Transcription Of Interleukin Research Articles

PRMT6 expression in lung of COPD mouse model and its relationship with inflammatory gene expression

Objective To explore the expression of protein arginine methyltransferases 6 (PRMT6) in chronic obstructive pulmonary disease (COPD) mouse model and its correlation with inflammation gene interleukin 6 (IL-6) and cyclooxygenase 2 (COX-2). Methods Sixteen C57BL/8J mice were randomly divided into 2 groups: control group and cigarette smoke extract (CSE)-induced COPD group. Each group was injected intraperitoneally with phosphate-buffered saline solutions (PBS) or CSE at days 1, 12, 23 and measured lung function and collected lung tissue at day 29. The morphology change of the lung tissue was determined by hematoxylin and eosin (HE) stainning. The protein expression of PRMT6, H3R2me2a and H3K4me3 were detected in lung homogenates by Western-blotting. The mRNA expression of PRMT6, IL-6 and COX-2 were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Results Comparing to control group, COPD group showed typical emphysema changes in the lung tissue, and significantly decreased lung function. The mRNA and protein expression of PRMT6 in the lung tissue of the mice with COPD were significantly decreased, following with the down-regulated signal level of H3R2me2a protein expression, while the increased level of IL-6 and COX-2 mRNA. Meanwhile, PRMT6 was negatively correlated with IL-6 and COX-2 mRNA expression. Conclusions PRMT6 was significantly reduced in CSE-induced COPD mouse model, following with decreased histone H3R2 dimethyla-tion and increased H3K4 trimethylation, negatively correlating with inflammatory gene IL-6 and COX-2 transcription expression. PRMT6 downregulation may activate the transcriptional expression of inflammatory genes involved in the development of COPD, through the regulation of histone methylation level. Key words: Pulmonary disease, chronic obstructive; Protein-arginine N-methyltransferase/ME; Interleukin-6/ME; Cyclooxygenase 2/ME; Disease models, animal

Essential Oil from Fructus Alpiniae Zerumbet Protects Human Umbilical Vein Endothelial Cells In Vitro from Injury Induced by High Glucose Levels by Suppressing Nuclear Transcription Factor-Kappa B Signaling.

BackgroundIn China, the essential oil of the fruit, Fructus Alpiniae zerumbet (FAZ), is used to treat cardiovascular diseases. Recent in vitro studies have shown that the essential oil of FAZ (EOFAZ) can protect endothelial cells from injury. Because of the prevalence of diabetes mellitus and its effects on the cardiovascular system, the aim of this study was to investigate the mechanism of the effects of EOFAZ on human umbilical vein endothelial cells (HUVECs) treated with high levels of glucose in vitro.Material/MethodsThe lactate dehydrogenase (LDH) leakage assay was used to detect HUVEC injury. Tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), and nuclear transcription factor-kappa B (NF-κB) p65 subunit DNA-binding activity was detected. The expression of NF-κB pathway-associated proteins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) was studied by Western blotting. The cellular location of NF-κB in HUVECs was evaluated using immunofluorescence.ResultsCell viability and LDH leakage assays showed that high glucose-induced HUVEC injury was reduced by EOFAZ. High glucose-induced secretion of IL-8, TNF-α, ICAM-1, and VCAM-1 was reduced, and translocation of the p65 subunit of NF-κB to the endothelial cell nucleus was inhibited by EOFAZ. Western blotting confirmed that EOFAZ blocked the activation of NF-κB induced by high glucose levels. EOFAZ reduced high glucose-induced p65/DNA binding to inhibit NF-κB activation.ConclusionsThe findings of this in vitro study showed that treatment of HUVECs with EOFAZ had a protective role against the effects of high glucose levels via the NF-κB signaling pathway.

MicroRNA-520c-3p negatively regulates EMT by targeting IL-8 to suppress the invasion and migration of breast cancer.

Interleukin-8 (IL-8), which is secreted by cancer cells undergoing epithelial-mesenchymal transition (EMT), can promote EMT in adjacent epithelial-like cells. MicroRNAs (miRNAs/miRs) can affect the expression of target genes via binding to their 3'-untranslated regions (3'-UTRs), which may subsequently affect the biological behaviors of cancer cells. In our previous study, miR-520c-3p was predicted to directly target the 3'-UTR of IL-8. Therefore, the present study was carried out to investigate whether miR-520c-3p can interact with the IL-8 gene and regulate the EMT of breast cancer cells. Web-based prediction algorithms were used to identify miRNAs that potentially target the IL-8 transcript. Luciferase reporter assays were used to confirm the targeting of IL-8 by miR-520c-3p. Reverse transcription-quantitative PCR and western blot analyses were used to examine the levels of IL-8 and EMT-related genes in breast cancer cells. The functional impact of miR-520c-3p on EMT phenotype was evaluated using Transwell and wound-healing assays, and rescue experiments were conducted by overexpressing IL-8 to determine its effect on cell properties. miR-520c-3p was predicted by all three databases, which strongly suggested its interaction with the 3'-UTR of IL-8. The relative Renilla luciferase activity of luciferase reporter construct containing the wild-type 3'-UTR of IL-8 was markedly decreased by miR-520c-3p transfection when compared with scrambled miRNA control transfection(P<0.001). In addition, compared with the scrambled miRNA control transfection, the overexpression of miR-520c-3p significantly reduced the expression of IL-8, and resulted in increased E-cadherin and decreased vimentin and fibronectin levels in MCF-7 and T47D cells (all P<0.001). Introduction of miR-520c-3p inhibited the invasion and migration of MCF-7 and T47D cells(all P<0.001). By contrast, the rescue of IL-8 expression led to the recovery of EMT-related protein expression patterns and cell motility and invasion capabilities. In conclusion, aberrant miR-520c-3p expression may lead to reduced IL-8 expression and promote the mesenchymal phenotype in breast cancer cells, thereby increasing invasive growth.

LAT is essential for the mast cell stabilising effect of tHGA in IgE-mediated mast cell activation

Mast cells play a central role in the pathogenesis of allergic reaction. Activation of mast cells by antigens is strictly dependent on the influx of extracellular calcium that involves a complex interaction between signalling molecules located within the cells. We have previously reported that tHGA, an active compound originally isolated from a local shrub known as Melicope ptelefolia, prevented IgE-mediated mast cell activation and passive systemic anaphylaxis by suppressing the release of interleukin-4 (IL-4) and tumour necrosis factor (TNF)-α from activated rat basophilic leukaemia (RBL)-2H3 cells. However, the mechanism of action (MOA) as well as the molecular target underlying the mast cell stabilising effect of tHGA has not been previously investigated. In this study, DNP-IgE-sensitised RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA. To dissect the MOA of tHGA in IgE-mediated mast cell activation, the effect of tHGA on the transcription of IL-4 and TNF-α mRNA was determined using Real Time-Polymerase Chain Reaction (qPCR) followed by Calcium Influx Assay to confirm the involvement of calcium in the activation of mast cells. The protein lysates were analysed by using Western Blot to determine the effect of tHGA on various important signalling molecules in the LAT-PLCγ-MAPK and PI3K-NFκB pathways. In order to identify the molecular target of tHGA in IgE-mediated mast cell activation, the LAT and LAT2 genes in RBL-2H3 cells were knocked-down by using RNA interference to establish a LAT/LAT2 competition model. The results showed that tHGA inhibited the transcription of IL-4 and TNF-α as a result of the suppression of calcium influx in activated RBL-2H3 cells. The results from Western Blot revealed that tHGA primarily inhibited the LAT-PLCγ-MAPK pathway with partial inhibition on the PI3K-p65 pathway without affecting Syk. The results from RNAi further demonstrated that tHGA failed to inhibit the release of mediators associated with mast cell degranulation under the LAT/LAT2 competition model in the absence of LAT. Collectively, this study concluded that the molecular target of tHGA could be LAT and may provide a basis for the development of a mast cell stabiliser which targets LAT.

Antarctic seals: Molecular biomarkers as indicators for pollutant exposure, health effects and diet

Weddell (Leptonychotes weddellii), Ross (Ommatophoca rossii) and crabeater seals (Lobodon carcinophaga) are phocid seals with a circumpolar distribution around Antarctica. As long-lived and large top predators, they bioaccumulate contaminants and are considered as sentinels of ecosystem health. Antarctic seals are increasingly exposed to climate change, pollution, shipping and fisheries. To reveal and understand possible anthropogenic impacts on their immune and health status, this study investigates sensitive biomarkers of the xenobiotic metabolism and immune system in relation to mercury (Hg) burden. Gene-transcription studies using minimally-invasive blood samples are useful to monitor physiological processes in wildlife that can be related to different stressors. Blood samples of 72 wild-caught seals (Weddell n=33; Ross n=12; crabeater n=27) in the Amundsen and Ross Seas in 2008–2011 were investigated. Copy numbers per μl mRNA transcription of xenobiotic biomarkers (aryl hydrocarbon receptor (AHR)), aryl hydrocarbon receptor nuclear translocator (ARNT) and peroxisome proliferator-activated receptor (PPARα) and immune relevant cell mediators (cytokines interleukin-2 (IL-2), interleukin-10 (IL-10) and heat-shock-protein 70 (HSP70)) were measured using reference genes Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and ribosomal protein L4 (RPL4) by real time RT-qPCR. Hg concentration was analysed in fur. Hg concentration increased with body weight and standard length in all species. Crabeater seals showed a lower Hg concentration than Ross and Weddell seals. Species-specific differences in gene-transcription were found between all species with highest levels of AHR, ARNT and PPARα in crabeater seals. Ross seals showed highest IL-10 and HSP70 transcription, while HSP70 was exceptionally low in crabeater seals. Between Hg and HSP70 a clear negative relationship was found in all species. The species-specific, age and sex-dependent gene-transcription probably reflect dietary habits, pollutant exposure and immune status.

Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators

Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1β. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.

Epigenetic Regulation of Interleukin 6 by Histone Acetylation in Macrophages and Its Role in Paraquat-Induced Pulmonary Fibrosis.

Overexpression of interleukin 6 (IL-6) has been proposed to contribute to pulmonary fibrosis and other fibrotic diseases. However, the regulatory mechanisms and the role of IL-6 in fibrosis remain poorly understood. Epigenetics refers to alterations of gene expression without changes in the DNA sequence. Alternation of chromatin accessibility by histone acetylation acts as a critical epigenetic mechanism to regulate various gene transcriptions. The goal of this study was to determine the impact of IL-6 in paraquat (PQ)-induced pulmonary fibrosis and to explore whether the epigenetic regulations may play a role in transcriptional regulation of IL-6. In PQ-treated lungs and macrophages, we found that the mRNA and protein expression of IL-6 was robustly increased in a time-dependent and a dose-dependent manner. Our data demonstrated that PQ-induced IL-6 expression in macrophages plays a central role in pulmonary fibrosis through enhanced epithelial-to-mesenchymal transition (EMT). IL-6 expression and its role to enhance PQ-induced pulmonary fibrosis were increased by histone deacetylase (HDAC) inhibition and prevented by histone acetyltransferase (HAT) inhibition. In addition, the ability of CRISPR-ON transcription activation system (CRISPR-ON) to promote transcription of IL-6 was enhanced by HDAC inhibitor and blocked by HAT inhibitor. Chromatin immunoprecipitation experiments revealed that HDAC inhibitor increased histones activation marks H3K4me3 and H3K9ac at IL-6 promoter regions. In conclusion, IL-6 functioning through EMT in PQ-induced pulmonary fibrosis was regulated dynamically by HDAC and HAT both in vitro and in vivo via epigenetically regulating chromatin accessibility.

Reciprocal androgen receptor/interleukin-6 crosstalk drives oesophageal carcinoma progression and contributes to patient prognosis.

Oesophageal squamous cell carcinoma (ESCC), a leading lethal malignancy of the digestive tract, is characterized by marked gender disparity. Clarifying the roles of the function and regulatory pathway of the androgen receptor (AR) will improve our understanding of oesophageal cancer progression, thereby facilitating the personalized management of ESCC. Here we report evidence to show that AR is a key mediator of inflammatory signals in ESCC cancer progression. High AR expression was associated with poor overall survival in tobacco-using ESCC patients but not in ESCC patients not using tobacco. A gain and loss of AR function enhanced and repressed ESCC cell growth, respectively, by altering cell cycle progression. In mice bearing human ESCC xenografts, silencing AR expression attenuated tumour growth, whereas AR overexpression promoted tumour growth in mice of different androgen statuses (male, female, and castrated male). Array assays revealed that the inflammatory cytokine interleukin-6 (IL6) is a prominent AR target gene in ESCC. By directly binding to the IL6 promoter, AR enhances IL6 transcription, and IL6 can in turn activate AR expression, thus forming a reciprocal regulatory circuit to sustain STAT3 oncogenic signalling in ESCC. Moreover, high expression levels of both AR and IL6 in human ESCC predict poor clinical outcome in tobacco users. Together, these data establish that AR promotes ESCC growth and is associated with poor patient prognosis. The discovery of a positive feedback loop between IL6 and AR bridges the knowledge gaps among lifestyle factor-associated inflammation, gender disparity, and oesophageal carcinoma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcription Factor Ets-2 Acts as a Preinduction Repressor of Interleukin-2 (IL-2) Transcription in Naive T Helper Lymphocytes

IL-2 is the first cytokine produced when naive T helper (Th) cells are activated and differentiate into dividing pre-Th0 proliferating precursors. IL-2 expression is blocked in naive, but not activated or memory, Th cells by the transcription factor Ets-2 that binds to the antigen receptor response element (ARRE)-2 of the proximal IL-2 promoter. Ets-2 acts as an independent preinduction repressor in naive Th cells and does not interact physically with the transcription factor NFAT (nuclear factor of activated T-cells) that binds to the ARRE-2 in activated Th cells. In naive Th cells, Ets-2 mRNA expression, Ets-2 protein levels, and Ets-2 binding to ARRE-2 decrease upon cell activation followed by the concomitant expression of IL-2. Cyclosporine A stabilizes Ets-2 mRNA and protein when the cells are activated. Ets-2 silences directly constitutive or induced IL-2 expression through the ARRE-2. Conversely, Ets-2 silencing allows for constitutive IL-2 expression in unstimulated cells. Ets-2 binding to ARRE-2 in chromatin is stronger in naive compared with activated or memory Th cells; in the latter, Ets-2 participates in a change of the IL-2 promoter architecture, possibly to facilitate a quick response when the cells re-encounter antigen. We propose that Ets-2 expression and protein binding to the ARRE-2 of the IL-2 promoter are part of a strictly regulated process that results in a physiological transition of naive Th cells to Th0 cells upon antigenic stimulation. Malfunction of such a repression mechanism at the molecular level could lead to a disturbance of later events in Th cell plasticity, leading to autoimmune diseases or other pathological conditions.

RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells: A HOMEOSTATIC RESPONSE?

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain- and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-κB activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2 Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.

Suppression of Inflammatory Arthritis by Human Gut-Derived Prevotella histicola in Humanized Mice.

The gut microbiome regulates host immune homeostasis. Rheumatoid arthritis (RA) is associated with intestinal dysbiosis. This study was undertaken to test the ability of a human gut-derived commensal to modulate immune response and treat arthritis in a humanized mouse model. We isolated a commensal bacterium, Prevotella histicola, that is native to the human gut and has systemic immune effects when administered enterally. Arthritis-susceptible HLA-DQ8 mice were immunized with type II collagen and treated with P histicola. Disease incidence, onset, and severity were monitored. Changes in gut epithelial proteins and immune response as well as systemic cellular and humoral immune responses were studied in treated mice. When treated with P histicola in prophylactic or therapeutic protocols, DQ8 mice exhibited significantly decreased incidence and severity of arthritis compared to controls. The microbial mucosal modulation of arthritis was dependent on regulation by CD103+ dendritic cells and myeloid suppressors (CD11b+Gr-1+ cells) and by generation of Treg cells (CD4+CD25+FoxP3+) in the gut, resulting in suppression of antigen-specific Th17 responses and increased transcription of interleukin-10. Treatment with P histicola led to reduced intestinal permeability by increasing expression of enzymes that produce antimicrobial peptides as well as tight junction proteins (zonula occludens 1 and occludin). However, the innate immune response via Toll-like receptor 4 (TLR-4) and TLR-9 was not affected in treated mice. Our results demonstrate that enteral exposure to P histicola suppresses arthritis via mucosal regulation. P histicola is a unique commensal that can be explored as a novel therapy for RA and may have few or no side effects.

HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib

AbstractActivating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88.

Transmembrane protein 147 (TMEM147): another partner protein of Haemonchus contortus galectin on the goat peripheral blood mononuclear cells (PBMC).

BackgroundRecombinant galectins of male and female Haemonchus contortus (rHco-gal-m/f) have been recognized as significant regulators of the functions of goat peripheral blood mononuclear cells (PBMC). In previous research, transmembrane protein 63A (TMEM63A) was identified as a partner protein in the regulation associated with H. contortus infection. However, in the identification of binding partners for galectins of male and female H. contortus (Hco-gal-m/f) by yeast two-hybrid (YTH) screening, it was found that the transmembrane protein 147 (TMEM147) could also bind to Hco-gal-m/f. In this study, the functions of TMEM147 in the regulations of H. contortus galectin on the goat PBMC were investigated.MethodsTo identify Hco-gal-m/f-interacting proteins, a yeast two-hybrid system to detect interactions was used. Co-immunoprecipitation and immunoblotting were used to validate the interaction between recombinant galectins of male H. contortus (rHco-gal-m) and candidate binding protein. The localization of TMEM147 in PBMC was explored by immunofluorescence in confocal imaging studies. Flow cytometry was used to determine the distribution of TMEM147 in T cells, B cells and monocytes in PBMC. The modulatory effects of rHco-gal-m and TMEM147 on cell proliferation, phagocytosis, nitric oxide production, migration, apoptosis and cytokine mRNA transcription were observed by co-incubation of rHco-gal-m and knockdown of the tmem147 gene.ResultsIn this research, it was demonstrated that TMEM147 could bind to rHco-gal-m/f. Immunofluorescence assays showed that TMEM147 was localized to the cell membrane and within the cell membrane in goat PBMC. Flow cytometric analysis revealed that TMEM147 was expressed in all B cells and monocytes in goat PBMC. However, 3.8 % of T cells did not express this protein. Knockdown of the tmem147 gene using RNA interference (RNAi) showed that the interaction of galectin with TMEM147 mainly mediated cell proliferation, cell apoptosis, transcription of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) of goat PBMC. This membrane protein, together with TMEM63A, was also related to the regulation of galectin on phagocytosis and nitric oxide production of goat PBMC. However, it might not be involved in the regulation of galectin on the migration and interferon-γ (IFN-γ) transcription of goat PBMC.ConclusionsOur results showed that TMEM147 was a binding partner of Hco-gal-m/f and mediated the immunological regulation of Hco-gal-m/f on goat PBMC in a manner different to that of TMEM63A.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1640-0) contains supplementary material, which is available to authorized users.

Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection.

Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side.

Interleukin-6 and rs1800796 locus single nucleotide polymorphisms in response to hypoxia/reoxygenation in hepatocytes.

Ischemia-reperfusion injury due to hypoxia/reoxygenation (H/R) is one of the main causes of liver damage during liver surgery. Donor interleukin-6 (IL-6) rs1800796 single nucleotide polymorphisms (SNPs) affect the metabolism of tacrolimus following liver transplantation-related hepatic H/R. This study investigated the response of IL-6 and its promoter polymorphisms to hepatic H/R in liver parenchymal cells. The association between IL-6 rs1800796 SNPs and IL-6 expression was measured in 84 disease-free liver tissues using tissue microarrays and immunohistochemistry. Subsequently, LO2G, LO2C and NC-LO2 cells were successfully constructed via stable lentivirus-mediated transfection. The effects of IL-6 and its SNPs on the biological function of LO2 cells were examined using a cell model of H/R. Our results revealed that IL-6 was mainly expressed in hepatocytes. The intermediate IL-6 expression rate in genotype CC carriers was higher than that in genotype CG/GG carriers (P=0.006), which was subsequently verified at the IL-6 mRNA level (P=0.002). The concentrations of alanine aminotransferase in the LO2G cells were significantly higher than those in the LO2C cells following H/R for 6 h and H/R for 24 h (P<0.05). The viability of the LO2C cells was higher than that of the LO2G cells (P<0.05). Furthermore, the expression of IL-6 and its downstream molecules was significantly increased in the LO2C cells compared with the LO2G cells (P<0.05). Therefore, the sequence variants of rs1800796 SNPs (G→C) exhibit an increased IL-6 transcription efficiency in liver parenchymal cells. In addition, the increased expression of IL-6 protects the hepatocytes following hepatic H/R injury.

Fenton Reaction-Generated Advanced Oxidation Protein Products Induces Inflammation in Human Embryonic Kidney Cells.

Fenton reaction is a new mechanism able to generate advanced oxidation protein products (AOPPs) by exposing the human serum albumin to the Fenton system. Here, we characterized the effects of Fenton reaction-generated advanced oxidation protein products (AOPP-FR) on the gene transcription of the nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) in human embryonic kidney cells (HEK 293). To investigate the effects of AOPP-FR and AOPP-HOCl on transcription of inflammatory genes, the NF-κB, COX-2, and IL-6 luciferase promoter activities were analyzed. AOPP-FR and AOPP-HOCl were able to induce the activation of the gene transcription of NF-κB, COX-2, and IL-6 in HEK 293 cells. However, the effects of AOPP-FR were significantly higher than the effects of AOPP-HOCl in relation to COX-2 and IL-6. AOPP-FR induces the activation of the gene transcription of NF-κB, COX-2, and IL-6 and may represent a novel pathogenic mediator of inflammation in kidney.

β-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how β-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. β-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. β-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that β-naphthoflavone (25nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. β-naphthoflavone (100nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested β-naphthoflavone concentrations alone. However, β-naphthoflavone (25 and 50nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to β-naphthoflavone concentrations ranging from 25 to 100nM, with and without LPS or poly I: C, expressed AhR protein. LPS or β-naphthoflavone (5-50nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that β-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.

MEK1/2 inhibitors induce interleukin-5 expression in mouse macrophages and lymphocytes

Uptake of oxidized low-density lipoprotein (oxLDL) by macrophages facilitates the formation of foam cells, the prominent part of atherosclerotic lesions. Interleukin-5 (IL-5) is a cytokine regulating interactions between immune cells. It also activates the production of T15/EO6 IgM antibodies in B-1 cells, which can bind oxLDL thereby demonstrating anti-atherogenic properties. We previously reported that inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by mitogen-activated protein kinase kinases 1/2 (MEK1/2) inhibitors can reduce atherosclerosis. In this study, we determined the effects of MEK1/2 inhibitors on IL-5 production both in vitro and in vivo. In vitro, MEK1/2 inhibitors (PD98059 and U0126) substantially inhibited phosphorylation of MEK1/2 and ERK1/2. Associated with inhibition of ERK1/2 phosphorylation both in vitro and in vivo, MEK1/2 inhibitors induced IL-5 protein expression in macrophages (RAW macrophages and peritoneal macrophages) and lymphocytes (EL-4 cells). In vivo, administration of mice with MEK1/2 inhibitors increased serum IL-5 levels, and IL-5 protein expression in mouse spleen and liver. At the mechanistic level, we determined that MEK1/2 inhibitors activated IL-5 mRNA expression and IL-5 promoter activity in the liver X receptor (LXR) dependent manner indicating the induction of IL-5 transcription. In addition, we determined that MEK1/2 inhibitors enhanced IL-5 protein stability. Taken together, our study demonstrates that MEK1/2 inhibitors induce IL-5 production which suggests another anti-atherogenic mechanism of MEK1/2 inhibitors.

Long non-coding RNA DILC regulates liver cancer stem cells via IL-6/STAT3 axis

Emerging evidence has demonstrated the aberrant expression of long non-coding RNAs (lncRNAs) in various malignancies including HCC. However, the knowledge of cancer stem cell-related lncRNAs remains limited. lnc-DILC (lncRNA downregulated in liver cancer stem cells (LCSCs)) was identified by microarray and validated by real-time PCR. The role of lnc-DILC in LCSCs was assessed both in vitro and in vivo. Pull down assay and oligoribonucleotides or oligodeoxynucleotides treatment were conducted to evaluate the interaction between lnc-DILC and interleukin-6 (IL-6) promoter. Depletion of lnc-DILC markedly enhanced LCSC expansion and facilitated HCC initiation and progression, whereas ectopic expression of lnc-DILC dramatically inhibited LCSC expansion. Mechanistically, lnc-DILC inhibited the autocrine IL-6/STAT3 signaling. The putative binding locus of lnc-DILC within IL-6 promoter was confirmed by pull down assay. Consistently, the oligoribonucleotide mimics and an oligodeoxynucleotide decoy of lnc-DILC abrogated the effects on IL-6 transcription, STAT3 activation and LCSC expansion triggered by lnc-DILC depletion and lnc-DILC overexpression. Moreover, our data suggested that lnc-DILC mediated the crosstalk between TNF-α/NF-κB signaling and IL-6/STAT3 cascade. Clinical investigation demonstrated the reduction of lnc-DILC in patient HCCs, and suggested the correlation between lnc-DILC levels and IL-6, EpCAM or CD24 expression. Decreased lnc-DILC expression in HCCs predicts early recurrence and short survival of patients, highlighting its prognostic value. lnc-DILC mediates the crosstalk between TNF-α/NF-κB signaling and autocrine IL-6/STAT3 cascade and connects hepatic inflammation with LCSC expansion, suggesting that lnc-DILC could be not only a potential prognostic biomarker, but also a possible therapeutic target against LCSCs.

Bclaf1 is an important NF-κB signaling transducer and C/EBPβ regulator in DNA damage-induced senescence.

Inducing senescence in cancer cells is an effective approach to suppress cancer growth, and it contributes significantly to the efficacy of therapeutic drugs. Previous studies indicated that transcription factors NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) and C/EBPβ (CCAAT/enhancer-binding protein-β) play a critical role in the establishment of senescence by upregulating proinflammatory cytokines, notably interleukin-6 (IL-6) and interleukin-8 (IL-8). However, it is not clear how these two factors are activated in response to senescence-inducing stimuli and subsequently regulate gene transcription. Here, we reveal Bcl-2-associated transcription factor 1 (Bclaf1) as a novel player in the therapeutic drug doxorubicin-induced senescence (TIS) in multiple cancer cells. Bclaf1 is upregulated through the ATM/Nemo/NF-κB pathway during TIS and is a direct target of p65 and c-Rel. The induction of Bclaf1 by NF-κB is essential for C/EBPβ upregulation and IL-6/IL-8 transcription during TIS. Bclaf1 can interact with the leucine zipper region of C/EBPβ and cooperate with C/EBPβ to upregulate IL-8. Furthermore, we show that Bclaf1 is required for the effectiveness of doxorubicin (Dox) treatment-induced tumor suppression in a xenograft tumor model. These finding suggest that Bclaf1 plays a crucial role in transducing the senescence-inducing signal from NF-κB to C/EBPβ during TIS, thus amplifying the signals for the establishment of senescence. Given the recent revelation that Bclaf1 is involved in tumorigenesis, our data indicate that the responsiveness of Bclaf1 to NF-κB may determine the effectiveness of therapeutic drugs.