After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6(+) IL-17(+) γδ T cells migrate into the epithelium. γδ T-cell-deficient (TCRδ(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mRNA increased 19-fold at 3 h, and protein increased ∼ 16-fold at 6 h after injury. Systemic or topical treatment of wild-type C57BL/6 mice with anti-CCL20 reduced γδ T-cell accumulation in the cornea by >50% with a concomitant decrease in epithelial healing and stromal inflammation. In addition to CCR6 and IL-17, corneal γδ T cells stained positively for RORγt, IL-23R, and IL-22. Anti-IL-22 reduced peak cell division of the healing epithelium by 52%. Treatment of TCRδ(-/-) mice with rIL-22 significantly promoted wound closure, with peak epithelial cell division increased >3-fold. In addition, rIL-22 restored neutrophil and platelet influx in the TCRδ(-/-) mice to wild-type levels and increased CXCL1 production by wounded corneal explants >2-fold. These results indicate that an important aspect of the healing response to corneal epithelial abrasion includes CCL20-dependent influx of CCR6(+) IL-17(+) IL-22(+) γδ T cells and that IL-22 contributes to the inflammatory response and promotes epithelial healing.

BACKGROUND

Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported.

OBJECTIVE

To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation.

METHODS

Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance.

RESULTS

LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier.

CONCLUSIONS

Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.

BACKGROUND

Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.

METHODS

Human chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR.

RESULTS

Antibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1-3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-kappaB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1-3, CXCL8, CCL20, CXCR4, IL-1beta, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2).

CONCLUSIONS

Differential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic-anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.

BACKGROUND

Regulatory T cells (Treg) and tumor-exosomes are thought to play a role in preventing the rejection of malignant cells in patients bearing nasopharyngeal carcinoma (NPC).

METHODS

Treg recruitment by exosomes derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients' plasma (Patient-Exo), and CCL20 were tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model (n = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA, and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). The Student's t test was used for group comparisons. All statistical tests were two-sided.

RESULTS

CCL20 allowed the intratumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34 fold increase, P < .001), which was statistically significantly inhibited (P < .001) by an anti-CCL20 blocking mAb. NPC-Exo also recruited conventional CD4(+)CD25(-) T cells and mediated their conversion into inhibitory CD4(+)CD25(high) cells. Moreover, NPC-Exo enhanced (P = .0048) the expansion of human Treg, inducing the generation of Tim3(Low) Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75 (P < .001). These results were consistent with a stronger suppression of responder cells' proliferation and the secretion of immunosuppressive cytokines (IL10, TGFB1).

CONCLUSIONS

Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.

OBJECTIVE

Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury.

METHODS

CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo.

RESULTS

CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs.

CONCLUSIONS

Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.

Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) encoding proteins capable of initiating signal transduction pathways. Among them is the K15 ORF, which consists of eight exons encoding a protein with 12 predicted transmembrane domains and a cytoplasmic C terminus. When transiently expressed, the 8-exon K15 transcript gives rise to a protein with an apparent molecular mass of 45 kDa. K15 interacts with cellular proteins, TRAF (tumor necrosis factor receptor-associated factor) and Src kinases, and activates AP-1, NF-kappaB, and the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase and extracellular signal-regulated kinase. This signaling activity of K15 is related to phosphorylation of Y(481) of the K15 SH2-B motif Y(481)EEV. In this study we demonstrate the expression of an endogenous 45-kDa K15 protein in KSHV BAC36-infected epithelial cells. This endogenous K15 protein shows the same intracellular localization as transiently expressed K15, and expression kinetic studies suggest it to be a lytic gene. We have further determined the downstream target genes of K15 signaling using DNA oligonucleotide microarrays. We demonstrate that K15 is capable of inducing expression of multiple cytokines and chemokines, including interleukin-8 (IL-8), IL-6, CCL20, CCL2, CXCL3, and IL-1alpha/beta, as well as expression of Dscr1 and Cox-2. In epithelial cells, K15-induced upregulation of most genes was dependent on phosphorylation of Y(481), whereas in endothelial cells mutation of Y(481) did not result in a complete loss of Dscr1 and Cox-2 expression and NFAT-activity. Our study establishes K15 as one of the KSHV lytic genes that are inducing expression of multiple cytokines, which have been shown to play an important role in KSHV-associated pathogenesis.

BACKGROUND

T-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. Significantly, a comparative genome-scale transcriptional analysis of two T-cell subsets (CD4+ and CD8+) against each other and against the naturally mixed population (CD3+ cells) remains unexplored.

RESULTS

Comparison of the microarray-based gene expression patterns between CD3+ T cells, and the CD4+ and CD8+ subsets revealed largely conserved, but not identical, transcriptional patterns. We employed a Gene-Ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel T-cell activation genes and cell-type-specific genes associated with the immune response. We identified potential genes involved in the communication between the two subsets (including IL23A, NR4A2, CD83, PSMB2, -8, MIF, IFI16, TNFAIP1, POU2AF1, and OTUB1) and would-be effector-function-specific genes (XCL2, SLAMF7, TNFSF4, -5, -9, CSF3, CD48 and CD244). Chemokines induced during T-cell activation, but not previously identified in T cells, include CCL20, CXCL9, -10, -11 (in all three populations), and XCL2 (preferentially in CD8+ T cells). Increased expression of other unexpected cytokines (GPI, OSM and MIF) suggests their involvement in T-cell activation with their functions yet to be examined. Differential expression of many receptors, not previously reported in the context of T-cell activation, including CCR5, CCR7, IL1R2, IL1RAP, IL6R, TNFRSF25 and TNFRSF1A, suggests their role in this immune process. Several receptors involved in TCR activation (CD3D, CD3G, TRAT1, ITGAL, ITGB1, ITGB2, CD8A and B (CD8+ T-cell specific) along with LCK, ZAP70 and TYROBP were synchronously downregulated. Members of cell-surface receptors (HLA-Ds and KLRs), none previously identified in the context of T-cell activation, were also downregulated.

CONCLUSIONS

This comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ populations made possible the identification of many immune-response genes not previously identified in the context of T-cell activation. Significantly, it made possible to identify the temporal patterns of many previously known T-cell activation genes, and also identify genes implicated in effector functions of and communication between CD4+ and CD8+ T cells.

Autophagy contributes to the pathogenesis of cancer, whereas toll-like receptors (TLRs) also play an important role in cancer development and immune escape. However, little is known about the potential interaction between TLR signaling and autophagy in cancer cells. Here we show that autophagy induced by TLR4 or TLR3 activation enhances various cytokine productions through promoting TRAF6 (TNF receptor-associated factor 6, E3 ubiquitin protein ligase) ubiquitination and thus facilitates migration and invasion of lung cancer cells. Stimulation of TLR4 and TLR3 with lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] respectively triggered autophagy in lung cancer cells. This was mediated by the adaptor protein, toll-like receptor adaptor molecule 1 (TICAM1/TRIF), and was required for TLR4- and TLR3-induced increases in the production of IL6, CCL2/MCP-1 [chemokine (C-C motif) ligand 2], CCL20/MIP-3α [chemokine (C-C motif) ligand 20], VEGFA (vascular endothelial growth factor A), and MMP2 [matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)]. These cytokines appeared to be necessary for enhanced migration and invasion of lung cancer cells upon TLR activation. Remarkably, inhibition of autophagy by chemical or genetic approaches blocked TLR4- or TLR3-induced Lys63 (K63)-linked ubiquitination of TRAF6 that was essential for activation of MAPK and NFKB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) pathways, both of which were involved in the increased production of the cytokines. Collectively, these results identify induction of autophagy by TLR4 and TLR3 as an important mechanism that drives lung cancer progression, and indicate that inhibition of autophagy may be a useful strategy in the treatment of lung cancer.

The parasitic protozoan Giardia lamblia is a worldwide cause of diarrhea, but the mechanism of disease remains elusive. The parasite colonizes the small intestinal epithelium, known to be a sensor for the presence of enteric pathogens, without invading or causing severe inflammation. In this study we investigated the epithelial cell response to G. lamblia. Differentiated Caco-2 cells were infected with G. lamblia isolate WB-A11, and the transcriptome of the intestinal cells was analyzed after 1.5, 6, and 18 h of interaction, using oligonucleotide microarrays. A large number of genes displayed changed expression patterns, showing the complexity of the interaction between G. lamblia and intestinal cells. A novel chemokine profile (CCL2, CCL20, CXCL1, CXCL2, and CXCL3) was induced that was different from the response induced by enteric pathogens causing intestinal inflammation. Several genes involved in stress regulation changed their expression. These findings indicate that the intestinal epithelium senses the G. lamblia infection, and this is important for induction of innate and adaptive immunity. The induced stress response can be important in the pathogenesis.

Ligands of the EGF family regulate autocrine keratinocyte proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like growth factor, transforming growth factor-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and human β-defensin (hBD)-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250 × more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7, and CCL20, three of the most upregulated transcripts in psoriatic plaques.

The success of using glycolytic inhibitors for cancer treatment depends on studying the individual role of frequently deregulated glycolytic genes in cancer. This report aims to study the prognostic implication, and determine the cellular role and action mechanism of glycolytic ENO1 overexpression in head and neck cancer. The relationship of ENO1 mRNA expression in 44-pair clinical specimens with patient clinicopathologic characteristics was analysed by semi-quantitative RT-PCR, Kaplan-Meier survival curve and Cox model analyses. Following ectopic ENO1 expression or knockdown, we studied the proliferative, migratory, invasive, colony-forming and tumourigenic abilities of ENO1-genetically altered cells. DNA microarray analysis was used to identify downstream targets responsible for the ENO1 action in the cells. The expression of ENO1 mRNA was increased in 68% of tumour (T) specimens when compared to their normal (N) counterparts, and positively associated with clinical progression (p<0.05). High ENO1 expression (T/N2) was frequently observed in the patients with large primary tumours, late clinical stages or advanced neck metastasis. Moreover, high ENO1 patients had significantly poorer clinical outcomes than low expressers (T/N<2). Ectopic ENO1 expression stimulated cell transformation, invasion and tongue tumour formation. ENO1 knockdown abrogated the stimulation. Suppression of ENO1-induced proinflammatory CCL20 chemokine expression significantly attenuated its stimulatory effects on cell transformation and invasion. A concordant expression of ENO1 and CCL20 was validated both in ENO1-expressing cells and in clinical specimens. Together, we demonstrate a prognostic role of ENO1 overexpression in head and neck cancer and ENO1-mediated promotion of cell transformation and invasion partly via induced CCL20 expression.

Dendritic cells (DCs) are potent antigen-presenting cells that likely play multiple roles in human immunodeficiency virus type 1 (HIV-1) pathogenesis. We used the simian immunodeficiency virus (SIV)/macaque model to study the effects of infection on homeostatic chemokine expression and DC localization directly in secondary lymphoid tissues. SIV infection altered the expression of chemokines (CCL19/MIP-3beta, CCL21/ 6Ckine, and CCL20/MIP-3alpha) and of chemokine receptors (CCR7 and CCR6) that drive DC trafficking. CCL19/MIP-3beta, CCL20/MIP-3alpha, CCR6, and CCR7 expression increased in lymph nodes during the early systemic burst of viral replication (acute infection), whereas CCL21/6Ckine expression progressively decreased throughout disease to AIDS. Parallel with the SIV-induced perturbations in chemokine expression were changes in the expression of the DC-associated markers, DC-SIGN, DC-LAMP, and DECTIN-1. During AIDS, DC-LAMP mRNA expression levels were significantly reduced in lymph nodes and spleen, and DC-SIGN levels were significantly reduced in spleen. These findings suggest that the disruption of homeostatic chemokine expression is responsible, in part, for alterations in the networks of antigen-presenting cells in lymphoid tissues, ultimately contributing to systemic immunodeficiency.

Liver-and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3alpha/CCL20 is a CC chemokine which is constitutively expressed by follicle-associated epithelial cells in the mucosa, and attracts cells expressing CCR6 such as immature dendritic cells and alpha(4)beta(7)(high) intestine-seeking memory T cells. Here, we examine LARC/CCL20 expression in the skin. LARC/CCL20 mRNA and protein were induced in primary human keratinocytes upon stimulation with proinflammatory cytokines such as IL-1alpha and tumor necrosis factor (TNF)-alpha. In mice, intradermal injection of IL-1alpha and TNF-alpha rapidly induced a local accumulation of transcripts for LARC/CCL20 and its receptor CCR6 with a lag of several hours in the latter. In humans, immunostaining of LARC/CCL20 was weak if any in normal skin tissues but strongly augmented in lesional skin tissues with atopic dermatitis. Furthermore, massive infiltration of cells with markers such as CD1a, CD3 or HLA-DR was present in atopic skin lesions. Many infiltrating cells were also found to be CCR6(+) by a newly generated monoclonal anti-CCR6. However, Langerhans cells residing within the epidermis were hardly stained by anti-CCR6 in normal and atopic skin tissues. Furthermore, plasma levels of LARC/CCL20 were found to be elevated in patients with atopic dermatitis. Collectively, our results suggest that epidermal keratinocytes produce LARC/CCL20 upon stimulation with proinflammatory cytokines such as IL-1alpha and TNF-alpha, and attract CCR6-expressing immature dendritic cells and memory/effector T cells into the dermis of inflamed skin such as atopic dermatitis. LARC/CCL20 may not, however, play a major role in homeostatic migration of Langerhans cells into the skin.

Macrophages have a central role in innate-immune responses to bacteria. In the present work, we show that infection of human macrophages with Gram-positive pathogenic Streptococcus pyogenes or nonpathogenic Lactobacillus rhamnosus GG enhances mRNA expression of inflammatory chemokine ligands CCL2/monocyte chemoattractant protein-1 (MCP-1), CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha), CCL5/regulated on activation, normal T expressed and secreted, CCL7/MCP-3, CCL19/MIP-3beta, and CCL20/MIP-3alpha and CXC chemokine ligands CXCL8/interleukin (IL)-8, CXCL9/monokine induced by interferon-gamma (IFN-gamma), and CXCL10/IFN-inducible protein 10. Bacteria-induced CCL2, CCL7, CXCL9, and CXCL10 mRNA expression was partially dependent on ongoing protein synthesis. The expression of these chemokines and of CCL19 was dependent on bacteria-induced IFN-alpha/beta production. CCL19 and CCL20 mRNA expression was up-regulated by IL-1beta or tumor necrosis factor alpha (TNF-alpha), and in addition, IFN-alpha together with TNF-alpha further enhanced CCL19 gene expression. Synergy between IFN-alpha and TNF-alpha was also seen for CXCL9 and CXCL10 mRNA expression. Bacteria-stimulated macrophage supernatants induced the migration of T helper cell type 1 (Th1) cells, suggesting that in human macrophages, these bacteria can stimulate efficient inflammatory chemokine gene expression including those that recruit Th1 cells to the site of inflammation. Furthermore, L. rhamnosus-induced Th1 chemokine production could in part explain the proposed antiallergenic properties of this bacterium.

In this study the development and evaluation of outer membrane vesicles (OMVs) obtained from Bordetella pertussis as vaccines against pertussis disease is described. SDS-PAGE, immunoblot techniques and gel electrophoresis associated to tandem mass spectrometry were used to describe the composition of the OMVs obtained from B. pertussis Tohama CIP 8132 strain. These techniques revealed the presence of the main well-known pertussis surface immunogens in the OMVs such as pertactin, adenylate cyclase-haemolysin, pertussis toxin, as well as the lipo-oligosaccharide (LOS). A total of 43 proteins were identified by mass spectrometry. Some of them were predicted to have outer membrane or periplasmic location and the others with cytoplasmic or unknown location. The characterized pertussis OMVs were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by different routes. Killed detoxified whole-cell B. pertussis bacteria were used as reference. For intraperitoneal (i.p.) immunization, aluminum hydroxide was used as adjuvant. Since i.n. treatment with OMVs as well as killed whole-cell bacteria enhanced markers of innate immune response such as TNFalpha, IL-6 and CCL20, i.n. immunizations were performed with no adjuvant added. Immunized BALB/c mice were intranasally challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p<0.001). Adequate elimination rates (p<0.005) were observed in mice immunized either with OMV or whole-cell bacteria. Comparable results were obtained with both types of immunization route. In view to their capacity to induce airways innate and protective immunity in the mouse model, OMVs obtained from B pertussis are candidates to be used to protect against pertussis.

BACKGROUND

Previous work has identified CD11c(+)CD1c(-) dendritic cells (DCs) as the major "inflammatory" dermal DC population in patients with psoriasis vulgaris and CD1c(+) DCs as the "resident" cutaneous DC population.

OBJECTIVE

We sought to further define molecular differences between these 2 myeloid dermal DC populations.

METHODS

Inflammatory and resident DCs were single-cell sorted from lesional skin biopsy specimens of patients with psoriasis, and the transcriptome of CD11c(+)CD1c(-) versus CD1c(+) DCs was determined. Results were confirmed with RT-PCR, flow cytometry, immunohistochemistry, and double-labeled immunofluorescence. Human keratinocytes were cultured for functional studies.

RESULTS

TNF-related apoptosis-inducing ligand (TRAIL), Toll-like receptors 1 and 2, S100A12/ENRAGE, CD32, and many other inflammatory products were differentially expressed in inflammatory DCs compared with resident DCs. Flow cytometry and immunofluorescence confirmed higher protein expression on CD1c(-) versus CD1c(+) DCs. TRAIL receptors, death receptor 4, and decoy receptor 2 were expressed in keratinocytes and dermal cells. In vitro culture of keratinocytes with TRAIL induced CCL20 chemokine.

CONCLUSIONS

CD11c(+)CD1c(-) inflammatory DCs in psoriatic lesional skin express a wide range of inflammatory molecules compared with skin-resident CD1c(+) DCs. Some molecules made by inflammatory DCs, including TRAIL, could have direct effects on keratinocytes or other skin cell types to promote disease pathogenesis.

OBJECTIVE

Interleukin-10 knockout (IL-10(-/-)) mice spontaneously develop colitis characterized by T-helper cell type 1-polarized inflammation. We tested the possible therapeutic activity of the peroxisome proliferator-activated receptor alpha (PPARalpha) ligand fenofibrate, and the PPARdelta ligand GW0742, in IL-10(-/-) mice and investigated the cellular/molecular mechanisms for fenofibrate action.

METHODS

The effect of fenofibrate or GW0742 on the progression of colitis in C3H.IL-10(-/-) mice was evaluated. Effects of fenofibrate on cytokine and chemokine gene expression were studied in cultured splenocytes, pathogenic T cells isolated from C3H/HeJBir mice, and HT-29 colorectal cancer cells.

RESULTS

Treatment of C3H.IL-10(-/-) mice with fenofibrate delayed the onset of colitis, decreased the colonic histopathology score, and decreased colonic expression of genes encoding the inflammatory cytokines interferon-gamma and interleukin (IL)-17. The target for fenofibrate, PPARalpha, was expressed in lymphocytes, macrophages, and crypt and surface epithelial cells of the colon. The mean number of lymphocytes was decreased by more than 75% in colonic sections of fenofibrate-treated as compared with control IL-10(-/-) mice, and fenofibrate repressed interferon-gamma and IL-17 expression in isolated T cells. Fenofibrate also repressed the expression of the genes encoding 3 chemokines, CXCL10, CCL2, and CCL20, and repressed CXCL10 gene promoter activity in tumor necrosis factor-alpha-treated HT-29 cells. In contrast to the beneficial effect of fenofibrate, the PPARdelta ligand GW0742 accelerated the onset of colitis in IL-10(-/-) mice.

CONCLUSIONS

The immunopathology observed in IL-10(-/-) mice resembles that seen in Crohn's disease. The novel therapeutic activity of fenofibrate in this mouse model suggests that it may also have activity in Crohn's disease.

In this study, we show that microRNA-150 (miR-150) is significantly downregulated in advanced cutaneous T-cell lymphoma (CTCL), and that this downregulation is strongly associated with tumor invasion/metastasis. Inoculation of CTCL cell lines into nonobese diabetic/Shi-scid interleukin 2γ (IL-2γ) null mice led to CTCL cell migration to multiple organs; however, prior transfection of the cells with miR-150 substantially reduced the invasion/metastasis by directly downregulating CCR6, a specific receptor for the chemokine CCL20. We also found that IL-22 and its specific receptor subunit, IL22RA1, were aberrantly overexpressed in advanced CTCL, and that production of IL-22 and CCL20 was increased in cultured CTCL cells. IL22RA1 knockdown specifically reduced CCL20 production in CTCL cells, suggesting that IL-22 upregulation may activate the production of CCL20 and its binding to CCR6, thereby enhancing the multidirectional migration potential of CTCL cells. CTCL cells also exhibited nutrition- and CCL20-dependent chemotaxis, which were inhibited by miR-150 transfection or CCR6 knockdown. From these findings, we conclude that, in the presence of continuous CCR6 upregulation accompanied by miR-150 downregulation, IL-22 activation leads to continuous CCL20-CCR6 interaction in CTCL cells and, in turn, autocrine metastasis to distal organs. This suggests miR-150, CCL20, and CCR6 could be key targets for the treatment of advanced CTCL.

Dendritic cells (DCs) are central cells in the development of antitumor immune responses, but the number and function of these cells can be altered in various cancers. Whether these cells are affected during the development of melanoma is not known. We investigated the presence, phenotype, and functionality of circulating myeloid DCs (MDCs) and plasmacytoid DCs (PDCs) in newly diagnosed melanoma patients, compared to controls. The frequencies of PDCs and MDCs were equivalent in melanoma patients as compared with normal subjects. Both circulating DC subsets were immature, but on ex vivo stimulation with R848 they efficiently upregulated their expression of costimulatory molecules. We found that circulating DCs from melanoma patients and controls displayed similar pattern of expression of the chemokine receptors CXCR3, CXCR4, CCR7, and CCR10. Strikingly, PDCs from melanoma patients expressed higher levels of CCR6 than control PDCs, and were able to migrate toward CCL20. Further data showed that CCR6-expressing PDCs were present in melanoma primary lesions, and that CCL20 was produced in melanoma tumors. These results suggest that PDCs and MDCs are functional in melanoma patients at the time of diagnosis, and that CCL20 may participate to their recruitment from the blood to the tumor.

Cerebral inflammatory responses may initiate secondary cascades following traumatic brain injury (TBI). Changes in the expression of both cytokines and chemokines may activate, regulate, and recruit innate and adaptive immune cells associated with secondary degeneration, as well as alter a host of other cellular processes. In this study, we quantified the temporal expression of a large set of inflammatory mediators in rat cortical tissue after brain injury. Following a controlled cortical impact (CCI) on young adult male rats, cortical and hippocampal tissue of the injured hemisphere and matching contralateral material was harvested at early (4, 12, and 24 hours) and extended (3 and 7 days) time points post-procedure. Naïve rats that received only anesthesia were used as controls. Processed brain homogenates were assayed for chemokine and cytokine levels utilizing an electrochemiluminescence-based multiplex ELISA platform. The temporal profile of cortical tissue samples revealed a multi-phasic injury response following brain injury. CXCL1, IFN-γ, TNF-α levels significantly peaked at four hours post-injury compared to levels found in naïve or contralateral tissue. CXCL1, IFN-γ, and TNF-α levels were then observed to decrease at least 3-fold by 12 hours post-injury. IL-1β, IL-4, and IL-13 levels were also significantly elevated at four hours post-injury although their expression did not decrease more than 3-fold for up to 24 hours post-injury. Additionally, IL-1β and IL-4 levels displayed a biphasic temporal profile in response to injury, which may suggest their involvement in adaptive immune responses. Interestingly, peak levels of CCL2 and CCL20 were not observed until after four hours post-injury. CCL2 levels in injured cortical tissue were significantly higher than peak levels of any other inflammatory mediator measured, thus suggesting a possible use as a biomarker. Fully elucidating chemokine and cytokine signaling properties after brain injury may provide increased insight into a number of secondary cascade events that are initiated or regulated by inflammatory responses.

BACKGROUND

House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation.

OBJECTIVE

The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo.

METHODS

Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model.

RESULTS

The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity.

CONCLUSIONS

This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The 'second' or 'late' MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.

Large vessel vasculitides, such as Takayasu arteritis and giant cell arteritis, affect vital arteries and cause clinical complications by either luminal occlusion or vessel wall destruction. Inflammatory infiltrates, often with granulomatous arrangements, are distributed as a panarteritis throughout all of the artery's wall layers or cluster in the adventitia as a perivasculitis. Factors determining the architecture and compartmentalization of vasculitis are unknown. Human macrovessels are populated by indigenous dendritic cells (DCs) positioned in the adventitia. Herein, we report that these vascular DCs sense bacterial pathogens and regulate the patterning of the emerging arteritis. In human temporal artery-SCID chimeras, lipopolysaccharides stimulating Toll-like receptor (TLR)4 and flagellin stimulating TLR5 trigger vascular DCs and induce T-cell recruitment and activation. However, the architecture of the evolving inflammation is ligand-specific; TLR4 ligands cause transmural panarteritis and TLR5 ligands promote adventitial perivasculitis. Underlying mechanisms involve selective recruitment of functional T cell subsets. Specifically, TLR4-mediated DC stimulation markedly enhances production of the chemokine CCL20, biasing recruitment toward CCL20-responsive CCR6(+) T cells. In adoptive transfer experiments, CCR6(+) T cells produce an arteritis pattern with media-invasive T cells damaging vascular smooth muscle cells. Also, CCR6(+) T cells dominate the vasculitic infiltrates in patients with panarteritic giant cell arteritis. Thus, depending on the original danger signal, vascular DCs edit the emerging immune response by differentially recruiting specialized T effector cells and direct the disease process toward distinct types of vasculitis.

The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8+ T cells (TRMs), resident natural killer cells (NKRs), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3+CD4+ and PD-1+CD8+ T cells. Interestingly, Tregs and TRMs isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and TRMs isolated from the NTME. We found PD-1+ TRMs were the predominant T-cell subset responsive to anti-PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8+ T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NKRs via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed.