IL-6 Promoter Research Articles

Proteasome Inhibition Increases Interleukin‐8 Expression in Triple Negative Breast Cancer Cells, Resulting in Their Increased Survival, Proliferation, and Migration

Interleukin‐8 (IL‐8, CXCL8) is a pro‐inflammatory and pro‐angiogenic chemokine that stimulates cancer progression by inducing tumor cell proliferation, survival, and migration. IL‐8 expression is increased in many types of advanced cancers, including triple negative breast cancer (TNBC), and correlates with poor prognosis. Because no targeted therapies are currently available, and majority of TNBC patients initially responding to cytotoxic chemotherapy become drug‐resistant, development of novel therapeutic strategies is essential. Proteasome inhibition by Bortezomib (BZ; Velcade; PS‐341) and Carfilzomib (CZ), developed for its ability to inhibit transcription of NFκB‐dependent anti‐apoptotic genes, has been effective in treating hematological malignancies. By contrast, as single agents, proteasome inhibitors (PI) have failed to show a significant clinical activity in solid tumors, including TNBC, but the mechanisms are not fully understood. Here, we demonstrate that proteasome inhibition increases expression of IL‐8, resulting in increased survival, proliferation, and migration of TNBC cells. The induced IL‐8 expression is mediated by IkB kinase (IKK), and associated with an increased nuclear accumulation of p65 NFκB, and IKK‐dependent p65 recruitment to IL‐8 promoter. Inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ‐treated TNBC cells. These results are the first to show that proteasome inhibition increases IL‐8 signaling in TNBC cells, suggesting that targeting IKK activity might increase PI effectiveness in TNBC treatment.Support or Funding InformationSponsor: Dr. Ivana Vancurova (ASBMB member ID: 32743) Funding : NIH CA202775 grant (to Dr. Ivana Vancurova)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epimagnolin A inhibits IL-6 production by inhibiting p38/NF-κB and AP-1 signaling pathways in PMA-stimulated THP-1 cells.

Epimagnolin A is a lignan obtained from the flower buds of Magnolia fargesii, which is traditionally used in Asian medicine for treating headache and nasal congestion. A herbal compound fargesin obtained from M. fargesii, has exerted anti-inflammatory effects in human monocytic THP-1 cells in the previous study. The anti-inflammatory effects of epimagnolin A, however, have been not elucidated yet. In this study, it was demonstrated that epimagnolin A reduced phorbol-12-myristate-13-acetate (PMA)-induced IL-6 promoter activity and IL-6 production in human monocytic THP-1 cells. Furthermore, it was investigated the modulating effects of epimagnolin A on mitogen-activated protein kinase, nuclear factor-kappa B (NF-κB), and activator protein 1 (AP-1) activities. Phosphorylation of p38 and nuclear translocation of p50 and c-Jun were down-regulated by epimagnolin A in the PMA-stimulated THP-1 cell. The results revealed that epimagnolin A attenuated the binding affinity of NF-κB and AP-1 transcription factors to IL-6 promoter and IL-6 production through p38/NF-kB and AP-1 signaling pathways in the PMA-stimulated THP-1 cells. These results suggest that epimagnolin A can be a useful drug for the treatment of inflammatory diseases.

Orphan Nuclear Receptor NR2F6 Suppresses T Follicular Helper Cell Accumulation Through Direct Regulation of IL-21

CD4 follicular helper T (Tfh) cells are specialized to help B cells during the germinal center reaction and are essential for affinity maturation and long-term humoral immunity. Here we report that loss of the nuclear orphan receptor NR2F6 causes enhanced survival and accumulation of Tfh, GC B and plasma cells following T cell-dependent immunization. Loss of Nr2f6 in CD4 T cells but not B cells is the primary driver of cell accumulation as adoptively transferred Nr2f6-deficient CD4 T cells maintain a larger wild-type host Tfh population through a paracrine effect. Cytokine expression in Nr2f6-deficient CD4 T cells is dysregulated in Tfh cells, in particular, Il21 expression is enhanced. Mechanistically, NR2F6, as a transcription factor, represses interleukin 21 (IL-21) production through direct binding of the Il21 promoter. Disruption of IL-21 signaling reduces Tfh accumulation in Nr2f6-deficient mice. Thus, NR2F6 is a critical negative regulator of Tfh cytokine secretion and prevents excessive Tfh accumulation.

DNAJB3 attenuates metabolic stress and promotes glucose uptake by eliciting Glut4 translocation

Failure of the heat shock response is a key event that leads to insulin resistance and type 2 diabetes. We recently showed that DNAJB3 co-chaperone is downregulated in obese and diabetic patients and that physical exercise restores its normal expression with a significant improvement of the clinical outcomes. In 3T3-L1 adipocytes, DNAJB3 has a role in improving the sensitivity to insulin and glucose uptake. In co-immunoprecipitation assays, DNAJB3 interacts with both JNK1 and IKKβ kinases. However, the functional impact of such interaction on their activities has not been investigated. Here, we assessed the effect of DNAJB3 on the respective activity of JNK1 and IKKβ in cell-based assays. Using JNK1- and IKKβ-dependent luciferase reporters, we show a marked decrease in luciferase activity by DNAJB3 in response to PMA and TNF-α that was consistent with a decrease in the translocation of p65/NF-κB to the nucleus in response to LPS. Furthermore, TNF-α-mediated IL-6 promoter activation and endogenous mRNA expression are significantly abrogated by DNAJB3 both in 3T3-L1 and C2C12 cells. The ability of DNAJB3 to mitigate ER stress and oxidative stress was also investigated and our data show a significant improvement of both forms of stress. Finally, we examined the effect of overexpressing and knocking down the expression of DNAJB3 on glucose uptake in C2C12 as well as the molecular determinants. Accordingly, we provide evidence for a role of DNAJB3 in promoting both basal and insulin-stimulated glucose uptake. Our finding reveals also a novel role of DNAJB3 in eliciting Glut4 translocation to the plasma membrane. These results suggest a physiological role of DNAJB3 in mitigating metabolic stress and improving glucose homeostasis and could therefore represent a novel therapeutic target for type 2 diabetes.

Cutting Edge: Core Binding Factor β Is Required for Group 2 Innate Lymphoid Cell Activation.

Group 2 innate lymphoid cells (ILC2) are tissue-resident, long-lived innate effector cells implicated in allergy and asthma. Upon activation, mature ILC2 rapidly secrete large amounts of type-2 cytokines and other effector molecules. The molecular pathways that drive ILC2 activation are not well understood. In this study, we report that the transcriptional controller core binding factor β (CBFβ) is required for ILC2 activation. Deletion or inhibition of CBFβ did not impair the maintenance of ILC2 at homeostasis but abolished ILC2 activation during allergic airway inflammation. Treatment with CBFβ inhibitors prevented ILC2-mediated airway hyperresponsiveness in a mouse model of acute Alternaria allergen inhalation. CBFβ promoted expression of key ILC2 genes at both transcriptional and translational levels. CBF transcriptional complex directly bound to Il13 and Vegfa promoters and enhancers, and controlled gene transcription. CBFβ further promoted ribosome biogenesis and enhanced gene translation in activated ILC2. Together, these data establish an essential role for CBFβ in ILC2 activation.

Regulation of platelet-activating factor-induced interleukin-8 expression by protein tyrosine phosphatase 1B

BackgroundPlatelet-activating factor (PAF) is a potent lipid mediator whose involvement in the onset and progression of atherosclerosis is mediated by, among others, the modulation of cytokine expression patterns. The presence of multiple potential protein-tyrosine phosphatase (PTP) 1B substrates in PAF receptor signaling pathways brought us to investigate its involvement in PAF-induced cytokine expression in monocyte-derived dendritic cells (Mo-DCs) and to study the pathways involved in this modulation.MethodsWe used in-vitro-matured human dendritic cells and the HEK-293 cell line in our studies. PTP1B inhibition was though siRNAs and a selective inhibitor. Cytokine expression was studied with RT-PCR, luciferase assays and ELISA. Phosphorylation status of kinases and transcription factors was studied with western blotting.ResultsHere, we report that PTP1B was involved in the modulation of cytokine expression in PAF-stimulated Mo-DCs. A study of the down-regulation of PAF-induced IL-8 expression, by PTP1B, showed modulation of PAF-induced transactivation of the IL-8 promoter which was dependent on the presence of the C/EBPß -binding site. Results also suggested that PTP1B decreased PAF-induced IL-8 production by a glycogen synthase kinase (GSK)-3-dependent pathway via activation of the Src family kinases (SFK). These kinases activated an unidentified pathway at early stimulation times and the PI3K/Akt signaling pathway in a later phase. This change in GSK-3 activity decreased the C/EBPß phosphorylation levels of the threonine 235, a residue whose phosphorylation is known to increase C/EBPß transactivation potential, and consequently modified IL-8 expression.ConclusionThe negative regulation of GSK-3 activity by PTP1B and the consequent decrease in phosphorylation of the C/EBPß transactivation domain could be an important negative feedback loop by which cells control their cytokine production after PAF stimulation.

HuR controls apoptosis and activation response without effects on cytokine 3’ UTRs

ABSTRACT RNA binding proteins regulate gene expression through several post-transcriptional mechanisms. The broadly expressed HuR/ELAVL1 is important for proper function of multiple immune cell types, and has been proposed to regulate cytokine and other mRNA 3′ UTRs upon activation. However, this mechanism has not been previously dissected in stable cellular settings. In this study, HuR demonstrated strong anti-apoptotic and activation roles in Jurkat T cells. Detailed transcriptomic analysis of HuR knockout cells revealed a substantial negative impact on the activation program, coordinately preventing the expression of immune response gene categories, including all cytokines. Knockout cells showed a significant defect in IL-2 production, which was rescued upon reintroduction of HuR. Interestingly, the mechanism of HuR regulation did not involve control of the cytokine 3′ UTRs: HuR knockout did not affect the activity of 3′ UTR reporters in 293 cells, and had no effect on IL-2 and TNF 3′ UTRs in resting or activated Jurkats. Instead, impaired cytokine production corresponded with defective induction of the IL-2 promoter upon activation. Accordingly, upregulation of NFATC1 was also impaired, without 3′ UTR effects. Together, these results indicate that HuR controls cytokine production through coordinated upstream pathways, and that additional mechanisms must be considered in investigating its function.

H3K4 Methylation Regulates LPS-Induced Proinflammatory Cytokine Expression and Release in Macrophages

Histone methylation is an important epigenetic mechanism that plays an essential role in regulating gene expression in mammalian cells. To understand its influence on inflammation, methylation of H3K4, H3K9, H3K36, H3K79, and H4K20, the most common histones methylated in the inflammatory response was analyzed in murine RAW264.7 cells and bone marrow-derived macrophages (BMDMs) upon lipopolysaccharide (LPS) stimulation. LPS stimulation resulted in enhanced methylation at H3K4 and H3K9 in both RAW264.7 and BMDMs. To further confirm whether LPS-stimulated H3K4me2 and H3K9me2 were responsible for subsequent proinflammatory cytokine expression, the recruitment of H3K4me2 and H3K9me2 at the promoters of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) was assessed. H3K4me2, but not H3K9me2, was enriched at the promoters of both IL-6 and TNF-α. Furthermore, LPS-stimulated gene expression and release of IL-6 and TNF-α were markedly suppressed in macrophages by MTA, a specific inhibitor of H3K4 methylation. These results demonstrate that histone methylation, in particular H3K4me2, plays a critical role in the regulation of LPS-induced expression and release of IL-6 and TNF-α.

Inhibition of TNFα-induced interleukin-6 gene expression by barley (Hordeum vulgare) ethanol extract in BV-2 microglia.

Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgare ethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. HVE inhibited TNFα-induced phosphorylation of IKKα/β, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.

Survivin facilitates T-helper 2-biased inflammation in the airway.

The biased T helper 2 (Th2) responses play a critical role in the pathogenesis of allergy. The underlying mechanism is not fully understood yet. Survivin can regulate multiple cellular activities. This study aims to elucidate the role of survivin in the development and maintenance of Th2 polarization. CD4+ T cells were isolated from blood samples collected from patients with allergic asthma (AS) and HS control (HS) subjects. Mice carrying CD4+ T cells with survivin knockout (KO mice) were employed to test the role of survivin in the development of the biased Th2 responses. KO mice failed to induce airway allergy. Peripheral CD4+ T cells expressed survivin, which was higher in the AS group than that in the HS group. Naive CD4+ T cells with higher expression of survivin were prone to differentiating into Th2 cells. Survivin bound to the Il4 promoter in CD4+ T cells to enhance Il4 gene transcription. The expression of Fas was lower in CD4+ T cells of the AS group than that in the HS group. Overexpression of survivin suppressed the expression of Fas and impaired the activation-induced cell death (AICD) of CD4+ T cells. Survivin facilitates the development of biased Th2 polarization through promoting expression of interleukin 4 (IL-4) and impairing the AICD machinery of CD4+ T cells. To modulate the expression of survivin in CD4+ T cells has the translational potential in the treatment of allergic diseases.

Curcumin Modulates Glycolytic Metabolism and Inflammatory Cytokines via Nrf 2 in Dalton's Lymphoma Ascites Cells In Vivo.

Warburg effect is characterized by the upregulation of HIF-1 and c-Myc regulated LDH-A, even aerobically owing to hypoxic environment and alterations in oncogenes or tumor suppressor genes in cancer. Reduced antioxidant defence system in transformed cells favors higher ROS production, which plays a significant role in carcinogenesis and acts as an important regulator of NF-κB. In addition, various proinflammatory cytokines play active roles in maintenance and progression of cancer. In continuation with our previous studies illustrating the long-term effect of curcumin using a liver tissue, present study was aimed to elucidate the anti-cancer effect of curcumin due to its long-term effect in the regulation of glycolytic metabolism, NF-κB activation, expression of proinflammatory cytokines in Dalton's lymphoma ascites cells in vivo. Spectrophotometric assays, RT-PCR and EMSA were performed to address the problems. Results revealed that curcumin-induced activation of antioxidant enzymes, Nrf2 and downstream signaling gene NQO1. Reduction of oxidative stress, down-regulation of NADPH: Oxidase, decline in ROS and H2O2 levels were also observed. Activation of NF-κB, expression of COX2, HIF-1α and cMyc, as well as expression and activity of LDH-A were significantly reduced by curcumin. Besides, expression of proinflammatory cytokines was significantly down-regulated via reducing binding of nuclear protein with AP-1, NF-IL6, ETS and NF-κB binding elements of IL-1α, IL-1β, TNF-α and IL-6 promoters, respectively. Curcumin downregulates glycolytic metabolism via modulation of stress-activated genes and reduces oxidative stress by enhancing antioxidant defence system, which inhibits activation of NF-κB signaling and expression of proinflammatory cytokines in Dalton's lymphoma ascites cells in vivo.

Association of common IL-10 promoter gene variants with the susceptibility to head and neck cancer in Tunisia

Background/aimWe investigated the association of three IL-10 promoter single-nucleotide polymorphisms and altered IL-10 plasma levels with the risk of head and neck cancer (HNC). Materials and methodsStudy subjects comprised 194 HNC patients [137 nasopharyngeal cancer (NPC) and 57 laryngeal cancer (LC)], and 263 healthy controls. Genotyping of rs1800896 (-1082A>G), rs1800871 (-819C>T), and rs1800872 (-592A>C) IL-10 variants was performed by real-time PCR; IL-10 levels were measured by enzyme amplified immuno sensitivity assay (EAISA). Results Carriage of rs1800896 A/A genotype was more frequent in the HNC and NPC cases, but was less frequent in the controls than the LC patients. Significant differences in IL-10 levels were observed between the rs1800896A/G genotype-carrying NPC patients and the controls. Positive association with NPC and LC was observed for rs1800871C/C, and carriage of rs1800872A/A genotype, and A allele were associated with higher risk of HNC and NPC, but not LC. GT rs1800896-rs1800871 haplotype was more frequent among the HNC and NPC patients than the controls in contrast to GC haplotype, which has a protective effect. Positive association was found between TA haplotype and LC. ConclusionOur results demonstrate that IL-10-1082, IL-10-819, and IL-10-592 variants, and haplotypes GC and GT constitute biomarkers for early detection of HNC, especially NPC subtype. IL-10 -819T/C and TA haplotype may be used as biomarkers for early detection of LC.

Inhibition of CREB-Mediated ZO-1 and Activation of NF-κB-Induced IL-6 by Colonic Epithelial MCT4 Destroys Intestinal Barrier Function

Background: Inflammatory bowel disease(IBD) is characterized by intestinal inflammation and impaired barrier function, associated with increased epithelial expression of monocarboxylate Transporter 4 (MCT4). Methods: we performed qPCR, IF, WB and utilized luciferase, and ChIP to analyze MCT4 on NF-κB and CREB activity and DNA binding in IECs. IP was used to analyze the effect of MCT4 on the interaction NF-κB-CBP or CREBCBP. In vivo assay were detected by IF, WB and to analyze the role and mechanism of MCT4 in IBD. Findings: Functional studies further showed that ectopic expression of MCT4 disrupted tight junction protein 1(ZO-1) expression and increased pro-inflammatory factors expression, leading to destroy intestinal epithelial barrier function in vitro and in vivo. Mechanistically, one hand, MCT4 led to nuclear translocation of p65 and increased the binding of NF-κB p65 to the promoter of IL-6, on the other hand, MCT4 suppressed cAMP-response element binding protein (CREB) activity and reduced CREBmediated ZO-1 expression. these effect were attributed to MCT4 contributed to NF-κB-CBP interaction, while dissolved CREB-CBP complex. Treatment of experimental colitis with MCT4 inhibitor by α-cyano-4- hydroxycinnamate (CHC) significantly ameliorated mucosal intestinal barrier function in vivo, which was attributed to attenuation of proinflammation factors expression and enhancement of ZO-1 expression. Interpretation: These findings suggested a novel MCT4 role in controlling development of IBD, which could serve as a new therapeutic target of IBD. Funding Statement: This work was supported by National Natural Science Foundation of China (No.81770552, No.81860101), Natural Science Foundation of Guangdong (No.2017A030313838, No.2018A0303130175), Medical Science and Technology Foundation of Guangdong (No.A2018395), Science and Technology plan program of Guangzhou (No.201804010148), General program of Guangzhou Health and plan commission (No.805150202068), Postdoctoral Research Funding of Guangzhou Women and Children’s Medical Center (NO.5001-3001075), Funding of Guangzhou Institute of Pediatrics/Guangzhou Women and Children’s Medical Center (NO.IP-2016-005, NO.IP2018-009). Declaration of Interests: The authors declared that they have no competing interests. Ethics Approval Statement: Human study: Based on the declaration of Helsinki as reflected in a prior approval by the institution’s human research committee, this study was conducted in a cohort of 54 patients with inflammatory bowel disease (IBD) and 16 healthy control in Guangzhou Women and Children’s Medical Center from 2016 to 2018 approved by the Medical Ethical Review Board(2017030602). Animal study: All animal experiments were approved by the Guangzhou Women and Children’s Medical Center animal care and use committee.

Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection.

The detection of microbial pathogens relies on the recognition of highly conserved microbial structures by the membrane sensor Toll-like receptors (TLRs) and cytosolic sensor NOD-like receptors (NLRs). Upon detection, these sensors trigger innate immune responses to eradicate the invaded microbial pathogens. However, it is unclear whether TLR and NOD signaling are both critical for innate immunity to initiate inflammatory and antimicrobial responses against microbial infection. Here we report that activation of both TLR and NOD signaling resulted in an augmented inflammatory response and the crosstalk between TLR and NOD led to an amplified downstream NF-κB activation with increased nuclear transactivation of p65 at both TNF-α and IL-6 promoters. Furthermore, co-stimulation of macrophages with TLR and NOD agonists maximized antimicrobial activity with accelerated phagosome maturation. Importantly, administration of both TLR and NOD agonists protected mice against polymicrobial sepsis-associated lethality with increased serum levels of inflammatory cytokines and accelerated clearance of bacteria from the circulation and visceral organs. These results demonstrate that activation of both TLR and NOD signaling synergizes to induce efficient inflammatory and antimicrobial responses, thus conferring protection against microbial infection.

A role for BATF3 in TH9 differentiation and T-cell-driven mucosal pathologies.

T helper 9 (TH9) cells are important for the development of inflammatory and allergic diseases. The TH9 transcriptional network converge signals from cytokines and antigen presentation but is incompletely understood. Here, we identified TL1A, a member of the TNF superfamily, as strong inducer of mouse and human TH9 differentiation. Mechanistically, TL1A induced the expression of the transcription factors BATF and BATF3 and facilitated their binding to the Il9 promoter leading to enhanced secretion of IL-9. BATF- and BATF3-deficiencies impaired IL-9 secretion under TH9 and TH9-TL1A polarizing conditions. In vivo, using a T cell transfer model we demonstrated that TL1A promoted IL-9-dependent, TH9 cell-induced intestinal and lung inflammation. Neutralizing IL-9 antibodies attenuated TL1A-driven mucosal inflammation. Batf3−/− TH9-TL1A cells induced reduced inflammation and cytokine expression in vivo compared to WT cells. Our results demonstrate that TL1A promotes TH9 cell differentiation and function and define a role of BATF3 in T cell driven mucosal inflammation.

Polymeric nanoparticles for the delivery of miRNA to treat Chronic Obstructive Pulmonary Disease (COPD)

RNA interference (RNAi) based therapeutics are considered an endogenous mechanism for modulating gene expression. In addition, microRNAs (miRNAs) may be tractable targets for the treatment of Chronic Obstructive Pulmonary Disease (COPD). In this study miR146a was adsorbed onto poly (glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, nanoparticles (NPs) to reduce target gene IRAK1 expression. NPs were prepared using an oil-in-water single emulsion solvent evaporation method incorporating cationic lipid dioleoyltrimethylammoniumpropane (DOTAP). This resulted in NPs of 244.80 ± 4.40 nm at 15% DOTAP concentration, zeta potential (ZP) of +14.8 ± 0.26 mV and miR-146a (40 µg/ml) maximum adsorption onto 15% DOTAP NPs was 36.25 ± 0.35 µg per 10 mg NP following 24 h incubation. Using the MTT assay, it was observed that over 75% at 0.312 mg/ml of A549 cells remained viable after 18 h exposure to cationic NPs at a concentration of 1.25 mg/ml. Furthermore, the in vitro release profile of miR-146a from loaded NPs showed a continuous release up to 77% after 24 h. Internalization of miR-146a loaded cationic NPs was observed in A549 cell lines using fluorescence and confocal microscopy. The miR146a delivered as miR-146a-NPs had a dose dependent effect of highest NPs concentrations 0.321 and 0.625 mg/ml and reduced target gene IRAK1 expression to 40%. In addition, IL-8 promoter reporter output (GFP) was dampened by miR-146a-NPs. In conclusion, miR-146a was successfully adsorbed onto PGA-co-PDL-DOTAP NPs and the miR-146a retained biological activity. Therefore, these results demonstrate the potential of PGA-co-PDL NPs as a delivery system for miR-146a to treat COPD.

Cellular Ca2+-Responding Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-palindromic NFAT-Response Elements.

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca2+ elevation. However, Ca2+ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca2+-calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca2+-mobilizing agonists independent of AP-1 activation.

Particulate matter induces inflammatory cytokine production via activation of NFκB by TLR5-NOX4-ROS signaling in human skin keratinocyte and mouse skin

Particulate matter (PM) increases levels of pro-inflammatory cytokines, but its effects on the skin remain largely unknown. We investigated the signal transduction pathway and epigenetic regulatory mechanisms underlying cellular inflammation induced by PM with a diameter of ≤ 2.5 (PM2.5) in vitro and in vivo. PM2.5-treated skin keratinocytes produced various inflammatory cytokines, including IL-6. The binding of PM2.5 to TLR5 initiated intracellular signaling through MyD88, and led to the translocation of NFκB to the nucleus, where it bound the NFκB site within IL-6 promoter. Furthermore, PM2.5 induced a direct interaction between TLR5 and NOX4, and in turn induced the production of ROS and activated NFκB-IL-6 downstream, which was prevented by siRNA-mediated knockdown of NOX4 or antioxidant treatment. Furthermore, expression of TLR5, MyD88, NOX4, phospho-NFκB, and IL-6 was increased in skin tissue of PM2.5-treated flaky tail mice. PM2.5-induced increased transcription of IL-6 was regulated via DNA methylation and histone methylation by epigenetic modification; the binding of DNA demethylase and histone methyltransferase to the IL-6 promoter regions resulted in increased IL-6 mRNA expression. Our findings provide deep insight into the pathogenesis of PM2.5 exposure and can be used as a therapeutic strategy to treat inflammatory skin diseases caused by PM2.5 exposure.

Obesity causes PGC-1α deficiency in the pancreas leading to marked IL-6 upregulation via NF-κB in acute pancreatitis.

Obesity is associated with local and systemic complications in acute pancreatitis. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator and master regulator of mitochondrial biogenesis that exhibits dysregulation in obese subjects. Our aims were: (1) to study PGC-1α levels in pancreas from lean or obese rats and mice with acute pancreatitis; and (2) to determine the role of PGC-1α in the inflammatory response during acute pancreatitis elucidating the signaling pathways regulated by PGC-1α. Lean and obese Zucker rats and lean and obese C57BL6 mice were used first; subsequently, wild-type and PGC-1α knockout (KO) mice with cerulein-induced pancreatitis were used to assess the inflammatory response and expression of target genes. Ppargc1a mRNA and protein levels were markedly downregulated in pancreas of obese rats and mice versus lean animals. PGC-1α protein levels increased in pancreas of lean mice with acute pancreatitis, but not in obese mice with pancreatitis. Interleukin-6 (Il6) mRNA levels were dramatically upregulated in pancreas of PGC-1α KO mice after cerulein-induced pancreatitis in comparison with wild-type mice with pancreatitis. Edema and the inflammatory infiltrate were more intense in pancreas from PGC-1α KO mice than in wild-type mice. The lack of PGC-1α markedly enhanced nuclear translocation of phospho-p65 and recruitment of p65 to Il6 promoter. PGC-1α bound phospho-p65 in pancreas during pancreatitis in wild-type mice. Glutathione depletion in cerulein-induced pancreatitis was more severe in KO mice than in wild-type mice. PGC-1α KO mice with pancreatitis, but not wild-type mice, exhibited increased myeloperoxidase activity in the lungs, together with alveolar wall thickening and collapse, which were abrogated by blockade of the IL-6 receptor glycoprotein 130 with LMT-28. In conclusion, obese rodents exhibit PGC-1α deficiency in the pancreas. PGC-1α acts as selective repressor of nuclear factor-κB (NF-κB) towards IL-6 in pancreas. PGC-1α deficiency markedly enhanced NF-κB-mediated upregulation of Il6 in pancreas in pancreatitis, leading to a severe inflammatory response. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory Tcells.

Whether human IL-10-producing regulatory Tcells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ Tcells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ Tcells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ Tcells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.