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. Author manuscript; available in PMC: 2026 Aug 29.
Published before final editing as: J Appl Toxicol. 2026 Aug 26:10.1002/jat.70415. doi: 10.1002/jat.70415

Toll-like Receptors in the Mechanism of Hexabromocyclododecane-induced Production of Pro-inflammatory Cytokines

Wenjun Zhu 1, Aleshia Seaton-Terry 1, Brian Townsend 1, Zinia Hunter 1, April Falconer-Turner 1, Syeda Raika Shahid 2, Margaret Whalen 2
PMCID: PMC13523799  NIHMSID: NIHMS2206146  PMID: 42648711

Abstract

Hexabromocyclododecane (HBCD), a brominated flame retardant, has raised increasing health concerns due to its bioaccumulation in human tissues and its ability to induce proinflammatory cytokines. Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are major mediators of inflammation, and their chronic elevation contributes to autoimmune disorders, cardiovascular disease, and cancer. Previous studies have implicated mitogen-activated protein kinases (MAPKs) as mediators of HBCD-induced IL-1β and IL-6 production. Because MAPKs act downstream of toll-like receptors (TLRs)-key inflammatory response regulators, identifying whether TLRs contribute to HBCD-driven inflammation is essential. This study examined the roles of TLR1/2, TLR2, TLR3, TLR4, and TLR8 in HBCD-induced IL-1β, IL-6, and TNF-α production in peripheral blood mononuclear cells (PBMCs). PBMCs were pretreated with selective inhibitors targeting TLR1/2 (CUCPT 22), TLR2 (C29), TLR3 (CUCPT 4a), TLR4 (TAK-242), TLR8 (CUCPT 9a), and MyD88 (TJ-M2010–5), followed by exposure to HBCD (1–5 μM). The results indicated that inhibition of TLR4 significantly reduced HBCD-induced production of all three cytokines, demonstrating its central roles in this response. Blocking TLR8 significantly reduced HBCD-induced IL-1β production. In contrast, inhibition of TLR1/2, TLR2 or TLR3 produced no consistent effects on HBCD-induced cytokine production. Inhibition of the TLR-associated adaptor protein MyD88 significantly attenuated HBCD-induced IL-1β and IL-6 production, whereas TNF-α production was not consistently affected. These results further elucidate the mechanism by which HBCD leads to elevation of pro-inflammatory cytokine production by human immune cells.

Keywords: Hexabromocyclododecane, TLR, IL-1β, IL-6, TNF-α, Inflammation, PBMCs, MAPKs

SHORT ABSTRACT

Hexabromocyclododecane (HBCD) increases production of IL-1β, IL-6, and TNF-α by immune cells in a MAP kinase (MAPK)-dependent manner. Toll-like receptors (TLRs) activate signaling pathways linked to MAPKs that lead to production of these cytokines. HBCD-induced production of these cytokines is dependent on TLR4, while IL-1β production also involves TLR8. TLR1/2, TLR2, and TLR3 do not consistently contribute to HBCD-induced cytokine production. Blocking TLR-associated adaptor protein MyD88 reduced HBCD-induced production of IL-1β and IL-6, but had no consistent effect on TNF-α.

INTRODUCTION

Chronic inflammation is a sustained inflammatory response that persists in the absence of infection or injury, playing a critical role in the development and progression of several major health conditions, such as autoimmune diseases, cancers, cardiovascular diseases, and metabolic syndrome (Libby, 2007; Mantovani et al., 2008). It is characterized by persistent elevation of key pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which leads to inflammatory responses, contributing to tissue damage, autoimmunity, and tumor progression (Adegbola et al., 2018; Balkwill & Mantovani, 2001; Barcia et al., 2011; Dinarello, 2000; Dinarello, 2004; Farrugia & Baron, 2016; Fischer & Maier, 2015; Gabay, 2006; Kumari et al., 2016; Macarthur et al., 2004; Scheller et al., 2011; Schneider et al., 2013). Toll-like receptors (TLRs) are integral components of the innate immune system, playing essential roles in the detection of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Duan et al., 2022; Santos-Sierra, 2021). Upon recognizing these molecular signatures, TLRs initiate signaling cascades that activate transcription factors such as nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1), which subsequently promote pro-inflammatory cytokine expression (Kawai & Akira, 2010). Activation of TLRs typically initiates downstream signaling cascades through adaptor proteins such as myeloid differentiation primary response 88 (MyD88) (Behzadi et al., 2021). This signaling subsequently activates mitogen-activated protein kinase (MAPK) pathways, particularly the extracellular signal-regulated kinase (ERK1/2) and p38 MAPK pathways (Arthur & Ley, 2013; Duan et al., 2022; Kawai & Akira, 2010; Kircheis & Planz, 2023; Kyriakis & Avruch, 2012; O’Neill & Bowie, 2007). The TLR-MAPK pathway induces potent inflammatory cytokines, IL-1β, IL-6, and TNF-α, which further reinforce the inflammatory response (Kawai & Akira, 2006). TLRs stimulate IL-1β and TNF-α transcription through NF-κB and MAPK activation, and both cytokines feedback to amplify inflammation via their own receptors (O’Neill, 2008). TNF-α binds TNFR1 and activates additional MAPK and NF-κB pathways through TRADD and RIP1, enhancing the expression of other cytokines and matrix-degrading enzymes involved in tissue destruction. Similarly, IL-1β binds to IL-1R, engaging MyD88-dependent signaling that converges on MAPK and NF-κB, sustaining chronic inflammation (Choy & Panayi, 2001; Giurini et al., 2022). IL-6, primarily induced downstream of TLR and TNFR signaling, acts systemically to promote acute phase protein production and supports the differentiation of Th17 cells, reinforcing adaptive immunity (Aluri et al., 2021; Farooq et al., 2021).

Hexabromocyclododecane (HBCD) is a bioaccumulative brominated flame retardant widely used in industrial materials including building insulation, furniture upholstery, and housing for appliances and electronics (Cao et al., 2018; Koch et al., 2015; Marques & Cairrao, 2023). It has been shown to increase IL-1β and IL-6 production in human peripheral blood mononuclear cells (PBMCs) through mitogen-activated protein kinase (MAPK)-dependent mechanisms (Anisuzzaman & Whalen, 2016; Falconer-Turner et al., 2025). HBCD can also increase TNF secretion from human immune cells in a MAPK dependent manner (Yasmin & Whalen, 2018). Recently, it was demonstrated that HBCD exposure increases both the secretion as well as the intracellular levels (production) of both IL-1β and IL-6. These increases were observed after 6 hours and more pronounced at 24 hours. The study identified that MAPKs play a crucial role in HBCD-induced cytokine production, (Falconer-Turner et al., 2025). However, while MAPK activation has been established, the roles of the upstream regulators of MAPK activation, TLRs, in HBCD-induced cytokine production remain unclear.

Environmental contaminants such as pentachlorophenol (PCP), Tributyltin (TBT) and dibutyltin dichloride (DBT) have been shown to increase IL-1β and IL-6 production in human peripheral blood mononuclear cells (PBMCs) in a MAPK-dependent manner (Brown et al., 2018; Martin, Gabure, et al., 2019; Martin, Maise, et al., 2019; Sushak et al., 2020), with TLR involvement demonstrated for PCP (TLR4, TLR8) (Seaton-Terry et al., 2025) and TBT (TLR4, TLR1/2, and TLR8) (Alcala et al., 2022). Like TBT and PCP, HBCD is structurally stable, lipophilic, and demonstrates persistent bioaccumulation (Covaci et al., 2006; Hong et al., 2005). Given that TLR activation is a major regulator of MAPKs and inflammatory cytokine production, and that structurally similar environmental contaminants use TLR-dependent mechanisms, it is critical to determine whether HBCD-induced inflammation is mediated through specific TLR pathways. To clarify the molecular mechanisms underlying HBCD-induced immunotoxicity, this study investigates the roles of TLR1/2, TLR2, TLR3, TLR4, and TLR8, and MyD88 in HBCD-induced production of IL-1β, IL-6, and TNF-α in human PBMCs,

MATERIALS AND METHODS

Preparation of Peripheral Blood Mononuclear Cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from leukocyte filters obtained from the Red Cross Blood Bank Facility in Nashville, TN. The filters were back-flushed with an elution solution containing phosphate-buffered saline (PBS) with 5 mM disodium EDTA and 2.5% [w/v] sucrose (Meyer et al., 2005). The collected eluate was then layered onto LymphoSep™ cell separation medium and centrifuged at room temperature for 30 minutes at 1200g. The granulocytes and red blood cells settled at the bottom, while the PBMCs remained at the LymphoSep interface. The PBMCs were then collected, washed with PBS, and resuspended after the supernatant was discarded. To remove platelets, the cells were overlaid on bovine calf serum (BCS) and centrifuged for five minutes at 450g, this process was repeated three times. The final cell pellet was suspended in cell culture media.

Chemical and Inhibitor Preparation

HBCD was sourced from Fisher Scientific (St. Louis, MO). A 50 mM stock solution was prepared by dissolving HBCD in dimethyl sulfoxide (DMSO), and working concentrations were obtained by diluting the stock solution in cell culture media. Stock solutions of TLR, MyD88, and MAPK pathway inhibitors were prepared at 50 mM concentrations in DMSO to ensure complete solubility and consistent application. Specific TLR inhibitors were CUCPT22 (TLR1/2 inhibitor); C29 (TLR2 inhibitor); CUCPT4a (TLR3 inhibitor); TAK242 (TLR4 inhibitor); and CUCPT9a (TLR8 inhibitor). The stock solutions were diluted in cell culture media to give final concentrations of 10 μM for C29, TAK242, and CUCPT9a (Bhattacharyya et al., 2018; Mistry et al., 2015; Zhang et al., 2018). CUCPT4A was used at a final concentration of 20 μM (Cheng et al., 2012), while that for CUCPT22 was 8 μM (Faksh et al., 2016). The MyD88 inhibitor, TJ-M2010–5 was diluted to give a final concentration of 10 μM (Miao et al., 2020)).

Cell Treatments

PBMCs were treated for 1 h with a specific inhibitor or appropriate control followed by a 24 h exposure to HBCD at different concentrations (1, 2.5 and 5 μM). The concentrations of HBCD were selected based on previous studies that showed consistent HBCD-induced increases in one or more pro-inflammatory cytokine at those exposures (Falconer-Turner et al., 2025). Those same studies showed increases in pro-inflammatory cytokine production down to 0.05 μM (50 nM), which was the lowest level tested (Falconer-Turner et al., 2025). The control treatment was prepared by diluting the vehicle (DMSO) to the same level as was seen in the 5 μM HBCD treatment (for a final DMSO concentration of less than 0.005%) The control for the presence of inhibitor was the DMSO vehicle plus the inhibitor. After incubation, cells were pelleted, and the supernatant was collected and stored at −80°C. The cell pellet was then washed with PBS, after which PBS was removed, and cells were lysed using M-PER™ mammalian protein extraction reagent (Thermo Scientific, Waltham, MA). The lysate was incubated on ice for 20 minutes with periodic mixing, followed by aliquoting into labeled tubes for storage at −80°C. Cell supernatants were analyzed to determine secreted cytokine levels using enzyme-linked immunosorbent assay (ELISA) and cell lysates were analyzed to determine cellular cytokine levels using western blot.

Measurement of secreted levels of IL-1β, IL-6, and TNF-α

IL-1β, IL-6, or TNF-α levels were measured using the OptEIA™ enzyme-linked immunosorbent assay (ELISA) kit for human IL-1β, IL-6, or TNF-α (BD-Pharmingen, San Diego, CA). A capture antibody diluted in coating buffer was added to the wells of a 96-well plate (Fisher, St. Louis, MO) and incubated overnight at 4°C. After incubation, the plate was washed three times with PBS containing 0.05% Tween-20. Wells were then blocked with a blocking buffer to minimize non-specific binding and incubated for one hour at room temperature. After washing away the blocking buffer, cell supernatants and IL-1β, IL-6, or TNF-α standards were added to the wells and incubated at room temperature for two hours. Following further washing, the wells were incubated with detection antibody for one hour. The excess antibody was removed through additional washes, and a substrate solution was added for 30 minutes at room temperature. The reaction was stopped, and the absorbance was recorded at 450 nm using a Thermo-Multi- scan plate reader (Fisher Scientific).

Western Blot

Cell lysates were analyzed by SDS-PAGE using 10% polyacrylamide gels, followed by protein transfer to a PVDF (polyvinylidene difluoride) membrane. The PVDF membranes were immunoblotted using specific primary antibodies against IL-1β (Cell Signaling Technology, Danvers, MA), IL-6, TNF-α (Cell Signaling Technology), and β-actin (Sigma). Detection was performed using the UVP Imaging Software. Protein band intensities were quantified via densitometric analysis using Image Station analysis software. Each experiment was performed with internal control, ensuring accurate comparisons across treatments. Normalization of protein band intensities was carried out using β-actin to adjust for variations in protein loading.

Molecular Docking

Receptor and Ligand Preparation:

The three-dimensional coordinates of the human Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) signaling complex were obtained from the RCSB Protein Data Bank (PDB accession number: 3FXI). Receptor preparation was performed using UCSF Chimera. Crystallographic water molecules, co-crystallized heteroatoms, native lipid ligands, magnesium ions not directly involved in ligand recognition, and N-linked carbohydrate residues located outside the MD-2 ligand-binding cavity were removed prior to docking analyses. The molecular structure of hexabromocyclododecane (HBCD) was retrieved from PubChem (CID: 33121). Ligand preparation was conducted using AutoDock Tools (v1.5.7), including assignment of atom types, addition of hydrogen atoms, definition of torsional parameters, and calculation of partial atomic charges using the Gasteiger–Marsili method. Non-polar hydrogen atoms were merged with their corresponding carbon atoms according to the AutoDock protocol. HBCD exhibited no rotatable bonds due to its rigid cyclic structure.

Charge and Grid Preparation:

Polar hydrogen atoms were added to the prepared TLR4/MD-2 receptor structure, and Kollman united atom charges were assigned using AutoDock Tools prior to conversion to PDBQT format. Due to the large size of the TLR4/MD-2 complex, a single docking grid could not adequately capture the full receptor surface while maintaining a grid spacing of 0.500 Å. Therefore, two overlapping grid boxes were generated to ensure complete coverage of potential binding regions. The first grid box was centered on the hydrophobic binding cavity of MD-2 and the adjacent region of the TLR4 ectodomain. The second grid box extended along the longitudinal axis of the complex to include the lower region of MD-2 and the distal portion of the TLR4 ectodomain. Docking simulations were performed independently within each grid, and binding poses were evaluated based on predicted binding energies and spatial location within the receptor.

Molecular Docking Simulation and Structural Clustering:

Molecular docking was performed using the Lamarckian genetic algorithm (LGA) implemented in AutoDock 1.5.7. The search parameters included 25,000,000 energy evaluations per run and a maximum of 27,000 generations. The initial population size was set to 150 individuals. A mutation rate of 0.02, crossover rate of 0.80, and elitism value of 1 were applied. One hundred independent docking runs were performed for each ligand. Docked conformations were clustered using a root-mean-square deviation (RMSD) cutoff of 2.0 Å. The lowest-energy pose in each cluster was selected as the representative binding mode. Binding poses were ranked based on predicted free energy of binding (ΔG, kcal/mol), cluster population, and lowest binding energy conformation. Docking results were visualized using PyMOL (v2.5).

Statistical Analysis

Significance was assessed using Student's t-test, with adjustments for Type I error performed via the Bonferroni-Holm method (Holm, 1979).

RESULTS

Effects of TLR1/2 inhibition on HBCD-Induced IL-1β, IL-6 and TNF-α production

Effects of TLR1/2 inhibition on the ability of HBCD to stimulate IL-1β, IL-6 and TNF-α production in PBMCs isolated from four distinct donors are shown in Figure1. Cells were pre-incubated with the TLR1/2 inhibitor CUCPT 22 (or appropriate control) for 1 h and then exposed to HBCD at concentrations of 5, 2.5, and 1 μM for 24 h. PBMCs from all donors exhibited increased IL-1β, IL-6, and TNF-α production in response to HBCD exposure, the magnitude of the response varied among donors and concentrations. Inhibition of TLR1/2 did not consistently attenuate HBCD-induced IL-1β, IL-6 and TNF-α production. For example, cells from donor F901 showed marked increases in IL-1β production at all HBCD concentrations, and inhibition of TLR1/2 enhanced the response at 5. μM HBCD (Figure 1A). Similarly, cells from donor F901 showed increases in IL-6 production following HBCD exposure, and inhibition of TLR1/2 did not diminish the HBCD-induced increases, however cells from all other donors showed a loss of HBCD-induced IL-6 production when TLR1/2 was blocked (Figure 1B). Additionally, cells from donor F901 demonstrated 1.5-, 1.4-, and 1.3-fold increases in TNF-α production when exposed to 5, 2.5, and 1 μM HBCD, respectively. With the inhibition of TLR1/2, TNF-α production was further increased to 2.3-, 2.0-, and 1.7-fold at the corresponding concentrations (Figure 1C). These results indicate that TLR1/2 is not consistently involved in mediating IL-1β, IL-6 and TNF-α production in response to HBCD exposure.

Figure 1.

Figure 1.

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Effects of TLR1/2 inhibition on HBCD-induced production of IL-1β, IL-6 and TNF-α. PBMCs were treated with the selective TLR1/2 inhibitor CUCPT22 (8 μM) or an appropriate control for 1 h prior to exposing the cells to 5.0, 2.5 and 1.0 μM HBCD for 24 h. A) Fold changes in production of IL-1β in human PBMCs (cells from donors F901, F902, F952, F954). B) Fold changes in production of IL-6 in human PBMCs C) Fold changes in production of TNF-α in human PBMCs. Fold changes in production are the average of the fold changes intracellular and secreted for the cytokine. Fold changes in intracellular levels were determined by western blot and fold changes for secreted levels were determined by ELISA. An increase in secretion or in intracellular levels is a number greater than 1; a decrease is a number less than 1. The control is arbitrarily set at 1, as all fold changes were determined relative to the control. Average of the intracellular and secreted fold changes greater than 1 indicates an increase in production. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

Effects of TLR2 Inhibition on HBCD-Induced IL-1β, IL-6 and TNF-α production

Figure 2 shows the effects of TLR2 inhibition on HBCD-stimulated IL-1β, IL-6 and TNF-α production in PBMCs isolated from four donors. Cells were treated, as described above for the TLR1/2 inhibitor, with the selective TLR 2 inhibitor C29. Inhibition of TLR2 did not consistently diminish HBCD-induced IL-1β, IL-6 and TNF-α production. For example, cells from donor F876 and F883 showed significant decreases in HBCD-stimulated IL-1β production when TLR2 was inhibited. In contrast, PBMCs from donor F884 and F886 showed enhanced IL-1β production at 5 μM HBCD in the presence of the inhibitor. PBMCs from donor F876 showed 1.5-, 1.5-, and 1.4-fold increases in IL-6 production when exposed to 5, 2.5, and 1 μM HBCD, respectively. In the presence of inhibitor, these HBCD-induced increases were completely blocked (Figure 2B). While cells from donor F886 exhibited increased IL-6 production following exposure to HBCD, inhibition of TLR2 resulted in increases in HBCD-induced IL-6 production at 5 and 2.5μM (Figure 2B). HBCD-stimulated TNF-α production in cells from donor F884 were 3.3-, 4.9-, and 2.2-fold at 5, 2.5, and 1 μM HBCD, respectively and the presence of TLR2 inhibitor enhanced these increases (Figure 2C). In contrast, PBMCs from donor F876 showed marked reductions in HBCD-induced TNF-α production when TLR2 was inhibited (Figure 2C). These data indicate that TLR2 does not consistently have a role in IL-1β, IL-6 and TNF-α induction by HBCD.

Figure 2.

Figure 2.

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Effects of TLR2 inhibition on HBCD-induced production of IL-1β, IL-6 and TNF-α. PBMCs were treated with the selective TLR2 inhibitor C29 (10 μM) or an appropriate control for 1 h prior to exposing the cells to 5.0, 2.5 and 1.0 μM HBCD. A) Fold changes in production of IL-1β in human PBMCs (cells from donors F876, F883, F884, F886). B) Fold changes in production of IL-6 in human PBMCs C) Fold changes in production of TNF-α in human PBMCs. Fold changes in production were determined as described in Figure 1. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

Effects of TLR3 Inhibition on HBCD-Induced IL-1β, IL-6 and TNF-α production

Figure 3 illustrates the effects of TLR3 inhibition on HBCD stimulation of IL-1β, IL-6 and TNF-α production in PBMCs. Cells were treated as previously described with the highly selective TLR3 inhibitor (CUCPT4a). Inhibition of TLR3 did not consistently alter HBCD-induced IL-1β production. In cells from donor F887, exposure to HBCD resulted in approximately 1.7-, 2.2-, and 2.3-fold increases in IL-1β production at 5, 2.5, and 1 μM HBCD, respectively. Inhibition of TLR3 significantly reduced these HBCD-stimulated increase (Figure 3A). However, cells from donors F888 and F893 showed enhanced HBCD-stimulated IL-1β production when TLR3 was inhibited (Figure 3A). Similarly, inhibition of TLR3 did not consistently inhibit the ability of HBCD to increase IL-6 production (Figure 3B) or TNF production (Figure 3C) in PBMCs.

Figure 3.

Figure 3.

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Effects of TLR3 inhibition on HBCD-induced production of IL-1β, IL-6 and TNF-α. PBMCs were treated with the selective TLR3 inhibitor CUCPT 4a (20 μM) or an appropriate control for 1 h prior to exposing the cells to 5.0, 2.5 and 1.0 μM HBCD. A) Fold changes in production of IL-1β in human PBMCs (cells from donors F886, F887, F888, and F893). B) Fold changes in production of IL-6 in human PBMCs. C) Fold changes in production of TNF-α in human PBMCs. Fold changes in production were determined as described in Figure 1. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

Effects of TLR4 Inhibition on HBCD-Induced IL-1β, IL-6 and TNF-α production

The effects of TLR4 inhibition on HBCD-stimulated IL-1β, IL-6 and TNF-α production in PBMCs are shown in Figure 4. Cells were treated as described above with the TLR4 inhibitor TAK-242. Inhibition of TLR4 consistently diminished HBCD-induced increases in IL-1β, IL-6 and TNF-α production across donors. For example, cells from donor F860 exhibited approximately 1.5- and 1.6- fold increases in IL-1β production when exposed to 5 and 2.5μM HBCD, respectively. When TAK-242 was present, these HBCD-induced increases were reduced to no-increase and 1.2- fold, respectively (Figure 4A). Likewise, donor F868 exhibited elevated IL-1β production following exposure to HBCD, with approximately 6.6-, 1.7-, and 1.4-fold increases at 5, 2.5, and 1 μM HBCD. Treatment with TAK-242 reduced IL-1β production to 1.1- at 5 μM and no-increase at both 2.5 μM and 1 μM (Figure 4B). Similarly, cells from donor F861 exhibited increases in IL-6 production of approximately 2.8-, 1.1-, and 1.2-fold at 5, 2.5, and 1 μM HBCD, respectively. Treatment with the TLR4 inhibitor significantly reduced IL-6 production at all concentrations (Figure 4C). Cells from 3 additional donors showed similar results (Figure 4D). Cells from donor F861 demonstrated 5.1-, 2.1-, and 1.2-fold increases in TNF-α production following HBCD exposure. Treatment with TLR4 inhibitor resulted in significantly reduced TNF-α production at 5 and 2.5 μM HBCD tested (Figure 4D). The results from experiments with 3 additional donors are shown in Figure 4 E These donors exhibited similar results. The observed reductions across multiple donors and concentrations support the involvement of the TLR4 in mediating HBCD-stimulated IL-1β, IL-6 and TNF-α production in PBMCs.

Figure 4.

Figure 4.

Figure 4.

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Effects of TLR4 inhibition on HBCD-induced production of IL-1β, IL-6 and TNF-α. PBMCs were treated with the selective TLR4 inhibitor TAK 242 (10 μM) or an appropriate control for 1 h prior to exposing the cells to 5.0, 2.5 and 1.0 μM HBCD. A) Blot from a representative experiment (F860) showing the effects of TLR4 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of IL-1β. B) Fold changes in production of IL-1β in human PBMCs (cells from donors F860, F861, F867, and F868) after. C) Blot from a representative experiment (F861) showing the effects of TLR4 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of IL-6. D) Fold changes in production of IL-6 in human PBMCs. E) Blot from a representative experiment (F861) showing the effects of TLR4 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of TNF-α. F) Fold changes in production of TNF-α in human PBMCs. Fold changes in production were determined as described in Figure 1. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

Effects of TLR8 Inhibition on HBCD-Induced IL-1β, IL-6 and TNF-α production

Figure 5 shows effects of inhibition of TLR8, using the selective inhibitor CUCPT9a, on HBCD-stimulated IL-1β, IL-6 and TNF-α production in PBMCs. Inhibition of TLR8 decreased HBCD-induced IL-1β production across donors. For example, PBMCs from donor F901 exhibited 2.6-, 1.7-, and 1.6-fold increases in IL-1β production when exposed to 5, 2.5, and 1 μM HBCD, respectively. With the inhibition of TLR8, these HBCD-induced increases were reduced to 1.3- and 1.1- fold at 5 and 2.5 μM exposures respectively, and no increase at the 1 μM exposure (Figure 5A). Similar results were seen in cells from all donors (Figure 5B). In contrast, inhibition of TLR8 did not consistently diminish the ability of HBCD to increase the production of either IL-6 or TNF-α (Figures 5C and 5D). Thus, TLR8 has a role in mediating HBCD-induced IL-1β production but does not appear to play a consistent role in HBCD-stimulated IL-6 and TNF-α production in human immune cells.

Figure 5.

Figure 5.

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Effects of TLR8 inhibition on HBCD-induced production of IL-1β, IL-6 and TNF-α. PBMCs were treated with the selective TLR8 inhibitor CUCPT 9a (10 μM) or an appropriate control for 1 h prior to exposing the cells to 5.0, 2.5 and 1.0 μM HBCD. A) Blot from a representative experiment (F932) showing the effects of TLR8 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of IL-1β. B) Fold changes in production of IL-1β in human PBMCs (cells from donors F860, F861, F867, F868). C) Fold changes in production of IL-6 in human PBMCs. D) Fold changes in production of TNF-α in human PBMCs. Fold changes in production were determined as described in Figure 1. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

Effects of MyD88 inhibition on HBCD-Induced IL-1β, IL-6 and TNF-α production

Figure 6 (A–E) illustrates the effect of MyD88 inhibition on HBCD-stimulated IL-1β, IL-6, and TNF-α production by PBMCs. Cells were pre-incubated with a MyD88 inhibitor TJ-M2010–5 or an appropriate control prior to 24-hour exposure to HBCD at concentrations of 5, 2.5, and 1 μM. Inhibition of MyD88 reduced HBCD-induced IL-1β and IL-6 production across donors, while TNF-α production was less consistently affected. For IL-1β production, HBCD exposure resulted in increased cytokine levels across all donors. Cells from donors F891, F892, F895, F897, and F899 exhibited 1.34-, 1.75-, 1.44-, 1.4-, 1.35-fold increases in IL-1β production at 2.5 μM HBCD respectively, which were reduced to 1.23-, 1.08-, 1.09-, and no increase (F897, and F899) in the presence of the MyD88 inhibitor (Figure 6B). A similar trend was observed for IL-6 production. Cells from donors F891, F892, F895, F897, and F899 exhibited 1.75-, 2.32-, 1.57-, 1.75-, 1.43- fold increase in IL-6 production at 5 μM HBCD respectively, which were reduced to 1.53-, 1.51-, 1.07-, 1.50-, 1.18- fold in the presence of the MyD88 inhibitor (Figure 6D). In contrast, TNF-α production was not consistently reduced by MyD88 inhibition (Figure 6E). MyD88 inhibition resulted in reductions in HBCD stimulation of TNF-α production in doner F892 and F895. However, these effects were not consistent across donors. For example, in cells from donor F891 inhibition of MyD88 caused no changes in HBCD-induced TNF-α production.

Figure 6.

Figure 6.

Figure 6.

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Effects of MyD88 inhibition on HBCD-induced production of IL-1β, IL-6 and TNF-α. PBMCs were treated with a selective MyD88 inhibitor (TJ-M2010–5, 10 μM) or an appropriate control for 1 h prior to exposing the cells to 5, 2.5, 1 μM HBCD. A) Blot from a representative experiment (F895) showing the effects of MyD88 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of IL-1β. B) Fold changes in production of IL-1β in human PBMCs (cells from donors F891, F892, F895, F897, and F899). C) Blot from a representative experiment (F895) showing the effects of MyD88 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of IL-6. D) Fold changes in production of IL-6 in human PBMCs. E) Fold changes in production of TNF-α in human PBMCs. Fold changes in production were determined as described in Figure 1. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

Effects of ERK1/2 pathway and p38 inhibition on HBCD-Induced TNF-α Production

Figure 7A illustrates the effect of inhibition of the ERK1/2 (MAPK1/2) pathway on HBCD-stimulated TNF-α production by PBMCs. Cells were incubated with MAPK/ERK kinase (MEK) inhibitor (PD98059) or appropriate control for 1 hour and subsequently exposed to HBCD at concentrations of 5, 2.5, and 1 μM for 24 hours. PBMCs from all four donors exhibited increases in TNF-α production at one or more HBCD concentration and inhibition of the ERK1/2 pathway did not consistently block these increases. This indicated that ERK1/2 pathway activation by HBCD is not a driver of HBCD-stimulated TNF-α production.

Figure 7.

Figure 7.

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Effects of inhibition of MEK and p38 on HBCD-stimulated TNF-α production. PBMCs were treated with MEK inhibitor (PD98059, 50 μM) or an appropriate control or with p38 inhibitor (SB202190, 50 μM) or an appropriate control for 1 h prior to exposing the cells to 5, 2.5, 1 μM HBCD. A) Fold changes in production of TNF-α in human PBMCs (cells from donors F869, F872, F874, F875). Fold changes in production were determined as described in Figure 1. B) Blot from a representative experiment (F941) showing the effects of p38 inhibition on HBCD-induced changes in intracellular levels, secreted levels, and production (average of intracellular and secreted fold changes compared to their control) of TNF-α. C) Fold changes in production of TNF-α in human PBMCs (cells from donors F876, F936, F937, F941). Fold changes in production were determined as described in Figure 1. An asterisk (*) indicates a significant decrease in fold production (P<0.0167 initially, using the Bonferroni-Holm method).

PBMCs were incubated with the p38 inhibitor (SB202190) or appropriate control for 1 hour followed by exposure to HBCD at concentrations of 5, 2.5, and 1 μM for 24 hours. PBMCs from all four donors exhibited marked increases in TNF-α production at one or more HBCD concentrations. Cells from donor F876 exhibited 5.1-, 3.2-, and 2.2-fold increases in TNF-α production when exposed to 5, 2.5, and 1 μM HBCD, respectively. With the inhibition of p38, these HBCD-induced increases were significantly reduced to 1.5-, 1.2-, and no-increase, respectively (Figure 7B). Inhibition of p38 consistently diminished HBCD-induced increases in TNF-α production across cells from all donors (Figure 7C). Results indicate that inhibition of p38 MAPK consistently suppresses HBCD-induced TNF-α production in human PBMCs. The significant reduction observed across multiple donors and concentrations supports a central role for p38 MAPK signaling in mediating HBCD-stimulated TNF-α production in PBMCs.

Molecular Docking of HBCD with TLR4

Molecular docking studies of HBCD with TLR4 indicated that HBCD is capable of interacting with TLR4 in the lipid binding pocket formed with the co-receptor MD2 (Figure 8 A–D). The dual-grid docking approach allowed sampling of the TLR4/MD-2 surface at a grid spacing of 0.500 Å. Docking results from both grid boxes converged on the same predicted binding site within the MD-2 co-receptor. In the upper-domain grid, the lowest-energy cluster contained 24 out of 100 docking runs, with a mean binding energy of −7.4 kcal/mol. The representative pose from this cluster showed a binding energy of −7.42 kcal/mol and an estimated inhibition constant (Ki) of 3.62 μM. In the lower-domain grid, docking results also converged to the same predicted binding site. The dominant cluster contained 30 out of 100 docking runs, with a mean binding energy of −7.4 kcal/mol. The top-ranked pose from this grid showed a binding energy of −7.43 kcal/mol and an estimated Ki of 3.59 μM. In both grid sets, predicted binding poses were located within the hydrophobic cavity of MD-2.

Figure 8. Structural visualization of hexabromocyclododecane (HBCD) bound to the Toll-like receptor 4 (TLR4) complex.

Figure 8.

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Structural modeling based on the crystal structure of the human TLR4–MD-2 complex (PDB ID: 3FXI). (A) Anterior (front) view and (B) posterior (back) view of the TLR4 ectodomain monomer (shown in gray) format illustrating the spatial distribution and predicted binding orientation of HBCD (represented as magenta sticks). The accessory myeloid differentiation factor 2 (MD-2) protein is shown in blue. (C and D) Enlarged view of the HBCD ligand within the primary cavity of the MD-2 accessory protein. Surrounding amino acid residues located within a 4 Å radius of the docked ligand—specifically Arg106, Asp99, Asp100, Lys72, and Gly97—are depicted as sticks and individually labeled to map the structural boundaries of the binding pocket Molecular graphics were generated using PyMOL v3.1.

DISCUSSION

TLRs, a family of pattern-recognition receptors expressed on immune and tissue cells, recognize conserved molecular motifs from pathogens (PAMPs) as well as endogenous danger signals released by damaged cells (DAMPs) (R. Chen et al., 2025; Li & Wu, 2021; Marongiu et al., 2019). Upon binding a PAMP or DAMP, TLR proteins undergo dimerization and recruit intracellular adaptor proteins like MyD88 or TRIF (TIR-domain-containing adapter-inducing IFN-β) (O’Neill & Bowie, 2007; Wicherska-Pawłowska et al., 2021). These adaptors initiate signaling cascades that activate downstream kinases and transcription factors (MAPKs and NF-κB), which in turn drive the expression of pro-inflammatory genes (Balka & De Nardo, 2019; Tartey & Takeuchi, 2017). Through this pathway, TLR activation rapidly triggers the production of key inflammatory cytokines such as IL-1β, IL-6, and TNF-α (Akhtar et al., 2020; Mogensen, 2009). Thus, aberrant TLR activity or overstimulation can contribute to pathological inflammation. Because TLRs are such crucial gatekeepers for inflammation, they represent a vulnerable interface through which environmental contaminants can disrupt immune homeostasis.

HBCD has been classified as a persistent organic pollutant, and its use has been restricted or banned in many countries (Abdallah et al., 2008; Koch et al., 2015). Nevertheless, existing contamination and the chemical’s long environmental-life mean that human exposure to HBCD is ongoing (Covaci et al., 2006). HBCD has been found in various human tissues and blood. It was detected in 30 out of 33 human milk samples, at concentrations ranging between 3 and 188 ng/g lipid weight (lw), with a median value of 27 ng/g lw (Eljarrat et al., 2009). Additionally, evidence indicates that HBCD crosses the placental barrier, impacting fetal development (Arnot et al., 2009; Covaci et al., 2006; Johnson-Restrepo et al., 2008; Meijer et al., 2008). Several studies have found HBCD in human serum of individuals with no occupational exposure at concentrations ranging from 0.5 to 11 ng/g lw (Covaci et al., 2006; Roosens et al., 2009; Thomsen et al., 2008). A study in Norway of workers in a plant producing expandable polystyrene with HBCD added as a flame retardant showed levels as high as 856 ng/g-lipid in the serum of these workers (approximately 13 nM) (Thomsen et al., 2007). Further, workers engaged in the direct production of HBCD may experience even higher levels, based on prediction models (Yi et al., 2016). While the levels of HBCD examined in this study are above those measured in human serum, HBCD-induced increases in pro-inflammatory cytokines have been seen at the lowest levels of HBCD tested of 50 nM (which approaches those measured in serum) (Falconer-Turner et al., 2025).

Pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α are key mediators of inflammation, and their sustained elevation has been linked to the development of cardiovascular disease, autoimmune disorders, neurodegenerative diseases, and cancer (Adegbola et al., 2018; Balkwill & Mantovani, 2001; Barcia et al., 2011; Dinarello, 2004; Farrugia & Baron, 2016; Fischer & Maier, 2015; Gabay, 2006; Kumari et al., 2016; Macarthur et al., 2004; Schneider et al., 2013). Recent immunotoxicological studies have shown that 24 h HBCD exposure can elevate the production of the pro-inflammatory cytokines IL-1β and IL-6 (Falconer-Turner et al., 2025). HBCD’s ability to induce IL-1β and IL-6 appears to involve changes at the gene expression level, HBCD-treated PBMCs show heightened mRNA levels for these cytokines, indicative of transcriptional upregulation (Falconer-Turner et al., 2025). Furthermore, consistent with the importance of MAPKs noted above, HBCD’s induction of IL-1β and IL-6 depends on MAPK signaling, specifically the p38 and ERK 1/2 MAPK pathways PBMCs (Falconer-Turner et al., 2025). Previous research reported that HBCD exposure of PBMCs increases TNF-α secretion, indicating that it can broadly activate the key cytokines of the innate inflammatory response (Yasmin & Whalen, 2018). The inappropriate elevation of IL-1β, IL-6, and TNF-α by a persistent toxicant like HBCD has the potential to initiate sterile inflammation (inflammation in the absence of infection) (G. Y. Chen & Nuñez, 2010; Rock et al., 2010) and contribute to chronic inflammatory pathologies (Adegbola et al., 2018; Balkwill & Mantovani, 2001; Barcia et al., 2011; Dinarello, 2004; Farrugia & Baron, 2016; Fischer & Maier, 2015; Gabay, 2006; Kumari et al., 2016; Macarthur et al., 2004; Schneider et al., 2013) in exposed populations.

MAPKs are critical downstream components of Toll-like receptor (TLR) signaling pathways, which regulate innate immune responses to PAMPs or DAMPs (Akira et al., 2001; Kawai & Akira, 2006). Based on this relationship, the current study investigated whether TLR activation contributes to HBCD-induced cytokine production in human PBMCs. Cell-surface TLRs (TLR1/2, TLR2, TLR4), intracellular TLRs (TLR3, TLR8), and TLR-associated adaptor protein MyD88 were examined due to their established roles in inflammatory signaling and cytokine regulation (Akira et al., 2001; Kawai et al., 2024).

The results demonstrate that TLR4 plays a central role in HBCD-induced production of IL-1β, IL-6, and TNF-α. Inhibition of TLR4 consistently reduced cytokine (IL-1β, IL-6, and TNF-α) production across experimental conditions, indicating that HBCD activates inflammatory signaling through pathways associated with this receptor. Additionally, TLR8 was found to contribute specifically to IL-1β production, suggesting a role for intracellular TLR signaling in modulating selective cytokine responses. In contrast, inhibition of TLR1/2, TLR2, and TLR3 did not consistently affect cytokine (IL-1β, IL-6, and TNF-α) production, indicating that these TLRs are not primary mediators of HBCD-induced inflammatory responses. MyD88 is the immediate downstream component of many TLRs including TLR4 and TLR8. Results showed that when MyD88 was blocked, HBCD was consistently impeded in its ability to stimulate IL-1β and IL-6 production. This is confirmatory of a role for MyD88 coupled TLRs, like TLR4 and TLR8, in causing HBCD elevation of these cytokines. In contrast, TNF-α production was less consistently affected by MyD88 inhibition, indicating that HBCD-induced TNF-α production may involve additional signaling mechanisms.

These findings are consistent with previous studies examining other environmental toxicants. Pentachlorophenol (PCP) has been shown to induce IL-1β and IL-6 production through TLR4- and TLR8-dependent mechanisms, while organotin compounds such as tributyltin (TBT) involve TLR4, TLR1/2, and TLR8 in cytokine induction (Alcala et al., 2022; Seaton-Terry et al., 2025). The similarity between the results for HBCD and those from other hydrophobic contaminants such as PCP and TBT (Arnold et al., 1997; Nowosielski & Fein, 1998). suggests that HBCD, which is also highly lipophilic (Hayward et al., 2006), may similarly localize to membrane environments where it can interact with TLRs and initiate receptor-mediated signaling. Although HBCD is capable of crossing cellular membranes and may directly influence intracellular signaling components such as MAPKs, the observed dependence on TLR4 suggests that receptor-mediated mechanisms are a major driver of cytokine induction. HBCD as well as other man-made hydrophobic compounds, such as PCP and TBT, may preferentially interact with and activate TLR4 due the fact that TLR4 activation requires the myeloid differentiation (MD)-2 protein co-receptor which creates a hydrophobic area required for activation of TLR4 by one of its physiological stimuli, bacterial lipopolysaccharide (Resman et al., 2009). Previous studies have shown that PCP interacts with TLR4 via the hydrophobic pocket formed by TLR4/MD2 interaction (Seaton-Terry et al., 2025). Molecular docking studies presented here suggest that hexabromocyclododecane (HBCD) also interacts with the TLR4/MD-2 receptor complex within the hydrophobic cavity of MD-2.

Activation of TLRs leads to downstream signaling through adaptor proteins such as MyD88, resulting in activation of MAPK pathways and transcription of pro-inflammatory cytokines (De Nardo, 2015; O’Neill, 2008). As mentioned above HBCD has been shown to use p38 in its stimulation of both IL-1β and IL-6 production in immune cells (Falconer-Turner et al., 2025) and in HBCD-stimulated TNF-α secretion (Yasmin & Whalen, 2018), but has not yet been examined in HBCD stimulation of TNF-α production. Here we show that, inhibition of p38 significantly reduced TNF-α production. In contrast, inhibition of ERK1/2 resulted in increased cytokine production, suggesting the presence of complex regulatory or compensatory mechanisms within MAPK signaling pathways.

Once TLRs are engaged, the MyD88 adaptor protein likely transduces the signal, leading to activation of downstream kinases including p38. These signaling events then drive the transcription and secretion of IL-1β, IL-6, and TNF-α. Results highlight that TLR4 is the receptor that mediates HBCD induced increases in IL-1β, IL-6, and TNF-α, adding an important piece to the puzzle of HBCD’s immunotoxic profile. These findings carry significant implications for chronic inflammation and public health. HBCD has the capacity to act as a chronic inflammatory stimulus by persistently elevating immune cell production of pro-inflammatory cytokines. This study shows that blocking TLR4 can significantly reduce upregulated cytokines (IL-1β, IL-6, and TNF-α) induced by HBCD and that blocking TLR8 can reduce HBCD induced IL-1β production. It also shows that the p38 MAPK pathway is needed for HBCD-induced TNF-α production. The consistent reduction of IL-1β and IL-6 following MyD88 inhibition across donors suggests that these cytokines are primarily regulated through MyD88-dependent TLR signaling pathways. This is consistent with the established role of MyD88 in activating downstream signaling cascades, including MAPK pathways, which are critical for the transcriptional regulation of pro-inflammatory cytokines. In contrast, TNF-α production was not consistently diminished by MyD88 inhibition, indicating that HBCD-induced TNF-α expression may be partially independent of MyD88. One possible explanation is the involvement of alternative adaptor proteins, such as TRIF, particularly in the context of TLR4 signaling, which is known to utilize both MyD88-dependent and MyD88-independent pathways. The persistence of TNF-α production despite MyD88 inhibition suggests that TRIF-mediated signaling or other parallel pathways may compensate for the loss of MyD88 function. These findings provide important insight into the molecular mechanisms underlying HBCD-induced immune activation and environmental contaminants can drive inflammation through toll-like receptors.

ACKNOWLEDEMENTS:

This work was supported by the National Institutes of Health grant 5U54CA163066

Footnotes

CONFLICT OF INTEREST STATEMENT: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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