C-X-C Motif Chemokine Receptor 2 Research Articles

A novel CX3CR1 inhibitor AZD8797 facilitates early recovery of rat acute spinal cord injury by inhibiting inflammation and apoptosis.

The present study aimed to evaluate the effect of the CX3CR1 inhibitor AZD8797 in early recovery after acute SCI and elucidate its potential mechanism in blocking inflammation and apoptosis. Adult rats were sacrificed after 3, 7, 10, or 14 days of SCI. The injured spinal tissues were collected for assessing C-X3-C motif chemokine ligand 1(CX3CL1)/C-X3-C motif chemokine receptor 1 (CX3CR1) expression at each time point via western blotting (WB) and quantitative PCR. The cellular localization of the proteins was detected by immunofluorescence. Another batch of rats (subdivided into sham, injury model, AZD8797 and methylprednisolone groups) were used to evaluate locomotive recovery with a Basso Beattie Bresnahan score. Based on the expression level of CX3CR1, these rats were sacrificed at the most prominent stage of CX3CR1 expression (10 days after SCI), for assessing the serum levels of tumor necrosis factor-α/interleukin (IL)-6/IL-1β and the expression of CX3CL1/CX3CR1/caspase 3/Bcl-2/Bax in the spinal cord tissues through WB and ELISA. Additionally, apoptosis and necrosis in the injured spinal cord were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining/fluoro-jade B staining. Expression levels of both CX3CR1 and CX3CL1 reached their peak 10 days after the injury, followed by a dramatic downward trend at 14 days. The enhanced expression of CX3CR1 was detected in astrocytes and microglia of the injured spinal cord. AZD8797 improved locomotive recovery after 10 days of SCI and was as effective as methylprednisolone. The effect of AZD8797 was mediated by suppressing apoptosis, necrosis and inflammatory responses, as assessed by WB/ELISA and morphological examinations. The current study has demonstrated that AZD8797 can effectively block overwhelming inflammation, apoptosis and necrosis after SCI and facilitate early recovery of locomotive function.

Abstract P5-04-03: Conversion of non-T cell-inflamed triple-negative breast cancer by in situ immunomodulation of induced intratumoral cross-presenting dendritic cells

Abstract Checkpoint inhibitor (CPI)-based immunotherapy such as PD-1/PD-L1 blockade has shown an unprecedented success for the treatment of a variety of malignancies; however, meaningful clinical activity is seen only in a subset of breast cancer patients. This is due, at least in part, to poor T-cell infiltration into the tumor prior to CPI therapy, which is negatively correlated with the treatment response (non-T cell-inflamed tumor). Effective priming and trafficking of tumor-specific T cells to the tumor microenvironment (TME) can theoretically overcome current limitations of immunotherapy for non-T cell-inflamed tumors such as breast cancer. Accumulating evidence indicates that tumor-residing Batf3-dependent CD103+ dendritic cells (DCs) play critical roles in not only priming of tumor-specific CD8+ T cells, but also trafficking of CD8+ T cells to the TME, thus enhancing responses to PD-1/PD-L1 blockade. In the present study, we hypothesized that mobilization and activation of cross-presenting CD103+ DCs in the TME would increase CD8+ T cell infiltrate to the tumor, and facilitate effective regression of breast cancer. To this end, we utilized two triple-negative syngeneic mammary cancer models, 4T1 on a H-2d background and AT3 derived from the PyMT-MMTV model on a H-2b background, and evaluated antitumor efficacy of a combinatorial therapeutic regimen comprised of intratumoral administration of Fms-like tyrosine kinase 3 receptor ligand (Flt3L) to mobilize CD103+ DCs to the TME, local radiotherapy (RT) to induce immunogenic cell death and maturation of DCs, and peritumoral administration of a toll-like receptor 3 (TLR3) agonist to activate DCs. We found intratumoral administration of Flt3L increased the number of CD103+ DCs in the tumor, blood and lymph nodes, and local RT induced upregulation of TLR3, CD40 and CD86 on the tumor-residing CD103+ DCs. Following intratumoral administration of Flt3L, local RT, and in situ delivery of a TLR3 agonist significantly increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), mediated effective regression of large established tumors, and significantly improved survival in multiple mouse tumor models. Phenotypic analysis of circulating T cells revealed that the combinatorial regimen increased the frequency of effector memory and central memory T cell subsets with upregulation of PD-1 and C-X3-C motif chemokine receptor 1 (CX3CR1), suggesting effective activation and differentiation of T cells. Interestingly, we found the frequency of macrophages significantly decreased in the tumor after combinatorial treatment, indicating remodeling of both the myeloid and lymphoid compartments in the TME. Furthermore, increased PD-1 expression on CD4+ and CD8+ TILs, and PD-L1 expression in the TME suggest that antitumor efficacy of the combinatorial regimen can be additionally enhanced by PD-1/PD-L1 blockade. Taken together, combinatorial in situ immunomodulation with intratumoral Flt3L administration, RT and, peritumoral administration of a TLR3 agonist can convert non-T cell-inflamed tumors to T-cell inflamed tumors, remodel an immunosuppressive TME, and potentially overcome primary resistance to PD-1/PD-L1 blockade for triple-negative breast cancer. Acknowledgement: This work was supported by Uehara Memorial Foundation (T.O.), NIH/NCI K08CA197966 (F.I.) and P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Flow Cytometry Core. Citation Format: Takaaki Oba, Tibor Keler, Henry C Marsh, Scott Abrams, Fumito Ito. Conversion of non-T cell-inflamed triple-negative breast cancer by in situ immunomodulation of induced intratumoral cross-presenting dendritic cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-03.

MMP9, CXCR1, TLR6, and MPO participant in the progression of coronary artery disease.

Coronary artery disease (CAD) is the most frequent cardiovascular disease, which is induced by the decreased myocardial blood supply. The present study is conducted to understand the mechanisms of CAD. The GSE98583, GSE69587, and GSE71226 datasets from the Gene Expression Omnibus database were obtained. The differentially expressed genes (DEGs) were analyzed by the limma package, then the DEGs appeared in two or three datasets were selected as the coregulated genes using the VENNY tool, followed by enrichment analysis using DAVID tool. Protein-protein interaction (PPI) network, microRNA-transcription factor-target regulatory network, and drug-gene network were visualized. Finally, quantitative PCR and dual-luciferase reporter assay were conducted to validate the expression of key genes and the target relationship. There were 221 coregulated genes in GSE98583, GSE69587, and GSE71226. Besides, four pathways and 23 functional terms for co-upregulated genes, and 11 functional terms for co-downregulated genes were enriched. The degrees of PPI network nodes matrix metallopeptidase 9 (MMP9), C-X-C motif chemokine receptor 1 (CXCR1), toll-like receptor 6 (TLR6), and myeloperoxidase (MPO) were relatively higher. Moreover, MPO could interact with MMP9, CXCR1, and TLR6 in the PPI network. In the regulatory network, TLR6 and MMP9 separately were targeted by miR-3960 and v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA). Additionally, MMP9, CXCR1, and MPO were involved in the drug-gene network. The expression of MMP9, CXCR1, TLR6, and MPO were significantly upregulated in CAD samples than control, and miR-3960 could bind to TLR6 to inhibit its expression. CXCR1 and MPO might be involved in the progression of CAD. Besides, miR-3960 might function in the pathogenesis of CAD through targeting TLR6, and RELA might exert its role in CAD via targeting MMP9.

Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5.

Necroptosis is a form of programmed cell death that is accompanied by release of intracellular contents, and reportedly contributes to various diseases. Here, we investigate the significance of necroptosis in pancreatic cancer. We used immunohistochemistry and western blot analysis to evaluate expression of the key mediators of necroptosis-receptor-interacting serine/threonine protein kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL)-in human pancreatic cancer. We also tested the effects of conditioned media (CM) from necroptotic cells on pancreatic cancer cells in Transwell migration and Matrigel invasion assays. Protein array analysis was used to investigate possible mediators derived from necroptotic cells. RIP3 and MLKL are highly expressed in human pancreatic cancer tissues compared with normal pancreas. MLKL expression was particularly intense at the tumor invasion front. CM derived from necroptotic cells promoted cancer cell migration and invasion, but not CM derived from apoptotic cells. C-X-C motif chemokine 5 (CXCL5) was upregulated in CM derived from necroptotic cells compared with CM derived from control or apoptotic cells. Moreover, expression of the receptor for CXCL5, C-X-C-motif chemokine receptor-2 (CXCR2), was upregulated in pancreatic cancer cells. Inhibition of CXCR2 suppressed cancer cell migratory and invasive behavior enhanced by necroptosis. These findings indicate that necroptosis at the pancreatic cancer invasion front can promote cancer cell migration and invasion via the CXCL5-CXCR2 axis.

Evaluation of CXCR1 as a possible diagnostic biomarker in acute appendicitis.

The present study was conducted to determine the genes with common expression in blood and appendix tissue samples in order to introduce them as possible diagnostic biomarkers. Diagnosis of acute appendicitis (AA) without applying computed tomographytomography (CT), subjecting the patient to significant radiation, can be surprisingly difficult. Blood circulation may have conscious alterations in its RNA, protein, or metabolite composition. The genes related to appendix tissue and blood samples of the patients with AA were extracted from public databases. Fold change (FC) ≥ 2 in blood and FC ≥ 5 in appendix tissue samples were considered to screen differentially expressed genes (DEGs). A protein-protein interaction network was organized using the search tool for retrieval of interacting genes and proteins (STRING) database as a plugin of Cytoscape software version 3.6.0. The main genes were enriched by DAVID Bioinformatics Resources to find the related biochemical pathways. Among the DEGs in blood and appendix tissue samples, C-X-C motif chemokine receptor 1(CXCR1), leukocyte immunoglobulin-like receptor A3 (LILRA3), low-affinity immunoglobulin gamma Fc region receptor III (FCGR3), and superoxide dismutase 2(SOD2) were common in both sources. CXCR1 was found as only hub gene upregulated in both blood and tissue of the patients with AA compared to controls and those with other abdominal pain. CXCR1, FCGR3, LILRA3, and SOD2 were determined as a suitable possible biomarker panel for diagnosis of AA disease.

Mild Traumatic Brain Injury Causes Nociceptive Sensitization through Spinal Chemokine Upregulation

High rates of acute and chronic pain are associated with traumatic brain injury (TBI), but mechanisms responsible for the association remain elusive. Recent data suggest dysregulated descending pain modulation circuitry could be involved. Based on these and other observations, we hypothesized that serotonin (5-HT)-dependent activation of spinal CXC Motif Chemokine Receptor 2 (CXCR2) may support TBI-related nociceptive sensitization in a mouse model of mild TBI (mTBI). We observed that systemic 5-HT depletion with p-chlorophenylalanine attenuated mechanical hypersensitivity seen after mTBI. Likewise, selective spinal 5-HT fiber depletion with 5,7-dihydroxytryptamine (5,7-DHT) reduced hypersensitivity after mTBI. Consistent with a role for spinal 5-HT3 serotonin receptors, intrathecal ondansetron administration after TBI dose-dependently attenuated nociceptive sensitization. Also, selective CXCR2 antagonist SCH527123 treatment attenuated mechanical hypersensitivity after mTBI. Furthermore, spinal CXCL1 and CXCL2 mRNA and protein levels were increased after mTBI as were GFAP and IBA-1 markers. Spinal 5,7-DHT application reduced both chemokine expression and glial activation. Our results suggest dual pathways for nociceptive sensitization after mTBI, direct 5-HT effect through 5-HT3 receptors and indirectly through upregulation of chemokine signaling. Designing novel clinical interventions against either the 5-HT3 mediated component or chemokine pathway may be beneficial in treating pain frequently seen in patients after mTBI.

Effect of sufentanil on chemokine receptors and p38 mitogen activated protein kinase in neuropathic pain rats

Objective To investigate the effect of sufentanil on chemokine receptors and p38 mitogen activated protein kinase(MAPK) in neuropathic pain (NP) rats. Methods Fifty-four adult Sprague Dawley (SD) rats were divided into the sham operation group (S group), chronic constriction injury (CCI) group (CCI group) and sufentanil group (Suf group) by random number table method, with 18 rats in each group. In CCI group and Suf group, NP rat models were made by CCI. Suf group received daily intraperitoneal injection of sufentanil (10 μg/kg) after operation, while S group and CCI group received the same volume of physiological saline. Paw mechanical withdrawal threshold (PMWT) and paw withdraw thermal latency (PWTL) were measured at 1 d before operation (T0), 3 d (T1), 7 d (T2), and 14 d (T3) after operation. The expression of C-C chemokine receptor type 2 (CCR2), C-X3-C motif chemokine receptor 1 (CX3CR1), total p38MAPK (t-p38), phosphorylated p38MAPK (p-p38) and nuclear factor-κB (NF-κB) p65 protein in the dorsal horn of the spinal cord were detected by Western blot. Results Compared with S group, PMWT and PWTL were significantly decreased in CCI group and Suf group at T1-T3 (P<0.05). The levels of CCR2, CX3CR1, p-p38 and NF-κB p65 protein were significantly increased (P<0.05). Compared with CCI group, PMWT and PWTL were significantly increased in Suf group at T1-T3 (P<0.05), while the levels of CCR2, CX3CR1, p-p38 and NF-κB p65 protein were significantly decreased in Suf group (P<0.05). Conclusions Sufentanil can reduce the expression of chemokine receptors CCR2 and CX3CR1, inhibit the activation of spinal dorsal horn p38MAPK and NF-κB, and alleviate the pain response of NP rats. Key words: Sufentanil; Neuropathic pain; Chemokine receptor; p38 mitogen activated protein kinase

CXCL1 and CXCL2 Inhibit the Axon Outgrowth in a Time- and Cell-Type-Dependent Manner in Adult Rat Dorsal Root Ganglia Neurons.

The ability to regrow their axons after an injury is a hallmark of neurons in peripheral nervous system which distinguish them from central nervous system neurons. This ability is influenced by their intrinsic capacity to regrow and by the extracellular environment which needs to be supportive of regrowth. CXCL1 [Chemokine (C-X-C motif) Ligand 1] and CXCL2 [Chemokine (C-X-C motif) Ligand 2] are two low-molecular-weight chemokines which can influence neuronal proliferation, differentiation and neurogenesis, but which are also upregulated by injury or inflammation. In this study we investigated the effects of long-term incubation (24, 48 and 72h) with different concentrations of CXCL1 (0.4, 4 or 40nM) or CXCL2 (0.36, 3.6 or 36nM) on the axon outgrowth of adult rat dorsal root ganglia neurons in culture. The results showed that both chemokines significantly inhibited the axon outgrowth, with large and medium NF200 (NeuroFilament 200) (+) dorsal root ganglia neurons affected quicker, compared to small IB4 (Isolectin B4) (+) dorsal root ganglia neurons which were affected after longer exposure. Blocking CXCR2 (C-X-C motif chemokine receptor 2) which mediates the effects of CXCL1 and CXCL2 prevented these effects, suggesting that CXCR2 may represent a new therapeutic target for promoting the axon outgrowth after a peripheral nerve injury.

The chemokine CXCL1 and its receptor CXCR2 contribute to chronic stress-induced depression in mice.

Depression is increasingly recognized as an inflammatory disease, with inflammatory crosstalk in the brain contributing its pathogenesis. Life stresses may up-regulate inflammatory processes and promote depression. Although cytokines are central to stress-related immune responses, their contribution to stress-induced depression remains unclear. Here, we used unpredictable chronic mild stress (UCMS) to induce depression-like behaviors in mice, as assessed through a suite of behavioral tests. C-X-C motif chemokine ligand 1 (CXCL1)-related molecular networks responsible for depression-like behaviors were assessed through intrahippocampal microinjection of lenti-CXCL1, the antidepressant fluoxetine, the C-X-C motif chemokine receptor 2 (CXCR2) inhibitor SB265610, and the glycogen synthase kinase-3β (GSK3β) inhibitor AR-A014418. Modulation of apoptosis-related pathways and neuronal plasticity were assessed via quantification of cleaved caspase-3, B-cell lymphoma 2-associated X protein, cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) protein expression. CXCL1/CXCL2 expression was correlated with depression-like behaviors in response to chronic stress or antidepressant treatment in the UCMS depression model. Intrahippocampal microinjection of lenti-CXCL1 increased depression-like behaviors, activated GSK3β, increased apoptosis pathways, suppressed CREB activation, and decreased BDNF. Administration of the selective GSK3β inhibitor AR-A014418 abolished the effects of lenti-CXCL1, and the CXCR2 inhibitor SB265610 prevented chronic stress-induced depression-like behaviors, inhibited GSK3β activity, blocked apoptosis pathways, and restored BDNF expression. The CXCL1/CXCR2 axis appears to play a critical role in stress-induced depression, and CXCR2 is a potential novel therapeutic target for patients with depression.-Chai, H.-H., Fu, X.-C., Ma, L., Sun, H.-T., Chen, G.-Z., Song, M.-Y., Chen, W.-X., Chen, Y.-S., Tan, M.-X., Guo, Y.-W., Li, S.-P. The chemokine CXCL1 and its receptor CXCR2 contribute to chronic stress-induced depression in mice.

Interleukin-35 Promotes Macrophage Survival and Improves Wound Healing After Myocardial Infarction in Mice.

Targeting inflammation has been shown to provide clinical benefit in the field of cardiovascular diseases. Although manipulating regulatory T-cell function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. IL (interleukin)-35, an immunosuppressive cytokine mainly produced by regulatory T cells, is a novel member of the IL-12 family and is composed of an EBI3 (Epstein-Barr virus-induced gene 3) subunit and a p35 subunit. However, the role of IL-35 in infarct healing remains elusive. This study aimed to determine whether IL-35 signaling is involved in healing and cardiac remodeling after myocardial infarction (MI) and, if so, to elucidate the underlying molecular mechanisms. IL-35 subunits (EBI3 and p35), which are mainly expressed in regulatory T cells, were upregulated in mice after MI. After IL-35 inhibition, mice showed impaired infarct healing and aggravated cardiac remodeling, as demonstrated by a significant increase in mortality because of cardiac rupture, decreased wall thickness, and worse cardiac function compared with wild-type MI mice. IL-35 inhibition also led to decreased expression of α-SMA (α-smooth muscle actin) and collagen I/III in the hearts of mice after MI. Pharmacological inhibition of IL-35 suppressed the accumulation of Ly6Clow and major histocompatibility complex IIlow/C-C motif chemokine receptor type 2- (MHC IIlow CCR2-) macrophages in infarcted hearts. IL-35 activated transcription of CX3CR1 (C-X3-C motif chemokine receptor 1) and TGF (transforming growth factor) β1 in macrophages by inducing GP130 signaling, via IL12Rβ2 and phosphorylation of STAT1 (signal transducer and activator of transcription family) and STAT4 and subsequently promoted Ly6Clow macrophage survival and extracellular matrix deposition. Moreover, compared with control MI mice, IL-35-treated MI mice showed increased expression of α-SMA and collagen within scars, correlating with decreased left ventricular rupture rates. IL-35 reduces cardiac rupture, improves wound healing, and attenuates cardiac remodeling after MI by promoting reparative CX3CR1+Ly6Clow macrophage survival.

Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients.

C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138+ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14+CD16+ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.

Identification of novel candidate indicators for assessing zinc status during pregnancy in mice from microarray data

BackgroundThis study aimed to identify potential zinc status indicators and to clarify the mechanisms underlying zinc deficiency-induced organ damage and mortality in mice.MethodsThe dataset GSE97112, including placental tissues of mice fed diets containing normal and low concentrations of zinc, was downloaded and preprocessed. Differentially expressed genes (DEGs) were calculated and identified for zinc deficiency-related gene clusters by using the weighed gene co-expression network analysis (WGCNA) algorithm. The Gene Ontology (GO)-Biological Process (BP) and KEGG pathway of genes in the zinc deficiency-related WGCNA modules were analyzed, and the protein-protein interaction (PPI) network was constructed. In addition, modules of the PPI network were identified, and transcription factors (TFs) and miRNAs regulating DEGs were predicted. Finally, drug-gene interactions were selected.ResultsA total of 1055 DEGs containing 586 up- and 469 down-regulated genes were obtained. Three modules based on WGCNA had high correlation with degree of zinc deficiency. Annexin A1 (ANXA1), C-C motif chemokine receptor 3 (CCR3), C-X-C motif chemokine receptor 2 (CXCR2), and interleukin 2 (IL-2) were hub nodes in the PPI network. Three modules in the PPI network were identified, including module 1 associated with olfactory conduction and module 2 associated with inflammatory response. ANXA1, CCR3, and IL-2 were regulated by TFs. In addition, CXCR2, ANXA, and IL-2 were drug targets.ConclusionCXCR2, ANXA1, and CCR3 as well as olfactory receptor-related genes (proteins) may be used as biomarkers to assess zinc status in mice.

CX3CL1 promotes lung cancer cell migration and invasion via the Src/focal adhesion kinase signaling pathway.

The present study investigated the role of C‑X3‑C motif chemokine ligand 1 (CX3CL1) in lung cancer cell migration and invasion and its potential mechanism. The expression levels of C‑X3‑C motif chemokine receptor 1 (CX3CR1) in six human lung cancer cell lines and one human bronchial epithelial cell line were assessed using reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell proliferation was assessed using the Cell Counting Kit‑8 assay. Cell migration and invasion were examined using the Transwell assay, with and without Matrigel, respectively. The signaling pathway activated by CX3CL1 was analyzed via western blotting and inhibitory migration and invasion assays. CX3CR1 was expressed in the six lung cancer cell lines and one normal lung cell line. The lung cancer cell line, H460, was selected for further study. CX3CL1 did not significantly affect H460 proliferation; however, CX3CL1 did significantly enhance the migration and invasion of H460 cells. The Src/focal adhesion kinase (FAK) signaling pathway was activated in a time‑dependent manner upon stimulation of CX3CL1. However, blocking Src activity with saracatinib prevented CX3CL1‑mediated cell migration and invasion. Therefore, the findings indicated that CX3CL1 promotes lung cancer cell migration and invasion in vitro, and the Src/FAK signaling pathway serves a vital role in this process.

The Relationship of CXCR1, IκBα and HIF-1α Expression Levels with Clinicopathological Parameters in Colorectal Cancer.

The role of C-X-C motif chemokine receptor 1 (CXCR1), inhibitor of kappa B (IκBα), and hypoxia-inducible factor 1 alpha (HIF-1α) have been reported to promote tumorigenesis and progression in colorectal cancer (CRC). This study is to evaluate the expression of CXCR1, IκBα, and hypoxia-inducible factor 1 alpha (HIF-1α), in combination, in CRC tissues. It also aims to analyze the relationship of these three factors with clinico-pathological characteristics. CRC and tumor-adjacent tissues were surgically collected from CRC patients. All patients were diagnosed by pathological examination, and none of the patients had received preoperative chemotherapy or radiotherapy. RNA extraction and cDNA synthesis were performed, and the transcription of CXCR1, IκBα, HIF-1α, and β-actin was quantified by RT-qPCR. The significant increase of CXCR1, HIF-1α, and decrease of IκBα mRNA expression level were observed in tumor samples of CRC patients (p < 0.05). In addition, CXCR1 expression level was correlated with lymph node metastasis (p = 0.013). Also, results demonstrated a relationship between HIF-1α expression and TNM stage and lymph node metastasis (p = 0.047 and p = 0.005, respectively). CXCR1 and HIF-1α simultaneous expression demonstrated the significant relationship with lymph node metastasis in CRC. Our findings indicated that CXCR1, HIF-1α, and IκBα can be used as potential prognostic factors indicating tumors in the advanced stage in patients with CRC.

Activation of CXCL5-CXCR2 axis promotes proliferation and accelerates G1 to S phase transition of papillary thyroid carcinoma cells and activates JNK and p38 pathways

ABSTRACTC-X-C motif chemokine ligand 5 (CXCL5) is initially identified to recruit neutrophils by interacting with its receptor, C-X-C motif chemokine receptor 2 (CXCR2). Our prior work demonstrated that the expression levels of CXCL5 and CXCR2 were higher in the papillary thyroid carcinoma (PTC) tumors than that in the non-tumors. This study was performed to further investigate how this axis regulates the growth of PTC cells. B-CPAP cells (BRAFV600E) and TPC-1 cells (RET/PTC rearrangement) expressing CXCR-2 were used as in vitro cell models. Our results showed that the recombinant human CXCL5 (rhCXCL5) promoted the proliferation of PTC cells. rhCXCL5 accelerated the G1/S transition, upregulated the expression of a group of S (DNA synthesis) or M (mitosis)-promoting cyclins and cyclin-dependent kinases (CDKs), and downregulated CDK inhibitors in PTC cells. The CDS region of homo sapiens CXCL5 gene was inserted into an eukaryotic expression vector to mediate the overexpression of CXCL5 in PTC cells. The phosphorylation of c-Jun N-terminal kinases (JNK) and p38, and the nuclear translocation of c-Jun were enhanced by CXCL5 overexpression, whereas attenuated by CXCR2 antagonist SB225002. Additionally, CXCL5/CXCR2 axis, JNK and p38 pathway inhibitors, SB225002, SP600125 and SB203580, suppressed the growth of PTC cells overexpressing CXCL5 in nude mice, respectively. Collectively, our study demonstrates a growth-promoting effect of CXCL5-CXCR2 axis in PTC cells in vitro and in vivo.

Long non‑coding RNA DILC is involved in sepsis by modulating the signaling pathway of the interleukin‑6/signal transducer and activator of transcription 3/Toll‑like receptor 4 axis.

Sepsis is characterized by systemic inflammatory responses. In the present study, the role of deleted in liver cancer 1 (DILC), interleukin (IL)‑6, signal transducer and activator of transcription 3 (STAT3), and Toll‑like receptor 4 (TLR4) in the pathogenesis of sepsis was investigated. Reverse transcription‑quantitative polymerase chain reaction analysis and western blotting were performed to evaluate the effects of lipopolysaccharide (LPS) on the expression of DILC, IL‑6, STAT3, and TLR4, in addition to the effects of DILC and IL‑6 on the synthesis of tumor necrosis factor (TNF‑α), chemokine ligand 5 (CCL5), E‑selectin and C‑X‑C motif chemokine receptor 1 (CXCR1). In addition, the regulatory association between DILC, IL‑6, STAT3 and TLR4 was investigated. LPS reduced the expression level of DILC, and enhanced the expression of IL‑6, STAT3 and TLR4. DILC directly and negatively regulated the synthesis of IL‑6, as demonstrated by the markedly decreased luciferase activity in cells transfected with a wild‑type DILC plasmid. On the other hand, compared with the scramble control, DILC and IL‑6 small interfering (si)RNAs significantly suppressed the expression of IL‑6, STAT3 and TLR4. In addition, DILC siRNA enhanced the expression of IL‑6, STAT3 and TLR4, whereas the expression levels of TNF‑α, CCL5, E‑selectin and CXCR1 in LPS‑treated THP‑1 cells were downregulated following transfection with DILC and IL‑6 siRNAs. In patients with sepsis, DILC expression was inhibited, although the expression levels of IL‑6, STAT3 and TLR4 were upregulated. In addition, the expression levels of TNF‑α, CCL5, E‑selectin and CXCR1 in patients with sepsis were higher compared with normal subjects. Therefore, DILC may mediate the crosstalk between the cascades of IL‑6/STAT3 and TNF‑α signaling, indicating that DILC may act as a prognostic biomarker of sepsis, and may serve as a potential therapeutic target for the treatment of sepsis.

The relevance of ceramides and their synthesizing enzymes for multiple sclerosis.

Ceramide synthases (CerS) synthesize chain length specific ceramides (Cer), which mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that the genetic deletion of CerS2 suppresses EAE pathology by interaction with granulocyte-colony stimulating factor (G-CSF) signaling and CXC motif chemokine receptor 2 (CXCR2) expression, leading to impaired neutrophil migration. In the present study, we investigated the importance of Cers and their synthesizing/metabolizing enzymes in MS. For this purpose, a longitudinal study with 72 MS patients and 25 healthy volunteers was performed. Blood samples were collected from healthy controls and MS patients over 1- or 3-year periods, respectively. Immune cells were counted using flow cytometry, ceramide levels were determined using liquid chromatography-tandem mass spectrometry, and mRNA expression was analyzed using quantitative PCR. In white blood cells, C16-LacCer and C24-Cer were down-regulated in MS patients in comparison with healthy controls. In plasma, C16-Cer, C24:1-Cer, C16-GluCer, and C24:1-GluCer were up-regulated and C16-LacCer was down-regulated in MS patients in comparison with healthy controls. Blood samples from MS patients were characterized by an increased B-cell number. However, there was no correlation between B-cell number and Cer levels. mRNA expression of Cer metabolizing enzymes and G-CSF signaling enzymes was significantly increased in MS patients. Interestingly, G-CSF receptor (G-CSFR) and CXCR2 mRNA expression correlated with CerS2 and UDP-glucose Cer glucosyltransferase (UGCG) mRNA expression. In conclusion, our results indicate that Cer metabolism is linked to G-CSF signaling in MS.

Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis.

Methamphetamine (METH), an extremely and widely abused illicit drug, can cause serious nervous system damage and social problems. Previous research has shown that METH use causes dopaminergic neuron apoptosis and astrocyte-related neuroinflammation. However, the relationship of astrocytes and neurons in METH-induced neurotoxicity remains unclear. We hypothesized that chemokine interleukin (IL) eight released by astrocytes and C-X-C motif chemokine receptor 1 (CXCR1) in neurons are involved in METH-induced neuronal apoptosis. We tested our hypothesis by examining the changes of CXCR1 in SH-SY5Y cells and in the brain of C57BL/6 mice exposed to METH by western blotting and immunolabeling. We also determined the effects of knocking down CXCR1 expression with small interfering ribonucleic acid (siRNA) on METH-exposed SH-SY5Y cells. Furthermore, we detected the expression levels of IL-8 and the nuclear factor-kappa B (NF-κB) pathway in U87MG cells and then co-cultured the two cell types to determine the role of CXCR1 and IL-8 in neuronal apoptosis. Our results indicated that METH exposure increased CXCR1 expression both in vitro and in vivo, with the effects obtained in vitro being dose-dependent. Silencing of CXCR1 expression with siRNAs reduced the expression of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), and other related proteins. In addition, IL-8 expression and release were increased in METH-exposed U87MG cells, which is regulated by NF-κB pathway. Neuronal apoptosis was attenuated by siCXCR1 after METH treatment in the co-cultured cells, which can be reversed after exposure to recombinant IL-8. These results demonstrate that CXCR1 plays an important role in neuronal apoptosis induced by METH and may be a potential target for METH-induced neurotoxicity therapy.Highlights –Methamphetamine exposure upregulated the expression of CXCR1.–Methamphetamine exposure increased the expression of interleukin-8 through nuclear factor-kappa B pathway.–Activation of CXCR1 by interleukin-8 induces an increase in methamphetamine-related neuronal apoptosis.

Bioinformatics approach reveals the key role of C‑X‑C motif chemokine receptor 2 in endometriosis development.

Endometriosis is a common gynecological disease, affecting 6–10% of women of reproductive age. The precise mechanisms underlying the development of endometriosis remain unclear. In the present study, a bioinformatics approach was applied to systematically identify the pathways and genes involved in the development of endometriosis and to discover potential biomarkers. The gene expression profiles of GSE6364, a microarray dataset of endometrial biopsies obtained from women with or without endometriosis, was downloaded from the Gene Expression Omnibus DataSets database that stores original submitter-supplied records (series, samples and platforms), as well as curated datasets. Differentially expressed gene (DEG) analysis was performed with GEO2R. DAVID was used to analyze the gene ontology enrichment of the DEGs. Gene Set Enrichment Analysis (GSEA) was conducted using the GSEA v3.0 software. Protein-protein interactions (PPI) were evaluated with the Search Tool for the Retrieval of Interacting Genes, and PPI network visualization was performed with Cytoscape. In addition, Cell Counting kit-8 and Transwell assays were performed on human endometrial stromal cells (HESCs). A total of 172 DEGs were extracted. Inflammatory response genes were significantly upregulated in the endometriosis tissues and C-X-C motif chemokine receptor 2 (CXCR2), was one of the most up-regulated genes according to DEG analysis. Cell-based experiments confirmed that CXCR2 promoted the proliferation, migration and invasion of HESCs. In conclusion, a bioinformatics approach combined with in vitro experiments in the present study revealed that CXCR2 may be associated with the development of endometriosis and has potential as a biomarker for the diagnosis of endometriosis.

Neutrophils in primary gastric tumors are correlated with neutrophil infiltration in tumor-draining lymph nodes and the systemic inflammatory response

BackgroundTumor-Associated Neutrophils (TANs) may be able to induce lymphangiogenesis and angiogenesis, although the detailed roles of TANs remain unclear. The Neutrophil-Lymphocyte Ratio (NLR) is an inflammation-based prognostic factor for gastric cancer. This study aimed to investigate the distribution of CD15+neutrophils in the primary tumor and Tumor-Draining Lymph Nodes (TDLNs), and to examine the association of TANs with the clinicopathological features (including NLR) of patients with gastric cancer.ResultsImmunohistochemical staining showed that the median number of CD15+TANs was 18 and 24 per high-power field (HPF) in primary tumors and TDLNs, respectively. Patients were divided into high and low infiltration groups based on the median number. A high number of infiltrating CD15+TANs in the primary tumors and in the TDLNs were associated with depth of invasion and lymph node metastasis. Kaplan-Meier analysis revealed that a poor overall survival was associated with high numbers of CD15+TANs, and the multivariate analyses revealed that a high number of CD15+TANs in the TDLNs was an independent prognostic factor. The numbers of CD15+TANs in the primary tumors and TDLNs showed weak positive correlation. The number of CD15+TANs in the primary tumors was positively correlated with the preoperative NLR, (P = 0.001, R = 0.327) and immunohistochemical staining revealed that C-X-C motif chemokine receptor 2 (CXCR2) +neutrophils might be the origin of the CD15+TANs. Flow cytometry analysis indicated that infiltrating neutrophils increased in the tumor and TDLN compared to non-cancerous tissue. Neutrophils treated with cancer supernatant upregulated TWIST and IL-6 genes in vitro.ConclusionOur findings suggested that local infiltration of CD15+TANs may be correlated with inflammation in TDLNs and systemic response to cause metastasis in gastric carcinoma.