IL-6 Promoter Research Articles

IFNγ induces JAK1/STAT1/p65 NFκB-dependent interleukin-8 expression in ovarian cancer cells, resulting in their increased migration.

Interferon-γ (IFNγ) is a pleiotropic cytokine that has a crucial role in immune response and tumor immunity. Because of its anti-tumor effects, IFNγ has been used in cancer treatment. However, IFNγ also has tumor-promoting functions that are less well understood. Here, we show that IFNγ induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) in ovarian cancer (OC) cells. The IFNγ-induced IL-8 expression is dependent on JAK1, STAT1, and p65 NFκB, and is associated with an increased occupancy of K314/315 acetylated p65 NFκB and Ser-727 phosphorylated STAT1 at the IL-8 promoter. Neutralization of IL-8 using anti-IL-8 antibody reduces IFNγ-induced migration of OC cells, and their invasion ability in 3D spheroids. Together, these findings identify IL-8 as a novel target induced by IFNγ/JAK1/STAT1/p65 NFκB signaling, and indicate that the IFNγ-induced IL-8 contributes to IFNγ pro-tumorigenic effects in ovarian cancer cells.

Interleukin – 6 Serum Level and Single Nucleotide Gene -174 G/C promoter Polymorphism in Patients with Rheumatoid Arthritis / Iraq

Objective: To 1) assess IL-6 levels in the serum of patients with rheumatoid arthritis (RA). 2) study IL-6 promoter -174 G>C “single nucleotide polymorphism (SNP)” as an imminent factor for the disease development. 3) find any relation between the level of serum IL-6 cytokine and other parameters such as age, gender, clinical severity of diseases and “disease activity scores (DAS28)”. Materials & methods: This research was carried out through a case – control approach at “Ibn – Senna Teaching Hospital” in Mosul city between November 2020 and July 2021. It included 61 RA patients diagnosed according to “ACR / EULAR 2010 criteria” and 50 healthy individuals. IL-6 serum levels were ascertained by ELISA and genotyping of IL-6 promoter was accomplished by “sequence-specific primer-polymerase chain reaction (SSP-PCR)”. Results: Mean IL-6 level in RA (69.42 ng /l ± 62.99) was elevated in comparison to healthy people (14.66 ng /l ± 23.58), P < 0.001. No age or gender effects on IL-6 concentration were noted. The ideal cut-off of IL-6 for discrimination of RA with best discriminative utility compared to healthy controls was 22.80 ng/l. At this value the IL-6 sensitivity was 91.8%, specificity 82.0% and accuracy rate 73.80%. G/G genotype was the most pervasive genotype in both RA patients and controls (70.5% in RA and 64% in healthy controls). However, it did not seem to be a risk factor for RA development compared to G/C or C/C genotypes “(OR = 1.3438, 95% CI=0.605-2.984,P=0.469)”. The mean IL-6 level in patients with GG genotype was (73.70 ng / l ± 71.09) compared to (58.37 ng /l ± 37.86) in patients with GC genotype. There was no significant difference in the IL-6 level between patients with GG and patients with GC genotypes (P = 0.2375). Although higher IL-6 mean concentration was reported in severe RA, however, no significant difference was found between patients with mild, moderate and severe RA respectively. No correlation of serum levels of IL-6 with genetic promoter polymorphism, clinical severity of diseases or DAS 28 score were reported. Conclusion: The concentration of serum IL-6 was elevated in RA in regard to healthy controls which confirmed its pivotal role in RA pathogenesis. Our data did not support the role of IL-6 promoter -174 G> C polymorphism as a risk factor for RA, nor seem to play a major role in the increase of IL-6 level among our patients with RA.

Biosynthesis of Silver Nanoparticles from Leaf Extract of Salvia coccinea and Its Effects of Anti-inflammatory Potential in Human Monocytic THP-1 Cells.

Inflammation is one of the basic pathophysiologically important components in many life-threatening diseases. Metallic nanoparticles play a crucial role in biomedical applications. The present study was aimed at investigating the ameliorative effect of biosynthesized silver nanoparticles (ScAgNPs) using Salvia coccinea leaf extracts and characterizing them using physical and chemical methods, followed by evaluation of their cytotoxic, anti-oxidant, and anti-inflammatory potentials in monocytic THP-1 cells. Luciferase reporter assays and qRT-PCRs were used for gene expression studies. As oxidative stress and inflammation are mutually induced by each other, inhibiting oxidative stress could subsequently lead to inhibition of inflammation. Spherical-shaped ScAgNPs with 24 nm average size were successfully synthesized. The DCF staining technique, in addition to DPPH and reducing power activity assays, showed that 100-400 μg/mL concentration of ScAgNPs decreased oxidative stress significantly, induced by high glucose, in THP-1 cells. Anti-inflammatory effects of ScAgNPs have corroborated inhibition of high-glucose-induced oxidative stress-sensitive transcription factor NF-κB-driven transcription of proinflammatory COX-2, MCP-1, IP-10, IL-17E, and IL-6 promoters significant in high-glucose-grown THP-1 cells, consistent with promoter inhibition, and the corresponding mRNA expression levels were also decreased, suggesting that ScAgNPs could be a potential anti-inflammatory agent, which could efficiently inhibit inflammation in THP-1 cells. Our initial in vitro studies suggested that ScAgNPs could serve as therapeutic candidates to alleviate inflammatory diseases by inhibiting oxidative stress and inflammation.

Long Non-Coding RNA NEAT1 Knockdown Alleviates Rheumatoid Arthritis by Reducing IL-18 through p300/CBP Repression.

Rheumatoid arthritis (RA) is chronic inflammatory autoimmune disease. The crucial role of long non-coding RNA (lncRNA) in the progression of RA has been highlighted. Hence, this study was designed to explore the specific downstream mechanism of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in RA. Initially, the expression of NEAT1, p-p65, p300, and IL-18 in clinical tissues and cells was determined. Then, interactions among p65, NEAT1, p300, CBP, and IL-18 were investigated by immunofluorescence staining, dual luciferase reporter gene assay, RT-qPCR assay ChIP assay, and RIP assay followed by the analysis of their effects on RA in vivo and in vitro after expression alteration. The expressions of NEAT1, p-p65, p300, and IL-18 were all upregulated in the synovial tissues from the mice and patients with RA. NEAT1 silencing reduced the infiltration of CD4+ T cells and macrophages in synovial tissues, downregulated expression of blood inflammatory factors, relieved RA severity, and lowered incidence of RA in mice. Further, p-p65 could increase the expression of NEAT1 by binding to the NEAT1 promoter region, NEAT1 could co-locate and interact with p300, thus regulating the expression of IL-18 by regulating histone acetylation modification in IL-18 promoter region. NEAT1 aggravated RA via p300/CBP/IL-18 axis, representing a promising therapeutic target in RA.

Selective Targeting of Immune Modulatory Proteins to Mitigate Fibrosis and Inflammation in Sclerodermatous Graft-Vs-Host Disease

Chronic graft-vs-host disease (cGVHD) is a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (HCT) in patients. This debilitating condition is characterized by chronic inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is one of the more severe forms of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy.Methods: To address the current gap in knowledge pertaining to the underlying pathophysiology of sclGVHD we used single cell RNA sequencing analyses on fresh patient biopsy specimens. In vivo studies were carried out by sub lethal irradiation of BALB.k recipients which underwent HCT from miAg-mismatched AKR/J donors. Recipient sclerodermatous-tissues were analyzed using FACS, IHC and IF staining. Human studies were conducted on (i) Primary samples from patients with severe sclGVHD using tissue microarrays (TMA) by Immuno-histofluorescence (IHF) and IF. (ii) Dermal fibroblasts from sclGVHD samples were subjected to ATACseq and ChiPseq CRISPR-Cas9 JUN deletion. (iii) Also, dermal fibroblasts from human scl-GVHD were implanted under the kidney capsule of NSG mice to study the effects of inhibiting pro-fibrotic pathways in vivo.Results: We show for the first time that in a mouse model of sclGVHD (male), recipients of female T-cell replete grafts developed severe scleroderma with massive skin thickening and collagen deposition. Fibroblasts strongly expressed JUN, which is part of AP-1, a transcription factor involved in the acute phase response that regulates gene expression in response to stimuli from cytokines, growth factors and pathogens. We have previously demonstrated JUN as a key player in the molecular pathogenesis of other fibrotic diseases (Wernig G et al. PNAS 2017, Cui et al. Nature comm. 2020, Lerbs et al. JCI i 2020). Likewise, CD47, an immune checkpoint protein that prevents removal of Mϕ, was strongly co-expressed in fibroblasts in sclGVHD - but not in the control mice (Fig A+B). Here we show that (i) In humans, (n = 45 sclGVHD patients), there is a strong expression and activation of JUN and CD47 in dermal fibroblasts which was not observed in control samples. Mixed inflammatory infiltrates were dominated by Mϕ and granulocytes. (ii) Isolated primary fibroblasts from fresh human sclGVHD skin biopsies analyzed for chromatin accessibility across the genome by ATAC-seq showed wide open accessibility to the JUN promoter, IL-6 promoter and CD47 enhancer and promoter indicating that they play a critical role in regulating the pathogenesis of sclGVHD. In contrast, normal fibroblasts displayed only minimal accessibility to the JUN promoter. We further validated our data using CRISPR-Cas9 knock-down studies on sclGVHD fibroblasts and show that the IL-6 promoter, enhancer and promoter of CD47 are regulated by JUN, with JUN deletion resulting in significant decrease in the promoter binding accessibility to IL-6 and CD47 (Fig C). Further, JUN activity appears to regulate key members of the Hh signaling pathway (GLI1, PTCH1 and PTCH2), as their chromatin accessibility was decreased with JUN deletion. These correlative findings were confirmed by JUN ChIP seq, an assay that identifies binding sites of DNA-associated proteins. (iii) To test our findings in vivo we established xenograft models of primary human sclGVHD by implanting cells under the kidney capsule of NSG mice.All treatments (except placebo) resulted in decreased fibrosis (Fig D), presumably by blocking the activation of JUN (pJUN) and its profibrotic downstream pathway members IL-6 and pSTAT3, as assessed by phospho flow. Conclusion: In our studies we demonstrate that in established SclGVHD, combinatorial therapy consisting of anti-CD47 antibody together with IL6 blockade has the highest potential to translate into a therapeutic intervention given its ability to be more effective than currently used antifibrotic and anti-inflammatory agents in clinic. The findings from our study are significant because we show an important mechanism underlying SclGVHD onset, identify a novel genetic signature that can be targeted, describe a new mouse model and a clinical assay that has a high throughput readout and suggest a treatment regimen for patients. [Display omitted] DisclosuresArai: Magenta Therapeutics: Research Funding. Shizuru: Forty seven Inc: Other: Inventor on a patent licenses by Forty Seven. Forty seven was acquired by Gilead in 2020; Jasper Therapeutics, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Chair of scientific advisory board.

The IL32/BAFF Axis Supports Prosurvival Dialog in the Lymphoma Ecosystem and Is Disrupted By NIK Inhibition

BackgroundAggressive B-cell lymphomas, such as Mantle cell lymphoma (MCL), are microenvironment-dependent tumors but, in contrast to tumoral intrinsic anomalies, complex interplays within their ecosystems are largely ignored. A better understanding of these dialogs could provide new perspectives integrating the key role of the microenvironment to increase treatment efficiency of this hard to cure B-cell malignancy.MethodsTo identify novel molecular regulations occurring in lymphoma protective ecosystems, we performed a transcriptomic analysis based on the comparison of publicly available datasets from circulating (PB, n=77) versus MCL lymph nodes (LN, n=107) together with deep RNA sequencing of purified CD19+CD5+ MCL (n=8) versus normal B-cells (n=6). This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of the cytokine IL32β by lymphoma cells. Using in situ multiplex immunohistochemistry , ex vivo models of primary MCL cells (n=23) and IL32 KO MCL cell lines (Crispr/Cas9), we studied the regulation and the functional impact of IL32β in the MCL context, especially in the dialog with tumor-associated macrophages.ResultsAmong the 6887 genes differentially expressed in MCL LN compared to PB in vivo, 70% were confirmed in CD19+ MCL cells cocultured ex vivo and 39% were tumor-specific, that is to say not upregulated in cocultured normal B cells (NBC). Top-genes scoring revealed that IL32 was the most upregulated genes within the “Tumor-specific” transcriptional program. Using ex vivo models of primary MCL cells, we demonstrated that the microenvironment-dependent secretion of IL32β was controlled by the CD40/NFKB2 axis whereas its tumor specificity was the consequence of IL32 promoter hypomethylation in MCL compared to NBC.IL32 protein expression was confirmed in MCL LN in situ by multiplex IHC. The latter allowed the concurrent detection of MCL cells, T cells, macrophages and IL32. Consistently with the microenvironment-dependent induction of IL32 in MCL, we observed that IL32 expression was enriched in situ in tumor zones infiltrated with T cells, compared to tumor-exclusive zones.Based on in vitro experiments using IL32 KO MINO cells (Crispr/Cas9), we demonstrated that, through the secretion of IL32β, the tumor was able to corrupt its microenvironment by polarizing monocytes into specific protumoral CD163 mid MCL-associated macrophages, which secrete both pro- (e.g. IL6, OSM, IL1a/b) and anti- inflammatory (e.g. IL10, IDO, IL18, IL4L1) soluble factors. We next highlighted that IL32β-stimulated macrophages supported tumor survival mostly through a soluble dialog, which is driven by BAFF.Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract both microenvironment-dependent induction of IL32β (RNA expression inhibition: 62 % ; n= 3) and BAFF-dependent survival of MCL cells (survival support inhibition : 47 % ; n=6).ConclusionsIn summary, our data uncovered the IL32β/BAFF axis as a previously undescribed pathway involved in MCL-associated macrophage polarization and tumor survival. Dependent on alternative-NFkB signaling, tumor-specific secretion of IL32β led to the corruption of the microenvironment through the polarization of monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. While IL32β-stimulated macrophages secreted several protumoral factors, they supported MCL survival through BAFF and consequent alternative-NFkB activation in tumor cells. Our data shows that targeting IL32b, BAFF or the alternative NFkB pathway through NIK inhibition could also be of major interest for counteracting the multiple cross-talks that occur in the MCL microenvironment and, especially, the CD40L + T-cell / MCL / CD163 + MCL-associated macrophage triad. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CLL-Derived Exosomes Turn Endothelial Cells into IL-6 Producing Cells

CLL is characterized by gradual accumulation of mature appearing long-lived lymphocytes that travel in blood and reside in lymph nodes, spleen and bone marrow. In these sites, pro inflammatory humoral factors support the survival and proliferation of the neoplastic cells. Previous studies showed that levels of the proinflammatory cytokine IL-6 are at least 10 folds higher in patients with CLL compared with healthy individuals. Yet, which cells produce and secrete IL-6 and what triggers this cellular activity in CLL is unknown.Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and carry a cargo that at least partially reflects the molecular makeup of its cell of origin. Exosomes, including those originating from neoplastic cells, function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts.Given the appropriate stimulation, endothelial cells produce IL-6 which provides CLL cells with a survival advantage. Therefore, we hypothesized that CLL-exosomes turn endothelial cells into “IL-6-secreting cells”.To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment naïve patients. We found that endothelial cells take-up CLL-exosomes in a dose- and time- dependent manner. Since CLL cells are protected from apoptosis in IL-6 rich environment, we wondered whether CLL-exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL-exosomes and found 50% increase in IL-6 levels, suggesting that the endothelial-exposed cells produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6 enriched medium. After 15 minutes, STAT3 became phosphorylated and there was 40% decrease in the rate of apoptosis, indicating that IL-6 activated STAT3-dependent anti-apoptotic pathway. Phosphor-proteomics analysis of endothelial cells that were loaded with CLL-exosomes revealed 23 phosphor-proteins that were upregulated. Network analysis unraveled the central role of phosphor-b-catenin. To test whether b-catenin induces IL-6 in these cells, we transfected HUVECs with a b-catenin containing plasmid. We found by ELISA 30% increase in the levels of IL-6 in the culture medium and by chromatin immunoprecipitation assay an increased binding of 3 transcription factors (NFkB, LEF/TCF, and CEBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through exosomes that they release. Once these exosomes are taken up by endothelial, they turn them into IL-6 producing cells, which in turn contributes to CLL cells' survival. DisclosuresNo relevant conflicts of interest to declare.

Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

IL10 promoter rs6667202 polymorphism is functional in health but not in grade c periodontitis patients: A pilot study.

Previous studies have demonstrated an association between the IL10 promoter rs6667202 (C > A) single-nucleotide polymorphism (SNP) and grade C, stage 3 or 4 periodontitis (Perio4C) in the Brazilian population, where the altered A allele was detected more frequently in these patients. However, no functional analysis of this variation has yet been performed. Thus, the objective of this preliminary study was to evaluate the functionality of rs6667202 in gingival fibroblasts (GFs) of individuals with Perio4C and with periodontal health (PH) stimulated with Aggregatibacter actinomycetencomitansprotein extract (AaPE). Patients with PH and Perio4C were segregated according to their genotype (AA, AC, or CC), and a biopsy was performed to establish the culture of the GFs. After GFs exposure to AaPE at 5µg/ml for 1.5h, RNA was extracted to analyze IL10 expression by qPCR. Aliquots of the cell's supernatant were subjected to immunoenzymatic analysis (MAGpix) to detect interleukin-10 (IL-10). In PH, the genotypes AA and AC are related to less expression of IL10 (p=0.027 and p<0.0001) and less production of IL-10 (p=0.002 and p=0.001), when compared to CC. In Perio4C, there was no statistical difference between the genotypes (p>0.05), although a lower IL-10 expression and release compared with PH CC was seen (p=0.033 and p<0.001). The rs6667202 SNP is functional in PH, as it decreases the expression and production of IL-10. In Perio4C, other factors may be masking its action by altering the IL-10's response.

Acetaminophen-Induced Liver Injury Exposes Murine IL-22 as Sex-Related Gene Product.

Gaining detailed knowledge about sex-related immunoregulation remains a crucial prerequisite for the development of adequate disease models and therapeutic strategies enabling personalized medicine. Here, the key parameter of the production of cytokines mediating disease resolution was investigated. Among these cytokines, STAT3-activating interleukin (IL)-22 is principally associated with recovery from tissue injury. By investigating paradigmatic acetaminophen-induced liver injury, we demonstrated that IL-22 expression is enhanced in female mice. Increased female IL-22 was confirmed at a cellular level using murine splenocytes stimulated by lipopolysaccharide or αCD3/CD28 to model innate or adaptive immunoactivation. Interestingly, testosterone or dihydrotestosterone reduced IL-22 production by female but not by male splenocytes. Mechanistic studies on PMA/PHA-stimulated T-cell-lymphoma EL-4 cells verified the capability of testosterone/dihydrotestosterone to reduce IL-22 production. Moreover, we demonstrated by chromatin immunoprecipitation that testosterone impairs binding of the aryl hydrocarbon receptor to xenobiotic responsive elements within the murine IL-22 promoter. Overall, female mice undergoing acute liver injury and cultured female splenocytes upon inflammatory activation display increased IL-22. This observation is likely related to the immunosuppressive effects of androgens in males. The data presented concur with more pronounced immunological alertness demonstrable in females, which may relate to the sex-specific course of some immunological disorders.

Inhibition of nuclear receptor ROR\u03b1 attenuates cartilage damage in osteoarthritis by modulating IL-6/STAT3 pathway

Osteoarthritis (OA) is characterized by cartilage destruction, chronic inflammation, and local pain. Evidence showed that retinoic acid receptor-related orphan receptor-α (RORα) is crucial in cartilage development and OA pathogenesis. Here, we investigated the role and molecular mechanism of RORα, an important member of the nuclear receptor family, in regulating the development of OA pathologic features. Investigation into clinical cartilage specimens showed that RORα expression level is positively correlated with the severity of OA and cartilage damage. In an in vivo OA model induced by anterior crucial ligament transaction, intra-articular injection of si-Rora adenovirus reversed the cartilage damage. The expression of cartilage matrix components type II collagen and aggrecan were elevated upon RORα blockade. RNA-seq data suggested that the IL-6/STAT3 pathway is significantly downregulated, manifesting the reduced expression level of both IL-6 and phosphorylated STAT3. RORα exerted its effect on IL-6/STAT3 signaling in two different ways, including interaction with STAT3 and IL-6 promoter. Taken together, our findings indicated the pivotal role of the RORα/IL-6/STAT3 axis in OA progression and confirmed that RORα blockade improved the matrix catabolism in OA chondrocytes. These results may provide a potential treatment target in OA therapy.

STAT5 Represses a STAT3-Independent Th17-like Program during Th9 Cell Differentiation.

IL-9-producing Th cells, termed Th9 cells, contribute to immunity against parasites and cancers but have detrimental roles in allergic disease and colitis. Th9 cells differentiate in response to IL-4 and TGF-β, but these signals are insufficient to drive Th9 differentiation in the absence of IL-2. IL-2-induced STAT5 activation is required for chromatin accessibility within Il9 enhancer and promoter regions and directly transactivates the Il9 locus. STAT5 also suppresses gene expression during Th9 cell development, but these roles are less well defined. In this study, we demonstrate that human allergy-associated Th9 cells exhibited a signature of STAT5-mediated gene repression that is associated with the silencing of a Th17-like transcriptional signature. In murine Th9 cell differentiation, blockade of IL-2/STAT5 signaling induced the expression of IL-17 and the Th17-associated transcription factor Rorγt. However, IL-2-deprived Th9 cells did not exhibit a significant Th17- or STAT3-associated transcriptional signature. Consistent with these observations, differentiation of IL-17-producing cells under these conditions was STAT3-independent but did require Rorγt and BATF. Furthermore, ectopic expression of Rorγt and BATF partially rescued IL-17 production in STAT3-deficient Th17 cells, highlighting the importance of these factors in this process. Although STAT3 was not required for the differentiation of IL-17-producing cells under IL-2-deprived Th9 conditions, their prolonged survival was STAT3-dependent, potentially explaining why STAT3-independent IL-17 production is not commonly observed in vivo. Together, our data suggest that IL-2/STAT5 signaling plays an important role in controlling the balance of a Th9 versus a Th17-like differentiation program in vitro and in allergic disease.

CLL-283: CLL-Derived Exosomes Turn Endothelial Cells into CLL-Supportive Cells

We hypothesized that via exosomes, CLL cells turn endothelial cells into "CLL-supportive cells." To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment-naïve patients. We found that endothelial cells take up CLL exosomes in a dose- and time-dependent manner. Since CLL cells are protected from apoptosis in an IL-6-rich environment, we wondered whether CLL exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL exosomes and found a 50% increase in IL-6 levels, suggesting that the endothelial cells exposed to CLL exosomes produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6-enriched medium. After 15 minutes, STAT3 became phosphorylated, and there was a 40% decrease in the rate of apoptosis among these cells, indicating that IL-6 activated the STAT3-dependent anti-apoptotic pathway. Phosphor-proteomic analysis of endothelial cells that were loaded with CLL exosomes revealed 23 phosphor-proteins that were upregulated. Annotation analysis unraveled the central role of phosphor-β-catenin. To test whether β-catenin enhances the generation of IL-6 in these cells, we transfected HUVECs with a β-catenin-containing plasmid. We found, by ELISA, a 30% increase in the levels of IL-6 in the culture medium, and, by ChIP, the increased binding of 3 transcription factors (NF-κB, LEF/TCF, and C/EBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through the exosomes that they release. Once these exosomes are taken up by endothelia, they turn them into IL-6-producing cells.

Genotyping of interleukins-18 promoters and their correlation with coronary artery stenosisin Saudi population.

Complex coronary atherosclerotic lesions often lead to coronary occlusion, clinically represented as a single-vessel disease(SVD) and multivessel disease(MVD). These occlusions could hinder the blood flow in coronary arteries that affects appropriate management of the CVD. The current study intended to genotype interleukin (IL)-18 promoter's hotspots (rs187238, rs1946518, and rs1946519) and their possible association with coronary artery stenosis. The IL-18 promoter genotyping was performed by the Sanger method along with the examination of biochemical parameters in 125 study subjects categorized into three groups, viz. controls, SVD and MVD. The current study observed a significant association of diabetes, hypertension, and dyslipidemia between the studied group's viz. healthy controls, SVD, and MVD. Fasting blood sugar and glycosylated hemoglobin (HBA1C) were also significantly enhanced from 4.82 vs. 8.01 and 4.33 vs. 8.27, inSVD, and MVD respectively. Despite the visible differences in the pattern of genotypic and allelic expressions, the current study did not show any statistically significant correlation with IL-18 promoter polymorphism at its hotspots with controls, SVD, and MVD subjects. The only exception of the above results was the distribution of allelic frequency at the rs1946519 hotspot, where a significant change (P < 0.05)was observed. This study is of additional value to our previous reports, revealing the pattern of genotypes and allelic frequency of IL-18 promoters in a small cohort of Saudi ethnicity. Further investigations on larger sample size are recommended to envisage the presence of functional mutations in the IL-18 gene that could establish or rule out the possible association of IL-18 polymorphism with SVD and MVD.

NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

BackgroundH. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models.MethodsTo investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays.ResultsFollowing infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models.ConclusionOur findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.

The Functional DNA Methylation Signatures Relevant to Altered Immune Response of Neonatal T Cells with l-Arginine Supplementation

l-Arginine is an important nutrient in the infant diet that significantly regulates the maturation of the immune system in neonates, including the maturation of CD4+ T cells. The biological activities of CD4+ T cells differ substantially between neonates and adults, and these differences may be governed by epigenetic processes. Investigating these differences and the causative processes may help understand neonatal and developmental immunity. In this study, we compared the functional DNA methylation profiles in CD4+ T cells of neonates and adults, focusing on the role of l-arginine supplementation. Umbilical cord blood and adult CD4+ T cells were cultured with/without l-arginine treatment. By comparing DNA methylation in samples without l-arginine treatment, we found that CD4+ T cells of neonatal cord blood generally showed higher DNA methylation than those of adults (average CpG methylation percentage 0.6305 for neonate and 0.6254 for adult, t-test p-value < 0.0001), suggesting gene silencing in neonates. By examining DNA methylation patterns of CpG dinucleotides induced by l-arginine treatment, we found that more CpG dinucleotides were hypomethylated and more genes appeared to be activated in neonatal T-cells as compared with adult. Genes activated by l-arginine stimulation of cord blood samples were more enriched regarding immune-related pathways. CpG dinucleotides at IL-13 promoter regions were hypomethylated after l-arginine stimulation. Hypomethylated CpG dinucleotides corresponded to higher IL-13 gene expression and cytokine production. Thus, DNA methylation partially accounts for the mechanism underlying differential immune function in neonates. Modulatory effects of l-arginine on DNA methylation are gene-specific. Nutritional intervention is a potential strategy to modulate immune function of neonates.

AhR Regulates Peptidoglycan-Induced Inflammatory Gene Expression in Human Keratinocytes

Bacterial peptidoglycan (PGN) stimulates toll-like receptor 2 (TLR2) on the surface of keratinocytes (KCs), triggering signaling pathways that promote an innate immune response. However, excessive TLR2 activation can lead to inappropriate inflammation, which contributes to skin conditions such as rosacea. To better treat these conditions, there is a need to understand the molecular mechanisms that regulate the cellular response to TLR2 activation in the skin. Aryl hydrocarbon receptor (AhR) is a transcription factor that modulates the immune response in KCs and is a promising therapeutic target for inflammatory skin diseases. Here, we investigated the role of the AhR in regulating the transcriptional response of human KCs to PGN. We performed whole-transcriptome sequencing in wild-type and AhR-depleted KCs after PGN stimulation. AhR depletion altered the expression of 72 genes in response to PGN, leading to increased expression of 48 genes and repression of 24 genes, including interleukin (IL)-1β. Chromatin immunoprecipitation showed that PGN stimulation resulted in AhR binding the promoters of IL-1β and IL-6 to activate them. More broadly, AhR promoted inflammatory gene expression by increasing JNK/mitogen-activated protein kinase signaling and FosB expression. Finally, we observed that AhR depletion increased TLR2 expression itself, raising the hypothesis that AhR may serve to restrain TLR2-mediated inflammation in KCs through negative feedback. Viewed together, our findings demonstrate a significant and complex role for AhR in modulating the expression of inflammatory genes in KCs in response to PGN.

Vitamin D Inhibits IL-22 Production Through a Repressive Vitamin D Response Element in the il22 Promoter.

Th22 cells constitute a recently described CD4+ T cell subset defined by its production of interleukin (IL)-22. The action of IL-22 is mainly restricted to epithelial cells. IL-22 enhances keratinocyte proliferation but inhibits their differentiation and maturation. Dysregulated IL-22 production has been associated to some inflammatory skin diseases such as atopic dermatitis and psoriasis. How IL-22 production is regulated in human T cells is not fully known. In the present study, we identified conditions to generate Th22 cells that do not co-produce IL-17 from naïve human CD4+ T cells. We show that in addition to the transcription factors AhR and RORγt, the active form of vitamin D3 (1,25(OH)2D3) regulates IL-22 production in these cells. By studying T cells with a mutated vitamin D receptor (VDR), we demonstrate that the 1,25(OH)2D3-induced inhibition of il22 gene transcription is dependent on the transcriptional activity of the VDR in the T cells. Finally, we identified a vitamin D response element (VDRE) in the il22 promoter and demonstrate that 1,25(OH)2D3-VDR directly inhibits IL-22 production via this repressive VDRE.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

Suggestive evidence of genetic association of −572G > C polymorphism with primary open angle glaucoma in a North Indian Punjabi population

BackgroundIL6 is an important candidate gene implicated in the pathogenesis of glaucoma. The present study assessed the genetic association of −174G > C and −572G > C polymorphisms in the IL6 promoter region with primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) in a north Indian Punjabi cohort. Methods910 subjects (313 POAG, 148 PACG cases and 449 controls) were recruited. Genotyping was done by TaqMan assays. Genetic association was tested under different genetic models using Plink. Diplotype and linkage disequilibrium (LD) analysis was done through Haploview. Association of clinical parameters with the genotypes was assessed by one-way ANOVA. Adjustment for potential confounding variables was done by binary logistic regression. IL6 levels were measured in POAG patients and controls. Results572G > C variant showed marginal difference in genotype frequency between pooled cases and POAG subgroup with respect to controls (p = 0.042; OR = 1.33; and p = 0.041; OR = 1.37). The GC genotype conferred 1.37-fold protection under codominant model in POAG cases (p = 0.034, OR = 1.37, 95% CI = 1.02–1.85; pcorr = 0.025, OR = 1.45, 95% CI = 1.04–2.02). The mean value for IOP was elevated among cases having ‘CC’ genotype at the −572G > C locus (p = 0.037).Lower levels of IL6 were detected in POAG patients in plasma samples (p = 0.0001). ConclusionThe study reports suggestive evidence for −572G > C variant in IL6 in affecting genetic susceptibility to POAG in the targeted North Indian Punjabi cohort. A correlation of IL6 levels in aqueous humor (AH) and systemic circulation in POAG was observed, the functional and diagnostic relevance of which may be further investigated.