Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

Introduction Although production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L). Material and Methods PBMCs isolated from healthy donors were pre-incubated with DE-71 at various concentrations and subsequently incubated with the monocyte stimulator LPS, or the T-cell activator PHA-L. Interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-17A, and IL-17F were quantified in the supernatants by Luminex kits. Results At non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71. Conclusions We demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.


DE-71 and Stimulation
DE-71, a gift kindly provided by Marta Axelstad, National Food Institute, Technical University of Denmark and Dr. Kevin Crofton of the U.S. Environmental Protection Agency, was dissolved in dimethyl sulfoxid (DMSO) (D2438 Sigma Aldrich, St. Louis, MO) and diluted in cell culture medium to final concentrations of 0.01, 0.1, 1, 5, and 10 μg/mL, respectively. Three standards in DMSO and two standards in cell culture medium were analysed to determine PBDE concentrations, as described below. The DMSO concentration in each well was 1‰. Cell cultures with DE-71 were pre-incubated for one hour at 37°C and 5% CO 2 . Subsequently, LPS from E. coli (100 pg/mL) or PHA-L (5 μl/mL, both with CAS 9008-97-3, from Sigma-Aldrich, St. Louis, MO) was added. Unstimulated PBMCs, i.e. cell cultures exposed to DE-71 but without LPS or PHA-L stimulation, were also investigated. Cultures were further incubated for 20 to 22 hours, centrifuged for 10 min. at 460 x G at 4°C, and supernatants were harvested and stored at -20°C. All measurements of DE-71 were performed in duplicate, except for two samples where only single determinations were done (one sample of 0.1 μg/mL DE-71; one DMSO control). All experiments included a culture medium-control and a culture medium-control with 1 ‰ DMSO added. The 1 ‰ DMSO control was used for statistical analysis.

Cytotoxicity Assessment
DE-71 induced cytotoxicity was analysed by assessing the content of lactate dehydrogenase (LDH) in cell culture supernatants as described [21]. For this purpose, a homogenous membrane integrity assay, CytoTox-ONE™ (Promega, Fitchburg, WI), was applied according to the manufacturer's protocol with the modification that LDH was not measured directly in cell cultures but in harvested supernatants. The CytoTox-ONE™ passes the following quality control, according to the manufacturer: average background < 20% of the average relative flouroscense unit generated by LDH equivalent to 50,000 cells, LDH activity equivalent to 1,562 cells, fluorescence produced by LDH activities ranging from 0 to 50,000 cells with an R2 ! 0.95. Briefly, frozen harvested cell culture supernatants were thawed, mixed by vortex, stimulated with LPS or PHA-L, and exposed to 0.01, 0.1, 1, 5, 10, and 50 μg/mL DE-71 (n = two blood cell cultures set up in triplicates). Harvested cell culture supernatants from the culture medium control with or without DMSO served as negative control. Fifty μl of each culture medium were transferred to a 96-well microwell plate (Th. Geyer, Renningen, Germany), followed by addition of 50 μl CytoTox-One reagent (Promega, Fitchburg, WI) to all wells. The microwell plate was mixed gently on a shaker and incubated at room temperature for 10 to 15 minutes. Hereafter, 25 μl stop solution was added, the plate was shortly shaken, and results were read on a fluorometer (Victor2, PerkinElmer, Waltham, MA). The LDH contents were proportional to the generated fluorescence (given in relative flourescense units), which was used to evaluate the toxicity of DE-71. The DMSO-concentration was 1 ‰ in the negative culture medium control and in cell cultures, to which 50, 10 or 5 μg/mL DE-71 had been added. In samples/cell cultures where 1, 0.1 or 0.01 μg/mL DE-71 had been added, the DMSO concentrations were 0.2‰, 0.02‰ and 0.002‰, respectively.

PBDE Analysis
Two supernatants and two cell samples (addition of DE-71 at concentrations of 10 μg/mL and 0.01 μg/mL respectively), two controls (1 ‰ DMSO), three stock solutions of DE-71 in DMSO, and two solutions of DE-71 in culture media were analysed for content and composition of BDE-congeners. This was done after the cytokine analysis using once-thawed samples. The analysis followed accredited methods for PBDEs in biota, as described elsewhere [22], and included 11 tri-to heptabrominated congeners (BDE-17, 28, 47, 49, 66, 85, 99, 100, 153, 154 and 183). Between batch analyses of the in house reference material (n = 18), sand eel oil, varied from 2.7% (BDE-47) to 14% (BDE-17), with a mean of 6.6% for all congeners. The detection limits of the instrument ranged between 0.05 and 0.25 pg. Briefly, the samples were spiked with recovery standards, dried with diatomaceous earth (Varian) and Soxhlet extracted with hexane:acetone (4:1). The extracts were cleaned up on a multilayer column consisting of aluminium oxide, silica, and acidified silica. After elution, volume reduction and addition of the internal standard (BDE-71, Cambridge Isotope Laboratories, Tewksbury, MA), the samples were analysed by gas chromatography-mass spectrometry (GC-MS) with electron capture negative ionization. Quantification was based on two calibrations of ten standards each (0.05-25 ng/mL). Three spiked control samples were extracted together with the samples. The DE-71 standards in DMSO, along with four spiked control samples, were evaporated to dryness in silicone vials [22], re-dissolved in iso-octane including the internal standard and analysed without further clean-up.

Ethics
The study was approved by The Danish committees on health research ethics, Capital region (Protocol number: H-1-2012-110 and additional protocol 44717), which in Denmark/Copenhagen also functions as the institutional review board. According to the committee law by the Danish Committees on Health Research Ethics, neither written nor oral informed consent is needed in studies of anonymous human blood samples, which was the case in this study. Blood samples of this study were drawn from anonymous healthy human volunteers, whose identity were unknown to the investigators, and thus the institutional review board waived the need for written informed consent from the participants.
The negative controls were compared by paired t-tests. No statistical analysis was performed for cytotoxicity assessment, since only two cell experiments were done. However, cytotoxicity data are presented in graphs as means ± SD. Results were considered statistically significant when p < 0.05.
Concentrations of INF-γ, IL-2, IL-4, IL-17A, and IL-17F were generally below the lowest standard, and therefore no further analyses of these data were performed (data not shown).
Post-hoc analysis showed a significant increase of IL-1β, IL-6, CXCL8, IL-10, and TNF-α secretion in cultures exposed to 5 and 10 μg/mL DE-71, compared to controls. A few other DE-71 concentrations also caused significant increases in TNF-α secretion (Fig 1, exact Table 1 in S1 File). Three out of six cultures showed a tendency towards an increase in IL-1β, IL-10, and TNF-α upon addition of 0.01 μg/mL DE-71, compared to relevant controls visualised in Fig 1. Increasing the concentration further to 0.1 μg/mL caused a slight, albeit non-significant decrease in cytokine production (data not shown).

Cytokine Secretion from PHA-L-Stimulated PBMC
Median and ranges for the DMSO controls with LPS stimulation are shown in Table 2. The differences found in the post hoc analysis are listed in Table B in S1 File. DE-71 significantly enhanced the PHA-L-elicited secretion of IFN-γ, TNF-α, IL-17A, and IL-17F (with p-values of 0.016, 0.043, < 0.001 and < 0.001, respectively; n = 6) (Fig 2), while secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71) (with p-values of 0.071, 0.19,  0.070, 0.14; n = 6 cultures in duplicates; data not shown and 0.40; n = 5 due to unmeasurable values in one experiment, respectively). DMSO did not seem to affect the cytokine secretion compared to medium alone (range of p-values, 0.06-0.66), except for that of IL-17A (p = 0.024) (n = 6 cultures in duplicates, data not shown). The concentration of IL-4 was generally below the detection limit in all experiments and therefore not subjected to further analysis (data not shown).
Although the post-hoc analyses did not show any significant differences (data not shown), and a true dose-response relationship could not be defined, lower concentrations of DE-71 seemed to stimulate IFN-γ secretion, while higher concentrations were associated with IFN-γ levels similar to those of the control (Fig 2). There was a tendency towards a dose-response increase of DE-71 on TNF-α release (Fig 2). However, this was only significant for 10 μg/mL DE-71, compared to the control in the post-hoc analysis (Fig 2, Table B in S1 File). The secretion of both IL-17A and IL-17F was inhibited by 5 and 10 μg/mL DE-71, compared to the lower DE-71 concentrations, and for IL-17A also to the control (Fig 2).). Conversely, the secretion of both IL-17A and IL-17F tended to increase upon exposure of PMBCs to the lower concentrations of DE-71, but thiswas not confirmed in the post-hoc analysis.
Cytotoxicity DE-71, at a concentration of 50 μg/mL, caused an increase in LDH-levels compared to the negative control, while no increase was observed at lower DE-71 concentrations. DMSO had no effect on cytotoxicity compared to culture medium controls (Fig 3 + Fig 4).

Endotoxin Test
All results were negative.

Discussion
The effect of PBDEs on the immune system is largely unknown. In this study, we have demonstrated an enhancing, non-cytotoxic effect of DE-71 (0.01-10 μg/mL) on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo. The cytokine pattern found for LPS-stimulated cells indicated that DE-71 enhanced proinflammatory responses by monocytes and macrophages, reflected by increasing secretion of TNF-α, IL-1β, IL-6, and IL-8. Secretion of the anti-inflammatory IL-10 was increased as well, possibly reflecting a subsequent compensatory down-regulation of an anti-inflammatory response [23,24]. Thus, DE-71 appeared to enhance pro-inflammatory responses by innate immune cells.
To examine the effect of DE-71 specifically on the adaptive immune system, PBMCs were stimulated with PHA-L. DE-71 tended to enhance PHA-L-induced IFN-γ responses at low concentrations (0.01-1 μg/mL) (Fig 2), supporting a promotion of Th1-cell responses. This conclusion was supported by an enhancing effect of DE-71 on TNF-α production, while not supported by statistical analyses of IFN-γ responses alone. High concentrations of DE-71 caused a reduction of the PHA-L-induced IL-17A and IL-17F production, indicating suppression of Th17-cell responses, in a greement with Th17 responses usually being suppressed by Th1 cytokines [25]. The apparent skewing of T-cell responses towards a Th1-response, and away from Th17 responses might indicate that defence against infection with extracellular bacteria and fungi, which normally requires Th17 responses [26,27], may be compromised by DE-71. It can further be speculated that inappropriate activation of the Th1 cells might facilitate development of autoimmune disease [28].
It is difficult to compare these findings with other studies, since studies concerning PBDE effects on immune cells in vitro and ex vivo are few, with a wide range of different endpoints and mixed conclusions. Only few studies have investigated cytokine responses: Koike et al. [29] found increased production of IL-4 (pg/mL) and increased antigen expression in mouse immune cells (splenocytes and bone marrow cells) after addition of five different flame retardant mixtures to final concentrations of 0.1-10 μg/mL. Koike et al. used concentrations of DE-71 that were comparable to ours, but stimulated the cells with granulocyte macrophage-stimulating factor rather than PHA-L or LPS, which may explain why their findings differed from ours. Hennigar et al. [13] found reduced TNF-α and IL-6 (pg/mL) secretion in porcine alveolar macrophages after addition of seven different concentrations of DE-71 (ranging from 0.0001 μg/mL to 2 μg/mL. These findings are in disagreement with our finding of a lack of effect on IL-4 (pg/mL), and enhancement of TNF-α and IL-6 responses. We used higher concentrations of DE-71 than Hennigar et al. (who used 0.1-2000 pg/mL), which may be one explanation for the diverging results.
Many protocols have been used to study the effects of PBDEs on human cells in vitro. One study investigated immunoglobulin synthesis and proliferation of lymphocytes incubated for 72 hours with two congeners, BDE-47 (4.85 Ã 10 −4 -4.85 μg/mL) and BDE-85 (5.65 Ã 10 −4 -5.65 μg/mL), i.e. at concentration ranges lower than those used in our study, and found no significant effect of these compounds [19]. Koike et al. used 24 hours incubation with DE-71 and two other commercial mixtures to examine the effect on ICAM-1, IL-6 and IL-8 expression in human bronchial epithelial cells pre-treated with protein kinase inhibitors or nuclear receptor antagonists [31]. As in the present study, they also diluted DE-71 in DMSO (1 ‰) and culture medium to final concentrations of 0.01-10 μg/mL. Neither study found any cytotoxicity at concentrations ranging from 0.01-10 μg/mL. Koike et al. measured the cytokine content by enzyme-linked immunosorbent assay, while in the current study a cytometric bead-array system was used. They found an increased production of IL-6, and IL-8, in accordance with our demonstration of an enhancement from DE-71 on the production of these mediators by LPSstimulated cells. Thus, a proinflammatory reaction similar to that of PBMCs occurred in bronchial epithelial cells. Finally, both data and the description above indicated a direct effect of PBDEs on cytokine production.
The composition of the DE-71 mixture used in this study was in good agreeement with the profile of DE-71 specifed by La Guardia et al. [32], and remained unchanged throughout dilutions. Consistent with the chemical analysis, which only showed unquantifiable traces of PBDEs in the control samples, no traces were observed in cell controls without addition of DE-71. Our analysis further showed a partitioning of the PBDEs between cell supernatants and remnants. For one of the samples, the detectable amounts were in agreement with the expected concentrations. However, an uncertain quantification in the second data set prevented an accurate mass balance calculation of this sample. Several factors are likely to influence the PBDE recovery, in particular sorption processes. Our findings underline that the quantification of actual contaminant concentrations in the assays is important in toxicological research.
The PBMCs in our experiment were incubated with DE-71 for 20-22 hours. In comparison, we investigated accumulation of DE-71 in human thyroid cells (unpublished) with an incubation time of 72 hours, where~100% of the added DE-71 was recovered fromthe cell remnants. Hence, it is plausible that DE-71 accumulates over time in human cells. The effect of different exposure times in other such experiments is unknown, and further experiments are needed to investigate these conditions.
The chosen concentrations of DE-71 were based on previous in vitro studies [29,33], which exceeded PBDE levels found in the indoor environment in Denmark [10], as well as in the blood of Danish donors [22,34]. These studies showed correlations between PBDEs in dust and blood, suggesting that dust was a primary exposure source in humans. However, it is not possible to extrapolate in vitro experiments to in vivo consequences. This might be due to reduced effective doses of biologically test chemicals in vitro, evaporation in vitro and/or nonspecific binding to extracellular matrices. Furthermore, a limited number of target sites in cells reduced the numberof toxicity pathways in vitro compared to pathways on a multi-organ level [35]. It is important to note that a comparison between environmental exposure sources and influences on cytokines in blood in vitro is not directly possible. The results, however, have contributed to the elucidation of the cellular effect mechanism of a commercial flame retardant mixture in PBMCs. Lower concentration ranges are suggested in future investigations considering our results. Consequences of the demonstrated specific cytokine response should be considered and compared to cytokine levels in normo-and pathophysiological conditions. Of major importance for the future, new flame retardant chemicals sharing structural and physico-chemical characteristics with the PBDEs have been found in dust samples in American and European homes, including samples from Denmark [36][37][38]. The same adverse effects as those observed after exposure to PBDEs may be expected for these new compunds but are largely unknown. Further studies should include investigations of any immunotoxic mode of action, as well as comparisons between the PBDEs and new flame retardants.

Conclusion
Previous studies on DE-71 have suggested that it can influence the immune system in various ways. Our results support this assumption: we showed that LPS-and PHA-L-induced cytokine secretions by innate immune cells and T cells, respectively, were influenced by DE-71, at noncytotoxic concentrations (0.01-10 μg/mL), in vitro. DE-71 appeared to have an enhancing effect on innate immune responses, as the production of all of the cytokines induced by LPS stimulation were increased. Moreover, our study indicates that DE-71 promotes Th1 responses and inhibits Th17 responses. These data thus add to earlier findings on cytotoxicity and proinflammatory responses in human and animal cells. However, cytokine measurements have been scarce and have in previous studies only dealt with few different cytokines per study. More studies on the cytokine secretion patterns after DE-71 exposure are therefore warranted.
Supporting Information S1 File. Table A and Table B. Post-hoc analyses of significant ANOVA-results. (DOCX)