CCL2 Promoter Research Articles

Helicobacter pylori Induces CCL20 Expression

CCL20 attracts immature dendritic cells and memory T cells and plays a role on mucosal surfaces in inflammation. However, whether Helicobacter pylori infection induces CCL20 in human gastric epithelial cells remains to be determined. The aim of this study was to analyze the molecular mechanism of H. pylori-induced CCL20 expression. Expression of CCL20 mRNA was assessed by reverse transcription-PCR. Five normal and five H. pylori-infected gastric tissue samples were stained immunohistochemically for CCL20. A luciferase assay was used to monitor activation of the CCL20 gene promoter, and an electrophoretic mobility shift assay was used to explore the binding of transcription factors to this promoter. The CCL20 expression in epithelial cells of H. pylori-positive tissues was higher than that in H. pylori-negative tissues. H. pylori induced CCL20 expression in gastric epithelial cell lines, and the induction was dependent on an intact cag pathogenicity island. Activation of the CCL20 promoter by H. pylori occurred through the action of NF-κB. Transfection of IκB kinase and NF-κB-inducing kinase dominant negative mutants inhibited H. pylori-mediated activation of CCL20. Treatment with an inhibitor of Hsp90 suppressed H. pylori-induced CCL20 mRNA due to deactivation of NF-κB. Collectively, these results suggest that H. pylori activates NF-κB through an intracellular signaling pathway that involves IκB kinase and NF-κB-inducing kinase, leading to CCL20 gene transcription, and that Hsp90 is a crucial regulator of H. pylori-induced CCL20 expression, presumably contributing to the immune response in H. pylori.

Opioid-induced chemokine expression requires NF-κB activity: the role of PKCζ

Opioid receptor agonists induce broad immunomodulatory activity, which substantially alters host defense and the inflammatory response. Previous studies have shown that the MOR selective agonist DAMGO has the capacity to increase the expression of the proinflammatory chemokines CCL2, CCL5, and CXCL10 in human PBMCs. NF-κB is a transcription factor that plays a pivotal role in innate and adaptive immune responses. We report that NF-κB is a vital player in the DAMGO-induced, MOR-mediated regulation of chemokine expression. Results show that NF-κB inhibitors prevent the induction of CCL2 expression in response to DAMGO administration and that the NF-κB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to MOR activation. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation, and this kinase is essential for NF-κB activation as well as CCL2 expression and transcriptional activity. Finally, ChIP analysis shows that DAMGO administration induces binding of p65 to the enhancer region of the CCL2 promoter. These data are consistent with the notion that MOR activation promotes a proinflammatory response, which involves NF-κB activation. Our results also suggest a significant and novel role for PKCζ as an essential participant in the MOR-mediated regulation of proinflammatory chemokine expression.

Cooperative Activation of CCL5 Expression by TLR3 and Tumor Necrosis Factor-α or Interferon-γ through Nuclear Factor-ĸB or STAT-1 in Airway Epithelial Cells

Background: CCL5/RANTES contributes to prolonged eosinophilic inflammation and asthma exacerbation after a viral infection. We studied the mechanism of CCL5 expression using viral product double-stranded RNA (dsRNA), a ligand of Toll-like receptor 3 (TLR3), and inflammatory cytokines in airway epithelial cells. Methods: The airway epithelial cell line BEAS-2B was used in our in vitro study, and the levels of CCL5 mRNA and CCL5 protein expression were determined using real-time PCR and ELISA. The activity of the CCL5 promoter region and nuclear factor (NF)-ĸB was assessed by dual luciferase assay using specific luciferase reporter plasmids. We used actinomycin D to assess the stability of mRNA. Phosphorylation of signal transducer and activator of transcription 1 (STAT-1) was analyzed by Western blot. Results: Synthetic dsRNA up-regulated the expression of CCL5 mRNA and CCL5 protein. Adding TNF-α or IFN-γ to dsRNA further increased the expression of CCL5. The combination of TNF-α and dsRNA cooperatively activated the CCL5 promoter region and the NF-ĸB-specific reporter. IFN-γ did not activate these reporters. However, it increased the stability of CCL5 mRNA induced by dsRNA. IFN-γ phosphorylated STAT-1, but dsRNA did not. The effects of IFN-γ were not evident in the cells transfected with short interfering RNA for STAT-1. Conclusions: Cross-talk between TLR3 signaling and inflammatory cytokines regulates the expression of CCL5 in airway epithelial cells. In this mechanism, TNF-α may activate NF-ĸB, in cooperation with TLR3 signaling. IFN-γ may stabilize CCL5 mRNA up-regulated by TLR3. This mechanism may depend on STAT-1.

Down-regulation of intestinal epithelial innate response by probiotic yeasts isolated from kefir

Kefir is obtained by milk fermentation with a complex microbial population included in a matrix of polysaccharide and proteins. Several health-promoting activities has been attributed to kefir consumption. The aim of this study was to select microorganisms from kefir able to down-regulate intestinal epithelial innate response and further characterize this activity. Caco-2 cells stably transfected with a human CCL20 promoter luciferase reporter were used to screen a collection of 24 yeast and 23 bacterial strains isolated from kefir. The Toll-like receptor 5 agonist, flagellin was used to activate the reporter cells, while pre-incubation with the selected strains was tested to identify strains with the capacity to inhibit cell activation. In this system, 21 yeast strains from the genera Saccharomyces, Kluyveromyces and Issatchenkia inhibited almost 100% of the flagellin-dependent activation, whereas only some lactobacilli strains showed a partial effect. K. marxianus CIDCA 8154 was selected for further characterization. Inhibitory activity was confirmed at transcriptional level on Caco-2/TC-7 and HT-29 cells upon flagellin stimulation. A similar effect was observed using other pro-inflammatory stimulation such as IL-1β and TNF-α. Pre-incubation with yeasts induced a down-regulation of NF-κB signalling in epithelial cells in vitro, as well as expression of other pro-inflammatory chemokines such as CXCL8 and CXCL2. Furthermore, modulation of CCL20 mRNA expression upon flagellin stimulation was evidenced in vivo, in a mouse ligated intestinal loop model. Results indicate kefir contains microorganisms able to abolish the intestinal epithelial inflammatory response that could explain some of the properties attributed to this fermented milk.

Transforming growth factor‐β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor‐κB in airway smooth muscle cells

Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling. We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4. These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

IL-9 induces CCL11 expression via STAT3 signalling in human airway smooth muscle cells.

BackgroundPrevious findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood.Methodology/Principal Findings In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3β over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression.Conclusion/SignificanceTaken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.

Abstract C19: High CCL2 protein expression is associated with poor outcome in ovarian cancer

Abstract Background: CCL2 is a potent chemoattractant for tumor-associated macrophages, which promotes tumor growth and metastasis. The CCL2/CCR2 interaction plays a key role in promoting tumor inflammation, angiogenesis, proliferation and metastasis. Ovarian tumors and tumor lines secrete CCL2, however, very little is known about the expression of CCR2 and macrophage markers in ovarian tumors. Using a Yale University ovarian cancer tissue microarray cohort with associated outcome data, we analyzed the expression of CCL2, CCR2, and macrophage markers CD68, iNOS (M1 marker) and Arginase-1 (M2 marker) using fluorescence immunohistochemistry methods. Methods: The Yale YTMA18-2 Ovarian Cancer tissue microarray cohort consists of 239 stage I–IV ovarian tumors of all major histological subtypes and also included 12 normal gynecologic tissue samples for comparison purposes. The expression levels were assessed using Automated QUantitative Analysis (AQUA® technology, HistoRx Inc.) and were correlated with clinical outcome. CCR2 and CCL2 were measured within pan-cytokeratin defined cytoplasmic regions, and CD68, iNOS and Arginase-1 were measured within the entire sample regions. These biomarkers were analyzed for relationships with each other by Spearman Rho analysis and unsupervised hierarchical clustering, and for association with outcome by Cox univariate analysis and Kaplan-Meier analysis. Punch cores from the tumor sample utilized in the ovarian cancer tissue microarray cohort were also analyzed for genotyping five SNPs in the CCL2 promoter region. Results: There was no significant difference in CCL2, CCR2, CD68, iNOS (M1 marker) and Arginase-1 (M2 marker) expression by age group, stage or major histology grouping (all p>0.05). When looking at all tumor tissues, there was a significant difference in patient outcome (10 year and full time period, p = 0.028 and p = 0.047, respectively) based on separation by CCL2 cluster-based expression groupings. High CCL2 expression patients have a worse outcome, with a 1.9 fold higher risk of death at 5 years than other patients in categorical Cox univariate analysis. Although there was no correlation between CCR2 and CCL2, Kaplan-Meier analysis of unsupervised cluster groupings based on both markers identified a subset of ovarian cancer patients with high expression of CCL2 and intermediate/high expression of CCR2 that have the poorest survival compared with other groupings. Additionally, ovarian tumor tissues expressed 3.5-fold higher level of CCL2 and 1.2 -fold higher levels of CCR2 (mean AQUA scores, respectively) compared to normal samples (p< .0001). There were no significant differences in patient outcome by CD68, iNOS (M1 marker) and Arginase-1 (M2 marker)cluster groupings. There was a statistically significant difference in patient outcome by Kaplan-Meier analysis between two SNPs but no correlation with CCL2 expression was observed. Conclusion: Taken together, these data suggest that there is a subset of ovarian cancer patients with elevated CCL2 protein expression levels associated with poor outcome. This is the first study to show a link between high CCL2 protein expression levels and poor survival in ovarian cancer. Thus, additional exploration of CCL2 expression levels as a predictive biomarker in ovarian cancer is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C19.

Macrophage-inflammatory protein-3α/CCL-20 is transcriptionally induced by the iron chelator desferrioxamine in human mononuclear phagocytes through nuclear factor (NF)-κB

Alterations in iron availability can trigger pro-inflammatory signals in various cell types. We demonstrate that desferrioxamine (DFX), an iron chelator used in clinics for the treatment of iron overload, neoplasias, and Alzheimer disease, stimulates the expression and secretion of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells, in primary human monocytes and monocyte-derived macrophages. Iron chelation was part of the mechanism by which DFX induced CCL20, because addition of iron sulfate counteracted its stimulatory effects. Functional studies of the CCL20 promoter, using a series of 5′-deleted and mutated reporter constructs, demonstrated that CCL20 mRNA induction was dependent on gene transcription activation and mediated by the NF-κB pathway. The NF-κB element located at position −92/−82 of the CCL20 promoter was required for gene transactivation by DFX because: (i) transcription was abrogated by a 3 bp mutation of the NF-κB-binding motif; (ii) treatment with DFX increased specific NF-κB binding to this sequence. Nuclear translocation of both NF-κBp65 and NF-κBp50 family members was increased in response to DFX and associated with I-κBα degradation, suggesting a role for these subunits in CCL20 promoter transactivation. In conclusion, this study provides the first evidence that iron chelation can transcriptionally induce CCL20 in mononuclear phagocytes and identify the NF-κB binding site as a regulatory sequence of the CCL20 promoter that is activated by iron deprivation. These results add new insights into our understanding of the mechanisms by which iron perturbations affect mononuclear phagocyte immune functions and regulate inflammatory responses.

Targeting Tumor Stroma Using Engineered Mesenchymal Stem Cells Reduces the Growth of Pancreatic Carcinoma

To analyze the efficacy of engineered mesenchymal stem cell based therapy directed towards pancreatic tumor stroma. Mesenchymal stem cells (MSC) are actively recruited to tumor stroma where they enhance tumor growth and metastases. Upregulation of chemotactic cytokine (CCL5) by MSCs within the tumor stroma has been shown to play a central role in this process. Murine MSCs were engineered to express reporter genes or therapeutic genes under control of the CCL5 promoter and adoptively transferred into mice with growing pancreatic tumors. The effect on tumor growth and metastases was then evaluated. MSCs isolated from bone marrow of C57/Bl6 p53 mice were stably transfected with red fluorescent protein (RFP), enhanced green fluorescent protein (eGFP), or herpes simplex virus (HSV) thymidine kinase (Tk) gene driven by the RANTES promoter. MSCs were intravenously applied once per week over 3 weeks to mice carrying an orthotopic, syngeneic pancreatic Panc02 tumor. eGFP and RFP signals driven by the CCL5 promoter were detected by fluorescence in treated pancreatic tumor samples. The HSV-Tk therapy group treated intraperitoneal with the prodrug ganciclovir 5 to 7 days after stem cell application lead to a 50% reduction of primary pancreatic tumor growth (P < 0.0003, student t test) and reduced liver metastases (0% vs. 60%). The active homing of MSCs into primary pancreatic tumor stroma and activation of the CCL5 promoter was verified using eGFP- and RFP-reporter genes. In the presence of ganciclovir, HSV-Tk transfected MSCs led to a significant reduction of primary pancreatic tumor growth and incidence of metastases.

Oncostatin M–induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT‐1 and STAT‐3 signaling: An implication for the pathogenesis of arthritis

To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA-protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis.

Critical Role for STAT3 in IL-17A-Mediated CCL11 Expression in Human Airway Smooth Muscle Cells

IL-17A has been shown to be expressed at higher levels in respiratory secretions from asthmatics and to correlate with airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanism remains unknown. We previously demonstrated that IL-17A mediates CC chemokine (CCL11) production from human airway smooth muscle (ASM) cells. In this study, we demonstrate that STAT3 activation is critical in IL-17A-mediated CCL11 expression in ASM cells. IL-17A mediated a rapid phosphorylation of STAT3 but not STAT6 or STAT5 in ASM cells. Interestingly, transient transfection with wild-type or mutated CCL11 promoter constructs showed that IL-17A-mediated CCL11 expression relies on the STAT6 binding site. However, STAT3 but not STAT6 in vivo binding to the CCL11 promoter was detected following IL-17A stimulation of ASM cells. Overexpression of DN STAT3 (STAT3beta) abolishes IL-17A-induced CCL11 promoter activity. This effect was not observed with STAT6 DN or the STAT3 mutant at Ser(727). Interestingly, disruption of STAT3 activity with the SH2 domain binding peptide, but not with control peptide, results in a significant reduction of IL-17A-mediated STAT3 phosphorylation and CCL11 promoter activity. IL-17A-mediated CCL11 promoter activity and mRNA were significantly diminished in STAT3- but not STAT6-silenced ASM cells. Finally, IL-17A-induced STAT3 phosphorylation was sensitive to pharmacological inhibitors of JAK2 and ERK1/2. Taken together, our data provide the first evidence of IL-17A-mediated gene expression via STAT3 in ASM cells. Collectively, our results raise the possibility that the IL-17A/STAT3 signaling pathway may play a crucial role in airway inflammatory responses.

JNK MAPK Pathway Regulates Constitutive Transcription of CCL5 by Human NK Cells through SP1

The MAPKs ERK, JNK, and p38 control diverse aspects of the immune response, including regulation of cytotoxin biology in NK cells and CTL. The chemokine CCL5 is coreleased with the cytotoxins, perforin, the granzymes, and granulysin, during the lethal hit administered by cytotoxic CD8+ T cells (CTL). CCL5 expression is up-regulated relatively late in CTL coincident with their functional maturation 3-7 days after activation. Unlike T cells, NK cells have the ability to kill virally infected or transformed cells when directly isolated from the peripheral circulation. In this study, we show that in contrast to T cells, peripheral blood NK cells express CCL5 constitutively. The use of specific inhibitors of the JNK, ERK, and p38 MAPK pathways showed that the JNK pathway controls expression of CCL5 by NK cells. Promoter-reporter assays identified a compact region of the CCL5 promoter responsible for the constitutive transcription of CCL5 by NK cells. EMSA, chromatin immune precipitation, the use of heterologous promoters, and site-directed mutagenesis demonstrated that transcription in NK cells is largely controlled through binding of the transcription factor specificity protein 1 to a region -75 to -56 upstream of the site of transcriptional initiation. Specificity protein 1 expression, and in turn the constitutive expression of CCL5, was found to be controlled through constitutive activation of the JNK/MAPK pathway in peripheral blood NK cells.

Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at -28/-10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.

Are There Specific Markers in Allografted Patients Blood Predicting GvHD?

The graft versus Host Disease (GvHD) is a major complication of allogenic hematopoietic stem cell transplantation (allo-HSCT) in term of frequency and/or mortality. The complex physiopathology is roughly characterized by 3 phases where several inflammation factors and apoptotic pathways are involved. Among them, the chemokines CCL5 and CXCL12 are mainly playing a role in the homing of hematopoietic stem cells (HSC) to bone marrow and the migration of lymphocytes to the target organs. This migration is mediated by adhesion markers like ICAM-1 (Inter Cellular Adhesion Molecule 1) or by growth factor like VEGF (Vascular Endothelial Growth Factor). The final necrotic changes in target organs due to inflammatory mechanisms involves Interleukin 2, 6 and 8 (IL2, IL6, IL8) and the activation of Nuclear Factor kappa B (NFkB). Different Single Nucleotide Polymorphisms (SNP) in several chemokines and cytokines were shown to modify the level of gene expression. The aim of this study was to identify, in the recipient before allo- HSCT, some predictive factors of occurrence of GvHD, among the molecules involved in the different steps of GvHD. After informed consent, 73 patients (26 children and 47 adults) were included. SNPs -C28G and -G403A in the CCL5 promoter and G801A in the 3′UTR region of CXCL12 were studied and expression of CCL5 was analysed by RT-QPCR. To verify their impact on corresponding gene expression, protein measurements of these chemokines (CCL5 and CXCL12) but also of their inflammatory partners IL2, IL6, IL8, VEGF, ICAM-1 and endoglin were done in 39 samples. Among the 73 patients, 41 have developed a GvHD (Grade II-IV) and 32 did not developed GvHD or developed only grade I GvHD. As shown in table 1, the global frequency of SNPs underlined an unbalanced distribution in this cohort.CCL5CXCL12-G403A-C28GG801AG/GG/AA/AC/CC/GG/GG/GG/AA/A43 (62.3%)20 (29%)6 (8.7%)69 (94.5%)4 (5.5%)036 (49.3%)34 (46.6%)3 (4.4%)Table1: Repartition of the different SNP of CCL5 and CXCL12However, we did not observed a link between the SNP of CCL5, its expression (p=0.812) or its protein concentration (p=0.993) and the occurrence of a GvHD. Serum concentration of VEGF, CXCL12 and Endoglin were different between adults and children. In fact, in adult, the VEGF and Endoglin serum concentrations were significantly higher than in pediatric samples, with, respectively, 30.66 pg/ml [6.92;100.53] vs 9.97 pg/ml [0;31.29] and 18831.49 pg/ml [17787.7;25190.86] vs 11104.52 pg/ml [7742.54;16231.12], in opposition to CXCL12, which is higher in pediatric patients (54,76 pg/ml [43,74 ; 70,82] vs 35,1 pg/ml [22,63 ; 52,12]), but the levels of these proteins were not associated with the occurrence of GvHD. However, when considering only the adult group, significant differences were observed ( table 2), between the group of patients who developed a GvHD (II-IV) and the group of patients with no or low grade GvHD. Adult with GvHDAdult with GvHD (Grade II-IV)Patients with no or GvHD Grade IICAM-1113,571.6 pg/mL [75,549.5 – 164,814.1]32,055.3 pg/mL [26,083.5 – 121,165.7]VEGF18.53 pg/mL [1.99 – 62.18]106.46 pg/mL [51.3 – 214.4]Table 2: Statistical difference (p<0.05) of serum concentrations (pg/mL) between adults who developped a GvHD (Grade II-IV) and adults who did not.In conclusion, two markers ICAM-1 and VEGF show a significant prognostic value for GvHD severity in adult, when quantify in recipient patient before allografting. Further studies on a wider cohort would help to confirm these results and to precise the role of CCL5 and CXCL12 SNPs.

PPARα and PPARγ effectively protect against HIV‐induced inflammatory responses in brain endothelial cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors which down-regulate inflammatory signaling pathways. Therefore, we hypothesized that alterations of PPAR functions can contribute to human immunodeficiency virus-1 (HIV-1)-induced dysfunction of brain endothelial cells. Indeed, treatment with HIV-1 transactivator of transcription (Tat) protein decreased PPAR transactivation in brain endothelial cells. We next stably over-expressed PPARalpha and PPARgamma in a newly developed cell line of human brain endothelial cells (hCMEC/D3 cells). Tat-induced up-regulation of inflammatory mediators, such as interleukin (IL)-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin were markedly attenuated in hCMEC/D3 over-expressing PPARalpha or PPARgamma. These results were confirmed in CCL2 and E-selectin promoter activity studies. Similar protective effects were observed in hCMEC/D3 after activation of PPARgamma by exogenous PPAR agonists (dPGJ(2) and rosiglitazone). PPAR over-expression also prevented Tat-induced binding activity and transactivation of nuclear factor-kappaB. Importantly, increased PPAR activity attenuated induction of IL-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin in hCMEC/D3 cells co-cultured with HIV-1-infected Jurkat cells. The protective effects of PPAR over-expression were reversed by the antagonists of PPARalpha (MK886) or PPARgamma (GW9662). The present data suggest that targeting PPAR signaling may provide a novel therapeutic approach to attenuate HIV-1-induced local inflammatory responses in brain endothelial cells.

Epigallocatechin‐3‐gallate diminishes CCL2 expression in human osteoblastic cells via up‐regulation of phosphatidylinositol 3‐Kinase/Akt/Raf‐1 interaction: A potential therapeutic benefit for arthritis

To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)-induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1)-CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis.

PKCβΙΙ Augments NF-κB-Dependent Transcription at the CCL11 Promoter via p300/CBP-Associated Factor Recruitment and Histone H4 Acetylation

The transcription factor NF-kappaB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) betaIotaIota in augmenting NF-kappaB-mediated TNF-alpha-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCbetaII activation was not involved in NF-kappaB translocation, but facilitated NF-kappaB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF's HAT activity. Furthermore, mouse embryonic fibroblasts from PKCbeta knockout mice showed markedly reduced TNF-alpha-induced CCL11 expression and NF-kappaB reporter activity that was restored on PKCbetaII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCbetaIotaIota in NF-kappaB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKɛ-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.

CCL2 expression in ovarian carcinoma is associated with chemotherapy response and survival but not with the -2518 A/A polymorphism

16509 Background: CCL2, a macrophage chemoattractant, is expressed in normal human ovarian epithelium but down-regulated in ovarian adenocarcinomas. The purpose of this study was to determine the association between quantitative expression of CCL2 with chemotherapy response and clinical outcome in ovarian cancer (OC) patients (pts) as well as its association with a known polymorphism (-2518 A/A) in the CCL2 promoter region. Methods: Solid or ascitic tumor cells from pts with advanced papillary serous OC were obtained at the time of primary surgery or neoadjuvant chemotherapy. RT-PCR with PCR-based amplification of the CCL2 promoter region containing the -2518 A/A polymorphism (PM) was performed on samples of genomic DNA from blood and tumor. The PM frequency was compared to the genotype frequency in control patients with renal cell carcinoma (RCC). Chemoresponse was correlated with genotype and CCL2 expression. Sensitive (S) tumors = complete response and no disease recurrence/progression 6 months after treatment. Resistant tumors (R) = progression or recurrence ≤6 months after treatment. Results: Sixty nine pt specimens were analyzed. The incidence of the PM in blood and matched tumor was 65 and 70%, respectively, a frequency significantly higher than that previously reported (∼50%) in population-based studies on CCL2 polymorphisms and higher than that in the RCC group (54%, p =0.02). 46/69 patients had clinical follow up (f/u) data available (median f/u = 36 months). RT-PCR analysis demonstrated that CCL2 was significantly down-regulated in the R versus the S tumors (p<0.001). Sensitivity was 89%. Lack of CCL2 mRNA expression correlated inversely with progression free survival (PFS, p=0.05) and overall survival (OS, p=0.01). Expression of the PM was not predictive of response or survival. On multivariate analysis, only debulking status and CCL2 expression were predictive of PFS and OS. Conclusions: Decreased expression of CCL2 is associated with poorer chemoresponse and survival. Although the CCL2 promoter-region PM -2518 A/A is commonly expressed in women with ovarian carcinoma, it is not predictive of PFS or OS. Further studies are needed to characterize the relationship between this PM, expression of CCL2 and clinical outcomes. No significant financial relationships to disclose.

Hypoxia transcriptionally induces macrophage-inflammatory protein-3α/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-κB

Hypoxia, a condition of low oxygen tension, occurring in many pathological processes, modifies the mononuclear phagocyte transcriptional profile. Here, we demonstrate hypoxic up-regulation of the CCL20 chemokine in primary human monocytes (Mn) and macrophages. mRNA induction was paralleled by protein secretion and dependent on gene transcription activation. Functional studies of the CCL20 promoter using a series of 5'-deleted and mutated reporter constructs demonstrated the requirement for the NF-kappaB-binding site located at position -92/-82 for gene transactivation by hypoxia, as 1) transcription was abrogated by a 3-bp mutation of the NF-kappaB motif; 2) three copies of the wild-type NF-kappaB-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-kappaB binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-kappaB family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-kappaB complexes binding the cognate kappaB site on the CCL20 promoter upon hypoxia exposure, and NF-kappaBp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-kappaBp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the -92/-82 kappaB site. Moreover, nuclear expression of the other NF-kappaB family members was inhibited in hypoxic Mn. In conclusion, this study characterizes a previously unrecognized role for hypoxia as a transcriptional inducer of CCL20 in human mononuclear phagocytes and highlights the importance of the NF-kappaB pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.