatocellular carcinoma.

This study aims to discover the therapeutic effect of chemokine (CXC motif) receptor 4 (CXCR4) antagonist AMD3100 combined with transcatheter arterial chemoembolization (TACE) in a rat model with hepatocellular carcinoma (HCC). An orthotopic model of HCC was established and treated with TACE (doxorubicin-lipiodol emulsion) with or without AMD3100. The tumor volume was measured by magnetic resonance imaging (MRI). Histopathological changes were detected by hematoxylin-eosin (HE) staining. HCC cell apoptosis was assessed by terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) staining. Immunohistochemistry was used to detect the expression of CD34, hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and Ki67. Gene and protein expressions were quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Both TACE and AMD3100 reduced the tumor volume in orthotopic rat model of HCC with the decreased CXCR4 expression in tumor tissues, and the combination had better effect. However, TACE increased the microvessel density (MVD) in HCC tissues of rats, while AMD3100 treatment reduced MVD in HCC tissues. AMD3100 reduced the TACE induced MVD in HCC tissues with the reduction of HIF-1α and VEGF expression. Either AMD3100 or TACE could promote HCC cell apoptosis accompanying by decreased cell proliferation, and their combined use had better therapeutic effects. CXCR4 antagonist AMD3100 enhance therapeutic efficacy of TACE in rats with HCC via promoting the HCC cell apoptosis, reducing cell proliferation, and inhibiting MVD, thus reducing tumor volume.

Analysis of the associations of chemokine receptors expression on circulating Tfh2 and Th2 cells with sIgE level and disease severity in patients with AR

Objective: To clarify the chemotactic characteristics of type 2 helper T cells (Th2 cells) and type 2 follicular helper T cells (Tfh2 cells) in peripheral blood of patients with allergic rhinitis (AR), and to explore the associations between the chemokine receptors expression and the levels of antigen-specific IgE (sIgE) and the severity of the disease. Methods: The peripheral blood mononuclear cells of 41 patients with AR (20 males and 21 females, aged 35.0 (24.5, 47.0) years) and 42 healthy controls (24 males and 18 females, aged 35.0 (24.8, 46.5) years) treated in Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from April 2017 to February 2018 were isolated. The expressions of chemokine receptor (CCR)2, CCR3, CCR4, CCR5, CCR7, CCR8, chemokine C-X3-C-motif receptor 1 (CX3CR1) and C-X-C motif receptor 4 (CXCR4) in Th2 and Tfh2 cells were explored by fluorescence activated cell sorting (FACs). The relationship between the expression of these chemokine receptors in Th2 cells and Tfh2 cells and the levels of serum sIgE and the scores of visual analogue scale (VAS) was analyzed. Graphpad prism 7.0 software was used for statistical analysis. Results: The significant differences in chemotactic characteristics between Th2 cells and Tfh2 cells in the control group were found: Th2 cells highly expressed chemokine receptors CCR2, CCR3, CCR5, CCR8 and CX3CR1, while Tfh2 cells highly expressed immune cell homing chemokine receptors CCR7 and CXCR4. AR patients, compared to the control, expressed higher levels of CCR2, CCR5 and CX3CR1 on peripheral Th2 cells(all P<0.01). At the same time, the proportion of CCR2+and CCR5+Th2 cells was positively correlated with VAS score (r value was 0.58 and 0.61, respectively, both P<0.01). In AR patients, higher expression levels of CCR7 on Tfh2 cells were detected (P<0.01), and the proportion of CCR7+Tfh2 cells was positively correlated with the level of serum sIgE (r=0.51, P<0.01). Conclusion: The percentage of CCR2+ and CCR5+ Th2 cells in peripheral of AR patients can reflect the severity of AR to some extent, while the percentage of CCR7+ Tfh2 cells is positively correlated with the level of serum sIgE.

Chemokine (C-X-C motif) ligand 1/chemokine (C-X-C motif) receptor 2 autocrine loop contributes to cellular proliferation, migration and apoptosis in cervical cancer

ABSTRACT Cervical cancer is the most common malignant tumor in gynecology with high mortality rate, so novel approaches for cervical cancer treatment are urgently needed. In this study, we analyzed the gene expression data and clinicopathological data of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) downloaded from University of California Santa Cruz (UCSC) Xena database. Chemokine (C-X-C motif) ligand 1 (CXCL1) was screened out as a key prognostic gene for cervical cancer. Revealed by the results of ELISA and Western blot, the expression of CXCL1 and chemokine (C-X-C motif) receptor 2 (CXCR2) in cervical cancer cell lines (HeLa and C33A) was significantly higher than that in the primary cervical epithelial cells. Cellular immunofluorescence was used in this study to observe CXCR2 localization. Through CCK8, clone formation assay, wound healing assay and Annexin V/PI staining, it was found that down-regulation of CXCL1 expression or treatment with CXCR2 antagonist (SB 225002) could reduce the cell viability, affect the proliferation, weaken the migration ability, and promote the apoptosis of cervical cancer cells; however, the effect of CXCR2 antagonist was improved after over-expressed CXCL1. CXCL1/CXCR2 chemokine system regulates the proliferation, migration, and apoptosis of cervical cancer cells in the form of an autocrine loop, thus affecting the development of cervical cancer. This study provides a theoretical basis for researching the molecular mechanism of cervical cancer deterioration and development, and brings forward a new idea for the prevention and treatment of cervical cancer.