Role of the CCL20/CCR6 axis in tubular epithelial cell injury: Kidney-specific translational insights from acute kidney injury to chronic kidney disease.

This study investigated the role of the axis involving chemokine receptor 6 (CCR6) and its ligand chemokine (C-C motif) ligand 20 (CCL20) in acute kidney disease (AKD) using an ischemia-reperfusion injury (IRI) model. The model was established by clamping the unilateral renal artery pedicle of C57BL/6 mice for 30 min, followed by evaluation of CCL20/CCR6 expression at 4 weeks post-IRI. In vitro studies were conducted to examine the effects of hypoxia and H2 O2 -induced oxidative stress on CCL20/CCR6 expression in kidney tissues of patients with AKD and chronic kidney disease (CKD). Tubular epithelial cell apoptosis was more severe in C57BL/6 mice than in CCL20 antibody-treated mice, and CCR6, NGAL mRNA, and IL-8 levels were higher under hypoxic conditions. CCL20 blockade ameliorated apoptotic damage in a dose-dependent manner under hypoxia and reactive oxygen species injury. CCR6 expression in IRI mice indicated that the disease severity was similar to that in patients with the AKD phenotype. Morphometry of CCL20/CCR6 expression revealed a higher likelihood of CCR6+ cell presence in CKD stage 3 patients than in stage 1-2 patients. Kidney tissues of patients with CKD frequently contained CCL20+ cells, which were positively correlated with interstitial inflammation. CCL20/CCR6 levels were increased in fibrotic kidneys at 4 and 8 weeks after 5/6 nephrectomy. These findings suggest that modulating the CCL20/CCR6 pathway is a potential therapeutic strategy for managing the progression of AKD to CKD.

Patched 1 and C-C Motif Chemokine Receptor 6 Distinguish Heterogeneous T Helper 17 Subsets in Colitic Lamina Propria.

T helper 17 (Th17) cells contribute to the pathogenesis of inflammatory bowel diseases (IBD). However, their heterogeneity and regulatory mechanisms in IBD are not completely disclosed. A mouse colitis model was established. Th17 cells were enriched from the mesenteric lymph nodes (mLN) and lamina propria (LP). The phenotypes and functions of Th17 subsets were analyzed by flow cytometry, Immunoblotting, and real-time RT-PCR. The contributions of the Th17 subsets to colitis pathogenesis were evaluated by histology, ELISA, and flow cytometry after adoptive transfer. Smoothened (SMO), GLI family zinc finger 1 (Gli1), and GLI family zinc finger 3 (Gli3) were markedly up-regulated while Patched 1 (PTCH1) was down-regulated in LP Th17 cells in colitic lamina propria. Based on the expression of PTCH1 and C-C motif chemokine receptor 6 (CCR6), LP Th17 cells were divided into a PTCH1lowCCR6low Th17 subset and a PTCH1highCCR6high Th17 subset. The former expressed higher T-bet, IFN-γ, TNF-α, IL-1ß, and GM-CSF but lower IL-17A, IL-22, IL-17F, and Gli3 than the latter. The PTCH1highCCR6high Th17 subset was more resistant to polarization towards T helper 1 (Th1) than the PTCH1lowCCR6low Th17 subset. Moreover, the PTCH1highCCR6high Th17 subset was more competent to maintain Th17 identity. The PTCH1highCCR6high Th17 subset induced less severe colitis than the PTCH1lowCCR6low Th17 subset. PTCH1highCCR6high Th17 cells are Th17 cells whereas PTCH1lowCCR6low Th17 cells are Th1-like Th17 cells. Our study deepens the understanding of Th17 heterogeneity and plasticity in colitis.

Molecular cloning, structure and expressional profiles of two novel single-exon genes (PoCCR6A and PoCCR6B) in the Japanese flounder (Paralichthys olivaceus).

CCR6 is an important binding receptor of CCL20 and beta-defensins, and has multiple functions in the innate and acquired immune responses. In this study, we cloned the PoCCR6A and PoCCR6B genes of the Japanese flounder and studied the gene structure and expression patterns of these two genes in bacterial infection. The full-length PoCCR6A cDNA is 1415 bp and the open reading frame (ORF) is 1113 bp, encoding a 370-amino-acid peptide. The full-length PoCCR6B cDNA is 2193 bp and the ORF is 1029 bp, encoding a 363-amino-acid peptide. The structures of PoCCR6A and PoCCR6B indicate that they are single-exon genes. The predicted proteins encoded by PoCCR6A and PoCCR6B have the typical G-protein-coupled receptor (GPCR) family signature of seven transmembrane domains and several conserved structural features. A tissue distribution analysis showed that PoCCR6A is predominately expressed in the intestine, gill, and blood, and PoCCR6B in the gill, spleen, and liver. The expression patterns of the two chemokine receptors were analyzed during bacterial infection. In spleen and kidney, the expression of PoCCR6A was significantly upregulated at 24 h after infection, whereas the expression of PoCCR6B was steady at these time points. While in intestine, both of them were upregulated at 6 h-12 h after infection, and in gill the expression levels of them were upregulated at 24 h. The patterns of expression suggested that PoCCR6A and PoCCR6B play an important role in the immune response of the Japanese flounder, especially in the mucosal tissues.

Association of CCR6 functional polymorphisms with Primary Biliary Cholangitis.

The biliary epithelial cells release CC chemokine receptor 6 (CCR6) ligand 20 (CCL20), leading to recruitment of CCR6+ T cells and subsequent infiltration into the biliary epithelium in primary biliary cholangitis patients. Previous genome-wide multi-national meta-analysis, including our Han Chinese cohort, showed significant association of CCR6 and CCL20 single nucleotide polymorphisms (SNP) with PBC. We report here that significantly associated SNPs, identified in the CCR6 locus based on our Han Chinese genome-wide association study, can be separated into "protective" and "risk" groups, but only "risk" SNPs were confirmed using a separate Han Chinese PBC cohort. Only weak association of CCL20 SNPs was observed in Han Chinese PBC cohorts. Fine-mapping and logistical analysis identified a previously defined functional variant that, leads to increased CCR6 expression, which contributed to increased genetic susceptibility to PBC in Han Chinese cohort.

Long non­coding RNA JPX promotes gastric cancer progression by regulating CXCR6 and autophagy via inhibiting miR­197.

Long non­coding RNAs (lncRNAs) serve a crucial role in gastric cancer (GC) progression. However, the molecular mechanism underlying lncRNA JPX transcript, XIST activator (JPX) in the tumorigenesis of GC is not completely understood. Reverse transcription­quantitative PCR (RT­qPCR) and western blotting were performed to detect gene expression. A luciferase reporter gene assay was conducted to determine the relationship between microRNA (miR)­197 and JPX or C­X­C motif chemokine receptor 6 (CXCR6). Cell viability, migration and invasion were determined by performing MTT, wound healing and Transwell assays, respectively. The Cancer Genome Atlas database and the RT­qPCR results indicated that JPX expression was upregulated and miR­197 expression was downregulated in patients with GC and in GC cells. Moreover, high JPX expression and low miR­197 expression in patients with GC indicated poor prognosis. miR­197 expression was directly inhibited by JPX. Compared with the short hairpin RNA (sh) negative control (NC) group, NCI­N87 and MKN­45 cells in the shJPX group displayed decreased cell viability and invasion, as well as a wider scratch width. NCI­N87 and MKN­45 cells in the shJPX + miR­197 inhibitor group had increased viability and invasion, but a narrower scratch width compared with the shJPX group. It was also identified that miR­197 directly inhibited CXCR6 expression. miR­197 inhibited Beclin1 protein expression and promoted p62 protein expression. Compared with the NC group, NCI­N87 and MKN­45 cells in the miR­197 mimic group had decreased cell viability and invasion, and a wider scratch width. Enhanced cell viability and invasion, and a narrower scratch width was also observed in the miR­197 mimic + CXCR6 and miR­197 mimic + Beclin1 groups, compared with the miR­197 mimic group. Collectively, the results indicated that lncRNA JPX promoted GC progression by regulating CXCR6 and autophagy via inhibiting miR­197. Furthermore, JPX knockdown regulated GC cell phenotype by promoting miR­197.

Chemokine CCL20 promotes the paclitaxel resistance of CD44+CD117+ cells via the Notch1 signaling pathway in ovarian cancer.

Studies have found that C­C motif chemokine ligand 20 (CCL20)/C­C 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 CD44­CD117­ 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 CD44­CD117­ 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 CD44­CD117­ groups. After treatment with PTX, apoptosis and ROS levels were decreased in the CD44+CD117+ groups compared with the CD44­CD117­ 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.

USP4 is pathogenic in allergic airway inflammation by inhibiting regulatory T cell response.

AIMS: Asthma is characterized by chronic inflammation and airway hyperresponsiveness (AHR). It is controllable, but not curable. Ubiquitin-specific peptidase 4 (USP4) has been verified as a regulator of regulatory T (Treg) cells and Th17 cells in vitro. In this study, we aim to investigate whether USP4 could serve as a therapeutic target for asthma. MAIN METHODS: Age-matched USP4 wild-type and knockout mice received an intraperitoneal injection of 100 µg ovalbumin (OVA) mixed in 2 mg aluminum hydroxide in 1 × PBS on days 0, 7 and 14. On days 21 to 27, the mice were challenged with aerosolized 1% OVA in 1 × PBS for 30 min. Tissue histology, ELISA and flow cytometry were applied 24 h after the last OVA challenge. KEY FINDINGS: USP4 deficiency protected mice from OVA-induced AHR and decreased the production of several inflammatory cytokines in T cells in vivo. Compared to the lung cells isolated from WT mice, Usp4-/- lung cells decreased secretion of IL-4, IL-13 and IL-17A upon stimulation in vitro. Meanwhile, the percentage of CD4+Foxp3+ Treg cells was elevated, with more CCR6+Foxp3+ Treg cells accumulating in the lungs of OVA-challenged USP4 deficient mice than in their wild-type counterparts. Treatment with the USP4 inhibitor, Vialinin A, reduced inflammatory cell infiltration in the lungs of OVA-challenged mice in vivo. SIGNIFICANCE: We found USP4 deficiency contributes to attenuated airway inflammation and AHR in allergen-induced murine asthma, and Vialinin A treatment alleviates asthma pathogenesis and may serve as a promising therapeutic target for asthma.

Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis.

Natural killer (NK) cells are innate immune cells that interface with the adaptive immune system to generate a pro-inflammatory immune environment. Primary Biliary Cholangitis (PBC) is a hepatic autoimmune disorder with extrahepatic associations including systemic sclerosis, Sjogren's syndrome and thyroiditis. Immunogenetic studies have identified polymorphisms of the IL-12/STAT4 pathway as being associated with PBC. As this pathway is important for NK cell function we investigated NK cells in PBC. Circulating NK cells from individuals with PBC were constitutively activated, with higher levels of CD49a and the liver-homing marker, CXCR6, compared to participants with non-autoimmune chronic liver disease and healthy controls. Stimulation with minimal amounts of IL-12 (0.005 ng/ml) led to significant upregulation of CXCR6 (p < 0.005), and enhanced IFNγ production (p < 0.02) on NK cells from PBC patients compared to individuals with non-autoimmune chronic liver disease, indicating dysregulation of the IL-12/STAT4 axis. In RNAseq studies, resting NK cells from PBC patients had a constitutively activated transcriptional profile and upregulation of genes associated with IL-12/STAT4 signaling and metabolic reprogramming. Consistent with these findings, resting NK cells from PBC patients expressed higher levels of pSTAT4 compared to control groups (p < 0.001 vs. healthy controls and p < 0.05 vs. liver disease controls). In conclusion NK cells in PBC are sensitive to minute quantities of IL-12 and have a "primed" phenotype. We therefore propose that peripheral priming of NK cells to express tissue-homing markers may contribute to the pathophysiology of PBC.

IL-12 and IL-15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells.

INTRODUCTION: Murine hepatic NK cells exhibit adaptive features, with liver-specific adhesion molecules CXCR6 and CD49a acting as surface markers. METHODS: We investigated human liver-resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood. RESULTS: Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver-resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver-resident double-positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single-positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL-12 and IL-15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver-resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression. CONCLUSION: IL-12 and IL-15 may be key for generating NK cells with a tissue-homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue-homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease.

High expression of CXC chemokine receptor 6 associates with poor prognosis in patients with clear cell renal cell carcinoma.

PURPOSE: Accumulating evidence indicates that CXC chemokine receptor 6 (CXCR6) has a crucial role in cancer development and progression, however, its role in clear cell renal cell carcinoma (ccRCC) remains obscure. The aim of this study is to investigate the prognostic value of CXCR6 expression in patients with ccRCC following surgery. MATERIALS AND METHODS: This study retrospectively included 239 patients with ccRCC who underwent nephrectomy and had paraffin tissue available at a single center. CXCR6 expression in tumor tissue was evaluated by immunohistochemistry and its associations with overall survival (OS) and recurrence-free survival (RFS) were investigated. RESULTS: A total of 47.3% tumors were considered as high expression of CXCR6, which was significantly associated with the male sex (P = 0.003) and high Fuhrman grade (P<0.001). A high expression of CXCR6 indicated a reduced OS (P<0.001) and RFS (P = 0.007). Multivariate analysis demonstrated that CXCR6 expression was an independent prognostic factor of OS (hazard ratio = 2.604; 95% CI: 1.338-5.068; P = 0.005) and RFS (hazard ratio = 1.957; 95% CI: 1.065-3.595; P = 0.031). Subgroup analysis found that CXCR6 expression could differentiate survival risks among patients with high-risk disease. Moreover, a nomogram integrating CXCR6 expression and traditional clinical and pathologic features was established and predicted postsurgical recurrence-risk well at 3- and 5-year. CONCLUSIONS: The expression of CXCR6 in tumor tissue may serve as a potential prognostic biomarker to refine clinical prognosis prediction combined with traditional clinical and pathological analysis for patients with ccRCC after surgery.

Expression of Membrane-Bound CC Chemokine Ligand 20 on Follicular T Helper Cells in T-B-Cell Conjugates.

The CC chemokine receptor 6 (CCR6) and its sole chemokine ligand CC chemokine ligand 20 (CCL20) display an emerging role in the coordination of humoral immune responses. Recent studies demonstrate a role of this chemokine axis in the migration of B cells to key immunological sites during an immune response, and facilitating the generation of high-quality antibodies. Very little, however, is known about CCL20 and its role in these functions. We undertook a preliminary investigation into the expression and function of CCL20 and demonstrate its well-noted upregulation in the spleen during immunization. Furthermore, we show that most follicular T helper (Tfh) cells can be CCR6+ and can produce CCL20. Surprisingly, CCL20 cannot only be found in the cytoplasm but also on the surface of these cells and their precursors. Analysis of T-B-cell conjugates revealed that mature Tfh cells, but not their precursors, are highly enriched in the conjugates. Further functional studies are needed to unravel the precise role of CCL20 in coordinating T and B cell interactions during the humoral immune response.

Role of CCL20/CCR6 and the ERK signaling pathway in lung adenocarcinoma.

Previous studies have revealed that carcinoma-associated fibroblasts communicate microenvironment-derived signals through chemokine/chemokine receptor interaction, resulting in carcinogenesis. C-C motif chemokine ligand 20 (CCL20)/C-C motif chemokine receptor 6 (CCR6) interactions are involved in the pathogenesis of colonic malignancies. The present study aimed to characterize the roles of CCL20/CCR6 and the extracellular signal-regulated kinase (ERK) signaling pathway in lung adenocarcinoma growth. Lung adenocarcinoma samples obtained at surgery were assessed for the expression, tissue localization and production of CCL20/CCR6. In addition, colony formation, ERK signaling and chemokine production were measured to assess the responsiveness of the A549 cell line to CCL20 stimulation. CCL20 and CCR6 were found to be highly expressed in the majority of samples in the recurrence group (76 and 66%, respectively). The staining indexes of CCL20 and CCR6 in the recurrence group were 149.3 and 134.4, respectively, which were significantly higher than those in the non-recurrence group (57.2 and 58.0, respectively); the protein and mRNA expression levels determined by western blot and reverse transcription-quantitative polymerase chain reaction were also found to be high in the recurrence group For A549 cells, the colony-forming capacity was increased by CCL20 stimulation, and this effect was dependent in part on ERK phosphorylation. Collectively, the findings suggest that CCR6 and CCL20 may serve a role in lung adenocarcinoma, leading to proliferation and migration via autocrine or paracrine mechanisms. The disruption of CCL20/CCR6 interactions may be a promising strategy for the treatment of cancer.

Expression of CCR6 in esophageal squamous cell carcinoma and its effects on epithelial-to-mesenchymal transition.

Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer associated with poor prognosis. We detected the expression of C-C motif chemokine receptor 6 (CCR6) and epithelial-to-mesenchymal transition (EMT) markers in esophageal tissues/cells, and evaluated the effects of CCR6 on ESCC cells proliferation, migration and invasion in response to C-C motif chemokine ligand 20 (CCL20) treatment. Our data showed CCR6 was highly expressed in ESCC cell lines (ECA-109 and TE-1), whereas kept in a low expression in normal cell lines HEEC (P < 0.001). CCL20 stimulus induced a significant decrease in the proliferation ability of ESCC (P < 0.05). The healing speed of CCL20 group was significantly higher than control in ECA-109 (P < 0.01), whereas significantly lower in αCCR6+CCL20 group than CCL20 group (P < 0.05).The number of cells permeabling through the polycarbonate membrane in CCL20 group was higher than control (P < 0.01). The cell number in αCCR6+CCL20 group was significantly reduced compared to CCL20 group in ECA-109 (P < 0.05). Moreover, after CCL20 stimulated in ECA-109, both mRNA and protein level of E-cadherin significantly decreased compared to control, while Vimentin was significantly higher. In αCCR6+CCL20 group, mRNA and protein level of E-cadherin significantly increased compared to CCL20 group, while Vimentin was much lower than CCL20 group. There was no significant difference in TE-1. In summary, high expression of CCR6 existed in the lymph node metastasis and TNM stage of ESCC. CCR6 play an important role in the regulation of tumor cell proliferation, invasion and migration. CCR6 may participate in regulating the occurrence of EMT in ESCC.

Production of CCL20 on nucleus pulposus cells recruits IL-17-producing cells to degenerated IVD tissues in rat models.

IL-17-producing cells play an important role in various autoimmune diseases. A higher level of IL-17-producing cells is observed in patients with intervertebral disc (IVD) degeneration. However, the mechanism of the accumulation of IL-17-producing cells remains to be elucidated. Our aim is to demonstrate whether the interaction of CC chemokine ligand (CCL) 20 and its specific receptor CCR6 is involved in the recruitment of IL-17-producing cells to degenerated disc tissues in rat models. The well-accepted needle puncture model and the autologous nucleus pulposus application model of rats were established. All of the animals were randomly divided into four groups: the sham surgery group, the needle puncture group (NP group), the sham surgery + autologous nucleus pulposus application group (sham-ANPA group) and the needle puncture + autologous nucleus pulposus application group (NP-ANPA group). Immunohistochemical staining, western blot and real-time PCR were used to evaluate the expression levels of CCL20, IL-17 and CCR6 in the IVD samples. Moreover, the IL-17 concentrations in the serum were detected by ELISA. Pearson correlations were performed to analyse the correlation among the expressions of CCL20, CCR6 and IL-17. The expressions of CCL20, IL17 and CCR6 were dramatically elevated in comparison with the control groups. The circulating IL-17 in the serum of the NP-ANPA group was elevated compared to the sham surgery group. In addition, there was a positive correlation among the expression levels of CCL20, CCR6 and IL-17. The results suggest a potential mechanism for the recruitment of IL-17-producing cells to degenerated intervertebral discs via a CCL20/CCR6 system in vivo.