The chemokine 20 (CCL20) is a member of the CC chemokine family and plays a role in tumor immunity and autoimmune disease. This work investigated the value of CCL20 as a serum diagnostic marker for primary hepatocellular carcinoma (HCC). Based on the data of hepatocellular carcinoma patients in the TCGA database, the up-regulated genes encoding secretory proteins were analyzed in each pathological stage, and the candidate marker CCL20 gene was selected. Serum concentrations of CCL20 in patients with primary HCC, benign liver disease, and healthy subjects were analyzed by enzyme-linked immunosorbent assay (ELISA). The ROC curve evaluated the efficacy of CCL20 alone or in combination with AFP in the diagnosis of HCC. It was found the expression of CCL20 in HCC patients was significantly higher than that in the benign liver disease group and healthy controls (P < 0.05); The AUC of ROC curve to distinguish HCC patients from healthy controls was 0.859, the sensitivity was 73.42%, and the specificity was 86.84%. After combination with AFP, the AUC increased to 0.968, the sensitivity was 88.16%, and the specificity was 97.37%. Although CCL20 was increased in the serum of patients with benign liver diseases, combined with AFP, the AUC to distinguish HCC patients from non-HCC cohorts (benign liver disease group and healthy control group) was 0.902, with a sensitivity of 91.67% and a specificity of 75.26%. Collectively, serum CCL20 is closely related to the occurrence of HCC, and detection of serum CCL20 can assist AFP in improving the diagnostic sensitivity of HCC.

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APCMIN/+ mice) to generate APCMIN/+ mice with CCR6 knocked out (CCR6KO-APCMIN/+ mice). CCR6KO-APCMIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APCMIN/+ also had normal sized spleens as compared to the enlarged spleens found in APCMIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APCMIN/+ as compared to APCMIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.

Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region.

Few studies have focused on investigating resistance mechanisms in myeloma immunotherapy. This study aimed to explore the relevant factor involved in the resistance of Elotuzumab and lenalidomide.

As deregulation of miRNAs and chemokine CCL20 was shown to play a role in colorectal cancer (CRC) pathogenesis, we analyzed the functional interactions of candidate miRNAs with CCL20 mRNA. After target prediction software programs indicated a role for miR-21 in CCL20 regulation, we applied the luciferase reporter assay system to demonstrate that miR-21 functionally interacts with the 3'UTR of CCL20 mRNA and down-regulates CCL20 in miR-21 mimic transfected CRC cell lines (Caco-2, SW480 and SW620). Thus, regulation of CCL20 expression by miR-21 might be a regulatory mechanism involved in progression of CRC.

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms.

Traumatic brain injury (TBI) evokes a systemic immune response including leukocyte migration into the brain and release of pro-inflammatory cytokines; however, the mechanisms underlying TBI pathogenesis and protection are poorly understood. Due to the high incidence of head trauma in the sports field, battlefield and automobile accidents identification of the molecular signals involved in TBI progression is critical for the development of novel therapeutics.

CCR6 is the receptor of chemokine CCL20. In the present study, we demonstrated that the surface expression of CCR6 was enhanced on the human HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially on HuH7 cells, but not on HLE or HLF cells. These HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially the HuH7 cells secreted a significant amount of CCL20 spontaneously, whereas HLE or HLF did not. Stimulation by CCL20 up-regulated the mRNA expression of CCR6 in HuH7 cells and significantly enhanced the growth of HuH7 cells. CCL20-stimulated growth of HuH7 cells was abrogated by the inhibition of downstream signal transduction pathway mediated by p44/42 MAPK, but not by p38 MAPK or SAPK/JNK. CCR6 expression in human HCC tissues was confirmed by RT-PCR. These results indicate that the growth of a proportion of human HCC cells may be mediated by CCL20-CCR6 axis, like HuH7 cells, in an autocrine or paracrine manner.

Studies have found that C‑C motif chemokine ligand 20 (CCL20)/C‑C motif chemokine receptor 6 (CCR6)/notch receptor 1 (Notch1) signaling serves an important role in various diseases, but its role and mechanism in ovarian cancer remains to be elucidated. The aim of the present study was to investigate the underlying mechanism of CCL20/CCR6/Notch1 signaling in paclitaxel (PTX) resistance of a CD44+CD117+ subgroup of cells in ovarian cancer. The CD44+CD117+ cells were isolated from SKOV3 cells, followed by determination of the PTX resistance and the CCR6/Notch1 axis. Notch1 was silenced in the CD44+CD117+ subgroup and these cells were treated with CCL20, followed by examination of PTX resistance and the CCR6/Notch1 axis. Furthermore, in nude mice, CD44+CD117+ and CD44‑CD117‑ cells were used to establish the xenograft model and cells were treated with PTX and/or CCL20, followed by proliferation, apoptosis, reactive oxygen species (ROS) and mechanism analyses. Higher expression levels of Oct4, CCR6, Notch1 and ATP binding cassette subfamily G member 1 (ABCG1), increased sphere formation ability, IC50 and proliferative ability, as well as lower ROS levels and apoptosis were observed in CD44+CD117+ cells compared with the CD44‑CD117‑ cells. It was found that CCL20 could significantly increase the expression levels of Oct4, CCR6, Notch1 and ABCG1, enhance the IC50, sphere formation ability and proliferation, as well as decrease the ROS and apoptosis levels in the CD44+CD117+ cells. However, Notch1 knockdown could markedly reverse these changes. Moreover, CCL20 could significantly increase the proliferation and expression levels of Oct4, CCR6, Notch1 and ABCG1 in the CD44+CD117+ groups compared with the CD44‑CD117‑ groups. After treatment with PTX, apoptosis and ROS levels were decreased in the CD44+CD117+ groups compared with the CD44‑CD117‑ groups. Collectively, the present results demonstrated that, via the Notch1 pathway, CCL20/CCR6 may promote the stemness and PTX resistance of CD44+CD117+ cells in ovarian cancer.

The incidence of cholangiocarcinoma (CCA) has risen in many countries, but there is still no appropriate screening and treatment available. The growing number of microarray data published todays can be a powerful resource for the discovery of biomarkers to tackle challenges in the management of CCA. This study analyzed multiple microarray datasets to identify the common transcriptional networks in CCA and select a possible biomarker for functional study in CCA cell lines. A systematic searching identified 4 microarray datasets from Gene Expression Omnibus (GEO) repository and PubMed articles. Differential expression analysis between tumor and normal tissues was performed in each dataset. In order to characterize the common expression pattern, differentially expressed genes (DEGs) from all datasets were combined and visualized by hierarchical clustering and heatmap. Gene enrichment analysis performed in each cluster revealed that over-expressed DEGs were enriched in cell cycle, cell migration and response to cytokines while under-expressed DEGs were enriched in metabolic processes such as oxidation-reduction, lipid, and drug. To explain tumor characteristics, genes enriched in cell migration and response to cytokines were further investigated. Among these genes, CCL20 was selected for functional study because its role has never been studied in CCA. Moreover, its signaling may be regulated by disrupting its only receptor, CCR6. Treatment with recombinant CCL20 induced higher cell migration and increased expression of N-cad. In contrast, knockdown of CCR6 by siRNA reduced cell migration ability and decreased N-cadherin level. Altogether, these results suggested the contribution of CCL20/CCR6 signaling in cell migration through epithelial-mesenchymal transition process. Thus, CCL20/CCR6 signaling might be a target for the management of CCA.

The CCL20/CCR6 chemokine/receptor axis has previously been shown to contribute to the initiation and progression of hepatocellular carcinoma (HCC) through the recruitment of CCR6-positive leukocytes to the tumor microenvironment. In particular, high serum levels of CCL20 are reported in patients with HCC induced by the hepatitis C virus (HCV). A potential non-immune role for the CCL20/CCR6 axis in HCC development has not yet been investigated. Microarray analysis (Benkheil et al., paper submitted for publication), revealed that CCL20 is highly upregulated in hepatoma cells infected with HCV compared with non-infected hepatoma cells. To determine the role of the CCL20/CCR6 axis in HCV-related HCC, we first explored which cell populations express CCR6 in human liver tissue with chronic disease or HCC. Immunohistochemical (IHC) analysis revealed that CCR6 is present on endothelial cells (ECs) of portal blood vessels in livers with chronic HCV infection and in HCV- and alcoholic-HCC tissue. In addition, we found CCR6 to be expressed on primary macrovascular (HUVECs) and microvascular ECs (HMVEC-ds) where it co-expressed with the endothelial marker CD31. In vitro angiogenesis experiments revealed that CCL20 is a direct pro-angiogenic molecule that induces EC invasion, sprouting and migration through CCR6. Moreover, using the angiogenesis matrigel plug assay in immunodeficient NMRI-nu mice, we clearly showed that CCL20 induces blood vessel formation, by attracting CCR6-positive ECs. Finally, we demonstrated that HCV-induced CCL20 protein expression and secretion in hepatoma cells could be abolished by antiviral treatment, indicating that CCL20 expression is dependent on HCV replication. In contrast to HCV, HBV-infection resulted in a decreased expression of CCL20, implying a virus-specific effect. Taken together, we identified HCV-induced CCL20 as a direct pro-angiogenic factor that acts on endothelial CCR6. These results suggest that the CCL20/CCR6 axis contributes to hepatic angiogenesis, promoting the hypervascular state of HCV-HCC.

The chemokine C-C motif ligand 20 (CCL20) is increased in the colonic mucosa during active inflammatory bowel disease (IBD) and can be found both in the epithelium and immune cells in the lamina propria. The present study investigated CCL20 and C-C motif Chemokine Receptor 6 (CCR6) in peripheral blood mononuclear cells (PBMCs) (n = 40) from IBD patients and healthy controls, to identify inductors of CCL20 release encountered in a local proinflammatory environment. CCL20 release from PBMCs was increased when activating TLR2/1 or NOD2, suggesting that CCL20 is part of a first line response to danger-associated molecular patterns also in immune cells. Overall, ulcerative colitis (UC) had a significantly stronger CCL20 release than Crohn's disease (CD) (+242%, p < 0.01), indicating that the CCL20-CCR6 axis may be more involved in UC. The CCL20 receptor CCR6 is essential for the chemotactic function of CCL20. UC with active inflammation had significantly decreased CCR6 expression and a reduction in CCR6⁺ cells in circulation, indicating chemoattraction of CCR6⁺ cells from circulation towards peripheral tissues. We further examined CCL20 induced release of cytokines from PBMCs. Stimulation with CCL20 combined with TNF increased IL-1β release from PBMCs. By attracting additional immune cells, as well as inducing proinflammatory IL-1β release from immune cells, CCL20 may protract the inflammatory response in ulcerative colitis.

CCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6).

CCL20 (CC chemokine ligand 20) is emerging as an important regulatory molecule in a pathway common to virus infection, alcoholic hepatitis, and non-alcoholic fatty liver disease (NAFLD) leading to the development of hepatic fibrosis. We previously observed upregulation of CCL20 in patients with NAFLD fibrosis and human hepatic stellate cells (LX-2 cells) in response to lipid loading. To date, the mechanisms mediating the relationship between CCL20 and hepatic fibrogenesis remain unknown. In this study, we sought to characterize the molecular mechanisms by which CCL20 may contribute to fibrogenesis in NAFLD. We observed that CCL20 levels increased with worsening severity of liver histology in NAFLD patients (normal < steatosis < inflammation < fibrosis) and during LX-2 cell activation in a time-dependent manner. We found that treatment of LX-2 cells with CCL20 corresponded with increased levels of CCL20 and ACTA2, and decreased levels of PLAU and SERPINE1, effects mitigated by CCL20 knockdown. We identified a putative binding site for miR-590-5p, which we previously reported to be downregulated in NAFLD fibrosis, in the CCL20 3' untranslated region (3'UTR), and found that exogenous miR-590-5p functionally interacted with the CCL20 3'UTR to downregulate its expression. Transfection of LX-2 hepatic stellate cells with miR-590-5p mimic or silencing RNA resulted in decreased or increased CCL20 levels, respectively. Our results indicate an association between CCL20 and hepatic stellate cell activation that includes modulation of key ECM components and functional interactions with a miRNA previously implicated in NAFLD fibrosis. Together, these findings support a novel mechanism by which CCL20 may promote fibrogenesis in NAFLD.

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.

Even though skin and oral mucosae are continuously in contact with commensal and opportunistic microorganisms, they generally remain healthy and uninflamed. Host defense peptides (HDPs) make up the body's first line of defense against many invading pathogens and are involved in the orchestration of innate immunity and the inflammatory response. In this study, we investigated the effect of two salivary HDPs, LL-37 and Hst1, on the inflammatory and antimicrobial response by skin and oral mucosa (gingiva) keratinocytes and fibroblasts. The potent antimicrobial chemokine CCL20 was investigated and compared with chemokines CCL2, CXCL1, CXCL8, and CCL27 and proinflammatory cytokines IL-1α and IL-6. Keratinocyte-fibroblast cocultures showed a synergistic increase in CCL20 secretion upon Hst1 and LL-37 exposure compared to monocultures. These cocultures also showed increased IL-6, CXCL1, CXCL8, and CCL2 secretion, which was IL-1α dependent. Secretion of the antimicrobial chemokine CCL20 was clearly IL-1α independent. These results indicate that salivary peptides can stimulate skin as well as gingiva cells to secrete antimicrobial chemokines as part of the hosts' defense to counteract infection.

Breast cancer is the leading cause of cancer-related death among women despite the significant improvement in diagnosis and treatment. Tumor-associated neutrophils have been shown to suppress antitumor functions of the host. However, how breast cancer tumor microenvironment influences the phenotype and functions of neutrophils to potentiate T cell immunosuppression is unknown. Herein, neutrophils isolated from peripheral blood of healthy donors were treated with supernatants from breast cancer cell lines or recombinant human CCL20. PD-L1 expression on neutrophils was then evaluated by immunofluorescence and flow cytometry. Neutrophils and Jurkat T cells were cocultured to evaluate the effect of tumor-associated neutrophils on T cell functions. Finally, immunohistochemical staining was performed to evaluate the clinical relevance of neutrophils infiltrating breast tumor tissues. Tumor-derived CCL20 activated and upregulated PD-L1 expression on neutrophils. A significant positive correlation was found between CCL20 and CD66b+ neutrophils in tumor tissues. Through in vitro experiment, tumor-associated neutrophils (TANs) effectively suppressed T cell immunity which was reversed upon PD-L1 blockade.Moreover, a high density of TANs was associated with short disease free survival in breast cancer patients. Furthermore, receiver operating curve showed that the density of TANs could accurately predict disease-free survival in breast cancer patients. Our findings suggest that targeting TANs via CCL20 immunosuppressive pathway may be a novel therapeutic strategy for breast cancer treatment.

The chemokine receptor CXCR4 and its ligand CXCL12 is overexpressed in the majority of tumors and is critically involved in the development and metastasis of these tumors. CXCR4 is expressed in malignant tumor cells whereas its ligand SDF-1 (CXCL12) is expressed mainly by cancer associated fibroblasts (CAF). Similarly to CXCR4, the chemokine CCL20 is overexpressed in variety of tumors; however its role and regulation in tumors is not fully clear. Here, we show that the chemokine receptor CXCR4 stimulates the production of the chemokine CCL20 and that CCL20 stimulates the proliferation and adhesion to collagen of various tumor cells. Furthermore, overexpression of CCL20 in tumor cells promotes growth and adhesion in vitro and increased tumor growth and invasiveness in vivo. Moreover, neutralizing antibodies to CCL20 inhibit the in vivo growth of tumors that either overexpress CXCR4 or CCL20 or naturally express CCL20. These results reveal a role for CCL20 in CXCR4-dependent and -independent tumor growth and suggest a therapeutic potential for CCL20 and CCR6 antagonists in the treatment of CXCR4- and CCL20-dependent malignancies.

The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production.

Recent evidence suggests that CC chemokine ligand 20 (CCL20) is upregulated after subarachnoid hemorrhage (SAH). Here, we investigated the functions of CCL20 in SAH injury and its underlying mechanisms of action. We found that CCL20 is upregulated in an SAH mouse model and in cultured primary microglia and neurons. CCL20-neutralizing antibody alleviated SAH-induced neurological deficits, decreased brain water content and neuronal apoptosis, and repressed microglial activation. We observed increased levels of CCL20, CC chemokine receptor 6 (CCR6), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), as well as of microglial activation in microglia treated with oxyhemoglobin (OxyHb). CCL20 or CCR6 knockdown reversed the effects of OxyHb on microglia. Conditioned medium from OxyHb-treated microglia induced neuronal apoptosis, while the percentage of apoptotic neurons in the conditioned medium from microglia transfected with CCL20 siRNA or CCR6 siRNA was decreased. We observed no decrease in OxyHb-induced apoptosis in CCL20-knockdown neurons. Conditioned medium from OxyHb-treated neurons led to microglial activation and induced CCR6, IL-1β and TNF-α expression, while CCL20 knockdown in neurons or CCR6 knockdown in microglia reversed those effects. Our results thus suggest CCL20 may be targeted to elicit therapeutic benefits after SAH injury.