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C-C motif chemokine ligand 20 (CCL20) participates in multiple oncogenic processes, but its role in lung adenocarcinoma (LUAD) is unclear. Herein, we explored the mechanism by which CCL20 works in LUAD progression. We performed bioinformatical analyses based on the complete transcriptome sequencing data from 1544 LUAD cases in 4 independent cohorts to evaluate signaling pathways regulated by CCL20. We established A549 and H358 cell lines with CCL20 knockdown to explore how CCL20 promotes tumor progression in vitro and in vivo experiments. Using another independent cohort of 348 urothelial carcinoma patients treated with the anti-PD-L1 agent (atezolizumab), we explored the synergistic effect of CCL20 and TGF-β on immunotherapy efficacy. High CCL20 expression is a poor prognostic marker for LUAD patients, and is associated with enhanced epithelial-mesenchymal transition (EMT), inflammatory response, and activated TNF pathway in LUAD. CCL20 knockdown restrained the EMT process and cell proliferation of LUAD cells in vitro and in vivo . Low CCL20 expression blocked the detrimental effects of high TGF-β on survival and effectively improved patients' response to anti-PD-L1 therapy. Collectively, we revealed the underlying mechanisms by which CCL20 promotes LUAD progression based on the largest sample size. The synergistic inhibitory effect of CCL20 and TGF-β on immune-checkpoint blockade therapy efficacy provides new views of immunotherapy resistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: CircRNA circ_0026344 was previously revealed as a tumour-suppressive gene in colorectal cancer (CRC) progression. The purpose of this research was to investigate the role of circ_0026344 in CRC cells metastasis induced by chemokines. Methods: Two human CRC cell lines SW480 and Caco-2 were treated by CCL20 and CXCL8. Cell proliferation, migration/invasion, expression of epithelial-mesenchymal transition (EMT) inducers and the expression of circ_0026344 were measured using sulforhodamine B assay, Transwell chamber, western blot and qRT-PCR, respectively. The effects of circ_0026344 on CRC cells migration/invasion and the expression of EMT inducers were evaluated. Moreover, the downstream miRNA and signalling pathways of circ_0026344 were studied. Results: CCL20 and CXCL8 synergized to facilitate the proliferation, migration and invasion of CRC cells. At the meantime, E-cadherin was downregulated, whereas N-cadherin, Vimentin and Snail were up-regulated by CCL20 and CXCL8 co-stimulation, which was accompanied by the mobilization of PI3K/AKT/ERK signalling. More interestingly, the expression of circ_0026344 was down-regulated by CCL20 and CXCL8 co-stimulation. Silence of circ_0026344 increased the migratory and invasive capacities of CRC cells and increased EMT process as well. Overexpression of circ_0026344 led to a contrary impact. miR-183 was negatively regulated by circ_0026344, and the inhibitory effects of circ_0026344 overexpression on Wnt/β-catenin pathway were reversed when miR-183 was overexpressed. Conclusion: Overexpression of circ_0026344 restrained CRC metastasis and EMT induced by CCL20 and CXCL8 synergistical treatment. miR-183 was a downstream effector of circ_0026344, and the anti-tumour function of circ_0026344 might be involved in the repressed Wnt/β-catenin signalling. Highlights CCL20 and CXCL8 synergize to decrease the expression of circ_0026344; Silence of circ_0026344 promotes CRC cells migration, invasion and EMT process; miR-183 is a downstream effector of circ_0026344.

Background: Acute-serum Amyloid A (A-SAA), one of the major acute-phase proteins, is mainly produced in the liver but extra-hepatic synthesis involving the skin has been reported. Its expression is regulated by the transcription factors NF-κB, C/EBPβ, STAT3 activated by proinflammatory cytokines., Objectives: We investigated A-SAA synthesis by resting and cytokine-activated Normal Human Epidermal Keratinocytes (NHEK), and their inflammatory response to A-SAA stimulation. A-SAA expression was also studied in mouse skin and liver in a model mimicking psoriasis and in the skin and sera of psoriatic and atopic dermatitis (AD) patients., Methods: NHEK were stimulated by A-SAA or the cytokines IL-1α, IL-17A, IL-22, OSM, TNF-α alone or in combination, previously reported to reproduce features of psoriasis. Murine skins were treated by imiquimod cream. Human skins and sera were obtained from patients with psoriasis and AD. A-SAA mRNA was quantified by RT qPCR. A-SAA proteins were dosed by ELISA or immunonephelemetry assay., Results: IL-1α, TNF-α and mainly IL-17A induced A-SAA expression by NHEK. A-SAA induced its own production and the synthesis of hBD2 and CCL20, both ligands for CCR6, a chemokine receptor involved in the trafficking of Th17 lymphocytes. A-SAA expression was increased in skins and livers from imiquimod-treated mice and in patient skins with psoriasis, but not significantly in those with AD. Correlations between A-SAA and psoriasis severity and duration were observed., Conclusion: Keratinocytes could contribute to psoriasis pathogenesis via A-SAA production, maintaining a cutaneous inflammatory environment, activating innate immunity and Th17 lymphocyte recruitment.

The CC-chemokine receptor 6 (CCR6) can be detected on naive and activated B cells. Counterintuitively, its absence accelerates the appearance of germinal centres (GCs) and increases the production of low-affinity antibodies. The detailed mechanism of CCR6 function during the humoral response has remained elusive, but previously we identified a distinct CCR6 high B-cell population in vivo early after antigenic challenge. In this study, we defined this population specifically as early, activated pre-GC B cells. In accordance, we show that CCR6 is upregulated rapidly within hours on the protein or mRNA level after activation in vitro. In addition, only activated B cells migrated specifically towards CCL20, the specific ligand for CCR6. Lack of CCR6 increased the dark zone/light zone ratio of GC and led to decreased antigen-specific IgG1 and IgG2a antibody generation in a B-cell intrinsic manner in mixed bone marrow chimeras. In contrast, antigen-specific IgM responses were normal. Hence, CCR6 negatively regulates entry of activated, antigen-specific pre-GC B cells into the GC reaction.

Targeting antigens encoded by DNA vaccines to the key antigen-presenting cells by chemotactic or growth factors, is an effective strategy for enhancing the potency of DNA vaccinations. Here, we report the effects of chemotactic or growth factors on a DNA vaccine against botulinum neurotoxin serotype A (BoNT/A) in a mouse model. We demonstrated that mice immunized with DNA constructs encoding the Hc domain of BoNT/A (AHc) fused with DC-stimulating Flt3L or MIP-3α cytokines failed to elicit an enhanced or efficacious AHc-specific humoral or protective response in mice. However, the potency of DNA vaccination was significantly modulated and enhanced by co-administration of AHc-expressing DNA with pFlt3L or pMIP-3α, which generated strong immune and protective responses against BoNT/A. Moreover, the enhanced potency was further boosted by co-administration of AHc-expressing DNA with the combination of pFlt3L and pMIP-3α in mice, but not with the Flt3L-MIP-3α fusion molecule, which indicated that co-immunization with both pFlt3L and pMIP-3α could synergistically enhance AHc-specific immune and protective responses against BoNT/A. In summary, our findings indicate that co-administration of plasmids encoding antigen and cytokine rather than administration of plasmids encoding cytokine-antigen fusion is effective to enhance the potency of AHc-expressing DNA vaccine., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications., Statement of Significance: Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site.

Background: The numbers of IL-27-producing CD4(+) T cells and the concentration of soluble IL-27 have been found to be increased in tuberculous pleural effusion (TPE). The objective of the present study was to explore the mechanism by which IL-27(+)CD4(+) T cells are recruited into the pleural space, and to explore the impact of IL-27 on pleural mesothelial cells (PMCs)., Methods: The expression profiles of chemokine receptor (CCR) were determined by flow cytometry. The chemoattractant activity of chemokines CCL20 and CCL22 for IL-27(+)CD4(+) T cells in vitro was observed. Effects of IL-27 on wound healing, proliferation and apoptosis of PMCs were also investigated., Results: IL-27(+)CD4(+) T cells in TPE expressed high level of CCR6, medium level of CCR4, and low levels of CCR2, CCR3, CCR5, CCR7, CCR10, and CXCR3. Recruitment of IL-27(+)CD4(+) T cells into TPE could be induced by pleural CCL20 and CCL22. By activating STAT3 signaling, IL-27 significantly improved wound healing and promoted proliferation of PMCs, and completely prevented apoptosis of PMCs induced by IFN-γ., Conclusions: After being recruited into pleural space by CCL20 or/and CCL22, these pleural IL-27-producing CD4(+) T cells may play important roles in tuberculosis immunity by affecting PMC functions.

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2-200 pg/ml) or CCL20 (5-500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5-2.7-fold), ALP (1.6-1.7-fold), and IL-6 protein production (1.3-1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3-5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3-5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.

Aim: To identify the mechanisms of chemokine ligand 20 (CCL20)-induced hepatocellular carcinoma (HCC) metastasis and evaluate it as a prognostic marker., Methods: Expression of CCL20 was evaluated by immunohistochemistry in HCC tissues from 62 patients who underwent curative resection. The relationship between CCL20 expression and clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate its predictive value for recurrence and survival of HCC patients. The expression levels of epithelial-mesenchymal transition (EMT)-and signaling pathway-related proteins were evaluated by Western blotting and immunocytochemistry. The effects of CCL20 on HCC cell proliferation and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium bromide (MTT) and Transwell assays., Results: CCL20 immunoreactivity was detected in all 62 patient specimens. CCL20 expression was associated with preoperative alpha-fetoprotein level (P = 0.043), tumor size (P = 0.000), tumor number (P = 0.008), vascular invasion (P = 0.014), and tumor differentiation (P = 0.007). Patients with high CCL20 expression had poorer recurrence-free and overall survivals compared to those with low CCL20 expression (both P < 0.001). CCL20 induced EMT-like changes in HCC cells and increased their proliferation and migration ability (P < 0.05). Western blotting and immunofluorescence staining showed that CCL20 induced an EMT-like phenotype in HCC cells, and increased expression of phosphorylated AKT, β-catenin and vimentin, and decreased E-cadherin expression (P < 0.05). The correlation analysis revealed that high CCL20 expression in HCC tissue specimens was negatively correlated with E-cadherin expression (13.33%, 4/30), and positively correlated with vimentin (90.0%, 27/30), β-catenin (96.67%, 29/30) and p-AKT (76.67%, 23/30) expression., Conclusion: CCL20 expression is associated with HCC recurrence and patient survival and promotes HCC cell proliferation and migration by inducing EMT-like changes via PI3K/AKT and Wnt/β-catenin pathways.

Chemokines and chemokine receptor-mediated effects are important mediators of the immunological response and cure in human leishmaniasis. However, in addition to their signalling properties for leukocytes, many chemokines have also been shown to act directly as antimicrobial peptides on bacteria and fungi. We screened ten human chemokines (CXCL2, CXCL6, CXCL8, CXCL9, CXCL10, CCL2, CCL3, CCL20, CCL27, CCL28) for antimicrobial effects on the promastigote form of the protozoan parasite Leishmania mexicana, and observed direct parasiticidal effects of several, CCL28 being the most potent. Damage to the plasma membrane integrity could be visualised by entrance of propidium iodide, as measured with flow cytometry, and by scanning electron microscopy, which showed morphological changes and aggregation of cells. The findings were in concordance with parasiticidal activity, measured by decreased mitochondrial activity in an MTT-assay. This is the first report of direct antimicrobial activity by chemokines on parasites. This component of immunity against Leishmania parasites identified here warrants further investigation that might lead to new insight in the mechanisms of human infection and/or new therapeutic approaches.

Newly discovered IL-9-producing CD4(+) helper T cells (Th9 cells) have been reported to contribute to tissue inflammation and immune responses, however, differentiation and immune regulation of Th9 cells in tuberculosis remain unknown. In the present study, our data showed that increased Th9 cells with the phenotype of effector memory cells were found to be in tuberculous pleural effusion as compared with blood. TGF-β was essential for Th9 cell differentiation from naïve CD4(+) T cells stimulated with PMA and ionomycin in vitro for 5 h, and addition of IL-1β, IL-4 or IL-6 further augmented Th9 cell differentiation. Tuberculous pleural effusion and supernatants of cultured pleural mesothelial cells were chemotactic for Th9 cells, and this activity was partly blocked by anti-CCL20 antibody. IL-9 promoted the pleural mesothelial cell repairing and inhibited IFN-γ-induced pleural mesothelial cell apoptosis. Moreover, pleural mesothelial cells promoted Th9 cell differentiation by presenting antigen. Collectively, these data provide new information concerning Th9 cells, in particular the collaborative immune regulation between Th9 cells and pleural mesothelial cells in human M. tuberculosis infection. In particular, pleural mesothelial cells were able to function as antigen-presenting cells to stimulate Th9 cell differentiation.

Absent in peripheral tissues during homeostasis, human plasmacytoid dendritic cells (pDCs) are described in inflamed skin or mucosa. Here, we report that, unlike blood pDCs, a subset of tonsil pDCs express functional CCR6 and CCR10, and their respective ligands CCL20 and CCL27are detected in inflamed epithelia contacting blood dendritic cell antigen 2(+) pDCs. Moreover, pDCs are recruited to imiquimod-treated skin tumors in WT but not CCR6-deficient mice, and competitive adoptive transfers reveal that CCR6-deficient pDCs are impaired in homing to inflamed skin tumors after intravenous transfer. On IL-3 culture, CCR6 and CCR10 expression is induced on human blood pDCs that become responsive to CCL20 and CCL27/CCL28, respectively. Interestingly, unlike myeloid DC, blood pDCs initially up-regulate CCR7 expression and CCL19 responsiveness on IL-3 ± CpG-B and then acquire functional CCR6 and CCR10. Finally, IL-3-differentiated CCR6(+) CCR10(+) pDCs secrete high levels of IFN-α in response to virus. Overall, we propose an unexpected pDCs migratory model that may best apply for mucosal-associated lymphoid tissues. After CCR7-mediated extravasation into lymphoid tissues draining inflamed epithelia, blood pDCs may be instructed to up-regulate CCR6 and/or CCR10 allowing their homing into inflamed epithelia (in mucosae or skin). At this site, pDCs can then produce IFN-α contributing to pathogen clearance and/or local inflammation.

Conclusion: The results of the present study indicate the potential of CCL20 as an effective mucosal adjuvant and suggest that nasal vaccination with P6 in combination with nasal CCL20 might be an effective regimen for the induction of nontypable Haemophilus influenzae (NTHi)-specific protective immunity., Objectives: Nasal vaccination is an effective therapeutic regimen for preventing upper respiratory infections. In the development of nasal vaccine, an appropriate adjuvant is required. In the present study we examined the efficacy of CCL20 as a mucosal adjuvant., Methods: CCL20 was administered intranasally to mice, which were then immunized intranasally with P6 protein of NTHi, and P6-specific immune responses were examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes., Results: Nasal application of CCL20 induced an increase in the number of dendritic cells in nasal-associated lymphoid tissue. P6-specific nasal wash immunoglobulin (Ig)A and serum IgG titers were elevated significantly after nasal immunization. Enhanced NTHi clearance from the nasopharynx was also observed.

Aims/hypothesis: We recently found that activation of the type III histone deacetylase sirtuin 1 suppresses T cell immune responses. Here we sought to determine the therapeutic potential of the sirtuin 1 activator resveratrol in the treatment of diabetes in the NOD mouse model of type 1 diabetes and the mechanisms underlying such potential., Methods: NOD mice were fed or subcutaneously injected with resveratrol and evaluated for development of diabetes. Splenocytes from resveratrol-treated and control mice were analysed by gene array. The altered expression of inflammatory genes induced by resveratrol was validated and the role of changed gene expression in prevention of diabetes was determined., Results: Resveratrol administration potently prevented and treated type 1 diabetes in NOD mice. Gene array analysis indicated a dramatic decrease in expression of Ccr6, which encodes chemokine (C-C motif) receptor (CCR) 6, in the splenocytes from resveratrol-treated mice. CCR6 abundance on IL-17-producing cells and CD11b(+)F4/80(hi) macrophages was inhibited by resveratrol treatment. Interestingly, CCR6(+) IL-17-producing cells and CD11b(+)F4/80(hi) macrophages accumulated in the spleens and pancreatic lymph nodes, but their presence in the pancreas was reduced, suggesting that resveratrol blocks their migration from peripheral lymphoid organs to the pancreas. Indeed, the migration of splenocytes toward media containing chemokine (C-C motif) ligand 20 (CCL20) was impaired by resveratrol treatment. CCL20 peptides, which block CCR6 binding to CCL20, inhibited development of type 1 diabetes., Conclusions/interpretation: Inhibition of CCR6-mediated migration of inflammatory cells by resveratrol may provide a powerful approach for treatment of type 1 diabetes and possibly of other inflammatory diseases.

Background: CD4(+)CD25(+) regulatory T cells (Tregs), a heterogeneous population, were enrichment in tumor mass and played an important role in modulating anti-tumor immunity. Recently, we reported a Treg subset, CCR6(+) Tregs but not CCR6(-)Tregs, were enriched in tumor mass and closely related to poor prognosis of breast cancer patients. However, the underlying mechanism remains elusive. Here, we carefully evaluate the enrichment of CCR6(+)Tregs in tumor mass during progression of breast cancer and explore its possible mechanism., Methodology/principal Findings: The frequency of CCR6(+)Tregs in tumor infiltrating lymphocytes (TILs ) was analyzed at early stage and at late stage of tumor in a murine breast cancer model by FACS respectively. The expansion of CCR6(+)Tregs and their CCR6(-) counterpart in tumor mass were determined by BrdU incorporation assay. The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated. The role of local expansion of CCR6(+)Tregs in their enrichment and suppression in vivo also was evaluated in adoptive cell transfer assay. We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ. Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-β dependent manner. Adoptive transfer of CCR6(+)Tregs was found to potently inhibit the function of CD8(+)T cells in vivo, which was dependent on their proliferation and subsequently enrichment in tumor mass., Conclusions/significance: Our finding suggested that CCR6(+) Tregs, a distinct subset of Tregs, exert its predominant suppressive role in tumor immunity through prior in situ expansion, which might ultimately provide helpful thoughts for the designing of Treg-based immunotherapy for tumor in the future.

CCL20 is a chemokine that attracts immature dendritic cells. We show that monocytes, cells characteristic of the innate immune response, infected with Mycobacterium tuberculosis express the CCL20 gene at a much higher level than the same cells infected with non-tuberculous mycobacteria. Interferon (IFN)-γ, a fundamental cytokine in the immune response to tuberculosis, strongly inhibits both the transcription and the translation of CCL20. We have also confirmed that dendritic cells are a suitable host for mycobacteria proliferation, although CCL20 does not seem to influence their intracellular multiplication rate. The chemokine, however, down-regulates the characteristic production of reactive oxygen species (ROS) induced by M. tuberculosis in monocytes, which may affect the activity of the cells. Apoptosis mediated by the mycobacteria, possibly ROS-dependent, was also inhibited by CCL20., (© 2010 British Society for Immunology.)

Subchondral bone remodeling in osteoarthritis (OA) and rheumatoid arthritis (RA) is mainly characterized by the formation of osteophytes/fibrosis and by the presence of infiltrating cells associated to bone resorption. In this study we analyzed CC (cysteine cysteine motif) chemokine ligand (CCL)20 and CC chemokine receptor (CCR)6 function in subchondral bone tissue and osteoblasts isolated from OA and RA patients. CCL20/CCR6 expression was evaluated by immunohistochemical techniques in bone tissue from OA and RA patients. CCL20-functional tests were performed on osteoblasts isolated from OA and RA patients to evaluate enzymatic response and cell proliferation. Moreover, we assessed Akt phosphorylation as the major signaling pathway for CCL20. In bone tissue biopsies we found that osteoblasts from both OA and RA patients expressed CCR6 while CCL20 was expressed only by RA osteoblasts. Both CCR6 and CCL20 were highly expressed in osteocytes and mononuclear cells from only RA patients. CCL20-stimulated OA osteoblasts showed a significant increase in beta-N-acetylhexosaminidase release compared to RA. Conversely, a significant increase in cellular proliferation was found only in CCL20-stimulated RA osteoblasts associated to Akt phosphorylation. These data were confirmed in bone tissue biopsies. This study demonstrates a different expression of CCL20-positive osteoblasts in OA versus RA disease that seem to be associated with the presence of infiltrating mononuclear cells. Moreover, CCL20 stimulation resulted in a greater proliferative response in RA osteoblasts compared to OA osteoblasts, mediated by Akt signaling, while OA osteoblasts showed increased enzymatic activity, thus suggesting a differential role of this chemokine in OA and RA., (Copyright 2009 Wiley-Liss, Inc.)

Problem: CCL20/MIP3alpha is a chemokine for immature dendritic cells as well as an antibacterial against gram-positive and gram-negative bacteria. The role of CCL20/MIP3alpha as an antiviral is unknown. In this study, we have examined the production of CCL20/MIP3alpha by epithelial cells from the upper female reproductive tract as well as its activity as an antiviral molecule., Method of Study: Primary uterine and Fallopian tube epithelial cells were treated with Poly(I:C) and CCL20/MIP3alpha mRNA and protein was measured by Realtime RT-PCR and ELISA assays. Anti-HIV activity was determined using an indicator cell line TZM-bl and quantified by using a luminometer., Results: Primary uterine and Fallopian tube epithelial cells produce CCL20/MIP3alpha constitutively and the production is enhanced following stimulation with viral double-stranded RNA mimic Poly(I:C). Recombinant CCL20/MIP3alpha was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 when virus was directly incubated with CCL20/MIP3alpha but not when CCL20/MIP3alpha was added to cells either prior to infection or post-infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and CCL20/MIP3alpha., Conclusion: This study demonstrates that CCL20/MIP3alpha is an important endogenous anti-HIV-1 microbicide of the female reproductive tract.

Stromal cell monolayers have been an important means of studying the regulation of hematopoiesis, because they produce cytokines. Cytosine arabinoside, vincristine, daunorubicin, and doxorubicin are common drugs for hematological cancer therapy, and they may have some effects on bone marrow stroma during chemotherapy. The aim of this study was to elucidate interactions between the bone marrow stromal microenvironment and leukemic cells after drug treatment. We tested the hypothesis that human HS-5 stromal cells, pretreated with anticancer drugs, affected the growth of leukemic K562 cells by changing the cytokines in the culture microenvironment. Thereafter, proliferation of K562 cells increased nearly 2.5-fold compared the co-cultivation with drugs-pretreated HS-5 stromal cells and drugs-untreated HS-5 stromal cells. The results indicated that co-cultivation with HS-5 stromal cells pretreated with drugs caused significant K562 cell proliferation. Cytokines in the microenvironment were detected via the RayBio((R))Human Cytokine Antibody Array Membrane. The levels of the cytokines CKbeta, IL-12, IL-13, IGFBP-2, MCP-1, MCP-3, MCP-4, MDC, MIP-1beta and MIP-1delta were decreased, with a particularly marked decrease in MIP-3alpha. In co-culture medium, there was a 20-fold decrease in MIP-3alpha in daunorubicin-pretreated HS-5 cells and at least a 3-fold decrease in Ara-C-pretreated cells. This indicated a significant effect of anticancer drugs on the stromal cell line. Using phosphorylated Erk and pRb proteins as cell proliferation markers, we found that phosphorylation of these markers in K562 cells was inhibited during co-cultivation with drug-pretreated stromal cells in MIP-3alpha-supplemented medium and restored by MIP-3alpha antibody supplement. In conclusion, anticancer drug pretreatment suppresses the negative control exerted by HS-5 cells on leukemic cell proliferation, via modulation of cytokines in the microenvironment, especially at the level of MIP-3alpha.

Objective: To identify interleukin-17 (IL-17)-producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis., Methods: Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17-producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)-positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22., Results: IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-gamma., Conclusion: This study is the first to define the frequency and characteristics of "Th17" cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

Human macrophage inflammatory protein 3alpha (MIP-3alpha), also known as CCL20, is a 70-amino-acid chemokine which exclusively binds to chemokine receptor 6. In addition, the protein also has direct antimicrobial, antifungal, and antiviral activities. The solution structure of MIP-3alpha was solved by the use of two-dimensional homonuclear proton nuclear magnetic resonance (NMR). The structure reveals the characteristic chemokine fold, with three antiparallel beta strands followed by a C-terminal alpha helix. In contrast to the crystal structures of MIP-3alpha, the solution structure was found to be monomeric. Another difference between the NMR and crystal structures lies in the angle of the alpha helix with respect to the beta strands, which measure 69 and approximately 56.5 degrees in the two structures, respectively. NMR diffusion and pH titration studies revealed a distinct tendency for MIP-3alpha to form dimers at neutral pH and monomers at lower pH, dependent on the protonation state of His40. Molecular dynamics simulations of both the monomeric and the dimeric forms of MIP-3alpha supported the notion that the chemokine undergoes a change in helix angle upon dimerization and also highlighted the important hydrophobic and hydrogen bonding contacts made by His40 in the dimer interface. Moreover, a constrained N terminus and a smaller binding groove were observed in dimeric MIP-3alpha simulations, which could explain why monomeric MIP-3alpha may be more adept at receptor binding and activation. The solution structure of a synthetic peptide consisting of the last 20 residues of MIP-3alpha displayed a highly amphipathic alpha helix, reminiscent of various antimicrobial peptides. Antimicrobial assays with this peptide revealed strong and moderate bactericidal activities against Escherichia coli and Staphylococcus aureus, respectively. This confirms that the C-terminal alpha-helical region of MIP-3alpha plays a significant part in its broad anti-infective activity.

The aim of this study was to investigate the roles of chemokine CCL20 in development of CD4(+)CD25(+) thymocytes by means of fetal thymus organ culture. Fetal mouse thymus lobes were removed at the fetus age of 14.5 days and cultured in complete RPMI 1640 with 20% FBS in vitro. Phenotypes of the thymocytes were analyzed by FACS and the number of cells per lobe was counted. The results revealed that from day 14.5 to day 19, the absolute and relative numbers of the CD4(+)CD25(+) thymocytes varied similarly as their development as in vitro culture at 6 days. Data showed that during the 6 days in vitro culture the CD4(+)CD25(+) cell percentage out of CD4(+) cells was 58.29%, 12.14%, 6.08%, 17.78%, 9.06%, 4.04% and the CD4(+)CD25(+) cell percentage out of CD25(+) cells was 3.75%, 10.81%, 17.20%, 51.93%, 61.64%, 80.06%. All these data indicated similar characters to their development in vivo. Moreover, at interference with CCL20, the percentage of CD4(+)CD25(+) T cells in thymocytes significantly decreased at the 3 and 6 days from 3.24+/-0.18 and 3.96+/-0.24 to 1.27+/-0.11 (p<0.001) and 1.76+/-0.22 (p<0.001) respectively. It is concluded that the development of CD4(+)CD25(+) thymocytes is similar both in vitro and in vivo, interfering with CCL20 significantly downregulate the expression of CD4(+)CD25(+) T cells. The above data may help to understand the development of naturally arising CD4(+)CD25(+) regulatory T cells.