Preparation of monocyte-depleted peripheral blood mononuclear cells (MD-PBMCs) and their treatment with Hexabromocyclododecane (HBCD) to study it effects on interleukin 1? (IL-?) secretionsAbstractHexabromocyclododecane (HBCD) is a hydrophobic/lipophilic brominated flame retardant that is used in a variety of applications including building insulation. In this study the effects of HBCD exposure on IL-1? secretion from human immune cells are studied. Monocyte- depleted peripheral blood mononuclear cells (MD-PBMCs) were exposed to different concentrations of HBCD ranging from 5 ?M to 0.05 ?M for 24 hours. The cells were then lysed and the secreted IL1-? levels were measured using enzyme-linked immunosorbent assay (ELISA). Results showed that HBCD induced increased secretions of IL-1? by human immune cells. The exact concentrations of HBCD that increased IL-1? secretion varied by donor. Alterations in IL1-? secretion of human immune cells following exposure to HBCD may potentially cause dysregulation and facilitate cancer metastasis.Introduction The environmental contaminant Hexabromocyclododecane (HBCD) is a hydrophobic, aliphatic cyclic brominated hydrocarbon with the chemical formula C12H18Br6. It is an additive mainly used as a flame retardant in a variety of applications. Away from its primary use as thermal insulation in the building and construction industry it is also used as a flame-retardant coating for furniture, upholstery, insulation blocks in trucks, textiles for motor vehicle interior, draperies, wall coverings, blinds, electronic appliances, cables, and others. Its ability to bestow flame retardant capabilities at low concentrations (0.5% – 1%) to materials without altering the physical or chemical properties of the end product made HBCD a highly desired commodity. HBCD became commercially available in the 1960s and its global demand grew rapidly in the 1990s and early 2000s (Marvin, 2011). China, Europe, and the United States were the top producers of HBCD at that time. According to a report issued by the United Nations Environment Program (UNEP), the global demand for HBCD in 2003 was estimated to be 43 million pounds (UNEP, 2011). In 2015, upwards of 66 million pounds were being produced worldwide with China being the dominant producer and consumer (Li et al. 2016). In 2016 it was estimated that the United States produced or imported 1-10 million pounds of HBCD (EPA, 2017).The high demand for HBCD over the years unfortunately produced a negative impact on the environment, wildlife, and humans. Recent studies have shown HBCD to be toxic to the environment and has been found in a large variety of environmental samples taken from marine organisms, bird eggs, sewage sludge soil and air (Covaci et al. 2006). The problems associated with HBCD stems from how it’s processed when conjugated with other materials. HBCD is not chemically bound to textiles or plastics and as a result it leaches from the surface of these products into the environment. The hydrophobicity and lipophilicity of HBCD further compounds the environmental and ecological issues since it greatly increases the potential for bioaccumulation (U.S. EPA 2014).Human exposure to HBCD is believed to be through dust inhalation and food ingestion (Harrad, 2009). Previous studies has shown that HBCD is an endocrine disruptor, developmental neurotoxicant, and causes alterations in the rat thyroid system (Palace et al. 2010). HBCD accumulation in mice liver, brain, blood, and fat tissues have also been observed after oral exposure to HBCD (Szabo et al. 2010).The human body’s defense against pathogens is the immune system. This system consists of two inherent defense systems, the innate (nonspecific) system and the adaptive (specific) system. Within the innate system the skin and mucous membranes is the first line of defense, while natural killer (NK) cells, phagocytes, antimicrobial proteins, inflammation and fever acts as the second line of defense. The adaptive defense system is the body’s third line of defense against pathogens. It consists of the humoral response (involving B cells) and the cellular response (involving T cells).  The innate and adaptive defense systems work both independently and cooperatively with each other in order to protect the body against foreign invaders. Communication between the immune cells occurs via cytokines. Cytokines are small proteins that are produced by a number of different cell and are involved in cell-to-cell communication. Interleukin-1? (IL-1?) and interleukin-6 are both pro-inflammatory cytokines produced by monocytes, NK lymphocytes, T lymphocytes and macrophages in response to an infection. IL-1? is involved with cell differentiation, cell proliferation and tissue repair, while IL-6 stimulates the production of acute phase proteins in response to an injury. Elevated levels of these pro-inflammatory cytokines have been associated with numerous disease states including cancer. Thus, synthesis and secretion of these regulators of inflammation in the absence of infection or injury can lead to chronic inflammation and those diseases that results, such as tumor growth, rheumatoid arthritis, Crohn’s disease, and multiple sclerosis.Previous studies have shown that HBCD decreases the ability of NK cells to destroy tumor cells by decreasing the binding function and cell-surface marker expression in NK cells (Hinkson and Whalen 2009, 2010). This modification of the NK cell’s function can in turn decrease the lytic capability of the NK cells. From these results it can be observed that HBCD has the capability to potentially disrupt normal immune cellular functions which can lead to various diseases and tumor growth. In this study the effects of HBCD exposures on IL-1? secretion was examined. As previously mentioned IL-1? is an important regulator of immune responsiveness, tissue growth and repair. Thus, if its levels are dysregulated, loss of proper immune function and increased invasiveness of tumors could ensue.Materials and MethodsPreparation of monocyte- depleted peripheral blood mononuclear cells (MD-PBMCs)Preparations of MD-PBMCs were isolated from Leukocyte filters were obtained from the Red Cross Blood Bank Facility in Nashville, TN. as described in Meyer et al., 2005.  Leukocytes were retrieved by back-flushing the filters with elution medium (PBS containing 5 mM disodium EDTA and 2.5% sucrose [w/v] and collecting the eluent. The eluent was then layered onto Ficoll-Hypaque and centrifuged for 30 minutes at room temperature. Granulocytes and red cells pelleted at the bottom of the tube while the MD-PBMCs floated on the Ficoll-Hypaque. MD-PBMCs were collected and washed with PBS. The supernatant was poured off and the pellet suspended in PBS. Cells were then layered on bovine calf serum (BCS) and centrifuged for 5 minutes. After successful platelet removal the pellet is suspended in complete media and placed in a glass petri dish at 37° C for 45 minutes in a CO2 incubator.Chemical preparation HBCD were purchased from (Sigma-Aldrich, St. Louis, MO). A stock solution was prepared by dissolving HBCD in dimethylsulfoxide (DMSO). Desired concentrations of HBCD were prepared by dilution of the stock into complete media.Cell treatments and lysateMD-PBMCs (at a concentration of 4 million cells/ mL) were treated with HBCD with appropriate control at concentrations of 0.05 – 5 µM for 24 hours. Following the incubation, the cells were pelleted, and the supernatants were collected and frozen at -80?C. The pellet was resuspended and washed in cold PBS that contains phosphatase inhibitor. The PBS is removed, and the cells lysed with Active motif buffer solution and placed on ice for 20 minutes. The cells were then aliquoted in appropriately labeled tubes and stored at -80?C.IL-1 ? secretion assay IL-1 ? levels were assessed using the OptEIA™ enzyme-linked immunosorbent assay (ELISA) human IL-1? kit (BD-Pharmingen, San Diego, CA). Capture antibody diluted in coating buffer was applied to the wells of a 96 well plate (Fisher, St. Louis, MO). The plate was incubated overnight at 4?C. Following the incubation, capture antibody was removed by washing the plate three times with wash buffer (PBS with 0.05% Tween-20). The wells were then treated with blocking buffer to prevent non-specific binding and the plate was sealed and incubated at room temperature for 1 h. The blocking buffer was then removed by washing the plate three times. The supernatants of the sample cell and the IL-1 ? standards were added to the plate and incubated at room temperature for 1 h. Following this incubation, the plate was washed five times and the detection antibody to IL-1? added to the plate and allowed to incubate for 1h. The plate was then washed seven times to remove excess detection antibodies. Next a substrate solution was added to the plate and incubated for 30 minutes at room temperature to produce a colored product. A stop solution was then added, and the absorbance was measured at 450 nm on a Thermo-Multiscan plate reader (Fisher Scientific).Statistical analysis Statistical analysis of the data was carried out utilizing ANOVA and Student’s t test. Data were initially compared within a given experimental setup by one way ANOVA. A significant ANOVA was followed by pair wise analysis of control versus exposed data using Student’s t-test, a p-value of less than 0.05 was considered significant.ResultsThe effects of exposures to HBCD on secretion of IL-1? from MD-PBMCs after 24 hours from five donors are shown in Table 1. There were significant increases in IL-1? secretion from MD-PBMC from all donors examined. Significant increases in IL-1? secretion was seen at 0.1, 2.5 and 5 ?M concentration for all donors. Additional concentrations of HBCD caused increases in IL-1? secretion depending on the donor and the magnitude of the increase at a given concentration of HBCD also varied among donors. For instance, the cells from the donor F521 treated with 0.05, 0.1, 0.25, 0.5, 1,2.5 and 5 ?M showed significant increases of 2.2, 2.0, 2.9, 3.1, 4.5, 5.1, and 9.0-fold respectively after 24 h while cells from donor F555 showed significant increases of 2.3, 1.7, 1.3, 1.9, 1.8, 1.8 and 2-fold at these same concentrations.Table 1. Effect of 24-hour exposure to HBCD on IL-1? secretion from MD-PBMCs.Interleukin 1? secreted in pg/mL (mean ± S.D.)HBCD (µM)F521F529F533F555F58601727±2194755±38217336±1009449±6618190±2930.053745±447*4761±78964090±1240*21501±55*22249±301*0.13359±159*11874±58*48308±918*16057±143*22645±817*0.254938±211*4361±7652592±1002*11967±528*23304±1176*0.55315±562*4995±11749379±1220*17830±295*16154±37617824±398*5495±21034057±1212*17089±583*21341±677*2.58947±755*7075±2121*29314±558*16898±313*26132±499*515508±319*7355±680*20308±601*18951±508*33941±509*Values are mean ± S.D. of triplicate determinations. * Indicates a significant change in secretion compared to control cells (cells treated with vehicle alone), p<0.05SummaryIn this study monocyte-depleted peripheral blood mononuclear cells (MD-PBMCs) were prepared and treated with the contaminant HBCD for 24 hours. The cells were then lysed and evaluated using ELISA. The preliminary results showed a significant increases in IL-1? secretion from MD-PBMC for all donors examined. These increases were observed at 0.1, 2.5 and 5 µM concentration for all donors. The exact concentrations of HBCD that increased IL-1? secretion could not be determined at this time since it varied by donor. For example MD-PBMCs from F521 showed maximum 9.0 fold increase in IL-1? secretion at 5 µM HBCD after 24 h, while cells from F555 showed maximum 2 fold increase at this same concentration.Future study will involve the preparation of more MD-PBMCs samples, the exposure of these samples to HBCD using different times (1hr, 6hr, 48hr and 6 days), SDS-PAGE analysis, evaluation the effects of HBCD on the production of IL-1? production, IL-6 secretion and production, and statistical evaluation of results collected. ReferencesAbdallah, M.; Harrad, S. Personal exposure to HBCDs and its degradation products via ingestion of indoor dust. Environ. 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