CXC Chemokine Ligand Research Articles

Roles of the CXCR1/CXCL8 axis in abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis

Objective: To investigate the role of the CXC chemokine receptor 1 (CXCR1)/CXC chemokine ligand 8 (CXCL8) axis in the abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis (PBC). Methods: 30 female C57BL/6 mice were randomly divided into the PBC model group (PBC group), reparixin intervention group (Rep group), and blank control group (Con group) in an in vivo experiment. PBC animal models were established after 12 weeks of intraperitoneal injection of 2-octanoic acid coupled to bovine serum albumin (2OA-BSA) combined with polyinosinic acid polycytidylic acid (polyI:C). After successful modelling, reparixin was injected subcutaneously into the Rep group (2.5 mg · kg(-1) · d(-1), 3 weeks). Hematoxylin-eosin staining was used to detect histological changes in the liver. An immunohistochemical method was used to detect the expression of cytokeratin 19 (CK-19). Tumor necrosis factor-α (TNF-α), γ-interferon (IFN-γ) and interleukin (IL)-6 mRNA expression were detected by qRT-PCR. Western blot was used to detect nuclear transcription factor-κB p65 (NF-κB p65), extracellularly regulated protein kinase 1/2 (ERK1/2), phosphorylated extracellularly regulated protein kinase 1/2 (p-ERK1/2), Bcl-2-related X protein (Bax), B lymphoma-2 (Bcl-2), and cysteine proteinase-3 (Caspase- 3) expression. Human intrahepatic bile duct epithelial cells were divided into an IL-8 intervention group (IL-8 group), an IL-8+Reparicin intervention group (Rep group), and a blank control group (Con group) in an in vitro experiment. The IL-8 group was cultured with 10 ng/ml human recombinant IL-8 protein, and the Rep group was cultured with 10 ng/ml human recombinant IL-8 protein, followed by 100 nmol/L Reparicin. Cell proliferation was detected by the EdU method. The expression of TNF-α, IFN-γ and IL-6 was detected by an enzyme-linked immunosorbent assay. The expression of CXCR1 mRNA was detected by qRT-PCR. The expression of NF-κB p65, ERK1/2 and p-ERK1/2 was detected by western blot. A one-way ANOVA was used for comparisons between data sets. Results: The results of in vivo experiments revealed that the proliferation of cholangiocytes, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the Con group compared with the PBC group. However, reparixin intervention reversed the aforementioned outcomes (P<0.05). In vitro experiments showed that the proliferation of human intrahepatic cholangiocyte epithelial cells, the expression of CXCR1 mRNA, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the IL-8 group compared with the Con group. Compared with the IL-8 group, the proliferation of human intrahepatic cholangiocyte epithelial cells, NF-κB and ERK pathway-related proteins, and inflammatory indicators were significantly reduced in the Rep group (P < 0.05). Conclusion: The CXCR1/CXCL8 axis can regulate the abnormal proliferation of bile duct epithelial cells in PBC, and its mechanism of action may be related to NF-κB and ERK pathways.

CCL26 in primary biliary cholangitis - Is it a novel disease mediator?

To verify the role of CX3C chemokine ligand 1 - CX3C chemokine receptor 1 (CX3CL1-CX3CR1) pathway in the pathogenesis of primary biliary cholangitis (PBC). To explore whether CCL26, a novel functional ligand to CX3CR1, participates in the immunological mechanism of PBC. Fifty-nine PBC patients and 54 healthy controls were recruited. Enzyme-linked immunosorbent assay and flow cytometry were used to measure CX3CL1 and CCL26 concentrations in plasma and CX3CR1 expression on peripheral lymphocytes, respectively. Chemotactic effects of CX3CL1 and CCL26 toward lymphocytes were detected by Transwell cell migration assays. CX3CL1 and CCL26 expressions in liver were assessed by immunohistochemical staining. Effects of CX3CL1 and CCL26 on stimulating cytokine production from lymphocytes were evaluated using intracellular flow cytometry. Significantly elevated CX3CL1 and CCL26 plasma concentration and CX3CR1 expression on CD4+ and CD8+ T cells were noted in PBC patients. CX3CL1 exhibited chemotactic activity toward CD8+ T, natural killer (NK) and NKT cells in a dose-dependent manner while such chemotactic effects were not detected for CCL26. In PBC patients, CX3CL1 and CCL26 were both increasingly expressed in biliary tracts and a concentration gradient of CCL26 in hepatocytes around portal areas was observed. Immobilized CX3CL1 could enhance interferon-γ production from T and NK cells while such effect was not exhibited by soluble CX3CL1 or CCL26. CCL26 expression is significantly elevated in plasma and biliary duct of PBC patients, yet does not appear to attract CX3CR1-expressing immune cells. CX3CL1-CX3CR1 pathway promotes the infiltration of T, NK and NKT cells into bile ducts and forms a positive feedback loop with T-helper 1 type cytokines in PBC.

NF-κB drives epithelial-mesenchymal mechanisms of lung fibrosis in a translational lung cell model.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α-smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

Early evolution of glial morphology and inflammatory cytokines following hypoxic-ischemic injury in the newborn piglet brain

Neuroinflammation is a hallmark of hypoxic-ischemic injury and can be characterized by the activation of glial cells and the expression of inflammatory cytokines and chemokines. Interleukin (IL)-1β and tumor necrosis factor (TNF)α are among the best-characterized early response cytokines and are often expressed concurrently. Several types of central nervous system cells secrete IL-1β and TNFα, including microglia, astrocytes, and neurons, and these cytokines convey potent pro-inflammatory actions. Chemokines also play a central role in neuroinflammation by controlling inflammatory cell trafficking. Our aim was to characterise the evolution of early neuroinflammation in the neonatal piglet model of hypoxic-ischemic encephalopathy (HIE). Piglets (< 24 h old) were exposed to HI insult, and recovered to 2, 4, 8, 12 or 24H post-insult. Brain tissue from the frontal cortex and basal ganglia was harvested for assessment of glial cell activation profiles and transcription levels of inflammatory markers in HI piglets with comparison to a control group of newborn piglets. Fluorescence microscopy was used to observe microglia, astrocytes, neurons, degenerating neurons and possibly apoptotic cells, and quantitative polymerase chain reaction was used to measure gene expression of several cytokines and chemokines. HI injury was associated with microglial activation and morphological changes to astrocytes at all time points examined. Gene expression analyses of inflammation-related markers revealed significantly higher expression of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin 1 beta (IL-1β), chemokines cxc-chemokine motif ligand (CXCL)8 and CXCL10, and anti-inflammatory cytokine transforming growth factor (TGF)β in every HI group, with some region-specific differences noted. No significant difference was observed in the level of C-X-C chemokine receptor (CCR)5 over time. This high degree of neuroinflammation was associated with a reduction in the number of neurons in piglets at 12H and 24H in the frontal cortex, and the putamen at 12H. This reduction of neurons was not associated with increased numbers of degenerating neurons or potentially apoptotic cells. HI injury triggered a robust early neuroinflammatory response associated with a reduction in neurons in cortical and subcortical regions in our piglet model of HIE. This neuroinflammatory response may be targeted using novel therapeutics to reduce neuropathology in our piglet model of neonatal HIE.

Lipopolysaccharide and lipoteichoic acid regulate the PI3K/AKT pathway through osteopontin/integrin β3 to promote malignant progression of non-small cell lung cancer.

Lung cancer (LC) is a malignancy with one of the highest mortality rates. Respiratory microbiota is considered to play a key role in the development of LC, but the molecular mechanisms are rarely studied. We used lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to study human lung cancer cell lines PC9 and H1299. The gene expression of CXC chemokine ligand (CXCL)1/6, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The Cell-Counting Kit 8 (CCK-8) was used to analyze cell proliferation. Transwell assays were performed to analyze cell migration ability. Flow cytometry was used to observe cell apoptosis. Western blot and qRT-PCR were used to analyze the expression of secreted phosphoprotein 1 (SPP1), toll-like receptor (TLR)-2/4, and NLR family pyrin domain containing 3 (NLRP3) to determine the mechanism of LPS + LTA. We evaluated the effect of LPS + LTA on cisplatin sensibility by analyzing cell proliferation, apoptosis, and caspase-3/9 expression levels. We observed the proliferation activity, apoptosis, and migration ability of cells in which SPP1 had been transfected small interfering (si) negative control (NC) and integrin β3 siRNA. Then the mRNA expression level and protein expression of PI3K, AKT, and ERK were analyzed. Finally, the nude mouse tumor transplantation model was conducted to verify. We studied that in two cell lines, the expression level of inflammatory factors in LPS+LTA group was significantly higher than that in single treatment group (P<0.001). We explored LPS + LTA combined treatment group significantly increased the expression of NLRP3 and genes and proteins. LPS + LTA + Cisplatin group could significantly reduce the inhibitory effect of LPS on cell proliferation (P<0.001), reduce the apoptosis rate (P<0.001) and significantly reduce the expression levels of caspase-3/9 (P<0.001) compared with Cisplatin group. Finally, we verified that LPS and LTA could increase osteopontin (OPN)/integrin β3 expression and activate the PI3K/AKT pathway to promote malignant progression of LC in vitro studies. This study provides a theoretical basis for further exploration of the influence of lung microbiota on NSCLC and the optimization of LC treatment in the future.

Icariside II potentiates the anti-PD-1 antitumor effect by reducing chemotactic infiltration of myeloid-derived suppressor cells into the tumor microenvironment via ROS-mediated inactivation of the SRC/ERK/STAT3 signaling pathways

BackgroundImmune checkpoint blockade agents, such as anti-PD-1 antibodies, show promising antitumor efficacy but only a limited response in patients with non-small cell lung cancer (NSCLC). Icariside II (IS), a metabolite of Herba Epimedii, is a COX-2 and EGFR inhibitor that can enhance the anti-PD-1 effect. This study aimed to evaluate the antitumor effect of IS in combination with anti-PD-1 and explore the underlying mechanism. MethodsTumor growth was assessed in Lewis Lung Cancer (LLC) tumor-bearing mice in seven groups (control, IS 20 mg/kg, IS 40 mg/kg, anti-PD-1, IS 20 mg/kg+anti-PD-1, IS 40 mg/kg+anti-PD-1, ERK inhibitor+anti-PD-1). Tumor-infiltrating immune cells were measured by flow cytometry. The mechanisms were explored by tumor RNA-seq and validated in LLC cells through molecular biological experiments using qRT‒PCR, ELISA, and western blotting. ResultsAnimal experiments showed that IS in combination with anti-PD-1 further inhibited tumor growth and remarkably reduced the infiltration of myeloid-derived suppressor cells (MDSCs) into the tumor compared with anti-PD-1 monotherapy. RNA-seq and in vitro experiments showed that IS suppressed the chemotactic migration of MDSCs by downregulating the expression of CXC chemokine ligands 2 (CXCL2) and CXCL3. Moreover, IS promoted reactive oxygen species (ROS) generation and inhibited the activation of SRC/ERK/STAT3 in LLC cells, which are upstream signaling pathways of these chemokines. ConclusionIS potentiates the anti-PD-1 anti-tumor effect by reducing chemotactic infiltration of the myeloid-derived suppressor cell into the tumor microenvironment, via ROS-mediated inactivation of SRC/ERK/STAT3 signaling pathways.

Reduced expression of CXCL16/CXCR6 is involved in the pathogenesis of hidradenitis suppurativa.

Mutations in the γ-secretase complex have been well-described in familial hidradenitis suppurativa (HS). No gene mutations have been identified in sporadic HS, which comprises 60%-70% of all HS cases. Obesity and smoking are risk factors for HS and are closely related to DNA methylation, an essential epigenetic phenomenon. Hence, we hypothesized that epigenetic modifications might be involved in sporadic HS. To investigate genes with aberrant methylation in sporadic HS cases and assess their expression in skin lesions and blood from patients with HS. Skin lesion samples and corresponding normal skin were obtained from three patients with HS and subjected to whole-genome DNA methylation sequencing. Blood samples were collected from 20 patients with HS and 20 healthy controls (HCs). The HS mouse model was established by applying tamoxifen to NcstnΔKC mice. Target gene expression was analysed by immunohistochemistry, immunofluorescence, western blotting, enzyme-linked immunosorbent assay (ELISA) and semiquantitative real-time polymerase chain reaction (RT-qPCR). Among 10807 differentially methylated genes, we filtered 2101 genes with hypermethylated promoter regions, and following bioinformatics analyses, we focused on CXC chemokine ligand 16 (CXCL16). Subsequent functional experiments confirmed the downregulation of CXCL16 and its receptor, CXC chemokine receptor (CXCR) 6, in skin tissue from HS patients and NcstnΔKC mice. Serum CXCL16 concentrations were also significantly decreased in patients with HS. Our data revealed the downregulation of CXCL16 and CXCR6 in HS.

Transcriptome sequencing analysis of the role of β-catenin in F-actin reorganization in embryonic palatal mesenchymal cells.

Palatogenesis is a highly regulated and coordinated developmental process that is coordinated by multiple transcription factors and signaling pathways. Our previous studies identified that defective palatal shelf reorientation due to aberrant mesenchymal β-catenin signaling is associated with Filamentous actin (F-actin) dysregulation. Herein, the underlying mechanism of mesenchymal β-catenin in regulating F-actin cytoskeleton reorganization is further investigated. Firstly, β-catenin silenced and overexpressed mouse embryonic palatal mesenchymal (MEPM) cells were established by adenovirus-mediated transduction. Subsequently, we compared transcriptomes of negative control (NC) group, β-catenin knockdown (KD) group, or β-catenin overexpression group respectively using RNA-sequencing (RNA-seq), and differentially expressed genes (DEGs) screened were further identified by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, in vivo experiments further verified the expression change of critical molecules. We discovered 184 and 522 DEGs in the knockdown and overexpression groups compared to the NC group, respectively (adjusted P<0.05; |fold change| >2.0). Among these, 106 DEGs were altered in both groups. Gene Ontology (GO) enrichment analysis relating to biological functions identified cytokine-cytokine receptor interaction, and positive modulation of cellular migration. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment assessment indicated that these DEGs were chiefly linked by the regulation of signaling receptor activity and chemokine signaling pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the similar expression trend of serum amyloid A3 (Saa3) and CXC-chemokine ligand 5 (Cxcl5) possibly involved in cytoskeletal rearrangement with RNA-seq data. Experiments in vivo displayed that no significant expression change of Saa3 and Cxcl5 was observed in β-catenin knockout and overexpressed mouse models. Our study provides an expression landscape of DEGs in β-catenin silenced and overexpressed MEPM cells, which emphasizes the important role of processes such as chemotactic factor and cell migration. Our data gain deeper insight into genes associated with F-actin reorganization that is regulated by β-catenin either directly or by another route, which will contribute to further investigation of the exact mechanism of mesenchymal β-catenin/F-actin in palatal shelf reorientation.

Abstract B022: The adaptor protein FRS2beta fashions a cytokine-rich inflammatory microenvironment that is associated with DCIS and promotes breast cancer carcinogenesis

Abstract Although it is commonly held that pro-inflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we report that FRS2beta, an adaptor protein with a highly restricted epithelial expression pattern, mediates the pro-inflammatory changes that alter the stroma in premalignant mammary tissues. We posit that these changes, which include lesions of ductal carcinoma in situ (DCIS), precede disease onset. In the MMTV-ErbB2 mouse model of mammary carcinoma, FRS2beta-deficiency markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2beta-deficient premalignant tissues. We found that the co-localization of FRS2beta and the NEMO subunit of the IkappaB kinase (IKK) complex in early endosomes activated nuclear factor-kappaB (NFkappaB), a master regulator of inflammation. Moreover, the inhibition of NFkappaB-induced cytokines CXC chemokine ligand (CXCL)12 and insulin-like growth factor (IGF1) abrogated tumorigenesis. In line with this, human breast cancer tissues that express higher levels of FRS2beta contain more stroma. The elucidation of the FRS2beta-NFkappaB axis uncovers a hitherto unknown molecular link between the pro-inflammatory changes in the DCIS and the disease onset. Citation Format: Noriko Gotoh. The adaptor protein FRS2beta fashions a cytokine-rich inflammatory microenvironment that is associated with DCIS and promotes breast cancer carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B022.

CX3CL1 deficiency ameliorates inflammation, apoptosis and accelerates osteogenic differentiation, mineralization in LPS-treated MC3T3-E1 cells via its receptor CX3CR1

BackgroundOsteoporosis is a devastating skeletal disease responsible for bone fragility and fracture. CX3C chemokine ligand 1 (CX3CL1) is an inflammatory chemokine which has been identified to possess increased expression in the serum of postmenopausal osteoporotic patients. This paper was to illuminate the impacts of CX3CL1 on inflammation, apoptosis and osteogenic differentiation, mineralization in LPS-treated osteoblasts and investigate the regulatory mechanism. MethodsThe viability of MC3T3-E1 cells exposed to elevating doses of LPS was detected by CCK-8 assay. CX3CL1 and C-X3-C motif chemokine receptor 1 (CX3CR1) expression were detected by RT-qPCR and western blot. CX3CR1 expression was examined again following CX3CL1 depletion. The binding of CX3CL1 with CX3CR1 was testified through Co-IP assay. In MC3T3-E1 cells co-transduced with CX3CL1 interference and CX3CR1 overexpression plasmids following LPS exposure, cell activity and inflammation were separately estimated via CCK-8 assay and RT-qPCR. Apoptosis was measured by TUNEL assay and western blot. Osteoblast differentiation was evaluated by ALP activity assay, RT-qPCR and western blot. Osteoblast mineralization was assessed by ARS staining, RT-qPCR and western blot. ResultsThe experimental data presented that LPS attenuated the viability and enhanced CX3CL1 and CX3CR1 expression in MC3T3-E1 cells in a dose-dependent manner. CX3CR1 interacted with CX3CL1 and was positively modulated by CX3CL1. The suppressive role of CX3CL1 absence in LPS-evoked viability decrease, inflammation and apoptosis in MC3T3-E1 cells was reversed by CX3CR1 elevation. Besides, CX3CR1 reversed the promoted osteoblast differentiation and mineralization imposed by CX3CL1 interference. ConclusionsCX3CL1 knockdown eased inflammation, apoptosis and promoted osteogenic differentiation, mineralization in MC3T3-E1 cells upon LPS exposure through down-regulating CX3CR1.

Comprehensive assessment of growth factors, inflammatory mediators, and cytokines in vitreous from patients with proliferative diabetic retinopathy

To assess alterations in growth factors, inflammatory mediators, and cytokines associated with vitreous-retinal diseases in vitreous humor from patients with proliferative diabetic retinopathy (PDR), and to identify potential new treatment targets and strategies. Control vitreous samples were collected from patients with macular hole, epiretinal membranes, or rhegmatogenous retinal detachments, and PDR samples from patients with complications of PDR, who required pars plana vitrectomy. Specimens were stored at -80°C and then investigated by Luminex multi-factor assay. Parametric and nonparametric analyses of demographic characteristics and cytokine expression levels were conducted using SPSS. There were no significant differences in demographic characteristics between patients with and without PDR. Expression levels of growth factors [platelet-derived growth factor (PDGF)-AA, glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor A (VEGFA)], inflammatory mediators [interleukin (IL)-8, IL-11, and tumor necrosis factor-α (TNF-α)] and cytokines [chemokine C-X-C ligand (CXCL)10, interferon-γ (IFN-γ), and granulocyte macrophage-colony stimulating factor (GM-CSF)] were significantly elevated in vitreous humor from patients with PDR compared with those in the control group (all P<0.05). Further, VEGFA levels were lower in patients with PDR treated with anti-VEGF injection than those who were not (P<0.05), and there was no difference between the PDR group treated with anti-VEGF and controls (P>0.05). This proof-of-concept study demonstrates the potential for combinational therapeutic strategies to ameliorate diabetic retinopathy progression by targeting growth factors, inflammatory factors, and cytokines, in addition to VEGFA.

Essential roles of the cytokine oncostatin M in crosstalk between muscle fibers and immune cells in skeletal muscle after aerobic exercise

Crosstalk between muscle fibers and immune cells is well known in the processes of muscle repair after exercise, especially resistance exercise. In aerobic exercise, however, this crosstalk is not fully understood. In the present study, we found that macrophages, especially anti-inflammatory (M2) macrophages, and neutrophils accumulated in skeletal muscles of mice 24h after a single bout of an aerobic exercise. The expression of oncostatin M (OSM), a member of the interleukin 6 family of cytokines, was also increased in muscle fibers immediately after the exercise. In addition, we determined that deficiency of OSM in mice inhibited the exercise-induced accumulation of M2 macrophages and neutrophils, whereas intramuscular injection of OSM increased these immune cells in skeletal muscles. Furthermore, the chemokines related to the recruitment of macrophages and neutrophils were induced in skeletal muscles after aerobic exercise, which were attenuated in OSM-deficient mice. Among them, CC chemokine ligand 2, CC chemokine ligand 7, and CXC chemokine ligand 1 were induced by OSM in skeletal muscles. Next, we analyzed the direct effects of OSM on the skeletal muscle macrophages, because the OSM receptor β subunit was expressed predominantly in macrophages in the skeletal muscle. OSM directly induced the expression of these chemokines and anti-inflammatory markers in the skeletal muscle macrophages. From these findings, we conclude that OSM is essential for aerobic exercise-induced accumulation of M2 macrophages and neutrophils in the skeletal muscle partly through the regulation of chemokine expression in macrophages.

CXC chemokine ligand-10 promotes the accumulation of monocyte-like myeloid-derived suppressor cells by activating p38 MAPK signaling under tumor conditions.

CXC chemokine ligand-10 (CXCL10) is a small (10kDa) secretory protein in the CXC subfamily of cytokines. CXCL10 has been reported to play an important role in antitumor immunity as a chemotactic factor. Tumor development is always accompanied by the formation of an immunosuppressive tumor microenvironment, and the role of CXCL10 in tumor immunosuppression remains unclear. Here, we reported that CXCL10 expression was significantly upregulated in mice with melanoma, and tumor cells secreted large amounts of CXCL10. Myeloid-derived suppressor cells (MDSCs) are an important part of the immunosuppressive tumor microenvironment. Our results showed that CXCL10 promoted the proliferation of monocyte-like (mo)-MDSCs by activating the p38 MAPK signaling pathway through CXCR3, which led to the abnormal accumulation of mo-MDSCs under tumor conditions. This finding provides a new understanding of the mechanism by which a tumor-induced immunosuppressive microenvironment forms and suggests that CXCL10 could be a potential intervention target for slowing tumor progression.

Arctigenin Attenuates Vascular Inflammation Induced by High Salt through TMEM16A/ESM1/VCAM-1 Pathway.

Salt-sensitive hypertension is closely related to inflammation, but the mechanism is barely known. Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in epithelial cells, smooth muscle cells, and sensory neurons. It can promote inflammatory responses by increasing proinflammatory cytokine release. Here, we identified a positive role of TMEM16A in vascular inflammation. The expression of TMEM16A was increased in high-salt-stimulated vascular smooth muscle cells (VSMCs), whereas inhibiting TMEM16A or silencing TMEM16A with small interfering RNA (siRNA) can abolish this effect in vitro or in vivo. Transcriptome analysis of VSMCs revealed some differential downstream genes of TMEM16A related to inflammation, such as endothelial cell-specific molecule 1 (ESM1) and CXC chemokine ligand 16 (CXCL16). Overexpression of TMEM16A in VSMCs was accompanied by high levels of ESM1, CXCL16, intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1). We treated VSMCs cultured with high salt and arctigenin (ARC), T16Ainh-A01 (T16), and TMEM16A siRNA (siTMEM16A), leading to greatly decreased ESM1, CXCL16, VCAM-1, and ICAM-1. Beyond that, silencing ESM1, the expression of VCAM-1 and ICAM-1, and CXCL16 was attenuated. In conclusion, our results outlined a signaling scheme that increased TMEM16 protein upregulated ESM1, which possibly activated the CXCL16 pathway and increased VCAM-1 and ICAM-1 expression, which drives VSMC inflammation. Beyond that, arctigenin, as a natural inhibitor of TMEM16A, can reduce the systolic blood pressure (SBP) of salt-sensitive hypertension mice and alleviate vascular inflammation.

Identification of cerebrospinal fluid biomarker candidates for anti-N-methyl-D-aspartate receptor encephalitis: High-throughput proteomic investigation.

Although the diagnosis is mainly dependent on the detection of anti-N-methyl-D-aspartate receptor (NMDAR) antibodies in cerebrospinal fluid (CSF) and/or serum, there was no direct correlations between anti-NMDAR antibody titers in CSF and disease severity and prognosis in anti-NMDAR encephalitis patients. Here, we aimed to extensively identify CSF biomarkers related to the occurrence, development, and prognosis of anti-NMDAR encephalitis using a high-throughput proteomic approach. A CSF cytokine antibody array containing 80 cytokines and inflammatory mediators related to immune and inflammatory responses was applied to identify biomarker candidates in individual CSF samples from a well-characterized cohort comprising patients with anti-NMDAR encephalitis (n = 6) and controls (n = 6). Validation and specific detection were performed in an extended cohort consisting of anti-NMDAR encephalitis patients (n = 13), controls (n = 13), and viral encephalitis (n = 13) by enzyme-linked immunosorbent assay (ELISA). Additionally, the levels of some inflammatory proteins in three groups in cohort 2 reported in previous literatures that may be involved in the development of anti-NMDAR encephalitis were also tested by ELISA. Correlations between candidate biomarkers and clinical characteristics of anti-NMDAR encephalitis patients were analyzed. Three differentially expressed cytokines and inflammatory mediators were screened from the 80-cytokine array in cohort 1. Functional enrichment analysis results suggested that these differentially expressed proteins were related to autophagy, immune/inflammatory responses, cell death, and other processes. In cohort 2, the elevations of cellular inhibitor of apoptosis protein-1 (cIAP-1), macrophage colony-stimulating factor (MCSF), CXC chemokine ligand 13 (CXCL13), and nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) in anti-NMDAR encephalitis were validated by ELISA. Linear regression revealed that the levels of CSF CXCL13 and cIAP-1 were positively correlated with the highest modified Rankin scale (mRS) score in the acute phase (p < 0.05). The level of cIAP-1 was positively correlated with the anti-NMDAR Encephalitis One-Year Functional Status (NEOS) score (p < 0.05). These biomarkers show promising functions to evaluate severity or prognosis of anti-NMDAR encephalitis. The biological processes of immune/inflammatory responses, altered levels of autophagy, and the tumornecrosis factor (TNF) signal pathway may be involved in the pathophysiology of anti-NMDAR encephalitis to some extent.

Discovery of Chemokine CXCL12 Inhibitors by Tandem Application of Virtual Screening and NMR Spectrometry.

The CXC chemokine ligand CXCL12 and its receptor CXCR4 play critical roles in stem-cell homing, infectious diseases, and cancer, which led the CXCL12/CXCR4 signaling axis to attract much attention in drug discovery. CXCR4 is regarded as the primary target while CXCL12 is considered too small to be a druggable target. In this paper, we employed virtual screening approaches and ligand-based NMR screening methods from a SPECS library and in-house natural products to discover new CXCR12 inhibitors. Four natural triterpene saponins were confirmed, and the triterpene sapogenin was identified as the main binding epitope by saturation transfer difference-nuclear magnetic resonance and molecular docking studies. The pentacyclic triterpene scaffold and its elucidated structure-activity relationships provide a new and valuable research direction for the development of novel CXCL12 inhibitors.

Mechanisms of electroacupuncture at "Zusanli"(ST36) in delaying colon "inflammation-cancer transformation"

To investigate the mechanism of electroacupuncture (EA) "Zusanli" (ST36) in delaying colon "inflammation-cancer transformation" in mice by anti-inflammatory. C57BL/6J mice were randomly divided into normal, model and EA groups, with 12 mice in each group. The mouse model of colorectal cancer (CRC) was established by intrape-ritoneal injection of azomethane (AOM) and feeding dextran sodium sulfate (DSS). At the beginning of the 2nd cycle, EA was applied to bilateral ST36 for 30 min once every other day for 12 times. The number of colon tumors in each group was observed, and the weight and length of colon were recorded. The contents of interleukin (IL)-1β, IL-6, IL-17A, IL-23, tumor necrosis factor (TNF)-α and CXC chemokine ligand 1 (CXCL1) of serum and colon tissue were detected by MSD multifactorial assay.The apoptosis of local cells in colon tumor was observed by TUNEL staining. Cell proliferation in colon tumor was observed by immunohistochemistry. Compared with the normal group, the colon length was significantly shortened (P<0.05) and the colon mass was significantly increased (P<0.001) in the model group, the contents of IL-1β, IL-6, TNF-α, IL-17A and CXCL1 of serum and colon tissue were significantly increased (P<0.05, P<0.01, P<0.001), and the content of IL-23 was increased in colon tissue (P<0.05) in the model group. Compared with the model group, the colon mass was decreased (P<0.05) and the contents of IL-1β, IL-6, TNF-α and IL-17A in serum were decreased (P<0.05), while the contents of IL-17A, CXCL1 and IL-23 in colon tissue were decreased (P<0.05) in the EA group, the percentage of local apoptotic cells in the EA group was increased (P<0.001), the percentage of PCNA positive cells was decreased (P<0.001), the number of tumors and the tumor volume were significantly decreased (P<0.01, P<0.05). The contents of IL-6, TNF-α, IL-17A and IL-23 in serum of CRC mice were positively correlated with tumor burden (P<0.05).The contents of IL-1β, TNF-α, CXCL1 and IL-23 in colon tissue of CRC mice were positively correlated with tumor burden (P<0.05). Electroacupuncture at ST36 can inhibit the inflammatory response of AOM/DSS inflammatory associated CRC mice and delay the "inflammation-cancer transformation" of colon.

Trans-interaction of risk loci 6p24.1 and 10q11.21 is associated with endothelial damage in coronary artery disease

Background and aimsSingle nucleotide polymorphism rs6903956 has been identified as one of the genetic risk factors for coronary artery disease (CAD). However, rs6903956 lies in a non-coding locus on chromosome 6p24.1. We aim to interrogate the molecular basis of 6p24.1 containing rs6903956 risk alleles in endothelial disease biology. Methods and ResultsWe generated induced pluripotent stem cells (iPSCs) from CAD patients (AA risk genotype at rs6903956) and non-CAD subjects (GG non-risk genotype at rs6903956). CRISPR-Cas9-based deletions (Δ63-89bp) on 6p24.1, including both rs6903956 and a short tandem repeat variant rs140361069 in linkage disequilibrium, were performed to generate isogenic iPSC-derived endothelial cells. Edited CAD endothelial cells, with removal of ‘A’ risk alleles, exhibited a global transcriptional downregulation of pathways relating to abnormal vascular physiology and activated endothelial processes. A CXC chemokine ligand on chromosome 10q11.21, CXCL12, was uncovered as a potential effector gene in CAD endothelial cells. Underlying this effect was the preferential inter-chromosomal interaction of 6p24.1 risk locus to a weak promoter of CXCL12, confirmed by chromatin conformation capture assays on our iPSC-derived endothelial cells. Functionally, risk genotypes AA/AG at rs6903956 were associated significantly with elevated levels of circulating damaged endothelial cells in CAD patients. Circulating endothelial cells isolated from patients with risk genotypes AA/AG were also found to have 10 folds higher CXCL12 transcript copies/cell than those with non-risk genotype GG. ConclusionsOur study reveals the trans-acting impact of 6p24.1 with another CAD locus on 10q11.21 and is associated with intensified endothelial injury.

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) positively regulates lipopolysaccharide-induced expression of CXC chemokine ligand 10 and 11 in mouse macrophages

TREM2 (Triggering receptor expressed on myeloid cells 2) is the causative gene for Nasu-Hakola disease, which is characterized by multiple bone cysts and leukoencephalopathy. In addition, mutations in this gene have been found to be correlated with the onset of Alzheimer's disease. TREM2 is an immunoreceptor expressed on dendritic cells, microglia, osteoclasts, and macrophages. TREM2 on the cell membrane is shed by some proteases and released as soluble TREM2 (sTREM2). Meanwhile, several TREM2 ligands have been reported, and lipopolysaccharide (LPS) is one of the candidates. Using RNA interference to examine TREM2-mediated LPS response in macrophages, we identified five chemokines whose expression was induced via TREM2. Furthermore, we showed that LPS-induced expression of CXC-motif chemokine ligand (Cxcl10) and Cxcl11 among the five chemokines was mediated in part through sTREM2. These results suggest that sTREM2 has cytokine-like functions in macrophages.

Relationship between cerebrospinal fluid biomarkers of inflammation and tissue damage in primary progressive multiple sclerosis

Background and ObjectivesIt is unclear to what extent intrathecal inflammation contributes to the pathogenesis in primary progressive multiple sclerosis (PPMS). We conducted an exploratory study to investigate the degree of intrathecal inflammation and its association with biomarkers of disease activity and severity in patients with PPMS. MethodsWe included patients with PPMS who participated in a randomized controlled trial conducted at the Danish Multiple Sclerosis Center, patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls. We analyzed concentrations of a panel of cytokines in CSF using electrochemiluminescence assays. We then explored the relationship between cytokines found in increased CSF concentrations in patients with PPMS (compared with healthy controls) with CSF concentrations of neurofilament light chain (NFL) and myelin basic protein (MBP), IgG-index, and magnetic resonance imaging (MRI) metrics (volume, magnetization transfer ratio and diffusion tensor imaging) from lesions, normal-appearing white matter, and cortical grey matter. ResultsWe included 59 patients with PPMS, 40 patients with RRMS, and 21 healthy controls. In patients with PPMS, CSF concentrations of CC chemokine ligand 3 (CCL-3), CXC chemokine ligand 8 (CXCL-8), CXCL-10, interleukin (IL)-10, IL-15, and vascular endothelial growth factor (VEGF)-A were increased compared with healthy controls and comparable with CSF concentrations in patients with RRMS. In addition, patients with PPMS had increased CSF concentrations of IL-12p40, IL-17A, tumor necrosis factor (TNF)-α, and lymphotoxin (LT)-α compared with healthy controls, but concentrations of these cytokines were even higher in patients with RRMS. For the remaining seven cytokines (CCL22, interferon-γ, IL-5, IL-7, IL-16, IL-22, IL-27), we found no difference between patients with PPMS and healthy controls. CSF concentrations of NFL and MBP correlated weakly with concentrations of IL-15, while the remaining proinflammatory cytokines were not associated with CSF concentrations of NFL or MBP. The IgG-index correlated with four cytokines (IL-10, IL-12p40, TNF-α, and LT-α). We did not observe any significant associations between MRI metrics and CSF biomarkers of inflammation. DiscussionIn this exploratory study, we found few and weak associations between intrathecal inflammation and the extent of neuroaxonal damage and demyelination, and no associations between intrathecal inflammation and MRI metrics, in patients with PPMS. Our findings suggest that, for patients with PPMS, these measures of intrathecal inflammation are not associated with the extent of neuroaxonal injury, demyelination, and disease severity, and these processes may therefore have less relevance in PPMS than in relapsing forms of MS.