IL-6 Promoter Research Articles

Adenovirus 7 Induces Interlukin-6 Expression in Human Airway Epithelial Cells via p38/NF-κB Signaling Pathway.

Human Adenovirus (AdV) infection is very common and usually has a significant impact on children. AdV-induced inflammation is believed to be one of the main causes of severe symptoms. However, an inflammatory response profile in the airway in AdV-infected children is still lacking, and the mechanism underlying AdV-induced inflammation in the airway is also poorly understood. In the current study, we determined the expression of a panel of inflammation cytokines in the airway samples from AdV 7 infected children and further investigated the molecular mechanism underlying AdV 7-induced cytokine expression. Our results showed that eight out of 13 tested inflammatory cytokines were significantly increased in nasal washes of AdV 7-infected children comparing to healthy control, with IL-6 showing the highest enhancement. AdV 7 infection of bronchial epithelial cell line and primary airway epithelial cells confirmed that AdV 7 increased IL-6 mRNA and protein expression in an infection dose-dependent manner. Promoter analysis revealed that AdV 7 infection transactivated IL-6 promoter and a NF-κB binding site in IL-6 promoter was involved in the transactivation. Further analysis showed that upon AdV 7 infection, NF-κB p65 was phosphorylated and translocated into nucleus and bound onto IL-6 promoter. Signaling pathway analysis revealed that p38/NF-κB pathway was involved in AdV 7 infection induced IL-6 elevation. Taken together, our study shows that AdV 7 infection triggers the expression of a range of inflammatory cytokines including IL-6 in the airway of infected children, and AdV 7 enhances IL-6 expression by transactivating IL-6 promoter via p38/NF-κB signaling pathway. Findings of our current study have provided more information toward a better understanding of AdV-induced airway inflammation, which might also benefit the development of intervention strategies.

Maternal exposure to soy diet reduces atheroma in hyperlipidemic F1 offspring mice by promoting macrophage and T cell anti-inflammatory responses

Background and aimsMaternal hypercholesterolemia has been implicated in earlier onset of atherosclerotic lesions in neonatal offspring. In this study, we investigated whether maternal exposure to soy protein isolate (SPI) diet attenuated the progression of atherosclerosis in F1 offspring. MethodPregnant apolipoprotein E knockout (Apoe−/−) female mice were fed SPI diet until postnatal day 21 (PND21) of the offspring (SPI-offspring). SPI-offspring were switched at PND21 to casein (CAS) diet until PND140. Mice fed CAS throughout their lifetime (gestation to adulthood) were used as controls (CAS-offspring). ResultsAtherosclerotic lesions in the aortic sinuses were reduced in SPI-offspring compared with CAS-offspring. Total serum cholesterol levels in CAS-offspring or dams were comparable to levels in their SPI-counterparts, suggesting that alternative mechanisms contributed to the athero-protective effect of maternal SPI diet. Aortic VCAM-1, MCP-1, and TNF-α mRNA and protein expression, and expression of macrophage pro-inflammatory cytokines was reduced in SPI-offspring. Interestingly, CD4+ T cells from SPI-offspring showed reduced IFN-γ expression (Th1), while the expression of IL-10 (Th2/Treg), and IL-13 (Th2) was increased. DNA methylation analyses revealed that anti-inflammatory T cell-associated Gata3 and Il13 promoter regions were hypomethylated in SPI-offspring. These findings suggest that anti-inflammatory macrophage and T cell response may have contributed to the athero-protective effect in SPI-offspring. ConclusionsOur findings demonstrate that gestational and lactational soy diet exposure inhibits susceptibility to atherosclerotic lesion formation by promoting anti-inflammatory responses by macrophages and T cells.

Cold stress promotes IL-33 expression in intestinal epithelial cells to facilitate food allergy development

BackgroundThe causative factors and pathogenesis of food allergy (FA) is not fully understood yet. Cold stress (CS) occurs frequently in human life that influences physiological activities in the body. In this study, we aimed to investigate the chronic CS (CS) effects on promoting the expression of IL-33 in intestinal epithelial cells. MethodsCS was carried out by placing mice at 4 °C for 1 h daily for 7 consecutive days. We developed a mouse model used to test the effects of CS on the FA development. ResultsWe found that, similar to conventional FA mouse model, CS induced the core body temperature to drop markedly in mice, increased intestinal epithelial barrier permeability and facilitated FA development. CS promoted interleukin (IL)-33 expression in intestinal epithelial cells through the adrenocorticotropic hormone (ACTH)/cortisol axis and via inducing the Il33 promoter methylation. CS facilitated the FA development in mice, that could be blocked by depletion of IL-33 expression in intestinal epithelial cells. ConclusionsCS induces IL-33 expression in intestinal epithelial cells to promote Th2 polarization in the intestinal tissues and facilitates FA development.

Cutting Edge: Inhibition of Glycogen Synthase Kinase 3 Activity Induces the Generation and Enhanced Suppressive Function of Human IL-10+ FOXP3+-Induced Regulatory T Cells.

IL-10 is critical for Foxp3+ regulatory T cell (Tregs)-mediated immune suppression, but how to efficiently upregulate IL-10 production in Tregs remains unclear. In this article, we show that human IL-10+ FOXP3+-induced regulatory T cell (iTreg) generation can be dramatically promoted by inhibiting GSK3 activity. IL-10+ FOXP3+ iTregs induced by GSK3 inhibition exhibit classical features of immune-suppressive T cells. We further demonstrate that IL-10+ iTregs exhibit enhanced suppressive function in both IL-10-dependent and -independent manners. The enhanced suppressive function of IL-10+ Tregs is not due to a single factor such as IL-10, although IL-10 may mediate this enhanced suppressive function to some extent. Mechanistically, the increased transcriptional activity of IL-10 promoter and the enhanced expression of C-Maf and BLIMP1 coordinately facilitate IL-10 expression in human iTregs under GSK3 inhibition. Our study provides a new strategy to generate human immune-suppressive IL-10+ FOXP3+ Tregs for immunotherapies.

Polymorphisms of IL-4 and IL-4R are associated to some demographic characteristics of differentiated thyroid cancer patients but are not determinants of risk in the Brazilian population.

IL-4 is known to present abnormal expression in thyroid tumors and SNPs in the IL-4 and its receptor IL-4R genes are associated to risk and mortality of various types of cancer. In order to evaluate their role in differentiated thyroid cancer (DTC), we investigated genetic frequencies of two IL-4 promoter SNPs (rs2070874 C>T, rs2243250 C>T) and four non-synonymous SNPs of the IL-4R gene (rs1805010 A>G, rs1805012 C>T, rs1805013 C>T, rs1801275 A>G) in 300 DTC patients matched to 300 controls. All patients were managed according to current guidelines and followed-up for a period of 12-252 months (69.20 ± 52.70 months). Although none of the six investigated SNPs showed association with risk of DTC, rs1805010 was associated with age of diagnosis and the SNPs rs1805012 and rs1801275 were associated to gender. Further, in-silico analysis showed that all these three SNPs were able to cause decreased stability of the protein. We were not able to demonstrate any other association to clinical features of aggressiveness or to patients' prognosis. These findings indicate that although genetic variants in IL-4 and IL-4R do not influence the risk or outcome of DTC patients, their influence on the behavior of thyroid tumors deserves further investigation.

Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity

Innate lymphoid cells (ILCs) and CD4+ T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4+ T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4+ T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4+ T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4+ T cells and ILCs to maintain intestinal homeostasis.

TAZ-regulated expression of IL-8 is involved in chemoresistance of hepatocellular carcinoma cells

Chemotherapy resistance is one of the major challenges for the treatment of hepatocellular carcinoma (HCC). In order to investigate the mechanisms involved in chemoresistance of HCC, we established cisplatin (CDDP) and doxorubicin (Dox) resistant HCC cells. The expression of transcriptional coactivator with PDZ-binding motif (TAZ), one of the major downstream effectors of Hippo pathway, was upregulated in chemoresistant HCC cells. Targeted inhibition of TAZ via its siRNAs can restore CDDP and Dox sensitivity of chemoresistant HCC cells. The upregulation of TAZ increased the expression of IL-8 in HCC/CDDP and HCC/Dox cells. Recombinant IL-8 (rIL-8) antagonized the increased chemosensitivity mediated by TAZ knockdown. Mechanistically, TAZ can directly bind with the promoter of IL-8 to activate its transcription in chemoresistant HCC cells. Collectively, our data showed that TAZ-regulated expression of IL-8 was involved in chemoresistance of HCC cells. It indicated that targeted inhibition of TAZ/IL-8 axis might be helpful to improve chemotherapy efficiency for HCC.

Ubiquitin ligase TRUSS augments the expression of interleukin-10 via proteasomal processing of NF-κB1/p105 to NF-κB/p50

The NF-κB/Rel family of transcription factors that play critical roles in a variety of cellular processes. Their production in the cell and physiological activation are tightly regulated. The proteasomal processing of inactive NF-κB1/p105 to active p50, with an anti-inflammatory role, is not well characterized. Here we show that ubiquitin ligase TRUSS is a mediator of transcriptional activation of anti-inflammatory cytokine IL-10 gene. Enforced expression of TRUSS led to enhanced IL-10 expression that could be inhibited in the presence of chemical inhibitors of NF-κB [BAY11–7082] and PI3K/Akt [LY249002] or after p65 overexpression. p50 was actively recruited on IL10 promoter in the presence of TRUSS but competed by p65 for binding. TRUSS facilitated the ubiquitination of NF-κB1/p105 and promoted its proteolytic processing to generate excess of p50. Our immune-histochemical studies confirmed enhanced expression of p105/p50 in the human HCC tumors. Further, the hepatic tumors of HCC patient as well as transgenic mice showed decreased levels of p50 as well as TRUSS and accumulation of p105. Thus, enhanced expression of IL-10 gene in the presence of TRUSS and regulation of NF-κB1/p105 processing could be an important regulatory mechanism for inflammatory response and tumorgenic transformation.

A reporter gene assay for measuring the bioactivity of anti-LAG-3 therapeutic antibodies.

Although enormous success has been achieved with anti-PD-1/PD-L1 and anti-CTLA-4 monoclonal antibodies (mAbs), their unsatisfactory response rate in cancer patients has been driving the research and development of novel immune checkpoint inhibitors (ICIs). Anti-LAG-3 mAbs, as one of the most promising candidates, are now being tested for various human cancers at different stages of clinical trials. Here, we describe the development and validation of a reporter gene assay (RGA) to measure the bioactivity of anti-LAG-3 mAbs. We established the bioassay based on parental Raji cells and a Jurkat cell line stably transfected with human LAG-3 gene and luciferase reporter elements controlled by nuclear factor of activated T cell (NFAT) from the IL-2 promoter. After optimization of key parameters, the established RGA showed excellent precision, specificity, accuracy, and stability. The mechanism of action (MOA) relatedness and the excellent assay performance make the RGA suitable for the characterization, lot release, and stability test of anti-LAG-3 mAbs.

Loss of Mef2D function enhances TLR induced IL-10 production in macrophages

Mef2 transcription factors comprise a family of four different isoforms that regulate a number of processes including neuronal and muscle development. While roles for Mef2C and Mef2D have been described in B-cell development their role in immunity has not been extensively studied. In innate immune cells such as macrophages, TLRs drive the production of both pro- and anti-inflammatory cytokines. IL-10 is an important anti-inflammatory cytokine produced by macrophages and it establishes an autocrine feedback loop to inhibit pro-inflammatory cytokine production. We show here that macrophages from Mef2D knockout mice have elevated levels of IL-10 mRNA induction compared with wild-type cells following LPS stimulation. The secretion of IL-10 was also higher from Mef2D knockout macrophages and this correlated to a reduction in the secretion of TNF, IL-6 and IL-12p40. The use of an IL-10 neutralising antibody showed that this reduction in pro-inflammatory cytokine production in the Mef2D knockouts was IL-10 dependent. As the IL-10 promoter has previously been reported to contain a potential binding site for Mef2D, it is possible that the binding of other Mef2 isoforms in the absence of Mef2D may result in a higher activation of the IL-10 gene. Further studies with compound Mef2 isoforms would be required to address this. We also show that Mef2D is highly expressed in the thymus, but that loss of Mef2D does not affect thymic T-cell development or the production of IFNγ from CD8 T cells.

The Influence of −174 G/C Interleukin 6 Promoter Gene Polymorphism to Interleukin 6 Concentration in the End Stage Renal Disease Patients with Dialysis: A Single-center Experience in Indonesia

BACKGROUND: Chronic kidney disease (CKD) is classified as a multifactorial disease as a combination of genetic and environmental factors that affect the onset and progression of end-stage renal disease (ESRD). In the last decades are recognized that inflammation, where the critical modulator is cytokines, can occur before the onset of kidney disease and can be a causative factor in the development of CKD. Interleukin (IL)-6 has several polymorphisms in the promoter region, such as 174 G-C, 634 C-G, 572 G-C, and 597 G-A. G/C single nucleotide polymorphism of the IL-6 gene at position 174 in the promoter region is reported to affect the level of IL-6 expression. Unfortunately, there is a lack of data about the genotype frequencies of -174 G/C IL-6 promoter gene polymorphism in Indonesian with ESRD.
 AIM: This study aimed to analyze whether −174 G/C IL-6 promoter gene polymorphism influences the concentration of IL-6 in ESRD patients with dialysis in Indonesia.
 METHODS: We recruited 95 outpatients who were undergoing regular hemodialysis for ≥3 months at the Rasyida Renal Hospital, Medan, in this cross-sectional study. IL-6 genotype polymorphism was analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The enzyme-linked immunosorbent assay method was performed to assess the quantitative IL-6 serum.
 RESULTS: PCR-RFLP examination showed the frequency distribution of the IL-6 genotype -174 G/C gene; 72 (75.8%) respondents had GG genotypes; 22.1% of respondents had the CG genotype, and (2.1%) had the CC genotypes. Patients with the CC genotype were statistically significant to have higher IL-6 concentration compared to other genotypes (p < 0.001). Likewise, with diabetic patients, statistics showed higher IL-6 concentration compared to non-diabetics patients (p < 0.001).
 CONCLUSION: This is the first study showing that -174 G/C IL-6 promoter gene polymorphism influences the IL-6 concentrations in ESRD patients with dialysis in Indonesia. Multicenter studies are needed to validate these findings.

Negative regulation of IL-8 in human astrocytes depends on β-catenin while positive regulation is mediated by TCFs/LEF/ATF2 interaction

Expression of cytokines/chemokines is tightly regulated at the transcription level. This is crucial in the central nervous system to maintain neuroimmune homeostasis. IL-8 a chemoattractant, which recruits neutrophils, T cells, and basophils into the brain in response to inflammation and/or injury is secreted predominantly by neurons, microglia, and astrocytes. Here, we investigated the mechanism by which astrocytes regulate IL-8 expression. We demonstrate that while β-catenin negatively regulated IL-8 transcription, its canonical transcriptional partners, members of the TCF/LEF transcription factors (TCF1, TCF3, TCF4 and LEF1) and Activating transcription factor 2 (ATF2) positively regulated IL-8 transcription. We further identified a putative TCF/LEF binding site at −175nt close to the minimal transcription region on the IL-8 promoter, mutation of which caused a significant reduction in IL-8 promoter activity. Chromatin immunoprecipitation demonstrated binding of TCF1, TCF4, LEF1 and ATF2 on the IL-8 promoter suggesting that TCFs/LEF partner with ATF2 to induce IL-8 transcription. These findings demonstrate a novel role for β-catenin in suppression of IL-8 expression and for TCFs/LEF/ATF2 in inducing IL-8. These findings reveal a unique mechanism by which astrocytes tightly regulate IL-8 expression.

Abstract 926: Binding of the novel inhibitory receptor KIR-X to its ligand HHLA2 negatively regulates T cell activation

Abstract Cancer immunotherapies blocking immuno-inhibitory pathways have become a successful treatment method in the last decade. HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) is a B7 family member and ligand that is often expressed on cancer cells and antigen presenting cells. HHLA2 has the ability to contribute to both T cell stimulation and inhibition. Previous results have shown that HHLA2 binding to TMIGD2 induces T cell activation, however, the inhibitory receptor was unknown. We identified KIR-X as an additional receptor for HHLA2. In order to test the functional consequence of HHLA2 binding to KIR-X, we set up a KIR-X receptor transfected Jurkat T cell model. Jurkat cells transfected with KIR-X or TMIGD2 and luciferase under the control of an NFAT-response element or IL-2 promoter were stimulated with CHO cells expressing scFv anti-human CD3 +/- HHLA2. The bioluminescence produced by the firefly luciferase reporter reflects NFAT or IL2 transcriptional activation. We confirmed that HHLA2 binding to TMIGD2 stimulates T cells in this Jurkat T cell model as we saw an increase in NFAT mediated transcription of luciferase. The goal of this project was to first test the immunological effect of HHLA2 binding to KIR-X on T cells, and second, test a panel of candidate therapeutic antibodies directed against KIR-X or HHLA2 for their ability to reverse effects observed in Jurkat cells. We found that HHLA2 binding to KIR-X inhibited a T cell antigen receptor/CD28 signal. Next, the antibodies were tested for their capacity to reverse HHLA2 mediated effects on T cell activation. I tested HHLA2 antibodies which block binding to KIR-X, TMIGD2 or both as well as KIR-X antibodies that block binding to HHLA2. I also tested antibodies known to not block binding to ligand. My results show that KIR-X antibodies 2F11 and 1G7 and the HHLA2 antibodies 2G2, 1C8, 6F10, and 2C4 enhanced activation of KIR-X Jurkat cells, consistent with the blockade of a KIR-X mediated inhibitory signal. The HHLA2 antibody 6F10 also reduced TMIGD2 Jurkat T cell activation while the 2C4 antibody did not, consistent with their ability or inability to block TMIGD2 binding, respectively. We found that antibodies that did not block the KIR-X/HHLA2 interaction did not have a functional consequence in the Jurkat T cell luciferase assay. Therefore, these assays showed that there was good correlation between blockade of receptor/ligand interactions and effects on T cell activation and that the binding sites on HHLA2 for KIR-X and TMIGD2 were close but distinguishable. We believe that blocking the KIR-X inhibitory receptor will be valuable for increasing T cell activation in cancer, while blocking TMIGD2 has potential to alleviate autoimmune disease.This work was supported by the Dana-Farber/Harvard Cancer Center Kidney Cancer SPORE P50CA101942, (GJF and KMM), Department of Defense Early Investigator Idea Development Award W81XWH1810500 (KMM), Kidney Cancer Association Advanced Discovery Award (KMM, GJF), and P01AI056299 (GJF). RSB was supported by NIH R01 CA196996. Citation Format: Alyssa N. Klee, Kathleen M. Mahoney, Antonio R. Arulanandam, Rupal Bhatt, Baogong Zhu, Gordon J. Freeman. Binding of the novel inhibitory receptor KIR-X to its ligand HHLA2 negatively regulates T cell activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 926.

Abstract 2600: Development of TCR-stimulating 300.19 and 300.19-hB7-1 cells using modified anti-CD3 scFv constructs

Abstract To facilitate immunological assays which require T cell activation, we made two membrane anchored anti-CD3 single chain fragment variable (scFv) constructs: one named Steinberger (St, which includes the hTIM-3 mucin domain as linker and medium strength human PGK promoter) and the other named Riley (Ri, which includes hCD8 stem as linker and weaker human UBC promoter). Isolating T cells activated using adherent CHO-TCR-Activator cells is labor intensive, results in variable amounts of CHO cells, and makes it difficult to examine early time points after TCR activation. To combat this issue, we established stable expressing cell lines by transfecting St and Ri into 300.19 cells (Abelson leukemia virus transformed murine pre-B cell line that grows as non-adherent single cells) as well as 300.19 cells stably transfected with hB7-1 and selected 300.19-hB7-1-St, 300.19-hB7-1-Ri, 300.19-St, and 300.19-Ri using the following procedures. We electroporated 300.19 and 300.19-hB7-1 cells with three different amounts of St and Ri linearized plasmid: 0.5µg, 5µg, and 50µg. After selection for resistant cells with hygromycin and puromycin, we stained each cell line with goat anti-mouse IgG (Fab2-specific)-Alexa647 and sorted into three levels of scFv expression (high, medium, and low) and cultured single cell clones. We tested the T cell-stimulatory capacities of our anti-CD3 cells in a Jurkat T cell model through assay of increased IL-2 promoter mediated transcription of luciferase compared to control and ranked the stimulatory capacity of each clone. We found that T cell ligation by 300-hB7-1-Ri and 300-hB7-1-St cells induced the highest IL-2 promoter luciferase reporter activity while the 300-Ri and 300-St induced significant but lesser amounts. Surprisingly, Ri construct cells were generally stronger than the St clones though the literature says the PGK promoter (St plasmid) is stronger than the UBC promoter (Ri plasmid). This difference in stimulatory capacity may be due to the Ri construct (scFv-CD8 linker) being more stable than the St construct (scFv-hTIM3 linker) or to different promoter activity in mouse pre-B cells. Our results also suggest that the amount of DNA transfected may be predictive of construct expression level. The TCR-engaging capacity of our cell lines makes them a widely applicable tool in cancer immunotherapy research. These cells improve the TCR-engaging assay technique by avoiding the use of adherent CHO-TCR-Activator cells and/or CD28 antibodies. Collectively, our anti-CD3 scFv construct-expressing cells may enable researchers to better understand the cascading processes involved in T cell proliferation and differentiation into effector T cells, assess the dynamics of co-stimulatory and co-inhibitory receptors under various controlled levels of T cell stimulation, and understand tumor microenvironments and cytokine production with and without CD28 co-stimulation. Citation Format: Melissa T. Bu, Baogong Zhu, Gordon J. Freeman. Development of TCR-stimulating 300.19 and 300.19-hB7-1 cells using modified anti-CD3 scFv constructs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2600.

Abstract 108: Elucidation of MLK3-induced signaling pathways in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal (GI) malignancies and a major cause of death worldwide, with limited therapeutic options. Evidence suggest that Mixed Lineage Kinase 3 (MLK3), a member of MAPK pathway, is a key regulator of tumorigenesis and metastasis in different cancer-types. Previous studies from our laboratory indicated that, knocking out MLK3 in mice alleviates hepatocarcinogenesis when treated with Diethylnitrosamine (DEN) in combination with phenobarbital (PB). While these suggested an involvement of MLK3 in HCC progression, the detailed mechanism by which this is accomplished is still unclear. To address the mechanism of MLK3-induced HCC, liver cancer cells stably overexpressing MLK3 wildtype (WT) or kinase dead (KA) in a Doxycycline (Dox)-inducible manner, were established. Our RT2 Profiler PCR array analysis with a cytokine and chemokine array (PAHS-150Z from QIAGEN) showed that MLK3-WT overexpression can significantly upregulate mRNA levels of inflammatory cytokines, such as IL11, BMP6, TNFα and IL6, which were absent with MLK3-KA overexpression. Q-PCR and ELISA analysis confirmed the increased IL11 mRNA levels and IL11 release following MLK3 overexpression. Interestingly, STAT3, the downstream signal transducer of Janus kinases (JAKs), which are activated by cytokine signals was also activated by MLK3 overexpression, suggesting that MLK3 may have a novel crosstalk with JAK-STAT3 signaling pathway. To explore this possibility, cells were treated with inhibitors of JAKs (AZD1480 and Ruxolitinib) in the presence of Dox (to induce MLK3), which significantly decreased STAT3 activation as well as IL11 release, suggesting the involvement of JAKs in MLK3-induced IL11 signaling. Furthermore, we also demonstrated a significant reduction of IL11 release following treatment with the JNK inhibitor, SP600125. To elucidate the detailed mecahnism and identify potential transcription factors that mediate the effect of MLK3 overexpression on IL11 promoter activity, deletion mutants of pGL4.16 IL11-luciferase reporter (-877/+169), (-249/+169), (-153/+169) and (-78/+169) were generated. Luciferase assays confirmed that MLK3-WT overexpression indeed increased IL11 promoter reporter activity, which required the fragment from -153 to -78. More studies are currently undergoing with site-directed mutagenesis to explore the detailed mechanism. In addition, our studies demonstrated an induction of epithelial to mesenchymal transition (EMT) during later stages of Dox treatment, suggesting that MLK3-WT overexpression may regulate EMT, cell migration and invasion. These results indicated that both JAKs, and MLK3/JNK signaling converge to mediate IL11 release in HCC, but the mechanism of crosstalk between MLK3 and JAK-STAT3 signaling pathway is still unclear. Taken together, our current studies indicate that overexpression of MLK3 may not only activate JNK/c-Jun pathway, but also regulate JAK-STAT3 signaling pathway to induce IL11 release and downstream signaling in hepatocellular carcinoma. Citation Format: Rong Ke, Navin Viswakarma, Kanchan Vishnoi, Sandeep Kumar, Ajay Rana, Basabi Rana. Elucidation of MLK3-induced signaling pathways in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 108.

Association between TNFA, IL10 and IL18 promoter gene variants and cognitive functions in patients with relapsing-remitting multiple sclerosis

ObjectivesTo investigate the relationship between TNFA-308G > A, IL10–1082A > G, IL18-607C > A, and cognitive functioning in relapsing-remitting multiple sclerosis (RRMS). ResultsIn the patients' group: AG genotype of TNFA-308G > A was associated with higher serum tumor necrosis factor-alpha (TNF-alpha) than GG genotype, and higher TNF-alpha levels correlated with poorer results on Symbol Digit Modalities Test; CC genotype of IL18-607C > A was related to lower score on Isaacs test, compared to AC variant; AA genotype of IL10–1082A > G was associated with abnormally low results on Paced Auditory Series Addition Test. ConclusionsTNFA-308G > A, IL10–1082A > G and IL18-607C > A gene variants may be associated with impaired cognitive functions in RRMS patients.

SETD1 and NF-κB Regulate Periodontal Inflammation through H3K4 Trimethylation

The inflammatory response to periodontal pathogens is dynamically controlled by the chromatin state on inflammatory gene promoters. In the present study, we have focused on the effect of the methyltransferase SETD1B on histone H3 lysine K4 (H3K4) histone trimethylation on inflammatory gene promoters. Experiments were based on 3 model systems: 1) an in vitro periodontal ligament (PDL) cell culture model for the study of SETD1 function as it relates to histone methylation and inflammatory gene expression using Porphyromonas gingivalis lipopolysaccharide (LPS) as a pathogen, 2) a subcutaneous implantation model to determine the relationship between SETD1 and nuclear factor κB (NF-κB) through its activation inhibitor BOT-64, and 3) a mouse periodontitis model to test whether the NF-κB activation inhibitor BOT-64 reverses the inflammatory tissue destruction associated with periodontal disease. In our PDL progenitor cell culture model, P. gingivalis LPS increased H3K4me3 histone methylation on IL-1β, IL-6, and MMP2 gene promoters, while SETD1B inhibition decreased H3K4me3 enrichment and inflammatory gene expression in LPS-treated PDL cells. LPS also increased SETD1 nuclear localization in a p65-dependent fashion and the nuclear translocation of p65 as mediated through SETD1, suggestive of a synergistic effect between SETD1 and p65 in the modulation of inflammation. Confirming the role of SETD1 in p65-mediated periodontal inflammation, BOT-64 reduced the number of SETD1-positive cells in inflamed periodontal tissues, restored periodontal tissue integrity, and enhanced osteogenesis in a periodontal inflammation model in vivo. Together, these results have established the histone lysine methyltransferase SETD1 as a key factor in the opening of the chromatin on inflammatory gene promoters through histone H3K4 trimethylation. Our studies also confirmed the role of BOT-64 as a potent molecular therapeutic for the restoration of periodontal health through the inhibition of NF-κB activity and the amelioration of SETD1-induced chromatin relaxation.

Histone deacetylase 5 regulates interleukin 6 secretion and insulin action in skeletal muscle

ObjectivePhysical exercise training is associated with increased glucose uptake in skeletal muscle and improved glycemic control. HDAC5, a class IIa histone deacetylase, has been shown to regulate transcription of the insulin-responsive glucose transporter GLUT4 in cultured muscle cells. In this study, we analyzed the contribution of HDAC5 to the transcriptional network in muscle and the beneficial effect of muscle contraction and regular exercise on glucose metabolism. MethodsHDAC5 knockout mice (KO) and wild-type (WT) littermates were trained for 8 weeks on treadmills, metabolically phenotyped, and compared to sedentary controls. Hdac5-deficient skeletal muscle and cultured Hdac5-knockdown (KD) C2C12 myotubes were utilized for studies of gene expression and glucose metabolism. Chromatin immunoprecipitation (ChIP) studies were conducted to analyze Il6 promoter activity using H3K9ac and HDAC5 antibodies. ResultsGlobal transcriptome analysis of Hdac5 KO gastrocnemius muscle demonstrated activation of the IL-6 signaling pathway. Accordingly, knockdown of Hdac5 in C2C12 myotubes led to higher expression and secretion of IL-6 with enhanced insulin-stimulated activation of AKT that was reversed by Il6 knockdown. Moreover, Hdac5-deficient myotubes exhibited enhanced glucose uptake, glycogen synthesis, and elevated expression levels of the glucose transporter GLUT4. Transcription of Il6 was further enhanced by electrical pulse stimulation in Hdac5-deficient C2C12 myotubes. ChIP identified a ∼1 kb fragment of the Il6 promoter that interacts with HDAC5 and demonstrated increased activation-associated histone marker AcH3K9 in Hdac5-deficient muscle cells. Exercise intervention of HDAC5 KO mice resulted in improved systemic glucose tolerance as compared to WT controls. ConclusionsWe identified HDAC5 as a negative epigenetic regulator of IL-6 synthesis and release in skeletal muscle. HDAC5 may exert beneficial effects through two different mechanisms, transcriptional control of genes required for glucose disposal and utilization, and HDAC5-dependent IL-6 signaling cross-talk to improve glucose uptake in muscle in response to exercise.

Association of cytokine gene polymorphisms with susceptibility to cutaneous leishmaniasis in a Turkish population.

The objective of this study was to determine the association of TNF-α -308 G/A, IFN-γ +874T/A, IL-12B+1188 A/C, IL-10 -1082 G/A and IL-4 -590 C/T polymorphisms with susceptibility to CL. A total of 55 CL patients and 110 controls from Sanlıurfa province of Turkey were included to this study. Polymorphisms were genotyped by 'polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)' and 'amplification refractory mutation system-PCR (ARMS-PCR)' methods. A statistically significant difference was noted in the allele (P<.001, P=.002) and genotype (P<.001, P=.001,) frequencies of TNF-α -308 G/A and IL-4 -590 C/T, respectively. TNF-α 308 GG versus GA genotype (OR=19.556 [95% CI 8.310-46.019] P<.001), GG versus GA+AA genotype (OR=20.444 [95% CI 8.707-48.004] P<.001) and G versus A allele (OR=6.968 [95% CI 3.903-12.440] P<.001) revealed significant association with CL. IL-4 -590 CC versus TT+CT genotype (OR=2.049 [95% CI 1.025-4.096], P=.041) and C versus T allele (OR=2.441 [95% CI 1.355-4.396], P=.002) revealed significant association with CL. Our study indicates that TNF-α 308 G/A and IL-4-590 C/T polymorphisms are significantly associated with susceptibility to CL. Individuals carrying A allele at TNF-α promoter -308 position and T allele at IL-4 promoter -590 position are at a higher risk for CL.

An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization.

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.