IL-1β Promoter Research Articles

Cigarette Smoke Condensate Extracts Induce IL-1-Beta Production from Rheumatoid Arthritis Patient-Derived Synoviocytes, but Not Osteoarthritis Patient-Derived Synoviocytes, Through Aryl Hydrocarbon Receptor-Dependent NF-Kappa-B Activation and Novel NF-Kappa-B Sites

Cigarette smoking is a major established environmental risk factor for rheumatoid arthritis (RA), and synoviocyte-derived proinflammatory cytokines are implicated in the pathogenesis of RA. We have reported that aryl hydrocarbon or cigarette smoke condensate (CSC) is able to upregulate the production of proinflammatory cytokines from an RA patient-derived synovial fibroblast cell line MH7A. In this study, we compared the effect of CSC on induction of interleukin-1β (IL-1β) from RA or osteoarthritis (OA) patient-derived synovial fibroblasts, and studied the mechanism of the effect of CSC. CSC induced IL-1β mRNA from RA patient-derived synoviocytes and MH7A, but not from OA patient-derived synoviocytes. CSC induced the mRNA and both precursor and mature forms of IL-1β, and caspase-1 activity in MH7A. The mechanism of CSC-induced IL-1β mRNA expression was investigated in MH7A. Reporter gene analyses and promoter pull-down assay indicated that 3 novel NF-κB sites at -3771 to -3762 bp, -3105 to -3096 bp, and -2787 to -2778 bp in the promoter region of the IL-1β gene, especially the far distal NF-κB site and NF-κB activation, are critical for the gene activation by CSC. CSC-induced NF-κB activation, IL-1β promoter activity, IL-1β mRNA upregulation, and CYP1A1 mRNA induction were all inhibited by an aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone. These results indicate that CSC induced IL-1β production from RA patient-derived synoviocytes, but not OA patient-derived synoviocytes, through AhR-dependent NF-κB activation and novel NF-κB sites.

Epidermal Growth Factor Protects Squamous Cell Carcinoma against Cisplatin-Induced Cytotoxicity through Increased Interleukin-1β Expression

The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR) are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression was confirmed by knockdown of RelA and Akt in cells or treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. The expression of dominant negative IκB also repressed the activation of NF-κB and inhibited EGF-induced IL-1β expression. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65) was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment.

Regulation of cooperative function of the Il12b enhancer and promoter by the interferon regulatory factors 3 and 5

The regulation of the Il12b gene, encoding the shared p40 subcomponent for IL-12 and IL-23, is critical for innate immune responses and subsequent T cell polarization. This gene is robustly induced upon Toll-like receptor (TLR) stimulation, wherein an enhancer located 10kb upstream of the transcription start site is required for promoter activity; however, the underlying mechanisms that regulate this enhancer in cooperation with the promoter has remained elusive. We show here that the Il12b enhancer contains functional ISREs for recognition by interferon regulatory factors (IRFs), and provide evidence that TLR-activated IRF5 mediates cooperativity of the enhancer with the promoter which also contains ISREs. By contrast, IRF3 activated by cytosolic RIG-I-like receptor (RLR) signaling binds to these ISREs and causes gene suppression. Consistently, IRF5 binding is accompanied with chromatin remodeling of both regulatory regions and the formation of a productive transcriptional complex containing other transcription factors, whereas these events are inhibited by IRF3 binding. We show that the ISREs embedded in the enhancer are indeed critical for its activation by IRF5. We also adduce evidence that the 5' sequences of the enhancer and promoter ISREs, all of which deviate from consensus ISREs, critically affect the function of IRF3. The dual commitment of these IRFs in the regulation of the Il12b enhancer and promoter is unique and may have implications for understanding the evolution of this gene.

Activation of IL-1β and TNFα genes is mediated by the establishment of permissive chromatin structures during monopoiesis

IL-1β and TNFα participate in a wide range of immunoregulatory activities. The overproduction of these cytokines can result in inflammatory and autoimmune diseases. Monocytes are the main producers of both cytokines. In contrast, studies with highly purified polymorphonuclear leukocytes (PMN) showed their inability to synthesize IL-1β and TNFα. Mature monocytes and PMN are derived from the same precursors. However, the reason for the differential IL-1β and TNFα expression is not elucidated. Our study investigates the epigenetic mechanisms that may explain this apparent discrepancy.The expression and promoter accessibilities of IL-1β and TNFα genes of primary and in vitro differentiated monocytes and PMN and their common precursors were compared. The effects of histone deacetylase (HDAC)-inhibition by trichostatin A (TSA) on IL-1β and TNFα expression and their promoter structures were measured in promyeloid HL-60 cells. Cytokine expression was assessed by real-time PCR and ELISA. Chromatin structures were analyzed using chromatin accessibility by real-time PCR (CHART) assay.The proximal IL-1β promoter was remodeled into an open conformation during monopoiesis, but not granulopoiesis. Although stimulation-dependent, remodeling of the TNFα promoter was again only observed in monocytes. TSA activated IL-1β and TNFα expression and supported chromatin remodeling of their promoters in HL-60 cells.The ability to express IL-1β and TNFα is linked to a cell type specific promoter structure, which is established during monocytic but not granulocytic differentiation. The participation of acetylation in IL-1β and TNFα promoter activation shed new light on the regulation of IL-1β or TNFα expression. These data may have implications for understanding the progression from normal to disease conditions.

DNA methylation at promoter regions of interleukin 1B, interleukin 6, and interleukin 8 in non-small cell lung cancer.

Epidemiologic and experimental evidences support the concept that inflammation promotes the development and progression of cancers. Interleukins (ILs) regulate the expression of several molecules and signaling pathways involved in inflammation. High expression of some ILs in the tumor microenvironment has been associated with a more virulent tumor phenotype. To examine the role of IL-1β, IL-6, and IL-8 in non-small cell lung cancer, we measured mRNA levels and promoter DNA methylation in a panel of cultured human lung cells (n = 23) and in matched pair lung tumor versus adjacent non-tumorous tissues (n = 24). We found that lung cancer cells or tissues had significantly different DNA methylation and mRNA levels than normal human bronchial epithelial cells or adjacent non-tumorous tissues, respectively. High DNA methylation of ILs promoters in lung cancer cells or tissues was associated with low mRNA levels. We found an inverse correlation between DNA methylation of IL1B, IL6, and IL8 gene promoters and their corresponding mRNA levels, such inverse correlation was more significant for IL1B (i.e., all cancer cell lines used in this study had a hypermethylated IL1B promoter which was associated with silencing of the gene). Our results underline for the first time the role of epigenetic modifications in the regulation of the expression of key cytokines involved in the inflammatory response during lung cancer development.

Zinc deficiency induces production of the proinflammatory cytokines IL-1β and TNFα in promyeloid cells via epigenetic and redox-dependent mechanisms

The deprivation of zinc, caused by malnutrition or as a consequence of aging or disease, strongly affects immune cell functions, causing higher frequency of infections. Among other effects, an increased production of reactive oxygen species (ROS) and proinflammatory cytokines has been observed in zinc-deficient patients, but the underlying mechanisms were unknown. The aim of the current study was to define mechanisms explaining the increase in proinflammatory cytokine production during zinc deficiency, focusing on the role of epigenetic and redox-mediated mechanisms.Interleukin (IL)-1β and tumor necrosis factor (TNF)α production was increased in HL-60 cells under zinc deficiency. Analyses of the chromatin structure demonstrated that the elevated cytokine production was due to increased accessibilities of IL-1β and TNFα promoters in zinc-deficient cells. Moreover, the level of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase-produced ROS was elevated under zinc deficiency, subsequently leading to p38 mitogen-activated protein kinase (MAPK) phosphorylation. The increased activation of p38 MAPK appeared to be necessary for posttranscriptional processes in IL-1β and TNFα synthesis.These data demonstrate that IL-1β and TNFα expression under zinc deficiency is regulated via epigenetic and redox-mediated mechanisms. Assuming an important role of zinc in proinflammatory cytokine regulation, this should encourage research in the use of zinc supplementation for treatment of inflammatory diseases.

IL-10 Regulates Il12b Expression via Histone Deacetylation: Implications for Intestinal Macrophage Homeostasis

To prevent excessive inflammatory responses to commensal microbes, intestinal macrophages, unlike their systemic counterparts, do not produce inflammatory cytokines in response to enteric bacteria. Consequently, loss of macrophage tolerance to the enteric microbiota plays a central role in the pathogenesis of inflammatory bowel diseases. Therefore, we examined whether the hyporesponsive phenotype of intestinal macrophages is programmed by prior exposure to the microbiota. IL-10, but not in vivo exposure to the microbiota, programs intestinal macrophage tolerance, because wild-type (WT) colonic macrophages from germ-free and specific pathogen-free (SPF)-derived mice produce IL-10, but not IL-12 p40, when activated with enteric bacteria. Basal and activated IL-10 expression is mediated through a MyD88-dependent pathway. Conversely, colonic macrophages from germ-free and SPF-derived colitis-prone Il10(-/-) mice demonstrated robust production of IL-12 p40. Next, mechanisms through which IL-10 inhibits Il12b expression were investigated. Although Il12b mRNA was transiently induced in LPS-activated WT bone marrow-derived macrophages (BMDMs), expression persisted in Il10(-/-) BMDMs. There were no differences in nucleosome remodeling, mRNA stability, NF-κB activation, or MAPK signaling to explain prolonged transcription of Il12b in Il10(-/-) BMDMs. However, acetylated histone H4 transiently associated with the Il12b promoter in WT BMDMs, whereas association of these factors was prolonged in Il10(-/-) BMDMs. Experiments using histone deacetylase (HDAC) inhibitors and HDAC3 short hairpin RNA indicate that HDAC3 is involved in histone deacetylation of the Il12b promoter by IL-10. These results suggest that histone deacetylation on the Il12b promoter by HDAC3 mediates homeostatic effects of IL-10 in macrophages.

RIGing the Antibacterial Response

Virus-induced activation of RIG-I–like receptors suppresses Toll-like receptor–dependent antibacterial responses.

No Association Between IL12B Gene Polymorphisms and Graves’ Disease in the Chinese Population

Background. Graves’ disease (GD) is one of the most common autoimmune thyroid disorders and has a striking characteristic of female preponderance. Objective. The main objective of this study was to investigate whether IL12B gene polymorphisms were associated with either GD itself or with gender bias in GD. Methods. GD patients (151 males, 97 females) and 211 healthy control subjects without antithyroid autoantibodies or a family history of autoimmune disorders were recruited for this study. The G/C polymorphism (rs6887695) of the IL12B gene was analyzed using the polymerase chain reaction–restriction fragment length polymorphism, and the deletion and insertion polymorphism (rs41292470) in the IL12B promoter was detected after polyacrylamide gel electrophoresis–silver staining method. Results. There was no significant difference between GD patients and normal subjects, and no differences in frequencies of genotypes or alleles of either polymorphism between male and female GD subjects. Conclusion. We conclude that these IL12B gene polymorphisms were not associated with sex bias in GD and do not confer susceptibility to Graves’ disease (GD) in the Chinese population.

Cross-interference of RLR and TLR signaling pathways modulates antibacterial T cell responses

Although the mechanisms by which innate pathogen-recognition receptors enhance adaptive immune responses are increasingly well understood, whether signaling events from distinct classes of receptors affect each other in modulating adaptive immunity remains unclear. We found here that the activation of cytosolic RIG-I-like receptors (RLRs) resulted in the selective suppression of transcription of the gene encoding the p40 subunit of interleukin 12 (Il12b) that was effectively induced by the activation of Toll-like receptors (TLRs). The RLR-activated transcription factor IRF3 bound dominantly, relative to IRF5, to the Il12b promoter, where it interfered with the TLR-induced assembly of a productive transcription-factor complex. The activation of RLRs in mice attenuated TLR-induced responses of the T helper type 1 cell (T(H)1 cell) and interleukin 17-producing helper T cell (T(H)17 cell) subset types and, consequently, viral infection of mice caused death at sublethal doses of bacterial infection. The innate immune receptor cross-interference we describe may have implications for infection-associated clinical episodes.

Hypercapnia Induces Cleavage and Nuclear Localization of RelB Protein, Giving Insight into CO2 Sensing and Signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-κB pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-κB family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3β or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-κB family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Molecular characterization of a cancer‐related single nucleotide polymorphism in the pro‐inflammatory interleukin‐1B gene

Interleukin-1β is a key pro-inflammatory cytokine that has been associated with chronic inflammation and inflammation-related cancer initiation and progression. There are inter-individual differences in IL1B expression which may be due to single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene. We have previously shown that a SNP located in the promoter of the IL1B gene (the IL1B T-31C SNP) was associated with lung cancer risk. Interestingly, the presence of the C allele was also associated with reduced IL1B expression in normal lung tissue of lung cancer patients. In the present study, we found that differential binding patterns of nuclear proteins to oligonucleotide probes containing the IL1B -31C allele compared to those with the T allele were due to specific binding of the transcription factor Yin Yang 1 (YY1). We further found evidence that specific recruitment of YY1 to the -31C region of the IL1B promoter regulated IL1B gene expression using siRNA directed towards YY1. The results indicate that the presence of a C allele at the -31 position may lead to decreased expression of the IL1B gene due to a specific binding of YY1 in lung epithelial cells. Our study provides functional significance of allelic variation at a single locus in the IL1B promoter and contributes to understanding the regulation of IL1B in inflammation-related carcinogenesis.

Oral administration of recombinant epinecidin-1 protected grouper (Epinephelus coioides) and zebrafish (Danio rerio) from Vibrio vulnificus infection and enhanced immune-related gene expressions

Immunostimulatory effects of the oral administration of the recombinant epinecidin-1 protein from BL21 Escherichia coli (containing the pET28a-epinecidin-1-dsRed plasmid) were studied in grouper (Epinephelus coioides) and zebrafish (Danio rerio). For this purpose, fish were fed diets for 30 days containing the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) at different bacterial numbers (104, 106, 108, and 1010 colony-forming units (cfu) of BL21 E. coli in 50 ml of LB medium) mixed with 50 g of eel powder as fodder. After 30 days of feeding, immune-related gene expressions for bacterial-infection responses and disease resistance against Vibrio vulnificus (204) were determined. The V. vulnificus (204) injected into the fish abdominal cavity mimicked gram-negative bacterial infections in culture ponds. Experimental results assessed whether the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) has up- (or down-) regulation immune-related genes expression. Results indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement significantly enhanced expressions several immune-related genes such as tumor necrosis factor (TNF)-1 in grouper and Toll-like receptor (TLR)4, interleukin (IL)-1β, nitric oxide synthase (NOS)2, and nuclear factor (NF)-κB in zebrafish. After being challenged with V. vulnificus (204) for 24, 48, 72, or 96 h, the percentage mortality was significantly reduced in treated fish, which indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement could bring about downregulation of TNF-1 expression and functioned like an antagonist for binding TLR4, which reduced the signal transduction pathway for inhibiting TNF and IL-1β expressions while reducing binding of the transcription factor, NF-κB, to TNF and the IL-1β promoter region. The experimental results indicated that dietary intake of the recombinant epinecidin-1 protein from BL21 E. coli modulated immune-related gene expressions and disease resistance of grouper and zebrafish after a V. vulnificus (204) infection.

Krüppel-like factor 4 inhibits the expression of interleukin-1 beta in lipopolysaccharide-induced RAW264.7 macrophages

RAW264.7 macrophages and human peripheral blood mononuclear cells were treated with LPS to determine the expression of KLF4 and release of IL-1β. A full-length cDNA or short interference RNA of KLF4 was transfected into RAW264.7 macrophages; the expression and release of IL-1β were analyzed. The transcription and DNA binding activities of KLF4 to the IL-1β promoter were detected further. The results showed LPS treatment resulted in the increase of KLF4 level and IL-1β release; KLF4 overexpression decreased the expression of IL-1β, while KLF4 inhibition increased the expression of IL-1β; overexpression of KLF4 promoted the DNA binding activity of KLF4 to the IL-1β promoter and attenuated the transcription of IL-1β promoter, indicating an important role of KLF4 in regulating expression of IL-1β.

Correlation between interleukin-1 and the obesity of polycystic ovary syndrome

To investigate the correlation between interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1ra) and the obesity of polycystic ovary syndrome (PCOS). (1) From Oct. 2006 to Jan. 2007, 118 PCOS patients were enrolled in this study in Peking University Third Hospital, which were divided into 56 patients in obese PCOS group and 62 patients in non-obese PCOS group according to the WHO International Obesity Task Force Asia-Pacific criteria [body mass index (BMI) 25 kg/m(2)]. The polymorphism of IL-1β gene promoter region, exon-5 and intron 2 of IL-1ra gene were detected by PCR. (2) Twenty-nine obese PCOS patients and 31 non-obese PCOS patients were selected randomizedly serum levels of IL-1β, IL-1ra were measured by ELISA, in the mean time, serum levels of fasting glucose, fasting insulin and the total white blood cell, hypersensitive C-reactive protein levels were measured. (1) Genetic test:the frequency of TT genotype and T allele of IL-1β promoter region (-511) in obese PCOS patients were significantly higher than those in non-obese patients (44.6% vs. 11.3%, 63.4% vs. 39.5%, all P < 0.05). The frequency of IL-1raI/V genotype and V allele of IL-1ra gene were 19.6% and 9.8% in obese PCOS patients, which were significantly higher than those in non-obese group (3.2% and 1.6%, P < 0.05). (2) Serological test:serum level of IL-1β and IL-1ra of (149 ± 36) and (284 ± 97) ng/L in obese PCOS group which were significantly higher than those in non-obese PCOS group [(96 ± 42) and (208 ± 84) ng/L, P < 0.05]. Fasting blood glucose, fasting insulin and hypersensitive C-reactive protein and white blood cell count were (5.1 ± 0.7) mmol/L, (17 ± 9) mU/L, (1.5 ± 0.6) mg/L and (7.0 ± 2.3) × 10(9)/L in obese PCOS group, which were significantly higher than in non-obese PCOS group [(4.9 ± 0.5) mmol/L, (11 ± 8) mU/L, (0.9 ± 0.4) mg/L and (5.9 ± 1.3) × 10(9)/L, P < 0.05]. (3)The correlation between interleukin and BMI: serum levels of IL-1β (r = 0.673) and IL-1ra (r = 0.557) were positively correlated with BMI in PCOS patients (P < 0.05). Inflammatory factors IL-1β and IL-1ra had correlation with obesity of PCOS patients, PCOS patients who carried T allele of IL-1β gene promoter region (-511) and V allele of IL-1ra gene were high risk of obesity.

Iκb‐ζ plays an important role in the ERK‐dependent dysregulation of malaria parasite GPI‐induced IL‐12 expression

Plasmodium falciparum glycosylphosphatidylinositols (GPIs) have been proposed as malaria pathogenic factors based on their ability to induce proinflammatory responses in macrophages and malaria-like symptoms in mice. Parasite GPIs induce the production of inflammatory cytokines by activating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. Importantly, inhibition of the extracellular-signal-regulated kinase (ERK) pathway upregulates a subset of cytokines, including IL-12. We investigated the role of nuclear transcription factor, IκB-ζ, in the GPI-induced dysregulated expression of IL-12 on inhibition of the ERK pathway. GPIs efficiently induced the expression of IκB-ζ in macrophages regardless of whether cells were pretreated or untreated with ERK inhibitors, indicating that ERK has no role in IκB-ζ expression. However, on ERK inhibition followed by stimulation with GPIs, NF-κB binding to Il12b promoter κB site was markedly increased, suggesting that the ERK pathway regulates Il12b transcription. Knockdown of IκB-ζ using siRNA markedly reduced the GPI-induced IL-12 production and abrogated the dysregulated IL-12 production in ERK inhibited cells. Together these results demonstrate that ERK modulates IL-12 expression by regulating IκB-ζ-dependent binding of NF-κB transcription factors to Il12b gene promoter. Additionally, our finding that IκB-ζ can be knocked down efficiently in primary macrophages is valuable for studies aimed at gaining further insights into IκB-ζ function.

Lipopolysaccharide(LPS)-Induction of the HTLV-1 LTR in Monocytes

Abstract 2167The human leukemia virus type 1 (HTLV-1) gene expression is regulated by the viral proteins and various cellular transcription factors. HTLV-1 genome encodes not only structural proteins, but also non-structural proteins such as Tax, a transcriptional activator for STAT5 p12I, and HTLV-1 bZIP factor (HBZ) encoded by the minus strand of the viral genome. The functional analysis of the viral proteins such as Tax has shed light on the pathogenesis of adult T cell leukemia/lymphoma (ATL). Expression of Tax is enhanced by T-cell activation stimuli such as phorbol ester (PMA), phytohemagglutinin (PHA) or sodium butyrate in chronically HTLV-1-infected CD4+T-cells. Transgenic mouse studies with Tax expression under the control of the granzyme B promoter or the proximal Lck promoter showed that disease progression is associated with infiltration of activated T- and inflammatory cells, and dysregulated inflammatory cytokine production. More recently, a transgenic mouse model with Tax expression regulated by the LTR (LTR-Tax) showed that LTR-Tax CD4 positive T-cells are hyper-proliferative and hyper-responsive to immune stimulation and strongly produce Th1-, Th2- and Th17-associated cytokines. In addition, HTLV-1 infection causes inflammatory disease of the central nerve system, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as ATL. Aberrant cytokine gene expression is the hallmark of HTLV-1-associated diseases.HTLV-1 infection is widely distributed among mammalian cells. We previously demonstrated that Tax transactivates the promoter of human proIL-1β gene (IL1B) gene through association with two transcription factors, NF-IL6 (C/EBPβ) and Spi-1 (PU.1) in monocytes. Tax synergized with lipopolysaccharide (LPS) to induce IL1B promoter activity. Spi-1 is an Ets family protein restricted in expression to monocytes/myelocytes, B cells, mast cells and erythrocyte stem cells, while NF-IL6 is widely expressed. LPS, a component of the gram-negative bacterial cell wall involved in the activation of monocytes, binds to TLR4, leading to activation of TRAF6, IRAK and MyD88. We now extend these studies to elucidate roles of LPS and Tax on HTLV-1 LTR promoter regulation in monocytes. When HTLV-1 LTR reporter was transfected into THP-1 monocytic cells, LPS dose-dependently induced HTLV-1 LTR. The mid-LTR of the HTLV-1 gene possesses three potential Ets binding elements centered on a GGAA motif (PuB1, pets and PuB2). Elf-1 has been shown to be the predominant protein binding to the HTLV-1 Ets sites in Jurkat and peripheral blood T-cells. In the present study, mutation of the Ets sites, especially pets and PuB2 caused significant inhibition of LPS-induced LTR activity in THP-1 cells. EMSA studies using THP-1 nuclear extracts showed binding of Spi-1 to the HTLV-1 Ets sites in THP-1 cells. Anti-Spi-1 Ab, but not anti-Elf-1 Ab or anti-ets-1 Ab supershifted the complex generated by THP-1 nuclear extract and HTLV-1 LTR Ets site. However, when migration pattern of the complex was compared with recombinant Spi-1 in vitro translated in a reticulocyte lysate system, the THP-1 complex migrated slower than recombinant Spi-1 protein. In this regard, anti-IRF-8 Ab further recognized the slow complex. Several reports recently showed that IRF-8 functions as a heterodimeric complex with spi-1 for expression of relevant genes.On the other hand, when Tax expression vector was cotransfected into THP-1 cells along with HTLV-1 LTR reporter, Tax synergized with LPS to activate LTR. Spi-1 protein has three independent transcriptional activation domains (TAD); a TBP binding region, a Q domain, and a PEST region. GST pull-down studies using GST-Tax and 35S-labeled recombinant Spi-1 revealed that mutant Spi-1 lacking the TADs still retains the ability to interact with Tax. In contrast to THP-1 cells, Jurkat T-cells showed only a marginal increase in IL1B promoter activity following Tax expression. Mutations of the Spi-1 binding site in the IL1B promoter did not affect Tax-induced activity in Jurkat cells. Any factors did not bind to the IL1B Spi-1 site in Jurkat cells. Thus, our data suggest that LPS cooperates with Tax to activate the viral and various cellular genes in HTLV-1-infected monocytes. Spi-1 is a key player in the monocyte-specific gene regulation in HTLV-1 infection. Disclosures:No relevant conflicts of interest to declare.

130-P Association of single nucleotide polymorphism in the INF-γ, IL1-β and TGF-β gene promoter with polyoma virus BK virus reactivation after kidney transplantation

130-P Association of single nucleotide polymorphism in the INF-γ, IL1-β and TGF-β gene promoter with polyoma virus BK virus reactivation after kidney transplantation

SIRT1 inhibits IL-1β mRNA transcription in lipopolysaccharide tolerant THP-1 cells

To explore the role of silent information regulation 2 homolog 1 (SIRT1) in the regulation of IL-1β mRNA transcription in lipopolysaccharide (LPS) tolerant THP-1 cells. THP-1 human promonocyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype was used. Chromatin immunoprecipitation assay (ChIP) and real-time PCR were applied to quantify the binding of SIRT1 and histone H3 lys9/H4 lys16 acetylation to IL-1β promoter. IL-1β mRNA transcription was studied after knocking down the SIRT1. The binding of SIRT1 to IL-1β promoter increased about 5 times in tolerant THP-1 cells (P < 0.05), which was accompanied by the low level of histone H3 lys9/H4 lys16 acetylation (P < 0.05, compared with normal cells). Knocking-down of SIRT1 increased the transcription of IL-1β mRNA up to the level of 68% of normal cells (P < 0.05), which was accompanied by the increase of histone H3 lys9/H4 lys16 acetylation (P < 0.05). However, there was no significant difference of p65 lys310 acetylation between normal and tolerant cells. SIRT1 inhibited the IL-1β mRNA transcription in tolerant THP-1 cells but had not related to p65 lys310 acetylation. However, it was related to IL-1β promoter acetylation.

NFIL3 Is a Regulator of IL-12 p40 in Macrophages and Mucosal Immunity

Regulation of innate inflammatory responses against the enteric microbiota is essential for the maintenance of intestinal homeostasis. Key participants in innate defenses are macrophages. In these studies, the basic leucine zipper protein, NFIL3, is identified as a regulatory transcription factor in macrophages, controlling IL-12 p40 production induced by bacterial products and the enteric microbiota. Exposure to commensal bacteria and bacterial products induced NFIL3 in cultured macrophages and in vivo. The Il12b promoter has a putative DNA-binding element for NFIL3. Basal and LPS-activated NFIL3 binding to this site was confirmed by chromatin immunoprecipitation. LPS-induced Il12b promoter activity was inhibited by NFIL3 expression and augmented by NFIL3-short hairpin RNA in an Il12b-bacterial artificial chromosome-GFP reporter macrophage line. Il12b inhibition by NFIL3 does not require IL-10 expression, but a C-terminal minimal repression domain is necessary. Furthermore, colonic CD11b(+) lamina propria mononuclear cells from Nfil3(-/-) mice spontaneously expressed Il12b mRNA. Importantly, lower expression of NFIL3 was observed in CD14(+) lamina propria mononuclear cells from Crohn's disease and ulcerative colitis patients compared with control subjects. Likewise, no induction of Nfil3 was observed in colons of colitis-prone Il10(-/-) mice transitioned from germ-free to a conventional microbiota. In conclusion, these experiments characterize NFIL3 as an Il12b transcriptional inhibitor. Interactions of macrophages with the enteric microbiota induce NFIL3 to limit their inflammatory capacity. Furthermore, altered intestinal NFIL3 expression may have implications for the pathogenesis of experimental and human inflammatory bowel diseases.