Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age-associated change is not fully understood. Here, we report the role of microRNA-125b (miR-125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T-cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age-related increase in CCL4. We then found the level of miR-125b was inversely correlated with the level of CCL4 in these cells, and the level of miR-125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock-down of miR-125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR-125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR-125b action requires the 'seed' sequence in 3'UTR of CCL4. Together these findings demonstrated that miR-125b is a negative regulator of CCL4 and its reduction is partially responsible for the age-related increase of CCL4.

Methylmercury (MeHg) is selectively toxic to the central nervous system, but mechanisms related to its toxicity are poorly understood. In the present study, we identified the chemokine, C-C motif Chemokine Ligand 4 (CCL4), to be selectively upregulated in the brain of MeHg-administered mice. We then investigated the relationship between CCL4 expression and MeHg toxicity using in vivo and in vitro approaches. We confirmed that in C17.2 cells (a mouse neural stem cell line) and the mouse brain, induction of CCL4 expression occurs prior to cytotoxicity caused by MeHg. We also show that the addition of recombinant CCL4 to the culture medium of mouse xprimary neurons attenuated MeHg toxicity, while knockdown of CCL4 in C17.2 cells resulted in higher MeHg sensitivity compared with control cells. These results suggest that CCL4 is a protective factor against MeHg toxicity and that induction of CCL4 expression is not a result of cytotoxicity by MeHg but is a protective response against MeHg exposure.

Methylmercury is an environmental pollutant that causes specific and serious damage to the central nervous system. We have previously shown that C-C motif chemokine ligand 4 (CCL4) protects cultured neural cells from methylmercury toxicity and expression of CCL4 is specifically induced in mouse brain by methylmercury. In this study, we examined the transcriptional regulatory mechanism that induces CCL4 expression by methylmercury using C17.2 mouse neural stem cells. The promoter region of the CCL4 gene was analyzed by a reporter assay, revealing that the region up to 50 bp upstream from the transcription start site was necessary for inducing expression of CCL4 by methylmercury. Nine transcription factors that might bind to this upstream region and be involved in the induction of CCL4 expression by methylmercury were selected, and the induction of CCL4 expression by methylmercury was suppressed by the knockdown of serum response factor (SRF). In addition, the nuclear level of SRF was elevated by methylmercury, and an increase in the amount bound to the CCL4 gene promoter was also observed. Furthermore, we examined the upstream signaling pathway involved in the induction of CCL4 expression by SRF, and confirmed that activation of p38 and ERK, which are part of the MAPK pathway, are involved. These results suggest that methylmercury induces the expression of CCL4 by activating SRF via the p38 and ERK signaling pathway. Our findings are important for elucidating the mechanism involved in the brain-specific induction of CCL4 expression by methylmercury.

Macrophage inflammatory protein-1 (MIP-1), MIP-1α (CCL3) and MIP-1β (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1α and MIP-1β form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1α and MIP-1β form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1α, thus depolymerization mutations enhance MIP-1α to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1α ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor with a poor prognosis and is a major public health burden in Taiwan. Angiogenesis, the formation of new blood vessels, promotes tumor proliferation, maintenance, and metastasis. Angiopoietin 2 (Angpt2), a mitogen with a strong angiogenic effect, is highly specific to endothelial cells and a key player in angiogenesis. The inflammatory chemokine (C-C motif) ligand 4 (CCL4) is also important in the pathogenesis and progression of cancer. In this study, an analysis of records from The Cancer Genome Atlas (TCGA) database found higher CCL4 expression in oral cancer tissue than in normal healthy tissue. CCL4 treatment of oral cancer cells upregulated Angpt2 expression and stimulated mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase 1/2 (ERK), and signal transducer and activator of transcription 3 (STAT3) phosphorylation. Transfection of oral cancer cells with MEK, ERK, and STAT3 inhibitors and their small interfering RNAs inhibited CCL4-induced promotion of Angpt2 expression and angiogenesis. In a mouse model of OSCC, CCL4-treated cells promoted neovascularization in implanted Matrigel plugs, whereas inhibiting CCL4 expression suppressed Angpt2 expression and angiogenesis. CCL4 shows promise as a new molecular therapeutic target for inhibiting angiogenesis and metastasis in OSCC.

The inflammatory chemokine (C-C motif) ligand 4 (CCL4) plays an important role in the pathogenesis and progression of cancer. In particular, higher serum CCL4 levels in patients with oral squamous cell carcinoma (OSCC) are associated with a more advanced stage of disease. OSCC accounts for approximately 95% of oral cancer in Taiwan and has a poor prognosis, due to aggressive local invasion and metastasis, leading to recurrence. OSCC spreads preferentially through lymphatic vessels and has the propensity to metastasize to the cervical lymph nodes even in the early stage of disease. Vascular endothelial growth factor C (VEGF-C) is an essential regulator of lymphangiogenesis. In particular, VEGF-C is specific to lymphatic vessel development, and VEGF-C expression levels have been found to directly correlate with lymph node metastasis in OSCC. However, it is unclear as to whether CCL4 correlates with VEGF-C expression and lymphangiogenesis in OSCC. We found that CCL4 increased VEGF-C expression and promoted lymphangiogenesis in oral cancer cells in vitro and in vivo. miR-195-3p mimic reversed CCL4-enhanced VEGF-C expression. CCL4 stimulation of oral cancer cells augmented JAK2 and STAT3 phosphorylation. Thus, CCL4 may be a new molecular therapeutic target for inhibition of lymphangiogenesis and metastasis in OSCC.

During neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown.

Eosinophilic airway inflammatory disease is associated with bronchial asthma, with eosinophilic chronic rhinosinusitis (ECRS) typical of refractory type 2 airway inflammation. CCL4 produced at local inflammatory sites is involved in them via the accumulation and activation of type 2 inflammatory cells, including eosinophils. The detailed mechanism of CCL4 production remains unclear, and also the possibility it could function as a biomarker of type 2 airway inflammation remains unresolved. In this study, we evaluated CCL4 mRNA expression and production via the TSLP receptor (TSLPR) and toll-like receptors (TLRs) or proteinase-activated receptor-2 (PAR2) in BEAS-2B bronchial epithelial cells co-incubated with purified eosinophils or eosinophil peroxidase (EPX). We examined serum chemokine (CCL4, CCL11, CCL26, and CCL17) and total IgE serum levels, fractionated exhaled nitrogen oxide (FENO), and CCL4 expression in nasal polyps in patients with severe ECRS and asthma. CCL4 was induced by TSLP under eosinophilic inflammation. Furthermore, CCL4 was released via TLR3 signaling, which was enhanced by TSLP. CCL4 was mainly located in nasal polyp epithelial cells, while serum CCL4 levels were reduced after dupilumab treatment. Serum CCL4 levels were positively correlated with FENO, serum IgE, and CCL17 levels. Thus, CCL4 released from epithelial cells via the innate immune system during type 2 airway inflammation may function as a useful biomarker for the condition.

Plasmacytoid dendritic cells (pDCs) are present in peripheral blood, leptomeninges and demyelinating lesions in patients with multiple sclerosis (MS). The ability of pDCs to produce chemokines and express the chemokine receptor CCR7 in MS is not known. We studied pDCs in MS patients and healthy subjects. The ability of pDCs to up-regulate CCR7 was significantly increased in untreated MS patients as compared to healthy subjects. IFN-beta treatment significantly inhibited TLR9 agonist-specific secretion of chemokines, which are ligands for CCR5-positive Th1 cells (CCL3, CCL4, and CCL5), and impaired TLR9 agonist-induced up-regulation of CCR7 and IFN-alpha in MS patients. This finding represents a new immunomodulatory effect of IFN-beta in patients with multiple sclerosis.

Breast cancer is a major cause of cancer mortality amongst women. Chemokine (C-C motif) ligand 4 is encoded by the CCL4 gene; specific CCL4 gene polymorphisms are related to the risks and prognoses of various diseases. In this study, we examined whether CCL4 gene single nucleotide polymorphisms (SNPs) predict the risk and progression of breast cancer. Between 2014 and 2016, we recruited 314 patients diagnosed with breast cancer and a cohort of 209 healthy participants (controls) without a history of cancer. Genotyping of the CCL4 rs1634507, rs10491121 and rs1719153 SNPs revealed no significant between-group differences for these polymorphisms. However, amongst luminal A and luminal B subtypes, compared with patients with the AA genotype, those carrying the AG genotype at SNP rs10491121 were less likely to develop lymph node metastasis. In addition, compared with AA carriers, those carrying the AG + GG genotype at SNP rs10491121 were at lower risk of developing distant metastasis, while the presence of the AT genotype at SNP rs1719153 increased the likelihood of pathologic grade (G3 or G4) disease. Variations in the CCL4 gene may help to predict breast cancer progression and metastasis.

Fibromyalgia (FM) is a chronic pain syndrome with a high incidence in females that may involve activation of the immune system. We performed exome sequencing on chemokine genes in a region of chromosome 17 identified in a genome-wide family association study.

Tribbles pseudokinase 1 (Trib1) is a negative regulator of CCAAT/enhancer binding protein α (C/EBPα) and is known to induce granulopoiesis while suppressing monocyte differentiation. Loss of Trib1 was previously shown to increase the neutrophil population in the spleen but lead to M2-like macrophage reduction. Because M2 macrophages are anti-inflammatory and promote tissue repair by producing fibrogenic factors, we investigated liver fibrosis in Trib1-deficient mice. Interestingly, loss of Trib1 suppressed fibrosis in the CCl4-induced chronic liver injury model. Trib1 knockout increased neutrophils but had a minimal effect on the macrophage population in the liver. Hepatic expressions of neutrophil matrix metalloproteinases (Mmp)8 and Mmp9 were increased, but the production of fibrogenic factors, including transforming growth factor β1, was not affected by loss of Trib1. These results suggest that neutrophils are responsible for the suppression of fibrosis in Trib1-deficient liver. Consistently, transplantation of Trib1-deficient bone marrow cells into wild-type mice alleviated CCl4-induced fibrosis. Furthermore, expression of chemokine (C-X-C motif) ligand 1 (Cxcl1) by adeno-associated viral vector in the normal liver recruited neutrophils and suppressed CCl4-induced fibrosis; infusion of wild-type neutrophils in CCl4-treated mice also ameliorated fibrosis. Using recombinant adeno-associated virus-mediated expression of Mmp8 and Mmp9 alleviated liver fibrosis. Finally, neutrophil depletion by infusion of Ly6G antibody significantly enhanced CCl4-induced fibrosis. Conclusion: While neutrophils are well known to exacerbate acute liver injury, our results demonstrate a beneficial role of neutrophils in chronic liver injury by promoting fibrolysis. (Hepatology Communications 2018;2:703-717).

Chemokine CCL4 (MIP-1β) is released from osteoblast cells to restore the homeostasis of hematopoietic stem cells during the activation of bone marrow. In this study, we investigated the function of CCL4 and its receptor CCR5 during osteoclastogenesis. CCL4 promoted the migration and viability of preosteoclast cells. However, CCL4 had no direct effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in mouse preosteoclast cells. In addition, CCR5 expression was rapidly reduced by RANKL treatment, which was recovered by IFN-γ during osteoclastogenesis. CCR5 downregulation by RANKL was mediated by MEK and JNK in preosteoclast cells and promoted osteoclastogenesis. These results suggest that CCL4 can enhance the recruitment of preosteoclasts to bone in the early stage, and the reduction of CCR5 promotes osteoclastogenesis when RANKL is prevalent.

Chronic inflammation in diabetes mellitus (DM) is the leading cause of non-healing wounds. Chemokine CC motif ligand 4 (CCL4) is enhanced in the circulation and in the wounds of DM patients. This study aimed to investigate the effect of endogenous CCL4 inhibition on diabetic wound healing. Endothelial progenitor cells (EPCs) and human dermal microvascular endothelial cells (HDMECs) were used. Mice were injected with streptozotocin to generate hyperglycemia. An enhanced CCL4 level as well as decreased tube formation and migration abilities were observed in high-glucose-treated HDMECs and in EPCs from type 2 DM patients. CCL4 inhibition by siRNA restored the damaged cell function by upregulating the Akt/endothelial nitric oxide synthase/vascular endothelial growth factor/stromal cell-derived factor-1α pathways. Wild-type diabetic mice had delayed wound repair, whereas the CCL4-knockout diabetic mice showed an accelerated rate of wound closure. In a Matrigel plug assay, CCL4-knockout diabetic mice showed higher blood vessel and hemoglobin levels. Higher CD31 and Ki67 expression in the wound area and Matrigel plugs was detected in the CCL4-knockout diabetic mice. CCL4-knockout mice had upregulated angiogenic factors and downregulated inflammatory factors. This study might provide the theoretical basis for CCL4 inhibition as a therapeutic option for clinical diabetic wound treatment.

Atherosclerosis is an arterial inflammatory disease. The circulating level of the C-C chemokine ligand (CCL4) is increased in atherosclerotic patients. This study aimed to investigate whether CCL4 inhibition could retard the progression of atherosclerosis. In ApoE knockout mice, CCL4 antibody treatment reduced circulating interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α levels and improved lipid profiles accompanied with upregulation of the liver X receptor. CCL4 inhibition reduced the atheroma areas and modified the progression of atheroma plaques, which consisted of a thicker fibrous cap with a reduced macrophage content and lower matrix metalloproteinase-2 and -9 expressions, suggesting the stabilization of atheroma plaques. Human coronary endothelial cells (HCAECs) and macrophages were stimulated with TNF-α or oxidized LDL (ox-LDL). The induced expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were attenuated by the CCL4 antibody or CCL4 si-RNA. CCL4 inhibition reduced the adhesiveness of HCAECs, which is an early sign of atherogenesis. CCL4 blockade reduced the activity of metalloproteinase-2 and -9 and the production of TNF-α and IL-6 in stimulated macrophages. The effects of CCL4 inhibition on down-regulating adhesion and inflammation proteins were obtained through the nuclear factor kappa B (NFκB) signaling pathway. The direct inhibition of CCL4 stabilized atheroma and reduced endothelial and macrophage activation. CCL4 may be a novel therapeutic target for modulating atherosclerosis.

Osteosarcoma is the most common type of primary malignant bone cancer, and it is associated with high rates of pulmonary metastasis. Integrin αvβ3 is critical for osteosarcoma cell migratory and invasive abilities. Chemokine (C-C motif) ligand 4 (CCL4) has diverse effects on different cancer cells through its interaction with its specific receptor, C-C chemokine receptor type 5 (CCR5). Analysis of mRNA expression in human osteosarcoma tissue identified upregulated levels of CCL4, integrin αv and β3 expression. Similarly, an analysis of records from the Gene Expression Omnibus (GEO) dataset showed that CCL4 was upregulated in human osteosarcoma tissue. Importantly, the expression of both CCL4 and integrin αvβ3 correlated positively with osteosarcoma clinical stages and lung metastasis. Analysis of osteosarcoma cell lines identified that CCL4 promotes integrin αvβ3 expression and cell migration by activating the focal adhesion kinase (FAK), protein kinase B (AKT), and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling pathways, which can downregulate microRNA-3927-3p expression. Pharmacological inhibition of CCR5 by maraviroc (MVC) prevented increases in integrin αvβ3 expression and cell migration. This study is the first to implicate CCL4 as a potential target in the treatment of metastatic osteosarcoma.

The purpose of this work was to analyze chemokine and chemokine receptor expression in untreated and in irradiated squamous cell carcinoma of the head and neck (SCCHN) tumor cell lines, aiming at the establishment of assays to test for the relevance of chemokine and chemokine receptor expression in the response of SCCHN to radiotherapy and radiochemotherapy. Five low passage and 10 established SCCHN lines, as well as two normal cell lines, were irradiated at 2 Gy or sham-irradiated, and harvested between 1 and 48 h after treatment. For chemokines with CC and CXC structural motifs and their receptors, transcript levels of target and reference genes were quantified relatively by real-time PCR. In addition, CXCL1 and CXCL12 protein expression was analyzed by ELISA. A substantial variation in chemokine and chemokine receptor expression between SCCHN was detected. Practically, all cell lines expressed CCL5 and CCL20, while CCL2 was expressed in normal cells and in some of the tumor cell lines. CXCL1, CXCL2, CXCL3, CXCL10, and CXCL11 were expressed in the vast majority of the cell lines, while the expression of CXCL9 and CXCL12 was restricted to fibroblasts and few tumor cell lines. None of the analyzed cell lines expressed the chemokines CCL3, CCL4, or CCL19. Of the receptors, transcript expression of CCR1, CCR2, CCR3, CCR5, CCR7, CCXR2, and CCXR3 was not detected, and CCR6, CXCR1, and CXCR4 expression was restricted to few tumor cells. Radiation caused up- and down-regulation with respect to chemokine expressions, while for chemokine receptor expressions down-regulations were prevailing. CXCL1 and CXCL12 protein expression corresponded well with the mRNA expression. We conclude that the substantial variation in chemokine and chemokine receptor expression between SCCHN offer opportunities for the establishment of assays to test for the relevance of chemokine and chemokine receptor expression in the response of SCCHN to radiotherapy and radiochemotherapy.

The massive infiltration of lymphocytes into the skin is a hallmark of numerous human skin disorders. By co-culturing murine keratinocytes with splenic T cells we demonstrate here that T cells affect and control the synthesis and secretion of chemokines by keratinocytes. While pre-activated CD8+T cells induce the synthesis of CXCL9 and CXCL10 in keratinocytes and keep in check the synthesis of CXCL1, CXCL5, and CCL20, keratinocytes dampen the synthesis of CCL3 and CCL4 in pre-activated CD8+T cells. One key molecule is IFN-γ that is synthesized by CD8+T cells under the control of NFATc1 and NFATc2. CD8+T cells deficient for both NFAT factors are unable to induce CXCL9 and CXCL10 expression. In addition, CD8+T cells induced numerous type I IFN-inducible "defense genes" in keratinocytes encoding the PD1 and CD40 ligands, TNF-α and caspase-1. The enhanced expression of type I IFN-inducible genes resembles the gene expression pattern at the dermal/epidermal interface in lichen planus, an inflammatory T lymphocyte-driven skin disease, in which we detected the expression of CXCL10 in keratinocytes in close vicinity to the infiltration front of T cells. These data reflect the multifaceted interplay of lymphocytes with keratinocytes at the molecular level.

In Taiwan, oral cancer has causally been associated with environmental carcinogens. CCL4 (C-C chemokine ligand 4), a macrophage inflammatory protein with a key role in inflammation and immune-regulation, was implicated in carcinogenesis by facilitating instability in the tumor environment. The purpose of this study was to identify gene polymorphisms of CCL4 specific to patients with oral squamous cell carcinoma (OSCC) susceptibility and clinicopathological characteristics. A total of 2,053 participants, including 1192 healthy people and 861 patients with oral cancer, were recruited for this study. Three single-nucleotide polymorphisms (SNPs) of the CCL4 gene were analyzed by a real-time PCR. We found that the T/T homozygotes of CCL4 rs1634507 G/T polymorphism and the GG haplotype of 2 CCL4 SNPs (rs1634507 and rs10491121) combined were associated with oral-cancer susceptibility. In addition, TA haplotype significantly decreased the risks for oral cancer by 0.118 fold. Among 1420 smokers, CCL4 polymorphisms carriers with the betel-nut chewing habit had a 15.476-20.247-fold greater risk of having oral cancer compared to CCL4 wild-type (WT) carriers without the betel-nut chewing habit. Finally, patients with oral cancer who had A/G heterozygotes of CCL4 rs10491121 A/G polymorphism showed a lower risk for an advanced tumor size (> T2) (p=0.046), compared to those patients with AA homozygotes. Our results suggest that the CCL4 rs1634507 SNP have potential predictive significance in oral carcinogenesis. Gene-environment interactions of CCL4 polymorphisms might influence oral-cancer susceptibility. CCL4 rs10491121 may be a factor to predict the tumor size in OSCC patients.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.