Chemokine/chemokine receptor interactions play a critical role in lymphocyte infiltration of tumors. Recent studies suggest that Th17 cells accumulate within many types of tumors, although the mechanisms that control this are unclear. We studied the distribution and phenotypic features of Th17 cells chemokine receptors, as well as the mRNA levels of CCL2, CCL17, CCL20, and CCL22 in tumors of patients with esophageal squamous cell carcinoma. We found that Th17 cells accumulated in tumors, and high expressions of CCR4, CCR6 were detected in Th17 cells. Levels of the chemokines CCL17, CCL20, and CCL22 in tumors were significantly higher than in tumor-free tissues, and were positively correlated with the distribution of Th17 cells in tumors. Furthermore, an in vitro migration assay showed that CCL17, CCL20 and CCL22 had chemotactic effects on tumor-derived Th17 cells. In conclusion, the CCR4-CCL17/22 and CCR6-CCL20 axis might play an important role in Th17 cell infiltration of tumors.

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

OBJECTIVE

To explore the effects of osmoprotectants on pro-inflammatory mediator production in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.

METHODS

HCECs cultured in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with 2-20 mM of l-carnitine, erythritol or betaine for different time periods. The mRNA expression and protein production of pro-inflammatory markers in HCECs were evaluated by RT-qPCR and ELISA.

RESULTS

Hyperosmolar media significantly stimulated the mRNA and protein expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokines, IL-8, CCL2 and CCL20 in HCECs in an osmolarity dependent manner. The stimulated expression of these pro-inflammatory mediators was significantly but differentially suppressed by l-carnitine, erythritol or betaine. l-Carnitine displayed the greatest inhibitory effects and down-regulated 54-77% of the stimulated mRNA levels of TNF-α (down from 12.3-5.7 fold), IL-1β (2.2-0.9 fold), IL-6 (7.3-2.9 fold), IL-8 (4.6-2.0 fold), CCL2 (15.3-3.5 fold) and CCL20 (4.1-1.5 fold) in HCECs exposed to 450 mOsM. The stimulated protein production of TNF-α, IL-1β, IL-6 and IL-8 was also significantly suppressed by l-carnitine, erythritol and betaine. l-carnitine suppressed 49-79% of the stimulated protein levels of TNF-α (down from 81.3 to 17.4 pg/ml), IL-1β (56.9-29.2 pg/ml), IL-6 (12.8-4.6 ng/ml) and IL-8 (21.2-10.9 ng/ml) by HCECs exposed to 450 mOsM. Interestingly, hyperosmolarity stimulated increase in mRNA and protein levels of TNF-α, IL-1β and IL-6 were significantly suppressed by a transient receptor potential vanilloid channel type 1 (TRPV1) activation inhibitor capsazepine.

CONCLUSIONS

l-carnitine, erythritol and betaine function as osmoprotectants to suppress inflammatory responses via TRPV1 pathway in HCECs exposed to hyperosmotic stress. Osmoprotectants may have efficacy in reducing innate inflammation in dry eye disease.

BACKGROUND

Recently, some strains of lactic acid bacteria (LAB) have been reported to prevent the development of atopic dermatitis and to improve allergic symptoms, especially in young children. However, the mechanisms involved in these effects are not fully understood. Intestinal microbiota play critical roles in the development of host immune development and are recognized and regulated by the host through intestinal epithelial cells (IECs). We thus hypothesized that LAB influence the host immune system through the activation of IECs. To begin testing this hypothesis, chemokine expression in IECs exposed to intestinal bacteria was investigated.

METHODS

Caco-2 cell monolayers were stimulated with different concentrations of various live or heat-killed intestinal bacteria or bacterial components for up to 3 h. Changes in the gene expressions of various chemokines were measured using quantitative real-time PCR.

RESULTS

The expressions of CCL20, CXCL8, CXCL10 and CX3CL1 were strongly induced by nonpathogenic Escherichia coli in a dose-dependent manner and were partially induced by some commensal LAB. In contrast, Lactobacillus rhamnosus GG (LGG) and Lactobacillus casei did not induce these chemokine expressions. In addition, LGG significantly suppressed the expressions of CCL20 and CXCL10 induced by E. coli, peptidoglycan or flagellin when cultured simultaneously.

CONCLUSIONS

LGG and L. casei markedly suppressed E. coli-induced chemokine expression, presumably through the suppression of the Toll-like receptor-mediated signal transduction pathway, at least in part. The clinical importance of this suppressive effect and the mechanisms involved require further investigation; however, such effects can be used as a marker to identify clinically useful LAB.

The maintenance of normal tissue homeostasis and the prevention of chronic inflammatory disease are dependent on the active process of inflammation resolution. In endothelial cells (ECs), proinflammation results from the activation of intracellular signaling responses and/or the inhibition of endogenous regulatory/pro-resolution signaling networks that, to date, are poorly defined. In this study, we find that DEP domain containing mTOR interacting protein (DEPTOR) is expressed in different microvascular ECs in vitro and in vivo, and using a small interfering RNA (siRNA) knockdown approach, we find that it regulates mammalian target of rapamycin complex 1 (mTORC1), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription 1 activation in part through independent mechanisms. Moreover, using limited gene arrays, we observed that DEPTOR regulates EC activation including mRNA expression of the T-cell chemoattractant chemokines CXCL9, CXCL10, CXCL11, CX3CL1, CCL5, and CCL20 and the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (P < .05). DEPTOR siRNA-transfected ECs also bound increased numbers of peripheral blood mononuclear cells (P < .005) and CD3+ T cells (P < .005) in adhesion assays in vitro and had increased migration and angiogenic responses in spheroid sprouting (P < .01) and wound healing (P < .01) assays. Collectively, these findings define DEPTOR as a critical upstream regulator of EC activation responses and suggest that it plays an important role in endogenous mechanisms of anti-inflammation and pro-resolution.

Environmental particulate matter (PM) exposure has been correlated with pathogenesis of acute airway inflammatory disease such as asthma and COPD. PM size and concentration have been studied extensively, but the additional effects of particulate components such as biological material, transition metals, and polycyclic aromatic hydrocarbons could also impact initial disease pathogenesis. In this study, we compared urban ambient particulate matter (APM) collected from Fresno, California with wildfire (WF) particulate matter collected from Escalon, California on early transcriptional responses in human bronchial epithelial cells (HBE). Global gene expression profiling of APM treated HBE activated genes related to xenobiotic metabolism (CYP 1B1), endogenous ROS generation and response genes (DUOX1, SOD2, PTGS2) and pro-inflammatory responses associated with asthma or COPD such as IL-1α, IL-1β, IL-8, and CCL20. WF PM treatments also induced a pro-inflammatory gene response, but elicited a more robust xenobiotic metabolism and oxidative stress response. Inhibitor studies targeting endotoxin, ROS, and trace metals, found endotoxin inhibition had modest selective inhibition of inflammation while inhibition of hydrogen peroxide and transition metals had broad effects suggesting additional interactions with xenobiotic metabolism pathways. APM induced a greater inflammatory response while WF PM had more marked metabolism and ROS related responses.

OBJECTIVE

Stomach cancer has a high mortality rate in East Asia, and is strongly associated with Helicobacter pylori (H. pylori) infection. H. pylori is known to express chemokine genes in the gastric mucosa, chemokines that are important host immune factors facilitating inflammation and tumor growth. To investigate the mechanism of carcinogenesis in the stomach, it is essential to determine which molecule of H. pylori is involved in induction of chemokines, but this has remained unclear. We previously reported that a tumor necrosis factor-alpha (TNF-alpha) inducing protein (Tipalpha) secreted from H. pylori acts as a tumor promoter in stomach cancer development, and thus started to investigate whether Tipalpha is involved in induction of chemokine genes.

METHODS

Comprehensive gene expression analysis was conducted using DNA microarray and KeyMolnet analyses. The gene expression was quantitatively analyzed by real-time RT-PCR.

RESULTS

Comprehensive and quantitative gene expression analyses revealed that Tipalpha induces gene expression of the chemokines Ccl2, Ccl7, Ccl20, Cxcl1, Cxcl2, Cxcl5 and Cxcl10 extensively and simultaneously in mouse stomach cancer cells, MGT-40. Tipalpha induced high levels of chemokine gene expression, whereas inactive deleted Tipalpha, del-Tipalpha, showed only marginal expression, suggesting a correlation between tumor promotion and chemokine gene expression by Tipalpha. MG-132, a proteasome inhibitor which represses NF-kappaB-activation, inhibited chemokine gene expressions.

CONCLUSIONS

We report here that Tipalpha of H. pylori gene product is a strong inducer of chemokine gene expressions, providing a new model for stomach cancer development.

Improved biomarkers are needed to facilitate clinical decision-making and as surrogate endpoints in clinical trials in multiple sclerosis (MS). We assessed whether neurodegenerative and neuroinflammatory markers in cerebrospinal fluid (CSF) at initial sampling could predict disease activity during 2 years of follow-up in patients with clinically isolated syndrome (CIS) and relapsing-remitting MS.

Using multiplex bead array and enzyme-linked immunosorbent assay, CXCL1, CXCL8, CXCL10, CXCL13, CCL20, CCL22, neurofilament light chain (NFL), neurofilament heavy chain, glial fibrillary acidic protein, chitinase-3-like-1, matrix metalloproteinase-9 and osteopontin were analysed in CSF from 41 patients with CIS or relapsing-remitting MS and 22 healthy controls. Disease activity (relapses, magnetic resonance imaging activity or disability worsening) in patients was recorded during 2 years of follow-up in this prospective longitudinal cohort study.

In a logistic regression analysis model, NFL in CSF at baseline emerged as the best predictive marker, correctly classifying 93% of patients who showed evidence of disease activity during 2 years of follow-up and 67% of patients who did not, with an overall proportion of 85% (33 of 39 patients) correctly classified. Combining NFL with either neurofilament heavy chain or osteopontin resulted in 87% overall correctly classified patients, whereas combining NFL with a chemokine did not improve results.

This study demonstrates the potential prognostic value of NFL in baseline CSF in CIS and relapsing-remitting MS and supports its use as a predictive biomarker of disease activity.

BACKGROUND

House dust mite (HDM) affects the immunological and physical barrier function of airway epithelium, leading to allergic sensitization, airway remodeling, and eosinophilic inflammation in mouse models, although the mechanisms are still largely unknown.

OBJECTIVE

Given the implications for adenosine triphosphate (ATP)-dependent Ca(2+) signaling in allergic sensitization in mice, we sought to determine the role of intracellular Ca(2+) concentration ([Ca(2+)](i)) in HDM-induced barrier dysfunction and pro-inflammatory activity of bronchial epithelium.

METHODS

We investigated the effect of HDM on accumulation of [Ca(2+)](i) levels, barrier function, and CCL20 release in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from healthy subjects and asthma patients. Involvement of ATP-dependent activation of purinergic receptors and downstream Ca(2+) influx was studied, using the ATP hydrolyzing agent apyrase, the purinergic receptor agonist PPADS, the calcium chelator BAPTA-AM, and calpain inhibitors.

RESULTS

Asthma PBECs were more susceptible to HDM-induced barrier dysfunction, CCL20 secretion, and Ca(2+) influx than healthy PBECs. Furthermore, we show that the HDM-induced increase in CCL20 in PBECs and 16HBE cells and the HDM-induced barrier dysfunction in 16HBE cells are dependent on [Ca(2+)](i) accumulation. Additionally, we demonstrate that [Ca(2+)](i) accumulation is initiated partly through the activation of purinergic receptors, which contributes to HDM-induced epithelial barrier dysfunction by disruption of cell-cell contacts, but not CCL20 secretion.

CONCLUSIONS

Our data show for the first time that Ca(2+) signaling plays a crucial role in barrier dysfunction and the pro-inflammatory response of bronchial epithelium upon HDM exposure and may thus have important implications for the development of allergic asthma.

Chemokines, which are basic proteins that exert their effects via G protein-coupled receptors and a subset of the cytokine family, are mediators deeply involved in leukocyte migration during an inflammatory reaction. Chemokine (C-C motif) ligand 20 (CCL20), also known as macrophage inflammatory protein (MIP)-3α, liver activation regulated chemokine (LARC), and Exodus-1, is a small protein that is physiologically expressed in the liver, colon, and skin, is involved in tissue inflammation and homeostasis, and has a specific receptor C-C chemokine receptor 6 (CCR6). The CCL20-CCR6 axis has long been known to be involved in inflammatory and infectious diseases, such as rheumatoid arthritis and human immunodeficiency virus infections. Recently, however, reports have shown that the CCL20-CCR6 axis is associated with several cancers, including hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, and kidney cancer. The CCL20-CCR6 axis promotes cancer progression directly by enhancing migration and proliferation of cancer cells and indirectly by remodeling the tumor microenvironment through immune cell control. The present article reviewed the role of the CCL20-CCR6 axis in cancer progression and its potential as a therapeutic target.

Keywords: CCL20-CCR6 axis; cancer progression; chemokines.

BACKGROUND

There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naive cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments.

RESULTS

We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known.

CONCLUSIONS

Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors.

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.

We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3alpha/CCL20 or Ser MIP-3alpha/CCL20. Both forms of MIP-3alpha/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3alpha/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3alpha/CCL20 and Ala MIP-3alpha/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known.

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (<em>CCL20</em>) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P<or=0.05) tissue specific effects for TLR5, TLR9, IL8, MIF and OPN. However, aside from STG stimulated increase in IL8 in MLN (approximately 10-fold increase relative to CTL; P<0.05), significant changes in other molecular targets were generally less than one-fold. Results suggest that transformed bacteria may be useful in modeling chronic oral exposure of pigs to economically important salmonellae serovars. However, although statistically significant effects of bacterial feeding were observed in selected tissues for some targets, most changes in mRNA were generally incremental in magnitude.

OBJECTIVE

Improvement of clinical diagnostics of idiopathic giant cell myocarditis (IGCM) and cardiac sarcoidosis (CS), two frequently fatal human myocardial diseases. Currently, IGCM and CS are diagnosed based on differential patterns of inflammatory cell infiltration and non-caseating granulomas in histological sections of endomyocardial biopsies (EMBs), after heart explantation or postmortem. We report on a method for improved differential diagnosis by myocardial gene expression profiling in EMBs.

RESULTS

We examined gene expression profiles in EMBs from 10 patients with histopathologically proven IGCM, 10 with CS, 18 with active myocarditis (MCA), and 80 inflammation-free control subjects by quantitative RT-QPCR. We identified distinct differential profiles that allowed a clear discrimination of tissues harbouring giant cells (IGCS, CS) from those with MCA or inflammation-free controls. The expression levels of genes coding for cytokines or chemokines (CCL20, IFNB1, IL6, IL17D; P < 0.05), cellular receptors (ADIPOR2, CCR5, CCR6, TLR4, TLR8; P < 0.05), and proteins involved in the mitochondrial energy metabolism (CPT1, CYB, DHODH; P < 0.05) were deregulated in 2- to 300-fold, respectively. Bioinformatic analyses and correlation of the gene expression data with immunohistochemical findings provided novel information regarding the differential cellular and molecular pathomechanisms in IGCM, CS, and MCA.

CONCLUSIONS

Myocardial gene expression profiling is a reliable method to predict the presence of multinuclear giant cells in the myocardium, even without a direct histological proof, in single small EMB sections, and thus to reduce the risk of sampling errors. This profiling also facilitates the discrimination between IGCM and CS, as two different clinical entities that require immediate and tailored differential therapy.

The aetiology of primary B-cell lymphomas of the thymus is enigmatic. Although thymic follicular lymphoid hyperplasia (TFH) is commonly associated with myasthenia gravis (MG), lymphoma is not a complication of this condition. The present paper reports a high frequency of monoclonal B-cell populations (6 of 18 cases; 33%) in micronodular thymoma (MNT), a peculiar thymic epithelial neoplasm with a B-cell-rich stroma, while B cells were consistently polyclonal in TFH (25 cases) and other types of thymomas (15 cases) (p < 0.001). An intratumoural lymphoma could be identified in three of the six monoclonal MNTs. Sequencing of the monoclonal IgH chain revealed partially overlapping VDJ gene usage in MNT and thymic mucosa-associated lymphoid tissue (MALT) lymphomas. The neoplastic epithelium of MNTs, but not of TFH and other types of thymoma, expressed high levels of dendritic cell, T-cell, and B-cell chemoattractants, such as CCL18, CCR6, and CCL20. It is concluded that abnormal chemokine expression in an epithelial tumour, MNT, can promote the recruitment of MALT, the emergence of monoclonal B cells, and, eventually, the subsequent development of mediastinal lymphomas. More generally, the concept that expression of a 'high-risk' spectrum of chemokines due to local or genetic factors may interfere with B-cell homeostasis and may contribute to MALT lymphoma development in chronic inflammatory states is proposed.

Chemotherapeutic resistance in triple-negative breast cancer (TNBC) has brought great challenges to the improvement of patient survival. The mechanisms of taxane chemoresistance in TNBC have not been well investigated. Our results illustrated C-C motif chemokine ligand 20 (CCL20) was significantly elevated during taxane-containing chemotherapy in breast cancer patients with nonpathologic complete response. Furthermore, CCL20 promoted the self-renewal and maintenance of breast cancer stem cells (BCSCs) or breast cancer stem-like cells through protein kinase Cζ (PKCζ) or p38 mitogen-activated protein kinase (MAPK)-mediated activation of p65 nuclear factor kappa B (NF-κB) pathway, significantly increasing the frequency and taxane resistance of BCSCs. Moreover, CCL20-promoted NF-κB activation increased ATP-binding cassette subfamily B member 1 (ABCB1)/multidrug resistance 1 (MDR1) expression, leading to the extracellular efflux of taxane. These results suggested that chemotherapy-induced CCL20 mediated chemoresistance via up-regulating ABCB1. In addition, NF-κB activation increased CCL20 expression, forming a positive feedback loop between NF-κB and CCL20 pathways, which provides sustained impetus for chemoresistance in breast cancer cells. Our results suggest that CCL20 can be a novel predictive marker for taxane response, and the blockade of CCL20 or its downstream pathway might reverse the taxane resistance in breast cancer patients.

IL-17-producing Th cells (Th17) are a distinct subset of effector cells that bridge the innate and adaptive immune system and are implicated in autoimmune disease processes. CD4(+) splenocytes from DO11.10 mice were activated with OVA peptide(323-339) and maintained under Th17 polarization conditions, resulting in significantly higher proportions of IL-17(+) T cells compared with nonpolarized (Th0) cells. Th17-polarizing conditions significantly increased the proportion of cells expressing the chemokine receptors CCR2, CCR6, and CCR9 when compared with Th0 cells. In contrast, there was a significant decrease in the proportion of cells expressing CXCR3 under Th17-polarizing conditions compared with nonpolarizing conditions. The respective chemokine agonists for CCR2 (CCL2 and CCL12), CCR6 (CCL20), and CCR9 (CCL25) elicited migration and PI-3K-dependent signaling events in Th17-polarized cells, thus indicating that all three receptors were functionally and biochemically responsive. Furthermore, postmigration phenotypic analysis demonstrated that the agonists for CCR2 and CCR6, but not CCR9, stimulated a modest enrichment of IL-17(+) cells compared with the premigration population. Pan-isoform inhibitors of PI-3K/Akt signaling prevented CCR2- and CCR6-mediated, polarized Th17 cell migration in a concentration-dependent manner. The unique chemokine receptor expression pattern of Th17 cells and their corresponding PI-3K-dependent migratory responses are important for understanding the pathogenesis of autoimmune diseases and may provide opportunities for the application of CCR2 and CCR6 antagonists and PI-3K isoform-selective inhibitors in defined inflammatory settings.

The mechanisms underlying the increased susceptibility of the elderly to respiratory infections are not well understood. The crosstalk between the dendritic cells (DCs) and epithelial cells is essential in maintaining tolerance as well as in generating immunity in the respiratory mucosa. DCs from aged subjects display an enhanced basal level of activation, which can affect the function of epithelial cells. Our results suggest that this is indeed the scenario as exposure of primary bronchial epithelial cells (PBECs) to supernatants from unstimulated DCs of aged subjects resulted in activation of PBECs. The expression of CCL20, CCL26, CXCL10, mucin, and CD54 was significantly increased in the PBECs exposed to aged DC supernatants, but not to young DC supernatants. Furthermore, aged DC supernatants also enhanced the permeability of the PBEC barrier. We also found that DCs from aged subjects spontaneously secreted increased levels of pro-inflammatory mediators, interleukin-6, tumor necrosis factor (TNF)-α, and metalloproteinase A disintegrin family of metalloproteinase 10, which can affect the functions of PBECs. Finally, we demonstrated that TNF-α, present in the supernatant of DCs from aged subjects, was the primary pro-inflammatory mediator that affected PBEC functions. Thus, age-associated alterations in DC-epithelial interactions contribute to chronic airway inflammation in the elderly, increasing their susceptibility to respiratory diseases.

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/ml tissue necrosis factor-α and 20 U/ml interferon-γ resulted in de novo expression of pro-inflammatory chemokines CCL2, CXCL9, CXCL11, and CCL20, with increased expression of CXCL2-3, CXCL8, and CXCL10 relative to basal levels. Cytokine treatment induced/enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, α(M)- and α(L)-integrin, with differential regulation of α(M) -integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human α(M)-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2%, respectively. Monoclonal antibodies against α(L)-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6%, respectively. This study demonstrates differential regulation of α(M)-integrin on circulating mononuclear cells in GBS, as well as an important role for α(M)-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro.

Chemokines play a critical role in the acute transplant rejection. In order to provide an overview of the chemokine expression during the course of acute allograft rejection, the intragraft expression profile of 11 chemokines representative of all four chemokine subfamilies was analyzed in a murine skin transplantation model of acute rejection. It was found that RANTES/CCL5, TARC/CCL17 and FKN/CX(3)CL1 were expressed at equivalent levels in iso- and allografts. However, the other eight chemokines expression was up-regulated to some extent in allograft compared with that in isograft. The levels of MIP-1alpha/CCL3, MIP-3alpha/CCL20 and CTACK/CCL27 were progressively increased from early stage (day 3 post-transplantation) to late stage (day 11). Mig/CXCL9, IP-10/CXCL10, I-TAC/CXCL11, CXCL16 and LTN/XCL1 expression was elevated at middle stage (day 7), and peaked at late stage. Among the up-regulated chemokines, I-TAC was the most obviously elevated chemokine. Therefore, the effect of I-TAC on the skin acute allograft rejection was evaluated. Block of I-TAC by the intradermal injection of anti-I-TAC monoclonal antibody (mAb) reduced the number of CXCR3(+) cells in skin allograft and significantly prolonged the skin allograft survival. The mAb treatment did not influence the proliferation of the intragraft infiltrating cells in response to the allogeneic antigens, but significantly decreased the number of the infiltrating cells and consequently lowered the secretion of IFN-gamma and TNF-alpha. These data indicate I-TAC might be a dominant chemokine involved in the intradermal infiltration and I-TAC-targeted intervening strategies would have potential application for the alleviation of acute transplant rejection.

OBJECTIVE

Secretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum.

METHODS

The gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 (hBD2) and chemokine (C-C motif) ligand 20 (CCL20).

RESULTS

We found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect.

CONCLUSIONS

Our results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.

Bronchiolitis obliterans syndrome (BOS) is one of the most important factors limiting the long-term survival of lung transplant recipients (LTR), however its pathogenesis still remains unclear. We hypothesized that an increased production of certain specific proinflammatory mediators in the first post-transplant year would predispose to BOS. We retrospectively evaluated temporal kinetics of some CC chemokines that have not yet been evaluated, including CCL3/MIP1-alpha, CCL4/MIP1-beta, CCL17/TARC, CCL19/MIP3-beta, CCL20/MIP3-alpha, CCL22/MDC and CCL26/eotaxin, in broncho-alveolar lavage fluid (BAL-f) in the first post-transplant year in a cohort of 8 LTR before the development of BOS (pre-BOS LTR) and 8 LTR with long-term stable clinical conditions (stable LTR). Chemokine levels were assayed by means of a multiplex sandwich ELISA. Furthermore, for those ligands which resulted significantly predictive of BOS onset, we analyzed the expression of specific receptors (CCR) on BAL cells. The proportion of CCR-expressing BAL cells was assessed by flow cytometry. We demonstrated that MIP3-beta/CCL19, MIP3-alpha/CCL20, MDC/CCL22 levels at 6 months post-transplant significantly predicted BOS onset. In addition, the temporal behavior of these factors resulted significantly different in pre-BOS patients as compared to stable LTR. Finally the expression of CCR was documented on BAL lymphocytes and macrophages, and, in some cases, their expression was found to vary between the two groups. Within the complexity of the chemokine network, these three CCL factors could play an additive role in the pathogenesis of the inflammatory process leading to bronchiolar fibro-obliteration.

OBJECTIVE

Inflammasome activation and IL-1β production have been proposed to have an important role in age-related macular degeneration (AMD). Growing evidence is emerging for involvement of interleukin-17A (IL-17A) in AMD pathogenesis. We investigated the effects of IL-17A on the activation of inflammasome and production of IL-1β in primary human RPE cells.

METHODS

Primary human RPE cells were isolated and cultured for the following experiments. Expression patterns of IL-17 receptor A (IL-17RA), IL-17 receptor C (IL-17RC), and ACT1 were analyzed by RT-PCR, flow cytometry, and immunofluorescence. IL-17A was added to the cell cultures, and cytokine expression, signaling pathways, and inflammasome machinery were investigated using real-time RT-PCR, ELISA, Western blot, flow cytometry, and small interfering RNA.

RESULTS

Retinal pigment epithelial cells constitutively expressed IL-17RA, IL-17RC, and ACT1. IL-17A upregulated the mRNA levels of pro-IL-1β, IL-8, CCL2, and CCL20, as well as the protein level of IL-1β. IL-17A induced the phosphorylation of Akt, Erk1/2, p38 MAPK, and NF-κB p65 in RPE cells. Blocking NF-κB attenuated IL-17A-induced expression of pro-IL-1β mRNA. IL-17A enhanced pro-caspase-1 and NLRP3 mRNA expression. Inhibiting caspase-1 activity and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the IL-17A-responsive inflammasome on the posttranscriptional level. The mechanism of IL-17A-triggered NLRP3 activation and subsequent IL-1β secretion was found to involve the generation of reactive oxygen species.

CONCLUSIONS

Our results suggest that IL-17A triggers a key inflammatory mediator, IL-1β, from RPE cells, via NLRP3 inflammasome activation, holding therapeutic potential for AMD.