CXC Chemokine Ligand Research Articles

Altered Expression of CXCL13 and Its Chemokine Receptor CXCR5 on B Lymphocytes during Active Graves’ Orbitopathy

ABSTRACT Purpose To characterize the phenotypic abnormalities of peripheral B cells in patients with Graves’ orbitopathy (GO) and explore the role of chemokine CXC ligand 13 and its receptor type 5 (CXCL13/CXCR5) in relation to B-cell homeostasis using specific neutralizing antibodies. Methods Adults with active GO (n = 22), inactive GO (n = 28), and healthy control subjects (n = 28) were included in the study. Peripheral B cells and B-cell subsets were quantified and analyzed for CXCR5 expression by flow cytometry. The serum CXCL13 concentration was measured by enzyme-linked immunosorbent assays. For chemotactic experiments, Transwell plates were used, and migrating B cells were further analyzed by flow cytometry. Results Compared to healthy subjects, patients with active GO had a significantly higher number of CD19+ B cells and the CD19+CD27+ memory B-cell subset (P = .041 and P = .019, respectively), whereas a marginal increase in the number of these cells was found in patients with inactive GO (P = .062 and P = .087, respectively). Serum CXCL13 levels were significantly higher in patients with active GO (86.9 ± 30.4 pg/mL) than in those with inactive GO (41.7 ± 18.1 pg/mL; P < .001) and in healthy subjects (36.2 ± 7.8 pg/mL; P < .001). The increased CXCL13 concentration was positively and significantly correlated with the clinical activity score (r = 0.757, P < .001). Finally, serum from patients with active GO exerted a stronger chemotactic activity towards B cells and the CD19+CD27+ memory B-cell subset. Blocking CXCL13 or CXCR5 with neutralizing antibodies reduced B-cell migration by a mean of 20%. Conclusions Our data suggest that aberrant CXCL13/CXCR5 expression may contribute to the deficits in B-lymphocyte homeostasis observed in active GO.

Circ_0000524/miR-500a-5p/CXCL16 axis promotes podocyte apoptosis in membranous nephropathy.

Podocytes apoptosis is a hallmark of membranous nephropathy (MN). Circ_0000524 has been reported to be associated with patients with MN, whereas the effect of circ_0000524 on podocytes apoptosis and the underlying mechanisms in MN have not been elaborated. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to detect the expressions of circ_0000524, microRNA-500a-5p (miR-500a-5p), and C-X-C chemokine ligand 16 (CXCL16) in MN tissues and podocytes. Podocyte injury was induced by angiotensin II (AngII). Cell apoptosis was detected by flow cytometry. Caspase-3 or caspase-9 activity was evaluated using a caspase-3 or caspase-9 activity assay kit, respectively. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and pull-down assay were used to address the relationship among circ_0000524,miR-500a-5p and CXCL16. Upregulation of circ_0000524 and CXCL16 and low expression of miR-500a-5p were observed in MN tissues. AngII treatment induced the overexpression of circ_0000524 and CXCL16, a decrease of miR-500a-5p, and induced cell apoptosis in podocytes. Circ_0000524 negatively modulated the expression of miR-500a-5p. Circ_0000524 depletion inhibited podocyte apoptosis, which was rescued by loss of miR-500a-5p. miR-500a-5p contained the binding sites with CXCL16. Circ_0000524 knockdown hampered CXCL16 expression by upregulating miR-500a-5p expression. Additionally, miR-500a-5p upregulation suppressed AngII-induced podocyte apoptosis, which was rescued by enhanced expression of CXCL16. Circ_0000524/miR-500a-5p/CXCL16 pathway regulated podocyte apoptosis in MN.

Menaquinone-7 protects astrocytes by regulating mitochondrial function and inflammatory response under hypoxic conditions.

Astrocytes play a key role in hypoxic brain injury. The aim of our research was to determine the effects of menaquinone-7 (MK-7), a subtype of vitamin K2 (VK2), on astrocytes during hypoxia and its potential mechanisms. Astrocytes from the palliums of newborn Sprague Dawley rats were cultured. An astrocyte-hypoxia model was established using a hypoxia workstation. Cell Counting Kit-8 (CCK-8) and BrdU assays were used to determine the effects of MK-7 on hypoxic astrocytes. 2',7'-Dichlorodihydrofluorescein diacetate (DCFDA) or dihydroethidium (DHE) assays were conducted to detect the levels of reactive oxygen species (ROS). An ATP assay was used to measure intracellular ATP production. The levels of proinflammatory cytokines and chemokines containing interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), CC-chemokine ligand 2 (CCL2), and CXC-chemokine ligand 10 (CXCL10), as well as vitamin K-dependent protein growth arrest-specific 6 (Gas6), were determined in hypoxia-induced astrocytes, in the presence or absence of MK-7 pretreatment. Small interfering RNA (siRNA) was used to knockdown Gas6 expression to determine its role in hypoxic astrocytes pretreated with MK-7. Hypoxia reduced astrocyte viability and proliferation significantly; however, when pretreated with MK-7, these conditions remarkably increased. MK-7 also inhibited hypoxia-induced ROS production and enhanced ATP generation of hypoxic astrocytes. Pretreatment with MK-7 effectively reduced the expression of IL-6, TNF-α, CCL2, and CXCL10 but enhanced the expression of Gas6 in hypoxic astrocytes. Gas6 inhibition markedly attenuated the decline in MK-7-induced ROS generation and IL-6 expression, and weakened MK-7-induced cell viability and ATP production in hypoxic astrocytes. Our study is the first to confirm that MK-7 can protect astrocytes from hypoxia-induced cytotoxicity, possibly by inhibiting mitochondrial dysfunction and the expression of proinflammatory cytokines. Gas6 may also participate in these protective effects.

Sequence characterization and expression pattern analysis of chemokines CXCL13 and CXCL14 in Asian swamp eel

In the present study, we analyzed and identified two CXC chemokine ligands in the swamp eel, Monopterus albus. The open reading frames of MaCXCL13 and MaCXCL14 were 375 bp and 300 bp, which encoded putative proteins of 124 aa and 99 aa, respectively. Phylogenetic and protein similarity analyses found that only MaCXCL14 has high homology with CXCL proteins from other vertebrates, while MaCXCL13 shared low homology with those of mammals. Tissue expression and in vitro expression analysis revealed different expression patterns of these MaCXC chemokines. MaCXCL13 was mainly expressed in immune tissues, such as the spleen and kidney, whereas MaCXCL14 was highly expressed in the brain, followed by the spleen. Upon induction with polyinosine-polycytidylic acid, the transcription levels of the two CXC chemokine genes were upregulated, while phorbol 12-myristate 13-acetate (PMA) only induced the upregulation of MaCXCL13. MaCXCL14 expression was downregulated after PMA stimulation. These results suggested that the two MaCXC chemokine ligands are associated with the immune response and homeostatic processes in the swamp eel.

The protective effects of umbilical cord-derived endothelial colony forming cells on hepatic veno-occlusive disease.

Hepatic veno-occlusive disease (HVOD) characterized by endothelial cell dysfunction is one of the serious complications after hematopoietic stem-cell transplantation or chemotherapeutic drug application. The mortality of HVOD patients with multiorgan dysfunction is as high as 80%. The primary aim of this study was to evaluate whether the infusion of human umbilical cord-derived endothelial colony forming cells (hUC-ECFCs) could mitigate HVOD injury and investigate the underlying mechanism. We found that the expression of chemokine C-X-C chemokine ligand 12 (CXCL12) was markedly increased in the livers of HVOD mice. Meanwhile, hUC-ECFCs infusion could significantly ameliorate liver injury in HVOD mice, which was accompanied by hUC-ECFCs recruitment in the liver, reduced liver pathological alterations, and decreased serum alanine aminotransferase and aspartate aminotransferase activity. Besides, CXCL12-induced migration in hUC-ECFCs was partly impeded by chemokine receptor type 7 (CXCR7) silence or CXCR4 blockage. In conclusion, our results demonstrated that hUC-ECFCs could mitigate HVOD through homing to the injured liver via the CXCL12-CXCR4/CXCR7 signaling pathway.

Semaphorin3A released from human dental pulp cells inhibits the increase in interleukin-6 and CXC chemokine ligand 10 production induced by tumor necrosis factor-α through suppression of nuclear factor-κB activation.

Human dental pulp cells (HDPCs) play an important role in pulpitis. Semaphorin3A (Sema3A), which is an axon guidance molecule, is a member of the secretory semaphorin family. Recently, Sema3A has been reported to be an osteoprotective factor and to be involved in the immune response. However, the role of Sema3A in dental pulp inflammation remains unknown. The aim of this study was to reveal the existence of Sema3A in human dental pulp tissue and the effect of Sema3A which is released from tumor necrosis factor (TNF)-α-stimulated HDPCs on production of proinflammatory cytokines, such as interleukin (IL)-6 and CXC chemokine ligand 10 (CXCL10), from HDPCs stimulated with TNF-α. Sema3A was detected in inflamed pulp as compared to normal pulp. HDPCs expressed Neuropilin-1(Nrp1) which is Sema3A receptor. TNF-α increased the levels of IL-6 and CXCL10 in HDPCs in time-dependent manner. Sema3A inhibited production of these two cytokines from TNF-α-stimulated HDPCs. TNF-α induced soluble Sema3A production from HDPCs. Moreover, antibody-based neutralization of Sema3A further promoted production of IL-6 and CXCL10 from TNF-α-stimulated HDPCs. Sema3A inhibited nuclear factor (NF)-κB P65 phosphorylation and inhibitor κBα degradation in TNF-α-stimulated HDPCs. These results indicated that Sema3A is induced in human dental pulp, and TNF-α acts on HDPCs to produce Sema3A, which partially inhibits the increase in IL-6 and CXCL10 production induced by TNF-α, and that the inhibition leads to suppression of NF-κB activation. Therefore, it is suggested that Sema3A may regulate inflammation in dental pulp and be novel antiinflammatory target molecule for pulpitis.

Functional analysis of CX3CR1 in human induced pluripotent stem (iPS) cell-derived microglia-like cells.

Microglia are the primary immune cells of the central nervous system and crucial to proper development and maintenance of the brain. Microglia have been recognized to be associated with neurodegenerative diseases and neuroinflammatory disorders. CX3C chemokine receptor 1 (CX3CR1), which is specifically expressed in microglia, regulates microglia homeostatic functions such as microglial activation and is downregulated in aged brain and disease-associated microglia in rodents, yet its role in human microglia is not fully understood. In this study, we investigated the function of CX3CR1 in human microglia using human induced pluripotent stem (iPS) cell-derived microglia-like cells. Human iPS cell-derived microglia-like cells expressed microglial markers and showed an activated state and phagocytic activity. Using CRISPR/Cas9 genome editing, we deleted CX3CR1 in human iPS cells and found increased inflammatory responses and phagocytic activity in mutant as compared to wild-type microglia-like cells. In addition, the CX3C chemokine ligand 1 (CX3CL1, a ligand for CX3CR1) significantly decreased the upregulation of IL-6 by lipopolysaccharide stimulation in human iPS cell-derived microglia-like cells. These results suggest that CX3CR1 in human microglia may contribute to microglial homeostasis by regulating inflammatory response and phagocytosis.

A Novel Predictive Model of Diabetic Macular Edema Based on Plasma Cytokines via Machine Learning Algorithms

Objective: The study aimed to investigate the change of plasma cytokines and to build a prediction model for diabetic macular edema (DME) in type 2 diabetes mellitus (T2DM). Methods: In pilot cohort, plasma samples were selected from 15 DM patients and 9 DME patients for screening changes of angiogenesis and inflammation related cytokines by the human cytokine antibody array. These screening cytokines were further validated by ELISA in validation cohort, which containing 100 T2DM with DME (DME group) and 100 T2DM without DME (T2DM group) patients. Pearson correlation were calculated to investigate relationship between cytokines and clinical characteristics. Finally, a prediction model based on plasma cytokines was built by machine learning algorithms. Results: In pilot cohort, 12 plasma cytokines were significant higher, among which there was obvious increase of platelet-derived growth factor-BB (PDGF-BB), tissue inhibitors of metalloproteinase 1 (TIMP-1), angiopoietin 1 (ANG-1), CXC-chemokine ligand 16 (CXCL16) and vascular endothelial cell growth factor receptor 2 (VEGF R2) in DME group (Foldchange > 6 and adjusted p < 0.05), comparing with T2DM group. PDGF-BB, TIMP-1, ANG-1, CXCL16 and VEGFR2 were further confirmed significant elevated in validation cohort. Machine learning algorithms employing random forests (RF) yielded the best performance. In the test set, accuracy, sensitivity, specificity, AUROC and f1 score of RF model were 83.3%, 86.2%, 80.6%, 0.83 and 0.83, respectively. Conclusion: Plasma cytokines were closely association with DME. The prediction model consisting of 4 plasma cytokines in the study was a novel tool for predicting DME in T2DM patients. Funding Statement: This study was funded by Science and Technology Committee of Tongzhou District (Number: KJ2019CX014-25). Declaration of Interests: The authors declare they have no conflict of interest. Ethics Approval Statement: In our research, all procedures complied with the Helsinki Declaration for investigation of human subjects. The study received ethical approval from the competent Institutional Review Boards of Beijing Luhe Hospital. All patient-related procedures were performed strictly following the approval of the internal review and ethics boards of the indicated hospitals.

Serum CXCL16 in Relation to Diabetic Nephropathy in Type 2 Diabetes Egyptian Patients

Abstract Background: CXC chemokine ligand 16 (CXCL16) is both an oxidized low density lipoprotein receptor (ox-LDL) and a chemokine with a potential role in the pathogenesis of Diabetic Nephropathy (DN). Aim of Study: The aim of this work is to evaluate the relationship between serum CXCL16 levels and (DN) in type 2 Egyptian diabetic patients. Methods: This case-controlled study was conducted on 80 T2DM patients (group A1: 24 patients with normoalbu-minuria; group A2: 28 patients with microalbuminuria, and group A3: 28 patients with macroalbuminuria) and 20 age-and sex-matched healthy controls. All were subjected to a complete clinical evaluation and laboratory investigations which included quantitative measurements of urinary albu-min/creatinine ratio and serum CXCL16 levels by enzyme-linked immunosorbent assays. Results: Serum CXCL16 levels were significantly higher in all T2DM groups compared with healthy controls. There was a positive correlation between CXCL16, creatinine and ACR (r=0.48, p:0.039 and r=0.53, p:0.019, respectively), whereas it was significantly negatively associated to eGFR (r=–0.46, p:0.05). Regression analysis indicated that CXCL16 levels were continued significantly correlated with creatinine, eGFR and ACR (p:<0.05, for all). Conclusion: Egyptian type 2 DM patients are characterized by increased serum CXCL16 levels. There was a statistically significant correlation between CXCL16 levels and conven-tional renal markers reflecting disease progression.

Promoting Role of Long Non-Coding RNA Small Nucleolar RNA Host Gene 15 (SNHG15) in Neuronal Injury Following Ischemic Stroke via the MicroRNA-18a/CXC Chemokine Ligand 13 (CXCL13)/ERK/MEK Axis.

BackgroundLong-non-coding RNA (lncRNA) SNHG15 has been reported to be an aberrantly expressed lncRNA in patients with ischemic stroke, but its role in neuronal injury following ischemic stroke remains unclear. We hypothesized that this lncRNA is associated with the pathogenesis of ischemic stroke.Material/MethodsA mouse model of ischemic stroke was established by middle cerebral artery occlusion (MCAO). A neurogenic mouse cell line Neuro-2a (N2a) was subjected to oxygen-glucose deprivation (OGD) for in vitro experiments. Expression of SNHG15, microRNA-18a (miR-18a), and CXCL13 in mouse brain and in OGD-treated N2a cells was determined. Altered expression of SNHG15 and miR-18a was introduced to detect their roles in N2a cell viability and apoptosis. Targeting relationships between miR-18a and SNHG15 or CXCL13 were validated by luciferase assays. Cells were treated with the ERK/MEK antagonist U0126 to assess the role of the ERK/MEK signaling pathway in N2a cell growth.ResultsSNHG15 and CXCL13 were overexpressed and miR-18a was underexpressed in MCAO-induced mice and OGD-treated N2a cells. Silencing of SNHG15 or overexpression of miR-18a promoted cell viability, while decreased cell apoptosis induced by OGD; however, subsequent disruption of the ERK/MEK signaling pathway reversed these effects. SNHG15 was found to bind to miR-18a, which could further target CXCL13.ConclusionsSilencing of SNHG15 led to CXCL13 upregulation through sequestering miR-18a and the following ERK/MEK activation, thus enhancing viability while reducing apoptosis of N2a cells. SNHG15 may serve as a novel target for ischemic stroke treatment.

Regulatory relationship between lncRNA KCNQ1OT1 and miR-146a-3p in preeclampsia

Objective: To observe the changes of the expression level of long non-coding RNA (lncRNA) KCNQ1OT1 and microRNA (miR)-146a-3p in placenta tissues of preeclampsia (PE) patients, as well as their effect and mechanism on the biological functions of trophoblast cells. Methods: A total of 45 cases of hospitalized PE patients in Hainan General Hospital from July 2017 to July 2018 were selected as the PE group, 55 normal pregnant women during the same period were chosed as the control group. The expression level of KCNQ1OT1 mRNA and miR-146a-3p in the placenta tissues between two groups were detected by using quantitative real time (qRT)-PCR. Pearson's test was furtherly analyzed the correlation between them. Human trophoblast cell line (HTR8/SVneo) were randomly divided into control and lipopolysaccharide (LPS) groups, and then LPS group were divide into four sub-groups,included LPS group, short hairpin RNA (sh)-KCNQ1OT1 (after silencing the expression of KCNQ1OT1), miR-146a-3p inhibitor and sh-KCNQ1OT1+miR-146a-3p inhibitor. The targeting relationship between KCNQ1OT1 and miR-146a-3p were predicted by bioinformatics software and confirmed by luciferase assay. The cell proliferation and invasion capacities were respectively detected by cell counting kit-8 (CCK-8) and transwell assay. The expression level of KCNQ1OT1 mRNA and miR-146a-3p were detected by qRT-PCR and the protein expression level of CXC chemokine ligand 12 （CXCL12） and CXC chemokine receptor type 4 (CXCR4) were tested by western blot. Results: (1) The mRNA expression level of KCNQ1OT1 in the placenta of PE group was lower than that of control group (0.23±0.03 vs 0.51±0.04, P<0.05), and the miR-146a-3p expression level was higher than that of the control group (0.49±0.03 vs 0.31±0.03, P<0.05), there were statistical significant differences between the two groups. (2) Luciferase assay showed that there was a targeting relationship between KCNQ1OT1 and mir-146a-3p. Compared with the control group, the mRNA expression level of KCNQ1OT1 in the LPS group were significantly decreased (0.91±0.03 vs 0.35±0.03, P<0.05), and the expression level of miR-146a-3p were significantly increased (0.22±0.03 vs 0.63±0.04, P<0.05). The cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 significantly reduced in the LPS group compared with control group (all P<0.05). The mRNA expression level of KCNQ1OT1 (0.23±0.03) in the sh-KCNQ1OT1 group were further decreased, the expression of miR-146a-3p (0.85±0.03) were further increased, and the cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 were all further reduced compared with control group，there were significant difference between two groups (all P<0.05). Comparing the miR-146a-3p inhibitor group, and sh-KCNQ1OT1+miR-146a-3p inhibitor group with the sh-KCNQ1OT1 group, respectively, the expression level of KCNQ1OT1 mRNA (0.78±0.04 vs 0.50±0.03) increased, and the expression level of miR-146a-3p (0.42±0.03 vs 0.46±0.03) decreased, the cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 were all increased ,there were statistically significant differences (all P<0.05). Conclusion: KCNQ1OT1 could target the regulation of miR-146a-3p through CXCL12/CXCR4 pathway in the proliferation, invasion an migration of HTR8/SVneo cells, which may be involved in the pathogenesis of PE.

Nile tilapia CXCR4, the receptor of chemokine CXCL12, is involved in host defense against bacterial infection and chemotactic activity

CXC chemokine receptor 4 (CXCR4), a member of seven-transmembrane (7-TM) G-protein-coupled receptor superfamily, is the receptor of the CXC chemokine ligand 12 (CXCL12), and plays important roles in host defense and inflammation. In the current study, we cloned and identified a homolog of CXCR4 from Nile tilapia (Oreochromis niloticus), designated as OnCXCR4. The open reading frame of OnCXCR4 is 1149 bp encoding a peptide of 382 amino acids, and the predicted molecular weight is 42.65 kDa OnCXCR4 shares common features of CXCR4 family, including a 7-TM domain and a characteristic CXC motif (containing CYC). Expression analysis showed that OnCXCR4 constitutively expresses in various tested tissues of Nile tilapia, with the highest level in the anterior kidney. When stimulated with Streptococcus agalactiae, Aeromonas hydrophila, Poly(I:C), or LPS in vivo and in vitro, the expression of OnCXCR4 was significantly regulated. AMD3100, a CXCR4 antagonist, could not only inhibit the chemotactic activity of the recombinant OnCXCL12 protein on the leukocytes from anterior kidney, but also reduce the expression of OnCXCR4 significantly. Taken together, these results of our study above indicate that OnCXCR4 may play important roles in host defense against bacterial infectionin in Nile tilapia, and being a receptor of OnCXCL12 to exert functions.

MMP-9 regulates CX3CL1/CX3CR1 in the early phase of neuropathic pain in chronic sciatic nerve constriction injury (CCI) rats.

To observe the effects of MMP-9 (matrix metalloproteases-9) on the mechanical allodynia and thermal hyperalgesia and the expression of CX3CL1 (CX3C chemokine ligand 1) protein in the spinal dorsal root ganglion (DRG) in rats with chronic sciatic nerve constriction injury (CCI). A total of 84 male SD rats were randomly divided into seven groups, namely the normal group , the sham operation group , the model group , the CCI + MMP-9 group, the CCI + TIMP-1 group , the CCI + siRNA group, and the CCI + MM-siRNA group. The CCI model was prepared 5 days after implantation of intrathecal catheter. The rat paw mechanical withdrawal threshold (PWMT) and paw thermal withdrawal latency (PWTL) were measured 1 day before CCI surgery and 1, 2, 3 and 5 days after CCI respectively. Western blot (WB) was used to detect the expressions of the MMP-9 and the CX3CL1 protein in the L5 DRG of the spinal cord 1 day after CCI operation. (I) Behavioral assessment of hyperalgesia: compared with the Sham group, the PWMT and PWTL of the CCI group were significantly reduced at each time point after CCI surgery; compared with the CCI group, the PWMT and PWTL of the CCI + MMP-9 group decreased 1 day after CCI; for the PWMT and PWTL of the CCI + TIMP-1 group and CCI + siRNA group, PWMT and PWTL increased 1 day after CCI; (II) The expressions of MMP-9 and CX3CL1 protein in the DRG of the spinal cord: compared with Sham group, the expressions of MMP-9 and CX3CL1 protein in the DRG of the CCI group increased significantly 1 day after CCI surgery; compared with the CCI group, the expressions increased in the CCI + MMP-9 group 1 day after CCI . However, the expressions of MMP-9 and CX3CL1 in the CCI + TIMP-1 group and CCI + siRNA group were reduced on the first postoperative day. The mechanism of MMP-9 participating in the early phase of neuropathic pain (NP) in CCI rats is related to the upregulation of CX3CL1.

High Level of IL-10 in Cerebrospinal Fluid is Specific for Diagnosis of Primary Central Nervous System Lymphoma.

PurposeThe pathological diagnosis of primary central nervous system lymphoma (PCNSL) by stereotactic brain biopsy and craniotomy is not often applicable due to the high cost and associated complications. In recent years, some biomarkers in cerebrospinal fluid (CSF), including interleukin 10 (IL-10), microRNAs, CXC chemokine ligand 13 (CXCL13), have been reported to be associated with PCNSL. However, this conclusion was controversial. Therefore, this study was to test whether Th17 cell-related cytokines could be used to distinguish PCNSL from other brain tumors.Patients and MethodsTh17 cell-related cytokines in CSF were measured in 108 patients with intracranial tumors, which included 66 PCNSL patients and 42 patients with other types of brain tumors. A receiver-operating characteristic (ROC) curve was utilized to analyze the diagnostic value of the cytokines based on the area under the curve (AUC).ResultsThe CSF IL-10 level and IL-10/IL-6 ratios were significantly higher in PCNSL than in the other brain tumors (58.2 pg/mL VS 1.5 pg/mL, p=0.001; 24.3 VS 0.6, p=0.001). When the cutoff level of IL-10 was set at 8.3 pg/mL, its sensitivity and specificity for diagnosing PCNSL were 59.0% and 98%, respectively. The CSF IL-10 levels over 5pg/mL (+LR 12.3) were of significant value for the diagnosis of PCNSL. These parameters are highly valuable in PCNSL diagnosis, but their sensitivity is less valuable. The sensitivity of IL-4 and IL-17A, the ratio of mature lymphocytes and the monocytes/macrophages ratio in CSF were relatively high. In combination, the sensitivity increased by 15% and the specificity remained above 85%. The best combination was IL-10 and IL-17A, whose sensitivity was 70% and specificity was 96%.ConclusionThe CSF level of IL-10 is a useful diagnostic biomarker in patients with PCNSL. The CSF levels of IL-4, IL-17A, mature lymphocytes and monocytes/macrophages can be used to increase the diagnostic value of CSF IL-10 level and IL-10/IL-6 ratio.

Carnosic acid inhibits inflammatory cytokines production in human periodontal ligament cells

Objectives Carnosic acid (CA), which is one of bioactive compounds from rosemary, has various biological activities. However, the effect of CA on periodontal ligament cells is still uncertain. The aim of this study was to examine the effects of CA on inflammatory cytokines production in human periodontal ligament cells. Methods Cytokine and chemokine levels were measured by ELISA. Activations of signal transduction pathway were determined by Western blotting. Results Treatment of CA decreased inflammatory cytokines such as interleukin (IL)-6, CXC chemokine ligand (CXCL)10, CC chemokine ligand (CCL)2, and CCL20 productions in IL-1β or tumor necrosis factor (TNF)-α-stimulated human periodontal ligament cells in a dose-dependent manner. Moreover, we found that CA could suppress Jun-N-terminal kinase (JNK) pathway, nuclear factor (NF)-κB pathway and signal transducer and activator of transcription (STAT)3 pathway activation in IL-1β or TNF-α-stimulated human periodontal ligament cells. Conclusion The results of this study suggest that CA has anti-inflammatory effects in human periodontal ligament cells by inhibiting JNK, NF-κB and STAT3 pathways.

Serum CXC chemokine ligand-12 as a potential predictor for in-hospital major adverse events after severe traumatic brain injury

BackgroundCXC chemokine ligand-12 (CXCL12) is associated with brain inflammation. We attempted to discern whether serum CXCL12 is a promising predictor for in-hospital major adverse events (IMAEs) after traumatic brain injury (TBI), including death, acute lung injury, acute traumatic coagulopathy, progressive hemorrhagic injury and posttraumatic cerebral infarction. MethodsIn this prospective, observational study, serum CXCL12 levels were quantified among 117 severe TBI patients. We investigated the relation of CXCL12 levels to IMAEs using a multivariate analysis. ResultsMedian value of serum CXCL12 concentrations was substantially higher in patients with IMAEs than in other remainders (21.1 vs. 11.6 ng/ml). With an increasing number of IMAEs, serum CXCL12 concentrations were significantly increased (r = 0.702). Serum CXCL12 independently predicted IMAEs (odds ratio, 1.253; 95% CI, 1.100–1.428). Serum CXCL12 concentrations discriminated risk of IMAEs with area under receiver operating characteristic curve of 0.759 (95% CI, 0.672–0.834), its concentrations >16.0 ng/ml distinguished IMAEs with 83.9% sensitivity and 67.2% specificity and its combination with Glasgow coma scale scores produced the best predictive ability compared with each one alone (p = 0.0116 or 0.0004). ConclusionSerum CXCL12 concentrations are independently associated with IMAEs following TBI, substantializing serum CXCL12 as a useful prognostic biomarker for head trauma patients.

CXCL13 Is Involved in the Lipopolysaccharide-Induced Hyperpermeability of Umbilical Vein Endothelial Cells

Sepsis is a disease that is characterized by a severe systemic inflammatory response to microbial infection and lipopolysaccharide (LPS) and is a well-known inducer of sepsis, as well as endothelial cell hyperpermeability. In the present study, we confirm the elevation of CXC chemokine ligand 13 (CXCL13) in sepsis patients. We also show that LPS exposure increases the release of CXCL13, as well as the mRNA and protein expression of CXCL13 and its receptor, CXC chemokine receptor 5 (CXCR5) in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. We also examined the effects of CXCL13 knockdown on LPS-mediated endothelial hyperpermeability and tight junction (TJ) protein expression in HUVECs. Our results show that HUVECs exposed to LPS result in a significant decrease in transendothelial electrical resistance (TER) and TJ protein (Zonula occluden-1, occludin, and claudin-4) expression, and a notable increase in fluorescein isothiocyanate (FITC)-dextran flux and p38 phosphorylation, which was partially reversed by CXCL13 knockdown. Recombinant CXCL13 treatment had a similar effect as LPS exposure, which was attenuated by a p38 inhibitor, SB203580. Moreover, the CXCL13-neutralizing antibody significantly increased the survival rate of LPS-induced sepsis mice. Collectively, our results show that CXCL13 plays a key role in LPS-induced endothelium hyperpermeability via regulating p38 signaling and suggests that therapeutically targeting CXCL13 may be beneficial for the treatment of sepsis.

Kinetic Analysis of the Early Signaling Steps of the Human Chemokine Receptor CXCR4

G protein–coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer–based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development.SIGNIFICANCE STATEMENTThe CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor–G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.

Exosomal MicroRNA-320a Derived From Mesenchymal Stem Cells Regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Activation by Suppressing CXCL9 Expression.

Rheumatoid arthritis (RA), a chronic systemic inflammatory disease, is a primary cause of disability worldwide. The involvement of fibroblast-like synoviocytes (FLSs) in the regulation of the pathogenesis of RA has been highlighted. Mesenchymal stem cells (MSCs) are important candidates for cell-based treatment in many inflammatory autoimmune diseases. Herein, we identify whether MSC-derived exosomes loaded with microRNA-320a (miR-320a) regulate RA-FLSs. Synovial tissues from 22 patients with RA and 9 patients with osteoarthritis were collected. RA-FLSs were obtained from patients with RA, and their functions were evaluated by determining levels of interleukin-1β (IL-1β), IL-6, and IL-8 and by transwell migration and invasion assays. Dual luciferase reporter gene assays were employed to identify interaction between miR-320a and CXC chemokine ligand 9 (CXCL9). A co-culture system of MSC-derived exosomes and RA-FLSs were performed. The collagen-induced arthritis (CIA) mouse models with arthritis and bone damage were developed. Our results revealed the existence of reciprocal expression of miR-320a and CXCL9 in the synovial tissues obtained from patients with RA. CXCL9 knockdown or miR-320a upregulation suppressed the activation, migration, and invasion of RA-FLSs. CXCL9 was confirmed to be a target of miR-320a, and CXCL9 overexpression restored RA-FLS function in the presence of miR-320a. MSC-derived exosomes containing miR-320a mimic significantly suppressed RA-FLS activation, migration, and invasion in vitro and attenuated arthritis and bone damage in mice with CIA in vivo. Our study uncovers that MSC-derived exosomes participate in the intercellular transfer of miR-320a and subsequently inhibit the progression of RA. These results provide a novel potential therapeutic approach for RA treatment by increasing miR-320a in exosomes.

CXCL5-mediated recruitment of neutrophils into the peritoneal cavity of Gdf15-deficient mice protects against abdominal sepsis

Sepsis is a life-threatening organ dysfunction condition caused by a dysregulated host response to an infection. Here we report that the circulating levels of growth and differentiation factor-15 (GDF15) are strongly increased in septic shock patients and correlate with mortality. In mice, we find that peptidoglycan is a potent ligand that signals through the TLR2-Myd88 axis for the secretion of GDF15, and that Gdf15-deficient mice are protected against abdominal sepsis due to increased chemokine CXC ligand 5 (CXCL5)-mediated recruitment of neutrophils into the peritoneum, leading to better local bacterial control. Our results identify GDF15 as a potential target to improve sepsis treatment. Its inhibition should increase neutrophil recruitment to the site of infection and consequently lead to better pathogen control and clearance.