2,2’4,4’-Tetrabromodiphenyl Ether (PBDE-47) Modulates the Intracellular miRNA Profile, sEV Biogenesis and Their miRNA Cargo Exacerbating the LPS-Induced Pro-Inflammatory Response in THP-1 Macrophages

Abstract

The 2,2’4,4’-tetrabromodiphenyl ether (PBDE-47) is one of the most prominent PBDE congeners detected in the environment and in animal and human tissues. Animal model experiments suggested the occurrence of PBDE-induced immunotoxicity leading to different outcomes and recently we demonstrated that this substance can impair macrophage and basophil activities. In this manuscript, we decided to further examine the effects induced by PBDE-47 treatment on innate immune response by looking at the intracellular expression profile of miRNAs as well as the biogenesis, cargo content and activity of human M(LPS) macrophage cell-derived small extracellular vesicles (sEVs). Microarray and in silico analysis demonstrated that PBDE-47 can induce some epigenetic effects in M(LPS) THP-1 cells modulating the expression of a set of intracellular miRNAs involved in biological pathways regulating the expression of estrogen-mediated signaling and immune responses with particular reference to M1/M2 differentiation. In addition to the cell-intrinsic modulation of intracellular miRNAs, we demonstrated that PBDE-47 could also interfere with the biogenesis of sEVs increasing their number and selecting a de novo population of sEVs. Moreover, PBDE-47 induced the overload of specific immune related miRNAs in PBDE-47 derived sEVs. Finally, culture experiments with naïve M(LPS) macrophages demonstrated that purified PBDE-47 derived sEVs can modulate macrophage immune response exacerbating the LPS-induced pro-inflammatory response inducing the overexpression of the IL-6 and the MMP9 genes. Data from this study demonstrated that PBDE-47 can perturb the innate immune response at different levels modulating the intracellular expression of miRNAs but also interfering with the biogenesis, cargo content and functional activity of M(LPS) macrophage cell-derived sEVs.

Impact of the flame retardant 2,2’4,4’-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles

“…Then, 10 ng/mL of LPS cells were added to the cells and incubated at 37°C and 5% CO 2 for a further 24 hours. Supernatants were collected and sEVs were separated according to the gold standard differential ultracentrifugation (dUC) method, as previously described by Longo and coworkers (16). Three independent preparations were used for the assays.…”

“…The cDNAs were retro-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Milan, Italy) using 2.5 µg/reaction of RNA template. The cDNA was diluted up to 100 ml and real time analyses were performed using a Step OnePlus ™ Real Time PCR System (Applied Biosystems, Monza, Italy) and SYBR Green technology as previously described (16). The primer sequences (Qiagen, Milan, Italy) used for human cytokines were: IL-6 (Interleukin 6, NM_000600), TNF-a (Tumor necrosis factor alpha, NM_000594) and TGF-b (Transforming Growth Factor-b, NM_000660); the housekeeping gene used in all analyses was ACT (human actin beta, NM_001101).…”

“…Furthermore, these chemicals can affect cellular homeostasis, and their presence has been linked to ROS production, DNA damage and apoptosis. PBDE-47 also affects immunity in a number of animal species (14,15), and in vitro assays have demonstrated that it can impair innate immune response (16)(17)(18).…”

“…Accumulating evidence has shown that M1 and M2 macrophage polarization participates in several innate immune responses but also shapes the adaptive immune response (28). Macrophage-derived EVs have been described, showing that they play an important role in the pathogenesis of inflammatory exacerbation and resolution by interacting with different cell types (16,(29)(30)(31). A paradigmatic example of this is the crosstalk between tumoral and immune cells that secrete EVs in the tumor immune microenvironment, modulating immunological activities including macrophage polarization, T cell regulation, and the inhibition of natural killer (NK) cell activity (32,33).…”

Environmental pollutants and essential elements as regulators of miR-30b, miR-223 and Let-7a microRNAs expression in maternal sera from the NEHO cohort

“…Recently, the International Centre of Advanced Study in Environment, Ecosystem and Human Health (CISAS) has developed a project (http://www.cisas.cnr.it/) aimed at investigating environmental pollution and its connection with the ecosystem and human health in highly contaminated areas of southern Italy monitoring pollutant concentrations in human tissues and also performing in vitro/ex vivo studies (Bocca et al 2020; Drago et al 2020; Longo et al 2019; Longo et al 2021; Montalbano et al 2020; Traina et al 2021).…”