Using cDNA microarray technology, the expression of chemokine genes in the elicitation site of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity (CHS) was examined in mice. Of the 33 genes analyzed, levels of 11 gene expressions changed, and these can be assigned to four groups based on their kinetic patterns; (1) LARC/CCL20 whose mRNA level increased rapidly at 3 h post-challenge and then gradually decreased, (2) JE/CCL2, MARC/CCL7, MIP-1gamma/CCL9, monocyte chemoattractant protein (MCP)-5/CCL12, ELC/CCL19 and BRAK/CXCL14 whose mRNA levels increased with time and reached the maximum at 6-9 h post-challenge, (3) LIX/CXCL5, Mig/CXCL9 and IP-10/CXCL10 whose mRNA levels increased gradually at least up to 12 h post challenge, and (4) SLC/CCL21 whose mRNA level decreased gradually with time after challenge. The findings suggest that sequential expression of chemokine genes is essential for orientating non-specific skin response to hapten-specific CHS response through the recruitment of inflammatory cells such as neutrophils, monocytes/macrophages and T-cells from the circulation into the tissue site.

Endothelial E- and P-selectins mediate lymphocyte trafficking in inflammatory processes by interacting with lymphocyte selectin ligands. These are differentially expressed among different T cell subsets and function alone or in cooperation to mediate T cell adhesion. In this study, we characterize the expression and functionality of E-selectin ligands in Th type 17 lymphocytes (Th17 cells) and report that CD43 functions as a Th17 cell E-selectin ligand in vitro that mediates Th17 cell rolling on the vascular endothelium and recruitment in vivo. We demonstrate Th17 cells express CD44, P-selectin glycoprotein ligand (PSGL)-1, and CD43. Few PSGL-1(-/-)CD43(-/-) Th17 cells accumulated on E-selectin under shear flow conditions compared with wild-type cells. CD43(-/-) Th17 cell accumulation on E-selectin was impaired as compared with wild-type and PSGL-1(-/-), and similar to that observed for PSGL-1(-/-)CD43(-/-) Th17 cells, indicating that CD43 alone is a dominant ligand for E-selectin. Notably, this finding is Th17 cell subset specific because CD43 requires cooperation with PSGL-1 in Th1 cells for binding to E-selectin. In vivo, Th17 cell recruitment into the air pouch was reduced in CD43(-/-) mice in response to CCL20 or TNF-α, and intravital microscopy studies demonstrated that CD43(-/-) Th17 cells had impaired rolling on TNF-α-treated microvessels. Furthermore, CD43(-/-) mice were protected from experimental autoimmune encephalomyelitis and had impaired recruitment of Th17 cells in the spinal cord. Our findings demonstrate that CD43 is a major E-selectin ligand in Th17 cells that functions independent of PSGL-1, and they suggest that CD43 may hold promise as a therapeutic target to modulate Th17 cell recruitment.

BACKGROUND

We aimed to investigate the impact of interleukin (IL)-17 on ventricular remodeling and the genesis of ventricular arrhythmia (VA) in an ischemic heart failure (HF) model. The expression of the proinflammatory cytokine IL-17 is upregulated during myocardial ischemia and plays a fundamental role in post-infarct inflammation. However, the influence of IL-17 on the genesis of VA has not yet been studied.

RESULTS

The level of inflammation and Th17 cell (CD4+IL-17+) expression in the rabbit model of ischemic HF were studied by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). The effect of IL-17 on VA induction following acute and chronic administration of IL-17 was determined using electrophysiological techniques and optical mapping. The expression of IL-17 target genes and related cytokines and chemokines in vivo and in vitro were measured using qPCR, ELISA, and immunoblotting. Th17 cells were markedly increased in the ischemic HF rabbit model. IL-17 directly induced VA in vivo and in vitro in a dose-dependent manner. IL-17 decreased conduction velocity, lengthened action potential duration, and increased the slope of the left ventricle (LV) restitution curve. IL-17 treatment led to fibrosis, collagen production and apoptosis in the LV. Furthermore, increased IL-17 signaling activated mitogen-activated protein kinase and increased the expression of downstream target genes, IL-6, TNF, CCL20, and CXCL1. An anti-IL-17 neutralizing antibody abolished the effects of IL-17.

CONCLUSIONS

The expression of IL-17 and its downstream target genes may play fundamental roles in inducing VA in ischemic HF.

Activated TCD4(+) cells are detected in human atherosclerotic plaques which indicate their participation in disease progression and destabilization. Among these cells, IFN-γ-producing T cells (TH1) are recognized as having a pro-atherogenic role. Recently, the IL-17-producing T helper lineage of cells (TH17) has been identified in atherosclerotic lesions. They have been linked to atheroma development through the production of pro-inflammatory mediators present in these lesions. Furthermore, IL-22 producing TCD4(+) cells (TH22) have been identified in the atheromatous environment, but their presence and function has not been investigated. The aim of this study was to analyze the immune response mediated by pro-inflammatory subtypes of TCD4(+) cells in atheromatous lesions. Atherosclerotic plaques of 57 patients with critical stenosis of carotid submitted to endarterectomy were evaluated. Three carotid fragments from organ donors were used as control. mRNA analysis showed expression of TH1 (IFN-γ, T-bet, IL-2, IL-12p35, TNF-α and IL-18); TH2 (GATA-3); TH17 (IL-17A, IL-17RA, Rorγt, TGF-β, IL-6, IL-1β, IL-23p19, CCL20, CCR4 and CCR6) and TH22 (IL-22 and Ahr) related markers. Asymptomatic patients showed higher expression of mRNA of IL-10, TGF-β, CCR4 and GATA-3 when compared to symptomatic ones. Immunohistochemistry analysis showed higher levels of IL-23, TGF-β, IL-1β and IL-18 in macrophages and foam cells in unstable lesions compared to stable and control ones. In vitro stimulation of atheroma cells induced IL-17 and IFN-γ production. Finally we were able to detect, the following subpopulations of TCD3(+) cells: TCD4(+) IFN-γ(+), TCD4(+)IL-17(+), TCD4(+)IL-4(+), TCD4(+)IL-22(+) and double positive cells (IFN-γ/IL-17(+), IFN-γ/IL-22(+) or IL-17/IL-22(+)). Our results showed the presence of distinct TCD4(+) cells subsets in human carotid lesions and suggest that interactions among them may contribute to the atheroma progression and destabilization.

γδ T cells continuously survey extralymphoid tissues, providing key effector functions during infection and inflammation. Despite their importance, the function and the molecules that drive migration of skin-recirculating γδ T cells are poorly described. Here we found that γδ T cells traveling in the skin-draining afferent lymph of sheep are effectors that produce IFN-γ or IL-17 and express high levels of the skin- and inflammation-seeking molecule E-selectin ligand. Consistent with a role for chemokine receptor CCR7 in mediating T cell exit from extralymphoid tissues, conventional CD4 and CD8T cells in skin-draining lymph were enriched in their expression of CCR7 compared to their skin-residing counterparts. In contrast, co-isolated γδ T cells in skin or lymph lacked expression of CCR7, indicating that they use alternative receptors for egress. Skin-draining γδ T cells were unresponsive to many cutaneous and inflammatory chemokines, including ligands for CCR2, CCR4, CCR5, CCR8, CCR10, and CXCR3, but showed selective chemotaxis toward the cutaneously expressed CCR6 ligand CCL20. Moreover, IL-17(+) γδ T cells were the most CCL20-responsive subset of γδ T cells. The data suggest that γδ T cells survey the skin and sites of inflammation and infection, entering via CCR6 and E-selectin ligand and leaving independent of the CCR7-CCL21 axis.

Traumatic brain injury (TBI) is complex and involves multiple processes that contribute to functional decline. Progressive neuropathies result from delayed cellular death following the initial impact. Although the precise mechanisms responsible for delayed injury are unknown, numerous data implicate a role for the peripheral immune system in perpetuating neuroinflammation after TBI. A previous report demonstrated that splenic CCL20 chemokine expression was upregulated 24 h after lateral fluid percussive impact (LFPI), prior to neuronal expression but consistent with neurodegeneration. Here, we expand on those data to report increased CCL20 protein expression in white matter 48 h after LFPI and demonstrate that CCL20 is directly toxic to primary neurons and oligodendrocytes subjected to oxygen glucose deprivation. The temporal expression profile of CCL20, coupled with in vitro toxicity to primary cells, suggests that this chemokine exerts deleterious effects on cell viability following TBI. These findings warrant further investigations into the use of CCL20 as a potential biomarker and/or therapeutic target.

We previously found that CCL20 induced primarily cultured healthy breast cell proliferation and migration. The objective of this study was to investigate the hypothesis that CCL20 modulated the epithelial-mesenchymal transition (EMT) of primarily cultured healthy breast epithelial cells and the angiogenesis in areas adjacent to the tumor. Key results showed that CCL20 (a) down-regulated E-cadherin and ZO-1; (b) up-regulated N-cadherin, vimentin, and Snail expressions; (c) increased mRNA and secretion of VEGF and (d) increased angiogenic micro vessel sprouting. Thus, the signal transduction pathways evoked by CCL20 were investigated. We showed that NF-kB p65 down-regulation (by small interfering RNA, siRNA) reversed CCL20-induced Snail and blocked the up-regulation of vimentin and N-cadherin mRNAs. Furthermore, PI3K/AKT inhibition (by LY294002) completely blocked CCL20-induced Snail and NF-kB activation. Inhibition of JNK1/2 (by SP60125) or PKC-α (by siRNA) or src (by PP1) blocked NF-kB activation and Snail expression suggesting that these kinases are all upstream of NF-kB/Snail. Inhibition of mTOR (by rapamycin) abolished the effects of CCL20 on N-cadherin and vimentin protein synthesis. Furthermore, siRNA of PKC-δ inhibited the phosphorylation of CCL20-induced mTOR and S6, increased vimentin and N-cadherin expressions and, finally, blocked the CCL20 induced-EMT. CCL20 increased mRNA and secretion of VEGF by healthy breast cells by using PKC-α, src, Akt, NF-kB, and Snail signalling. In summary, tumor cells signal to the surrounding healthy cells through CCL20 inducing the modulation of the expression of molecules involved in EMT and promoting angiogenesis directly and indirectly through the secretion of VEGF, a major contributor to angiogenesis. © 2015 Wiley Periodicals, Inc.

We have recently demonstrated that polymorphonuclear neutrophils (PMN), when cultured with LPS or TNF-alpha, have the capacity to release CCL20, a chemokine primarily chemotactic for immature dendritic cells and specific lymphocyte subsets. Here, we report that the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), as well as the immunoregulatory cytokine IFN-gamma, can significantly up-modulate the production of neutrophil-derived CCL20 through entirely unrelated mechanisms. We found that fMLP dramatically up-regulates CCL20 mRNA expression and synthesis in neutrophils stimulated with LPS for 2-3 h, and that its effect takes place through CCL20 mRNA stabilization. In contrast, IFN-gamma potentiates CCL20 gene expression and production only after 21 h of LPS treatment, its effect being mediated by endogenous TNF-alpha in an autocrine fashion, as revealed using neutralizing anti-TNF-alpha antibodies added to IFN-gamma plus LPS-treated PMN. Finally, we demonstrate that activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in mediating the production of CCL20 induced by LPS (with or without IFN-gamma), whereas activation of p42/44 extracellular signal-regulated kinases (ERK) is involved in the enhancing effect of fMLP. Taken together, these findings identify novel biological actions exerted by fMLP and IFN-gamma, potentially involved in the orchestration of inflammatory and immune responses within epithelial and mucosal tissue.

Large population-based registry studies have shown that breast cancer prognosis is inherited. Here we analyse single-nucleotide polymorphisms (SNPs) of genes implicated in human immunology and inflammation as candidates for prognostic markers of breast cancer survival involving 1,804 oestrogen receptor (ER)-negative patients treated with chemotherapy (279 events) from 14 European studies in a prior large-scale genotyping experiment, which is part of the Collaborative Oncological Gene-environment Study (COGS) initiative. We carry out replication using Asian COGS samples (n=522, 53 events) and the Prospective Study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) study (n=315, 108 events). Rs4458204_A near CCL20 (2q36.3) is found to be associated with breast cancer-specific death at a genome-wide significant level (n=2,641, 440 events, combined allelic hazard ratio (HR)=1.81 (1.49-2.19); P for trend=1.90 × 10(-9)). Such survival-associated variants can represent ideal targets for tailored therapeutics, and may also enhance our current prognostic prediction capabilities.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed β-galactomannan (βGM) prebiotic compared to the proven probiotic Saccharomyces cerevisiae var. boulardii on porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) cocultured in vitro with Salmonella. We observed that both S. cerevisiae var. boulardii and βGM inhibited the association of Salmonella with IECs in vitro. Our data indicated that βGM has a higher ability than S. cerevisiae var. boulardii to inhibit Salmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, βGM and S. cerevisiae var. boulardii induced some effects on DCs that were not observed on IECs: βGM and S. cerevisiae var. boulardii showed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of βGM or S. cerevisiae var. boulardii on DCs cocultured with Salmonella showed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control with Salmonella. In conclusion, the addition of βGM inhibits Salmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.

OBJECTIVE

To compare levels of the chemokine CCL20 and its receptor CCR6 in donor, osteoarthritic (OA), and rheumatoid arthritis (RA) synovium; and to determine the molecular mechanism of cellular activation induced by chemokine/receptor ligation in human fibroblast-like synoviocytes (FLS).

METHODS

Synovia and isolated FLS from donor, OA, and RA joints were analyzed for CCL20 and CCR6 expression by RT-PCR and immunohistochemistry. The effect of CCL20 on cytokines and mediators of cartilage degradation was examined by PCR for mRNA expression levels and ELISA, and Western blotting for protein. CCL20-dependent transcriptional and posttranscriptional activation of target genes was monitored using reporter constructs and luciferase assays in transfected donor FLS.

RESULTS

CCL20 and CCR6 proteins were abundantly expressed in RA synovial lining cells compared to donor or OA synovia as judged by immunohistochemistry. RT-PCR of synovial extracts confirmed the predominance of CCL20/CCR6 mRNA expression in RA synovium. CCL20 mRNA expression was low in donor FLS, but increased dramatically after stimulation with recombinant human (rh) interleukin 1ß (IL-1ß). rhCCL20 increased mRNA and protein expression of COX-2, IL-1ß, tumor necrosis factor-α, IL-6, and the matrix-destructive metalloprotease MMP-3 in donor FLS cultures. High constitutive levels of IL-6 were released from RA synovia; CCL20-induced expression of IL-6 occurred through an NSAID/COXIB-sensitive process. CCL20-induced expression of COX-2 was mediated by a PLCP1/PKCα/MEK1/2/ERK1/2-dependent pathway involving both transcriptional and posttranscriptional mechanisms.

CONCLUSIONS

CCL20/CCR6 may play an important role in the pathogenesis of RA by assembling the molecular and cellular components orchestrating synovitis.

BACKGROUND

Genetic and environmental factors influence susceptibility to Crohn's disease (CD): NOD2 is the strongest individual genetic determinant and smoking the best-characterised environmental factor. Carriage of NOD2 mutations predispose to small-intestinal, stricturing CD, a phenotype also associated with smoking. We hypothesised that cigarette smoke extract (CSE) altered NOD2 expression and function in intestinal epithelial cells.

RESULTS

Intestinal epithelial cell-lines (SW480, HT29, HCT116) were stimulated with CSE and nicotine (to mimic smoking) ±TNFα (to mimic inflammation). NOD2 expression was measured by qRT-PCR and western blotting; NOD2-RIPK2 interactions by co-immunoprecipitation (CoIP); nuclear NFκB-p65 by ELISA; NFκB activity by luciferase reporter assays and chemokines (CCL20, IL8) in culture supernatants by ELISA. In SW480 and HT29 cells the TNFα-induced NOD2 expression at 4 hours was reduced by CSE (p = 0.0226), a response that was dose-dependent (p = 0.003) and time-dependent (p = 0.0004). Similar effects of CSE on NOD2 expression were seen in cultured ileal biopsies from healthy individuals. In SW480 cells CSE reduced TNFα-induced NFκB-p65 translocation at 15 minutes post-stimulation, upstream of NOD2. Levels of the NOD2-RIPK2 complex were no different at 8 hours post-stimulation with combinations of CSE, nicotine and TNFα, but at 18 hours it was increased in cells stimulated with TNFα+CSE but decreased with TNFα alone (p = 0.0330); CSE reduced TNFα-induced NFκB activity (p = 0.0014) at the same time-point. At 24 hours, basal CCL20 and IL8 (p<0.001 for both) and TNFα-induced CCL20 (p = 0.0330) production were decreased by CSE. CSE also reduced NOD2 expression, CCL20 and IL8 production seen with MDP-stimulation of SW480 cells pre-treated with combinations of TNFα and CSE.

CONCLUSIONS

CSE delayed TNFα-induced NOD2 mRNA expression and was associated with abnormal NOD2/RIPK2 interaction, reduced NFκB activity and decreased chemokine production. These effects may be involved in the pathogenesis of small-intestinal CD and may have wider implications for the effects of smoking in NOD2-mediated responses.

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.

CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1β. We have determined that IL-1β-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-β. TGF-β is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin αvβ8 (itgb8). Here we confirm correlative increases in αvβ8 and IL-1β with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1β- and αvβ8-mediated TGF-β activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5'-flanking region of CCL20 to determine that IL-1β-driven CCL20 expression is dependent on αvβ8-mediated activation of TGF-β. We identify a TGF-β-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1β-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-κB on a NF-κB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which αvβ8-dependent activation of TGF-β regulates IL-1β-dependent CCL20 expression in COPD.

Connective tissue growth factor (CTGF), also known as CCN2, is a key proinflammatory mediator. In the present study, the involvement of the CTGF signaling pathway in human knee osteoarthritis (OA) fibroblast-like synoviocytes (FLSs) was investigated. FLSs were isolated from human OA synovium and incubated with CTGF in the absence or presence of interleukin‑1β (IL‑1β). The expression of relevant genes and proteins was analyzed by qPCR, western blotting and enzyme-linked immunosorbent assay (ELISA). Matrix metalloproteinase (MMP) activity and nuclear factor (NF)-κB activation were also evaluated. CTGF stimulation resulted in the significant production of IL-6, IL-8, C-C motif ligand 2 (CCL2), CCL20, MMP-1 and MMP-3 in FLSs in the presence, but not in the absence, of IL-1β. CTGF also enhanced the levels of phosphorylated extracellular signal-related kinase 1/2 (ERK1/2) and p38. In addition, CTGF at 25 ng/ml, in the presence of IL‑1β, significantly potentiated NF-κB activation. The results indicated that CTGF interacted with IL‑1β in FLSs to promote the inflammatory response in the synovium, leading to the initiation of the inflammatory cascade. These results support the proinflammatory role of CTGF in synovitis and joint destruction in OA.

BACKGROUND

Nonalcoholic fatty liver disease (NAFLD) is a prevalent complication of extreme obesity. Loading of the liver with fat can progress to inflammation and fibrosis including cirrhosis. The molecular factors involved in the progression from simple steatosis to fibrosis remain poorly understood.

METHODS

Gene expression profiling using microarray, PCR array, and RNA sequencing was performed on RNA from liver biopsy tissue from patients with extreme obesity. Patients were grouped based on histological findings including normal liver histology with no steatosis, lobular inflammation, or fibrosis, and grades 1, 2, 3, and 4 fibrosis with coexistent steatosis and lobular inflammation. Validation of expression was conducted using quantitative PCR. Serum analysis was performed using ELISA. Expression analysis of hepatocytes and hepatic stellate cells in response to lipid loading were conducted in vitro using quantitative PCR and ELISA.

RESULTS

Three orthogonal methods to profile human liver biopsy RNA each identified the chemokine CCL20 (CC chemokine ligand 20 or MIP-3 alpha) gene as one of the most up-regulated transcripts in NAFLD fibrosis relative to normal histology, validated in a replication group. CCL20 protein levels in serum measured in 224 NAFLD patients were increased in severe fibrosis (p < 0.001), with moderate correlation of hepatic transcript levels and serum levels. Expression of CCL20, but not its cognate receptor CC chemokine receptor 6, was significantly (p < 0.001) increased in response to fatty acid loading in LX-2 hepatic stellate cells, with relative increases greater than those in HepG2 hepatocyte cells.

CONCLUSIONS

These results suggest that expression of CCL20, an important inflammatory mediator, is increased in NAFLD fibrosis. CCL20 serves as a chemoattractant molecule for immature dendritic cells, which have been shown to produce many of the inflammatory molecules that mediate liver fibrosis. These data also point to hepatic stellate cells as a key cell type that may respond to lipid loading of the liver.

Two approaches have been pursued to elicit antitumour immunity: (i) induce recruitment of immature dendritic cells or their precursors at a site of antigen delivery, and (ii) induce activation of tumour-infiltrating dendritic cells (DCs). The recruitment of selected DC subtype conditions the class of the immune response. Each immature DC population displays a unique spectrum of chemokine responsiveness. For examples, Langerhans cells (LCs) migrate selectively in response to CCL20/MIP-3alpha (through CCR6), blood CD11c+ DC to MCP chemokines (through CCR2). All these chemokines are inducible in response to inflammatory stimuli. CCL20/MIP-3alpha in particular is only detected within inflamed epithelium, at the site of antigen entry, which is infiltrated by immature DCs. Furthermore, to reach the site of injury, sequential responsiveness might operate, blood DC precursors are recruited by a set of chemokines (MIP, MCP) while within the tissue other chemokines will direct their navigation (CCL20/MIP-3alpha). Of interest, when injected in vivo together with antigen, MCP-4/CCL13, but not CCL20/MIP-3alpha, recruits blood monocytes or blood DC precursors that promptly differentiate into typical DCs and that improve antitumour immune responses. After antigen uptake, DCs acquire, upon maturation, responsiveness to CCR7 ligands (CCL21/SLC/6Ckine, CCL19/ELC/MIP-3beta) due to receptor up-regulation. In particular, in the periphery, CCL21/SLC/6Ckine expressed by lymphatic vessels may direct into the lymph stream, antigen-loaded maturing DCs leaving the site of infection; while within lymph-node, CCL21/SLC/6Ckine plays a critical role in the entry of naïve T cells from the blood through HEV. In regard to its central role, we decided to investigate whether the expression of CCL21/SLC/6Ckine in tumour may lead to antitumour immune responses. C26 colon carcinoma tumour cell line transduced with CCL21/SLC/6Ckine showed reduced tumorigenicity when injected in vivo into immunocompetent mice. The protection was CD8 dependent and associated with an important intratumoral infiltration of DCs. Most tumour infiltrating DCs (TIDCs) had an immature phenotype, were able to present TAA in the context of MHC class I, but were refractory to stimulation with the combination of LPS, IFNgamma and anti-CD40 antibody. TIDC paralysis could be reverted, however, by in vitro or in vivo stimulation with the combination of a CpG immunostimulatory sequence and an anti-interleukin 10 receptor (IL10R) antibody. CpG or anti-IL10R alone were inactive in TIDC, while CpG triggered activation in normal DC. In particular, CpG plus anti-IL10R enhanced the TAA-specific immune response and triggered de novo IL-12 production. Subsequently, CpG plus anti-IL10R treatment showed robust antitumour therapeutic activity exceeding by far that of CpG alone, and elicited antitumour immune memory.

In the present study, monocyte-derived human macrophages were differentiated from buffy coats. Naïve CD4⁺ T-cells enriched from peripheral blood mononuclear cells using anti-CD4 magnetic beads and the autoMACS separation system were polarized under T-helper 17 (Th17)-promoting conditions for 6 days to get Th17 cells. The frequency of Th17 cell differentiation and the expression of C-C chemokine receptor type 6 (CCR6) on Th17 cells were investigated by flow cytometry. Plasmin-triggered induction of macrophage inflammatory protein-3alpha/C-C chemokine ligand 20 (CCL20) genes in macrophages was assessed by reverse transcription-polymerase chain reaction, and secreted protein levels were measured by enzyme-linked immunosorbent assay. Th17 cell migration induced by CCL20 secreted from plasmin-stimulated macrophages was tested in vitro by chemotaxis using a transwell system. These results demonstrate that plasmin triggers the expression of chemokine CCL20 messenger RNA and the release of CCL20 protein in human monocyte-derived macrophages, which critically depend on the proteolytic activity of plasmin and activation of p38 mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways. Expression of CCR6 was detected on 87.23 ± 8.6% of Th17 cells in vitro. Similar to chemotaxis triggered by recombinant human CCL20, supernatants collected from plasmin-stimulated macrophage-induced chemotactic migration of Th17 cells, which could be inhibited by an anti-CCL20 neutralizing antibody. These results suggest that plasmin generated in inflamed tissues might elicit production of chemokine CCL20 by human macrophages leading to the recruitment of CCR6 positive Th17 cells to the inflammatory sites.

Chemokines are key mediators of leukocyte recruitment during immunoregulatory and proinflammatory responses. CCL20 is a cysteine-cysteine chemokine that was originally shown to be chemotactic for immature dendritic cells, effector or memory CD4(+) T lymphocytes, and B lymphocytes. Additionally, CCL20 and its only receptor (CCR6) are exploited by cancer cells for migration and metastatic spread and play important roles in the development and progression of cancer. However, it still remains unclear how the activity of the CCL20/CCR6 axis is controlled and regulated at the transcriptional level. The CCL20 promoter region contains a transcription start site, a nuclear factor (NF)-κB binding site, a CCAAT/enhancer-binding proteins binding site, an activator protein-1 binding sites, and a specificity protein 1 (Sp1)-binding site. In this review, we outline recent advances in our understanding of the structure of the CCL20 promoter region and discuss the transcriptional regulation of the CCL20 promoter.

T helper (Th) 17 cells were reported to have the property of proinflammation and profibrosis. We first investigate the levels of Th17 cells in primary biliary cirrhosis (PBC) patients, and then explore their distribution and fibrotic role in the disease.We compared the circulating Th17 and hepatic interleukin (IL)-17-positive cells between patients and healthy controls (HCs) at different disease stages by flow cytometry and immunohistochemistry, respectively. The levels of chemokine (c-c motif) ligand (CCL) 20 were then measured. For exploration of the reason why Th17 cells increased, CD4CD161 populations were sorted and cultured with IL-23 and IL-1β to analyze their proliferation and IL-17 secretions. The serum IL-23 and IL-1β were tested by enzyme-linked immunosorbent assay. The proliferation and expressions of α-smooth muscle actin and IL-8 of hepatic stellate cells (HSCs) were identified after stimulated by different concentrations of IL-17.Circulating and hepatic Th17 cells were elevated in PBC patients compared with HCs. Early PBC patients presented with more Th17 cells in periphery blood and less in the liver than advanced PBC patients. Accordingly, the levels of both serum and hepatic CCL20 for Th17 cells were higher, especially in those with advanced disease. The progenitor of Th17, CD4CD161 cell was increased in PBC. Moreover, the percentage of Th17 cells was positively related with CD4CD161 cell. After stimulation with IL-23 and IL-1β which were improved in PBC patients, CD4CD161 cells from PBC patients expressed more IL-17, although their proliferation were not different between 2 groups. IL-17 can promote the proliferation of HSCs at a dose-dependent method, and also increase the IL-8 expression in a dose/time-dependent way. Anti-IL-17 can neutralize the above reactions.CD4CD161 cells are a source of increased Th17 in PBC patients. With disease progression, Th17 population decreased in the circulation, accompanied by greater accumulation in the liver, which is regulated by CCL20 in advanced patients. IL-17 may be involved in the process of PBC fibrosis.

Interleukin-17A (IL-17A) is a newly emerging player in the pathogenesis of chronic lung diseases that amplifies inflammatory responses and promotes tissue remodeling. Stimulation of lung epithelial cells with IL-17A leads to activation of the transcription factor nuclear factor κB (NF-κB), a key player in the orchestration of lung inflammation. We have previously demonstrated the importance of the redox-dependent posttranslational modification S-glutathionylation in limiting activation of NF-κB and downstream gene induction. Under physiological conditions, the enzyme glutaredoxin 1 (Grx1) acts to deglutathionylate NF-κB proteins, which restores functional activity. In this study, we sought to determine the impact of S-glutathionylation on IL-17A-induced NF-κB activation and expression of proinflammatory mediators. C10 mouse lung alveolar epithelial cells or primary mouse tracheal epithelial cells exposed to IL-17A show rapid activation of NF-κB and the induction of proinflammatory genes. Upon IL-17A exposure, sulfenic acid formation and S-glutathionylated proteins increased. Assessment of S-glutathionylation of NF-κB pathway components revealed S-glutathionylation of RelA (RelA-SSG) and inhibitory κB kinase α (IKKα-SSG) after stimulation with IL-17A. SiRNA-mediated ablation of Grx1 increased both RelA-SSG and IKKα-SSG and acutely increased nuclear content of RelA and tended to decrease nuclear RelB. SiRNA-mediated ablation or genetic ablation of Glrx1 decreased the expression of the NF-κB-regulated genes KC and CCL20 in response to IL-17A, but conversely increased the expression of IL-6. Last, siRNA-mediated ablation of IKKα attenuated nuclear RelA and RelB content and decreased expression of KC and CCL20 in response to IL-17A. Together, these data demonstrate a critical role for the S-glutathionylation/Grx1 redox axis in regulating IKKα and RelA S-glutathionylation and the responsiveness of epithelial cells to IL-17A.

Chlamydia trachomatis is an obligate intracellular epitheliotropic bacterial pathogen of humans. Infection of the eye can result in trachoma, the leading cause of preventable blindness in the world. The pathophysiology of blinding trachoma is driven by multiple episodes of reinfection of conjunctival epithelial cells, producing an intense chronic inflammatory response resulting in submucosal tissue remodeling and scarring. Recent reports have shown that infection with trachoma organisms lacking the cryptic chlamydial plasmid is highly attenuated in macaque eyes, a relevant experimental model of human trachoma infection. To better understand the molecular basis of plasmid-mediated infection attenuation and the potential modulation of host immunity, we conducted transcriptional profiling of human epithelial cells infected with C. trachomatis plasmid-bearing (A2497) and plasmid-deficient (A2497P(-)) organisms. Infection of human epithelial cells with either strain increased the expression of host genes coding for proinflammatory (granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage colony-stimulating factor [MCSF], interleukin-6 [IL-6], IL-8, IL-1α, CXCL1, CXCL2, CXCL3, intercellular adhesion molecule 1 [ICAM1]), chemoattraction (CCL20, CCL5, CXCL10), immune suppression (PD-L1, NFKB1B, TNFAIP3, CGB), apoptosis (CASP9, FAS, IL-24), and cell growth and fibrosis (EGR1 and IL-20) proteins. Statistically significant increases in the levels of expression of many of these genes were found in A2497-infected cells compared to the levels of expression in A2497P(-)-infected cells. Our findings suggest that the chlamydial plasmid plays a focal role in the host cell inflammatory response to infection and immune avoidance. These results provide new insights into the role of the chlamydial plasmid as a chlamydial virulence factor and its contributions to trachoma pathogenesis.

BACKGROUND

T(H)17 responses have recently been implicated to play a role in allergic airway diseases, but their local expression in the setting of allergic rhinitis (AR) and their regulation in allergic airway diseases remain unclear.

OBJECTIVE

We sought to investigate the regulatory role of Clara cell 10-kDa protein (CC10), an endogenous regulator of airway inflammation, on T(H)17 responses in the setting of AR.

METHODS

Wild-type and homozygous CC10-null mice were used to establish an ovalbumin (OVA)-induced AR model. Human recombinant CC10 was given during sensitization or challenge. T(H)17 responses in human subjects and mice were examined by using flow cytometry, quantitative RT-PCR assay, immunohistochemistry, and ELISA. The direct effect of CC10 on T(H)17 cells and CD11c(+) dendritic cells (DCs) was studied by means of cell culture. Adoptive transfer was used to examine the influence of CC10-conditioned DCs on airway inflammation. The regulatory effect of CC10 on the expression of the CCL20 gene was tested by using the BEAS-2B cell line.

RESULTS

Compared with those of control subjects, T(H)17 responses were enhanced in the nasal mucosa of patients with AR. CC10-null mice with AR showed enhanced T(H)17 responses, and CC10 treatment significantly decreased T(H)17 responses. CC10 had no direct effect on in vitro T(H)17 cell differentiation. CC10 could significantly decrease the expression of OX40 ligand, IL-23, and IL-6 but enhance CD86 and TGF-β expression in DCs. Importantly, CC10 was able to inhibit T(H)17 cell polarization in the presence of OVA-pulsed DCs. CC10 pretreatment inhibited T(H)17 responses elicited by adoptive transfer of OVA-pulsed DCs. Furthermore, CC10 decreased the expression of CCL20 in BEAS-2B cells induced by inflammatory cytokines.

CONCLUSIONS

T(H)17 responses are enhanced in patients with AR, and CC10 inhibits T(H)17 responses through modulation of the function of DCs.

The purpose of this study was to investigate whether baicalin, a Chinese herbal extract, down-regulates the expression of macrophage migration inhibitory factor (MIF), an inflammatory factor that regulates the function of macrophages (MΦ), in rats with trinitrobenzene sulphonic acid (TNBS)-induced ulcerative colitis (UC). The results showed that baicalin simultaneously down-regulated the expression of MIF, the quantity of MΦs and the amount of MΦ-related cytokines, including macrophage chemotactic factor-1 (MCP-1, CCL2) and macrophage inflammatory protein-3α (MIP-3α, CCL20), in rats with UC. There was no statistical difference between baicailin and mesalazine in down-regulating the expression of MIF. Our study demonstrated that baicalin, an inexpensive but effective monomer, could be a new and alternative pharmaceutical for UC.