Secreted Embryonic Alkaline Phosphatase Research Articles

Broad Antagonistic Activity of a Fatty Acid Binding Protein of Fasciola hepatica (Fh12) in the Presence of Whole Bacteria Extract Prelude a Broad Spectrum of Action

Inflammation process could be initiated by activation of single or multiple toll‐like receptors on antigen presenting cells via molecular pattern recognition of molecules of foreign pathogens like Gram positive and negative bacteria. The exacerbated immune response to these molecules can lead to septic shock syndrome that could be fatal. The identification of TLR‐antagonists that could block or diminish the activation of these receptors and suppress the inflammatory mediators has become in an excellent therapeutic alternative against sepsis. In the last decade, scientists have shown interest in the strong anti‐inflammatory role that some helminth parasites exhibit. Helminths use several immunomodulatory strategies to modify immune responses in order to survive into the mammalian host. Fasciola hepatica is able to induce long‐lasting disease in the host associated to Th2 responses, which is linked to an anti‐inflammatory phenotype of immune responses. This parasite secretes and expresses proteins on their surfaces that are essential either, its metabolism or immunomodulating the host's immune response. One of these proteins is a 12kDa fatty acid binding protein (Fh12). We had previously demonstrated that Fh12 could suppress the TLR4 activation. The current study aimed to ascertain whether Fh12 could also suppress other TLRs. THP1‐Blue CD14 cells is a cell line of human monocytes that express NF‐κB/AP‐1 inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were cultured for 18h, 37°C, 5% CO2 with 10μg Fh12 in the presence or absence of specific ligands for each TLR as well as with or without heat attenuated whole extracts of (1 × 108 cells) Escherichia coli, Klebsiella pneumonia or Enterococcus faecalis. Cells cultured only with RPMI‐medium, TLR‐ligands, Fh12 or whole bacteria extracts alone served as controls. Results demonstrated that Fh12 was capable to significantly suppress the activation of NF‐κB to Gram negative and Gram‐positive bacteria by 65% and 90%, respectively. In consistency with this finding Fh12 also suppressed the NF‐κB activation induced by specific ligands for TLR2, TLR4, TLR5, TLR6 and TLR8 by 80% to 89% respectively. FACS analysis also revealed that Fh12 also induced significant reduction in the expression of co‐stimulatory molecules like CD40, CD80 and CD86, which play crucial role in the initiation and maintenance of inflammatory immune response. Our results suggest that Fh12 not only impacts TLR4, but also could impact multiple TLRs and which prelude a broader anti‐inflammatory effect for Fh12. Further studies directed to explore the potential of Fh12 as a new class of drug against septic shock caused by bacteria and also others inflammatory diseases are under study.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, and NIH grants G12MD007600 and 2P40OD012217.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14.

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40–50 μg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP’s ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.

Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

Disturbances in redox equilibrium in tissue can lead to inflammatory state, which is a mediatory factor in many human diseases. The mechanism(s) by which exogenous oxidants may activate an inflammatory response is not fully understood. Emerging evidence suggests that oxidant-induced Toll-like receptor 4 (TLR4) activation plays a major role in “sterile” inflammation. In the present study, we used murine macrophage RAW-Blue cells, which are chromosomally integrated with secreted embryonic alkaline phosphatase (SEAP) inducible by NF-κB. We confirmed the expression of TLR4 mRNA and protein in RAW-Blue cells by RT-PCR and Western blot, respectively. We showed that oxidants increased intracellular reactive oxygen species production and lipid peroxidation, which resulted in decreased intracellular total antioxidant capacity. Consistent with the actions of TLR4-specific agonist LPS-EK, exogenous oxidants increased transcriptional activity of NF-κB p65 with subsequent release of NF-κB reporter gene SEAP. These effects were blocked by pretreatment with TLR4 neutralizing pAb and TLR4 signaling inhibitor CLI-095. In addition, oxidants decreased the expression of IκBα with enhanced phosphorylation at the Tyr42 residue. Finally, oxidants and LPS-EK increased TNFα production, but did not affect IL-10 production, which may cause imbalance between pro- and anti-inflammatory processes, which CLI-095 inhibited. For biological relevance, we confirmed that oxidants increased release of TNFα and IL-6 in primary macrophages derived from TLR4-WT and TLR4-KO mice. Our results support the involvement of TLR4 mediated oxidant-induced inflammatory phenotype through NF-κB activation in macrophages. Thus exogenous oxidants may play a role in activating inflammatory phenotypes that propagate and maintain chronic disease states.

Reconstruction of Toll-like receptor 9-mediated responses in HEK-Blue hTLR9 cells by transfection of human macrophage scavenger receptor 1 gene

We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-κB and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-κB and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle.

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Abstract 2732: Myelosuppression in patients treated with the telomerase inhibitor imetelstat is not mediated through activation of toll-like receptors

Abstract Imetelstat is a covalently lipidated 13-mer N3′-P5′ thio-phosphoramidate (NPS) oligonucleotide (5′-TAGGGTTAGACAA-3′) that is complementary to the RNA template region (hTR; 3′-AUCCCAACCUGUU-5′) of telomerase. Imetelstat is believed to act as a direct competitive inhibitor of telomerase enzyme activity and to not have an antisense mechanism of action (MOA) typical of other oligonucleotides. The purpose of this study was to assess whether the thrombocytopenia observed in some patients with myeloproliferative neoplasm treated with imetelstat can be attributed to activation of toll-like receptors (TLRs) by imetelstat. Imetelstat and related control oligonucleotides (Mis-match and Sense) were tested using 7 different HEK-Blue™ TLR cell lines, each stably coexpressing a human TLR gene (TLR2, 3, 4, 5, 7, 8, and 9) and a NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. After 20 hours’ incubation, the cells were measured for absorbance at 650 nm for SEAP activity. Each TLR experiment was performed twice, with 3 replicates per experiment. The results showed that the positive control for each individual TLR was recognized by the corresponding TLR, leading to a strong SEAP activity, whereas imetelstat at a clinically relevant concentration range (10 μM-60 μM) and the control oligonucleotides related to imetelstat yielded no induction of SEAP activity (similar level to untreated). These data demonstrate that imetelstat has no stimulatory effect on the majority of the tested TLRs. Imetelstat yielded slightly higher induction on TLR8 than the untreated control, but significantly lower than the positive control. Although thrombocytopenia has been observed in patients with myeloproliferative neoplasms treated with imetelstat, these data show that it cannot result from imetelstat acting as a ligand for TLRs. TLR9, associated with thrombocytopenia, was not activated by imetelstat and the slight activity of imetelstat on TLR8 is likely irrelevant as no link between TLR8 activation and thrombocytopenia has been reported. These findings are consistent with 13-mer sequence of imetelstat, differing from oligonucleotides known to stimulate TLR9, which comprise 2 CpG islands separated by 6-10 nucleotides and are longer than 21 nucleotides. Ex vivo studies have provided evidence that imetelstat may induce thrombocytopenia in patients with myeloproliferative neoplasms by blocking the terminal maturation of normal megakaryocyte precursors: megakaryocytic differentiation requires upregulation of telomerase activity which is inhibited by imetelstat. Correct understanding of the MOA of imetelstat is vital to guide its clinical use and strategies to mitigate thrombocytopenia in patients with myeloproliferative neoplasms. Citation Format: Gabriela M. Baerlocher, Joshua Rusbuldt, Fei Huang, Jacqueline Bussolari. Myelosuppression in patients treated with the telomerase inhibitor imetelstat is not mediated through activation of toll-like receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2732.

Characterization of the inflammatory effects of λ-carrageenan at the genomic and proteomic levels

Abstract λ-Carrageenan (λCGN) is often used to model local inflammation, but the mechanism by which this compound functions at the genomic and proteomic level has not been delineated. Whole blood from swine was stimulated with λCGN to determine inflammatory biomarkers. We also utilized a HEK-293 (human embryonic kidney) cell line transfected with a SEAP (secreted embryonic alkaline phosphatase)-release plasmid to determine which TLR (Toll-Like Receptor; TLR) signaling cascade was stimulated. Results indicated that Monocyte Chemotactic Protein-1 gene expression and protein were increased by λCGN stimulation while Serum Amyloid A gene expression and protein remained unchanged. λCGN stimulation increased mRNA levels of Phosphatidylinositol 3-Kinase and Alveolar Macrophage Chemotactic –II, decreased mRNA levels of Thrombospondin-2 Precursor, and had no effect on Interleukin-6 mRNA levels. λCGN stimulation increased protein levels of Interleukin-8 and had no effect on protein levels of Tumor Necrosis Factor-α. SEAP release from HEK-293 cells stimulated with λCGN indicated that both TLR2 and TLR4 signaling cascades were initiated. λCGN stimulation did not activate TLR3, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, Dectin-1a, or Dectin-1b. Experiments silencing either TLR1 or TLR6 to determine which TLR2 heterodimer is activated by λCGN are currently underway. In conclusion, while λCGN stimulation appears to signal through both the TLR2 and TLR4 pathways, significant differences exist between the genes and proteins stimulated by λCGN and pure TLR4 agonists such as E. coli-derived lipopolysaccharide.

Organophosphorus Pesticides Promote Cytokine Production in Human Macrophages

Macrophages are sentinel immune cells that reside in most if not all tissues and play a critical role in innate immunity. We previously established that exposure to organophosphorus pesticides (OPs) increases airway hyperreactivity in animal models. OPs are neurotoxicants that cause acute cholinergic toxicity via inhibition of the enzyme acetylcholinesterase (AChE); however, our studies demonstrated that the OP parathion causes airway hyperreactivity independent of inhibition of AChE activity. Rather, parathion‐induced airway hyperreactivity is mediated by dysfunction of autoinhibitory muscarinic M2 receptors expressed on the parasympathetic nerves that innervate airway smooth muscle. Recently, we reported that the airway hyperreactivity triggered by parathion requires macrophages and immune cytokines. Here, we test the hypothesis that OPs interact directly with macrophages to stimulate them to express cytokines that modulate neuronal M2 receptors. Using the human THP‐1 monocyte line, we evaluated cytokine expression, NFkB activation through a secreted embryonic alkaline phosphatase (SEAP) reporter expression and nuclear localization of the NFkB p65 subunit and intracellular calcium mobilization in PMA‐differentiated THP‐1 cells exposed to different OPs and their metabolites. Preliminary experiments indicate that in differentiated THP‐1 macrophages, OPs significantly increase the mRNA expression of the pro‐inflammatory cytokines TNFα and IL‐1β. OPs also significantly increased NFkB activation but did not increase intracellular calcium levels. Therefore, our in vitro data strongly indicate that OPs interact directly with macrophages to provoke release of pro‐inflammatory mediators via activation of the NFkB signaling pathway. Ongoing studies are exploring the action of carbamates and pyrethrins on macrophages and further evaluating the mechanism by which these pesticides stimulate macrophages.Support or Funding InformationThis work was supported by funding from the National Institutes of Health grant R01 ES017592.

Production of Human papillomavirus pseudovirions in plants and their use in pseudovirion-based neutralisation assays in mammalian cells.

Human papillomaviruses (HPV) cause cervical cancer and have recently also been implicated in mouth, laryngeal and anogenital cancers. There are three commercially available prophylactic vaccines that show good efficacy; however, efforts to develop second-generation vaccines that are more affordable, stable and elicit a wider spectrum of cross-neutralising immunity are still ongoing. Testing antisera elicited by current and candidate HPV vaccines for neutralizing antibodies is done using a HPV pseudovirion (PsV)-based neutralisation assay (PBNA). PsVs are produced by transfection of mammalian cell cultures with plasmids expressing L1 and L2 capsid proteins, and a reporter gene plasmid, a highly expensive process. We investigated making HPV-16 PsVs in plants, in order to develop a cheaper alternative. The secreted embryonic alkaline phosphatase (SEAP) reporter gene and promoter were cloned into a geminivirus-derived plant expression vector, in order to produce circular dsDNA replicons. This was co-introduced into Nicotiana benthamiana plants with vectors expressing L1 and L2 via agroinfiltration, and presumptive PsVs were purified. The PsVs contained DNA, and could be successfully used for PBNA with anti-HPV antibodies. This is the first demonstration of the production of mammalian pseudovirions in plants, and the first demonstration of the potential of plants to make DNA vaccines.

Exploring the Effects of Omega-3 and Omega-6 Fatty Acids on Allergy Using a HEK-Blue Cell Line.

Background: Allergic reactions can result in life-threatening situations resulting in high economic costs and morbidity. Therefore, more effective reagents are needed for allergy treatment. A causal relationship has been suggested to exist between the intake of omega-3/6 fatty acids, such as docosahexanoic acid (DHA), eicosapentanoic acid (EPA), docosapentanoic acid (DPA) and arachidonic acid (AA), and atopic individuals suffering from allergies. In allergic cascades, the hallmark cytokine IL-4 bind to IL-4 receptor (IL-4R) and IL-13 binds to IL-13 receptor (IL-13R), this activates the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE antibody production by B cells. The overall aim of this study was to characterize omega-3/6 fatty acids and their effects on STAT6 signaling pathway that results in IgE production in allergic individuals. Methods: The fatty acids were tested in vitro with a HEK-Blue IL-4/IL-13 reporter cell line model, transfected with a reporter gene that produces an enzyme, secreted embryonic alkaline phosphatase (SEAP). SEAP acts as a substitute to IgE when cells are stimulated with bioactive cytokines IL-4 and/or IL-13. Results: We have successfully used DHA, EPA and DPA in our studies that demonstrated a decrease in SEAP secretion, as opposed to an increase in SEAP secretion with AA treatment. A statistical Student’s t-test revealed the significance of the results, confirming our initial hypothesis. Conclusion: We have successfully identified and characterised DHA, EPA, DPA and AA in our allergy model. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors of IL-4R/IL-13R signalling, which regulates the STAT6 induced pathway in allergic cascades. Such findings are significant in the future design of dietary therapeutics for the treatment of allergies.

Damage-associated molecular patterns in the adsorbed protein layer on biomaterial surfaces

Event Abstract Back to Event Damage-associated molecular patterns in the adsorbed protein layer on biomaterial surfaces Laura Mckiel1* and Lindsay E. Fitzpatrick1 1 Queen's University, Chemical Engineering, Canada Introduction: The innate immune response is the body’s primary defense against infection and injury, and is facilitated largely by white blood cells such as macrophages. Toll-like receptors (TLRs) play a critical role in innate immunity by recognizing evolutionarily conserved pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively). DAMPs are released upon tissue injury and cell death, and are known to bind to TLRs and initiate inflammatory responses via activation of NF-κB and AP-1 transcription factors[1]. We hypothesize that TLRs contribute to biomaterial-induced inflammation by binding DAMPs created at the implant site and adsorbed to the surface of the biomaterial. The objective of this research is to create an in vitro model of macrophage response to DAMP-adsorbed biomaterials. Materials and Methods: Borosilicate coverslips (12 mm) and microscope slides (25 x 75 mm) were spin coated (3000 rpm, 2 min) with 20 mg/mL poly(methyl methacrylate) (PMMA) in chloroform, then UV sterilized for 30 min. 3T3 fibroblasts were lysed by freeze/thaw cycling to yield a lysate containing DAMPs. Fetal bovine serum (FBS) was used as a source of serum proteins. RAW-Blue cells were seeded onto serum or lysate-adsorbed PMMA and/or TCPS surfaces. RAW-Blue cells are a macrophage reporter line that stably express an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) that can be detected using QUANTI-Blue reagent. Prior to cell seeding, surfaces were exposed to lysate (30 min) or serum (HI-FBS or FBS, 20 hr) then rinsed with PBS. Lipopolysaccharide (LPS, 1.5 µg/mL), a known TLR agonist, was the positive control. HI-FBS-treated TCPS without cells was the negative control. Results and Discussion: Water contact angles for PMMA-coated compared to uncoated glass increased (71° vs 62°, p<0.05), showing complete coverage of PMMA. 3D AFM images of the surfaces show the changes in surface morphology and height following protein adsorption (Figure 1). Figure 1. 3D AFM images of PMMA films before and after protein adsorption. PMMA films alone (a) or following incubation in lysate (30 min) (b), or 10% HI-FBS (20 hr) (c). Lysate yielded the highest NF-кB/AP-1 activation compared to the other conditions (p<0.01) (Figure 2). The results suggest that PMMA activates these factors more than TCPS (p<0.01). NF-кB/AP-1 activity in FBS is higher than HI-FBS due to alkaline phosphatases in serum. Figure 2. NF-кB/AP-1-dependent SEAP activity in the supernatant of RAW-Blue cells cultured in HI-FBS or FBS on TCPS or PMMA surfaces. Relative SEAP activity was calculated as a fold-change relative to the control (TCPS with HI-FBS, no cells). (Mean ± SD, n=3; ** p<0.01, *** p<0.001) Conclusion: Molecules in cell lysate adsorb to polymer surfaces and strongly activate NF-кB/AP-1 in macrophages that are grown on PMMA. Future research will include TLR-signaling inhibition assays to define if increased NF-кB/AP-1 activity is attributed to TLR activation. Canada Foundation for Innovations (CFI) John R. Evans Leaders Fund (JELF); Senate Advisory Research Committee (SARC) grant; The R. Samuel McLaughlin Fellowship

An in vitro model of foreign body giant cell formation mediated by damage-associated molecular patterns

Event Abstract Back to Event An in vitro model of foreign body giant cell formation mediated by damage-associated molecular patterns Jeremy Antonyshyn1, 2 and Lindsay E. Fitzpatrick1 1 Queen's University, Department of Chemical Engineering, Canada 2 University of Toronto, Institute of Biomaterials & Biomedical Engineering, Canada Introduction: Protein adsorption to biomaterials is a well-known phenomenon thought to contribute to the progression of the foreign body reaction (FBR). While the contribution of serum proteins has been studied extensively, those derived from tissues damaged during implantation have received little attention. Toll-like receptors (TLRs) may mount an inflammatory response against these damage-associated molecular patterns (DAMPs) through their activation of NF-κB and AP-1 transcription factors[1]. The purpose of this study was to determine whether TLR-mediated macrophage recognition of DAMPs adsorbed to biomaterial surfaces contributes to the progression of the FBR. Methods: Fetal bovine serum (FBS) and 3T3 fibroblast lysate were used as sources of serum proteins and DAMPs, respectively. Total protein concentrations were determined by microBCA assay. Tissue culture polystyrene (TCPS) was incubated in lysate and/or FBS, then rinsed with phosphate-buffered saline (PBS). Activation of NF-κB/AP-1 in RAW-Blue macrophages induces expression of secreted embryonic alkaline phosphatase (SEAP), the enzymatic activity of which can be quantified with the QUANTI-Blue assay. These cells, cultured in 10% heat-inactivated FBS, were exposed to TCPS alone (negative control), lipopolysaccharide (LPS; positive control), and lysate/FBS-adsorbed TCPS. Culture was ended after 24hrs. Morphology of May-Grünwald-Giemsa –stained cells was evaluated. IL-4 and IFN-γ concentrations were determined by ELISAs. NF-κB/AP-1 –dependent SEAP activity was quantified. All experiments were run in triplicate, ANOVAs with Tukey’s post-hoc analyses used, and p<0.05 accepted as statistically significant. All values reported as mean±SD. Results and Discussion: Lysate induced significantly greater macrophage fusion than TCPS (39.2±13.9% vs 0.5±0.3%, respectively; p<0.05), yielding multinucleated cells (27.0±19.9 nuclei/cell; 9068.0±6548.0µm2) of irregular morphology and random nuclear arrangement (Fig 1). Previous models have relied on IL-4 and IFN-γ to induce fusion[2], but neither was detected suggesting an alternative mechanism. Lysate induced NF-κB/AP-1 activation to the same extent as known TLR-agonist LPS (11.4±0.1 vs 11.9±1.1 fold greater than TCPS, respectively), which was significantly greater than that induced by FBS (0.3±0.1 fold; p<0.001). While this suggests that TLRs mount an inflammatory response against DAMPs but not serum proteins, both compete for adsorption as evidenced by the dose-dependent suppression of NF-κB/AP-1 activity with dilution of lysate in FBS but not PBS (p<0.0001; Fig 2). Importantly, lysate continued to elicit significant activation compared to FBS when comprising as little as 0.06% of total protein added (p<0.05), supporting the relevance of DAMPs to the FBR in vivo. Conclusion: These findings indicate that DAMPs-adsorbed biomaterials elicit an inflammatory response in macrophages and promote their fusion to yield foreign body giant cells, the hallmark of the FBR. Canada Foundation for Innovation’s (CFI) John R. Evans Leaders Fund (JELF); Queen’s University Senate Advisory Research Committee (SARC) Grant

Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation

Necrotic cells passively release HMGB1, which can stimulate TLR4 in an autocrine fashion to potentially initiate “sterile” inflammation that maintains different disease states. We have shown that prooxidants can induce NF-κB activation through TLR4 stimulation. We examined whether prooxidants enhance HMGB1-induced TLR4 signaling through NF-κB activation. We used LPS-EK as a specific agonist for TLR4, and PPC and SIN-1 as in situ sources for ROS. As model systems, we used HEK-Blue cells (stably transfected with mouse TLR4), RAW-Blue™ cells (derived from murine RAW 264.7 macrophages) and primary murine macrophages from TLR4-KO mice. Both HEK-Blue and RAW-Blue 264.7 cells express optimized secreted embryonic alkaline phosphatase (SEAP) reporter under the control of a promoter inducible by NF-κB. We treated cells with HMGB1 alone and/or in conjunction with prooxidants and/or inhibitors using SEAP release as a measure of TLR4 stimulation. HMGB1 alone and/or in conjunction with prooxidants increased TNFα and IL-6 released from TLR4-WT, but not from TLR4-KO macrophages. Pro-oxidants increased HMGB1 release, which we quantified by ELISA. We used both fluorescence microscopy imaging and flow cytometry to quantify the expression of intracellular ROS. TLR4-neutralizing antibody decreased prooxidant-induced HMGB1 release. Prooxidants promoted HMGB1-induced NF-κB activation as determined by increased release of SEAP and TNF-α, and accumulation of iROS. HMGB1 (Box A), anti-HMGB1 and anti-TLR4-neutralizing pAbs inhibited HMGB1-induced NF-κB activation, but HMGB1 (Box A) and anti-HMGB1 pAb had no effect on prooxidant-induced SEAP release. The present results confirm that prooxidants enhance proinflammatory effects of HMGB1 by activating NF-κB through TLR4 signaling.

Abstract 3941: Recurrent IL4R mutations in primary mediastinal large B cell lymphoma

Abstract Introduction: Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of aggressive B cell lymphoma that arises from thymic medullary B cells and characteristically presents as a mass in the anterior mediastinum. A proportion of PMBCL patients suffer from refractory or relapsing disease, and subsequent salvage therapies are frequently ineffective. Development of targeted therapies is impeded by the lack of knowledge about the mutational landscape in the lymphoma genomes and mutation-associated phenotypes. We recently reported somatic mutations in the transcriptome of 7 PMBCL patients and 3 cell lines by next-generation sequencing and found a hotspot mutation in exon 8 of IL4R (Gunawardana et al, Nat Genet, 2014). Interleukin (IL)-4 is a type II cytokine that regulates immune responses through binding to a receptor complex consisting of the IL4 receptor alpha (IL4Rα) chain and IL13Rα or the common gamma chain (γc). Activation of IL4R by IL-4 and/or IL-13 initiates intracellular signal transduction mediated by the phosphorylation of JAnus Kinase-Signal Transducer and Activation of Transcription (JAK-STAT) pathway. We hypothesize that constitutively active JAK-STAT observed in PMBCL is in part due to gain-of-function IL4R mutations signaling through this oncogenic pathway. Methods and samples: A sequencing cohort consisting of fresh-frozen pretreatment lymph node biopsies from 62 PMBCL cases were selected from the BCCA tissue archive. DNA from PMBCL tumors and 3 PMBCL-derived cell lines were extracted for IL4R exonic PCR amplification and Sanger sequencing or high-throughput sequencing on an Illumina MiSeq instrument. WT and mutant (I242N) IL4R cDNA were cloned into mammalian expression vectors and expressed in the lymphoma cell line DEV and in engineered HEK293 cells expressing STAT6. Supernatant from cultured cells were used to measure the activity of STAT6-dependent expression of secreted embryonic alkaline phosphatase (SEAP). Extracted RNA and protein from transfected cells were used for qRT-PCR and Western blotting, respectively. Results: Somatic IL4R mutations were found in 18 of 65 (28%) cases confirming a hotspot mutation (I242N) in exon 8 in 11 of 18 (61%) mutated cases. Ectopic expression of the mutant in HEK293 cells showed increased SEAP levels compared to WT (534% vs. 100%) indicating STAT6 activation independent of cytokine stimulation. Introduction of the mutant into DEV cells showed IL4 independent hyperphosphorylation of JAK2, STAT5 and STAT6 proteins and upregulation of the B cell activation marker CD23 (963% vs. 100% in WT). Conclusions: IL4R is frequently mutated in PMBCL with a recurrent hotspot affecting the transmembrane domain of the protein. Functional analyses characterize these mutations as gain-of-function leading to constitutive activation of the JAK-STAT pathway and downstream target genes. These data suggest IL4R mutations as novel driver alterations in PMBCL and may provide a rational therapeutic target. Citation Format: Jay Gunawardana, Tessa Van Tol, Katina Mak, David Twa, Elizabeth Chavez, Bruce Woolcock, Robert Kridel, Anja Mottok, Shannon Healy, Adele Telenius, Merrill Boyle, Susana Ben-Neriah, Stacy Hung, Christoffer Hother, Randy Gascoyne, Christian Steidl. Recurrent IL4R mutations in primary mediastinal large B cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3941. doi:10.1158/1538-7445.AM2015-3941

147. Transcription Factor Binding Site in Plasmid Regulates Persistence of Transgene Expression in Mouse Liver

Persistent expression of transgene is most desirable for treatment of genetic diseases. It is primarily controlled by genetic elements in the promoter region where transcription factors bind. The objective of this study is to assess the role of the transcription factor binding sites (TFBS) present in the most commonly used CMV and albumin promoters containing the promoter core sequence and several TFBS for ATF1, NFκB, SRF, AP1, Sp1, NF1α, CEBPA, HNF4α, and HNF1 in determining persistency of transgene expression in mice. Secreted embryonic alkaline phosphatase (SEAP) and interleukin 10 (IL-10) were used as a reporter to monitor transgene expression. Variousdeletion mutation plasmids at the promoter region were made to examine the function of each of the TFBS in regulating reporter gene expression. Similarly, a series of new plasmids containing only one TFBS inserted at the 5’ end of the TATA-box region was made to assess its unique activity in affecting transgene expression. Our results showed that TFBSs are essential for effective transgene expression in mouse liver after a hydrodynamic tail vein gene delivery. Plasmid with a TATA-box with all TFBS deleted resulted in minimal level of gene product, 700-fold lower than the original plasmids. Reporter gene expression appears transient reaching the background level in less than 2 weeks. The more TFBS the plasmid has, the higher level of gene product was seen. Plasmid containing a single TFBS either NFκB, ATF1, CEBPA, HNF4α, or HNF1 exhibits persistent transgene expression for more than 2 months, at which animals injected with plasmids containing NFkB, ATF1, CEBPA, HNF4α, or HNF1 exhibited gene product level 60, 130, 1020, 2250, and 2076 folds higher than that of animals injected with plasmids without TFBS, respectively. Mechanism study employing real time PCR, Western Blotting, and ChIP reveals involvement of histone acetylation and the binding of acetylated histones to plasmid DNA sequences in TFBS-mediated enhancement in persistency of reporter gene expression. TFBSs for HNF4α appear most potent in recruiting H4AC and TBP proteins to the plasmids. Our results suggest that TFBSs for NFkB, ATF1, CEBPA, HNF4α, and HNF1 are important elements to include into the promoter for achieving longer-term gene expression. In addition, histone acetylation on plasmid is a probable pathway that TFBS regulate the duration of transgene expression.

Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1.

BackgroundWe have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.MethodsGC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.ResultsTGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.ConclusionsOur results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.

Mesenchymal Stem Cells Secrete Immunologically Active Exosomes

Mesenchymal stem cells (MSCs) have been shown to secrete exosomes that are cardioprotective. Here, we demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active. MSC exosomes induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue, a THP-1 reporter cell line with an NFκB-SEAP reporter gene. In contrast to lipopolysaccharide, they induced high levels of anti-inflammatory IL10 and TGFβ1 transcript at 3 and 72 h, and much attenuated levels of pro-inflammatory IL1B, IL6, TNFA and IL12P40 transcript at 3-h. The 3-h but not 72-h induction of cytokine transcript was abrogated by MyD88 deficiency. Primary human and mouse monocytes exhibited a similar exosome-induced cytokine transcript profile. Exosome-treated THP-1 but not MyD88-deficient THP-1 cells polarized activated CD4(+) T cells to CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) at a ratio of one exosome-treated THP-1 cell to 1,000 CD4(+) T cells. Infusion of MSC exosomes enhanced the survival of allogenic skin graft in mice and increased Tregs.

Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the β-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-κB/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.