Evaluating impacts of the trichloroethylene metabolite S-(1,2-dichlorovyinyl)-L-cysteine on transcriptomic responses and cytokine release in a macrophage model: implications for pregnancy outcomes.

Significant cytotoxicity was observed at 50 μM of DCVC or above, with no significant cytotoxicity observed at 20 μM or less of DCVC ( Figure 3 ). DCVC reduced release of TNFα in a dose-dependent manner with a significant decrease observed at 1 and 5 μM across LPS-, LTA-, and zymosan-induced inflammation. DCVC reduced the release of IL-1β in a dose- dependent manner with a significant decrease observed at 1 and 5 μM across LPS- and LTA-induced inflammation, and 5 μM for zymosan-induced inflammation. DCVC reduced the release of IL-18 in a dose- dependent manner with a significant decrease observed at 1 and 5 μM across LPS- and LTA-induced inflammation, with no significant effects observed with zymosan-induced inflammation. Lastly, DCVC reduced the release of CD163 in a dose-dependent manner with a significant decrease observed at 1 and 5 μM with LPS- induced inflammation, but there were significant effects with LTA and zymosan-induced inflammation ( Figure 4 ). DCVC did not significantly impact the expression of IL-6 or IFNα. Benchmark concentration modeling showed minimal differences in calculated benchmark concentrations for DCVC suppression of cytokine release, regardless of the inflammatory stimulator used ( Figure 5 , Table 1 ). For TNFα release, the benchmark concentrations were 0.21 μM, 0.20 μM, and 0.22 μM across LPS, LTA, and zymosan-stimulated inflammation, respectively. For IL-1β release, the benchmark concentrations were 0.13 μM, 0.35, μM, and 0.42 μM DCVC across LPS, LTA, and zymosan-stimulated inflammation, respectively. Additionally, the benchmark concentration of DCVC with no immune stimulation was 0.0008 μM for TNFα release, and 0.0006 μM for IL-1β release, and the cytotoxic benchmark concentration of DCVC was 24.17 μM ( Table 1 ). Several common and unique transcription factors were enriched among differentially- expressed genes for DCVC, LPS, or DCVC + LPS treatment groups relative to control samples ( Figure 6A ). Four transcription factors were enriched in the DCVC+LPS comparison that were not enriched in the LPS-only gene set (CEBPD, VDR, TRERF1, and EGR1). NF-κB1 and RELA, key transcription factors that mediate inflammation and immune responses, were enriched for all treatment conditions. Figure 6B shows gene expression changes for genes that are targeted by these transcription factors. Supplemental Table 1 shows the p- values and adjusted p-values for enriched transcription factors across each treatment group. Since previous Gene Set Enrichment Analysis showed that the Gene Ontology term “Phagocytosis” (GO:0006909) was enriched for LPS+DCVC vs. LPS-only treatment, we queried the transcriptomic dataset for differentially-expressed genes in Gene Ontology terms corresponding to the major steps of phagocytosis, including, 1) phagocytosis recognition, 2) phagocytosis engulfment, 3) phagolysosome assembly, and 4) phagosome maturation. Individual phagocytosis genes down-regulated by DCVC included: TNF (p < 0.05, fold- change = −3.49), TLR7 (p < 0.05, fold-change = −4.42), SYT7 (p < 0.05, fold-change = −4.77), and SELE (p < 0.05, fold-change = −4.02) ( Figures 7A and 7B ).

For this study, we use a combination of in vitro experiments using a macrophage cell model (THP-1) and bioinformatic analysis of a publicly available transcriptomic ( Harris et al. 2022 ). The experimental design is shown in Figure 1 . THP-1 cells were used as an in vitro macrophage cell model. While this cell line was originally isolated from a male leukemia patient, it replicates certain important anti-microbial functions performed by macrophages in the uterus and fetal membranes, which we sought to model in this study (e.g., pro-inflammatory cytokine release in response to microbial stimulators like lipopolysaccharide) ( Mason et al. 2013 , Lu et al. 2022 , Mohd Yasin et al. 2022 ) THP-1 cells have also been shown to respond similarly to toxicants as primary placental cells, suggesting that they replicate key functions of macrophages in the gestational compartment ( Tetz et al. 2015 ). THP-1 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (pen/strep) (all from Gibco ™ ; Waltham, MA) and incubated at 37°C at 5% CO 2 . Twenty-four hours prior to DCVC treatment, cells were plated into 96-well plates (for cytotoxicity experiments) or 12-well plates (for cytokine assays) at a concentration of 2 × 10 5 cells/mL. Phorbol 12-myristate 13-acetone (PMA; Millipore-Sigma; Burlington, MA) at 100 nM was included in cell culture media to differentiate cells to macrophage phenotype. To confirm that DCVC was not causing overt cytotoxicity in THP-1 cells at the doses tested for cytokine assays and to generate a dose-response curve for benchmark concentration modeling, we assessed cytotoxicity using CytoTox 96 ® Non-Radioactive Cytotoxicity Assay kit (Promega; Madison, Wisconsin), following kit protocols. In brief, after treatment with medium only (control) or DCVC (1, 5, 10, 20, 50, 100, 200, or 500 μM) for 24 hr, cell culture media was collected and LDH activity measured by the addition of a color-changing substrate, followed by quantification of 490/492 absorbance using an Eon ™ Microplate Spectrophotometer plate reader (BioTek, Winooski, VT). DCVC was synthesized by the University of Michigan Medicinal Chemistry Core Synthesis Lab (purity >98%) according to previously published methods ( McKinney et al. 1959 ). Following differentiation with PMA, cells were washed twice with PBS and then treated with medium only (no treatment) or DCVC (0.001, 0.05, 0.01, 0.5, 1, 5 μM) for another 20 hr. LPS (100 ng/ml), lipoteichoic acid (LTA, from Staphylococcus aureus ; [Sigma-Aldrich, St. Louis, MO], 1 μg/ml), or zymosan (from Saccharomyces cerevisiae; [Sigma-Aldrich, St. Louis, MO], 1 μg/ml) was added to selected wells for 4 hr. Treatment conditions included: 1) culture medium only (control), 2) LPS, 3) LTA, 4) zymosan, 5) DCVC (0.001, 1, 5 μM), or 6) DCVC (0.001, 1, 5 μM) + LPS/LTA/zymosan. LPS, LTA and zymosan concentrations were selected based on manufacturer recommendations and previous publications ( Boldenow et al. 2015 , Dahlman et al. 2021 , Harris et al. 2022 ). After 24 hr of total exposure to DCVC and/or microbial toxin, cell media was collected for subsequent quantification of pro- inflammatory cytokines. These microbial toxins were selected due to their unique pathways to elicit cytokine release, as shown in Figure 2 , adapted from ( Noh et al. 2020 ). After treatment with DCVC (with or without microbial toxins), cytokine concentrations in cell culture media were quantified using a Luminex Multiplex Assay Kit (R&D Systems, Minneapolis, MN), following the kit protocols. The kits measured M1 pro-inflammatory cytokines (TNFα, IL-1β, IL-6, and IL-18, and interferon [IFN]-α) and M2 anti-inflammatory marker (CD163). Samples below the limit of detection (LOD) were transformed to LOD/2 prior to statistical analysis. For the cytotoxicity and cytokine data, values were averaged for all replicates (n = 4–5/experiment). Data are presented as mean ± SEM and were analyzed using Prism software (Graphpad, La Jolla, CA). To identify significant differences between treatment groups, we conducted an analysis of variance (ANOVA) test. Following a significant difference with an ANOVA, a Tukey’s post-hoc test was conducted comparing every treatment to every other treatment group. Data were considered significant if the corrected p-value was < 0.05. Benchmark concentration modeling of cytotoxicity and cytokine data was performed using the EPA Benchmark Dose Software (BMDS 3.2) to quantify the relationship between DCVC concentration and the release of pro-inflammatory cytokines TNFα and IL-1β ( Gift et al. 2020 ). One standard deviation from the mean of controls was used as the benchmark response. Dose-response data were modeled using nine different statistical models and the model with the best fit, as indicated by the lowest Akaike information criterion score, was used to identifying benchmark concentrations. To investigate molecular mechanisms mediating the transcriptomic response to DCVC in a macrophage cell model, we conducted an analysis of an existing transcriptomic dataset from a previous study ( Harris et al. 2022 ) in which THP-1 cells (ATCC, Manassas, VA) were treated with DCVC (5 μM) for 20 hr (or non-treated controls) followed by a 4-hr treatment with the microbial toxin LPS (from Escherichia coli O55:B5 [Sigma-Aldrich, St. Louis, MO]; 100 ng/ml), or no LPS treatment. This resulted in four total treatment conditions: 1) medium only (Control), 2) LPS (100 ng/ml), 3) DCVC (5 μM), and 4) DCVC (5 μM) + LPS (100 ng/ml) ( n = 6 per treatment group). In this study, Harris, et al. found that DCVC inhibited LPS-stimulated inflammatory pathways, including phagocytosis. However, no analysis was conducted on the effect of DCVC on specific phagocytosis genes or on potential transcription factors that mediate the effects of DCVC in THP-1 cells ( Harris et al. 2022 ). The transcriptomic profiles used for this analysis are publicly available (Gene Expression Omnibus Accession Number: GSE183141 ). The enrichment software ENRICHR was used to identify enriched transcription factors for the GSE183141 transcriptomic dataset. Lists of differentially- expressed genes (FDR 2.5) for the four treatment comparisons (DCVC vs Control, LPS vs Control, DCVC+LPS vs Control, and DCVC+LPS vs LPS) were uploaded to ENRICHR and differentially-expressed genes were tested for enrichment of transcription factors on the ENRICHR platform ( Chen et al. 2013 , Kuleshov et al. 2016 , Xie et al. 2021 ) using the TRRUST database (Han et al. 2018). In the previously published study for the transcriptomic dataset ( Harris et al. 2022 ), the Gene Ontology term “phagocytosis” (GO: 0006909) was a significantly enriched pathway comparing the LPS + DCVC vs. LPS- only treatment groups, suggesting that DCVC interferes with the ability of macrophages to activate phagocytosis in response to pro-inflammatory stimulation. To gain further insight into DCVC impacts on genes that mediate phagocytosis, an assessment of gene expression data for genes in the following child terms of the “phagocytosis” parent term that corresponded to the major steps in process of macrophage phagocytosis: “phagocytosis, recognition (GO:0006910)”, “phagocytosis, engulfment (GO:0006911)”, “phago-lysosome assembly (GO:0001845)”, as well as the phagocytosis-associated term “phagosome maturation (GO:0090382)”. This analysis identified all differentially expressed genes (FDR < 0.05) for LPS + DCVC vs. LPS-only treatment groups for genes in one or more of these terms and plotted gene expression changes for all treatment group comparisons.

TCE immunotoxicity is well documented ( USEPA 2020 ), however important gaps remain in our understanding of the molecular mechanisms mediating these effects. Macrophages are a key immune cell type in virtually all human tissues ( Epelman et al. 2014 ). Therefore, TCE immunotoxicity in macrophages has potential implications for an array of tissues and organ systems. Moreover, TCE immunotoxicity in gestational tissues is also poorly understood, particularly during unique life events like pregnancy. Pregnant women are exposed to a wide variety of bacterial and fungal pathogens, and immune cells, including macrophages, at the maternal-fetal interface must maintain a delicate balance between active host defense functions against intrauterine pathogens and maternal immune tolerance of the semi-allogenic fetus ( Megli and Coyne 2022 ; Weng et al. 2023 ). Macrophages are a critical phagocytic immune cell type in the fetal membranes ( Aronoff 2020 ), placenta ( Mezouar et al. 2021 ), and uterus ( Brown et al. 2022 ). They are essential for host defense against infections ( Jena et al. 2019 ), tissue remodeling ( Brown et al. 2022 ), and the regulation of reactive oxygen species ( Mezouar et al. 2021 ). Thus, toxicant-induced suppression of immune responses and macrophage function could lead to increased susceptibility to intrauterine infection in vulnerable populations, which can contribute to multiple adverse pregnancy outcomes, such as preterm birth ( Goldenberg et al. 2000 ), neonatal sepsis ( Shane et al. 2017 ), spontaneous abortion (Cao et al. 2018), pre-eclampsia ( Nourollahpour Shiadeh et al. 2017 ), and fetal growth restriction ( Enderle et al. 2023 ). Therefore, it is critical to understand how immuno- toxicants like TCE and its metabolites may impact immune processes during pregnancy. In this study, we used a combination of in vitro methods and bioinformatics to identify the impacts of a relevant TCE metabolite: DCVC in a macrophage cell model (THP-1 cells). It should be noted that the cell model used in this study has limitations in replicating immune signaling during pregnancy. For example, THP-1 cells in vitro are genetically male and lack the influence of dynamic hormonal signaling changes (e.g. progesterone mediated anti-inflammatory signaling) that occur throughout pregnancy ( Raghupathy and Szekeres-Bartho 2022 ). However, The THP-1 model does replicate certain important anti-microbial functions performed by macrophages in the uterus and fetal membranes, such as the release of pro-inflammatory cytokines and phagocytosis. We first examined DCVC cytotoxicity on THP-1 cells to make sure concentrations used in subsequent experiments would not cause cell death. Significant cytotoxicity in THP-1 cells was observed at 50 μM DCVC ( Figure 3 ). Previous studies have demonstrated that DCVC impacts pathogen-stimulated inflammation at concentrations as low as 5 μM ( Boldenow et al. 2015 ). In addition, DCVC as low as 13 μM has been detected in blood samples from people occupationally exposure to 100 ppm TCE ( Lash et al. 1999 ). Therefore, we selected concentrations for subsequent experiments at or below 5 μM DCVC. In this study, we sought to assess DCVC impacts on immune responses to three distinct PAMPs, i.e., LPS, LTA, and zymosan, all of which utilize different pathways to stimulate the release of inflammatory cytokines ( Figure 2 ). LPS is a structural component of the outer membrane of gram-negative bacteria, such as Escherichia coli ( E. coli ), and evidence suggests that vaginal and intrauterine E. coli infections during pregnancy play a role in the pathogenesis of preterm labor and low fetal birthweight ( Krohn et al. 1997 , Cappelletti et al. 2021 , Kwon et al. 2022 ). LPS-induced inflammation occurs through a series of binding interactions with several proteins, including Toll-like receptor 2 and Toll-like receptor 4, which then trigger the release of pro-inflammatory cytokines ( Long et al. 2009 ). LTA is a structural component of the cell wall of gram-positive bacteria, such as GBS, and incidence of GBS colonization is present in 15% of preterm births ( Anthony et al. 1978 ). LTA functions as an inflammatory stimulator by binding to CD14 and Toll-like receptor 2, which triggers inflammatory cytokine release ( Long et al. 2009 ). Lastly, zymosan is an insoluble β-1,3-glucan polysaccharide that models an immune response to fungal pathogens, including Candida ( Brown 2006 ). Vaginal candidiasis is one of the most common fungal diseases reported in pregnant women, which can lead to systemic infections in the neonate, low birth weight, and preterm birth ( Rasti et al. 2014 , Farr et al. 2015 ). β-1,3-glucan acts through the dectin-1 receptor, and production of inflammatory cytokines due to beta-glucan containing pathogens is a result of synergism between dectin-1, Toll-like receptor 2, and Toll-like receptor 6 ( Gantner et al. 2005 ). DCVC significantly suppressed pro-inflammatory cytokine release regardless of the microbial stimulator used, suggesting that DCVC immunosuppression is a broad-based response that is relevant for a variety of intrauterine pathogens. This is significant because successful pregnancy outcomes are reliant upon complex coordination between the maternal immune system and the developing fetus, including immune cells and cytokine signaling pathways that mediate these communications throughout the pregnancy ( Yockey and Iwasaki 2018 , Mezouar et al. 2021 , Nakamura 2025 ). Thus, dysfunction of immune system regulation in the context of infection or toxicant exposure could result in detrimental impacts on the developing fetus and the outcome of the pregnancy. There are three stages of pregnancy that have important immunological features that are necessary for a healthy pregnancy: (1) implantation requires inflammatory responses including pro-inflammatory cytokines IL-6, IL-1β, and TNFα as well as adequate functioning of macrophages for maintenance of the corpus luteum within the ovary; (2) placentation requires immune cells, including macrophages and natural killer cells, for remodeling of the spiral arteries; and, (3) parturition is mediated by an inflammatory response that is characterized by macrophages infiltrating the smooth muscle of the uterus and IL-1β secretion for induction of muscle contraction ( Yockey and Iwasaki 2018 ). Conversely, many adverse obstetric outcomes have been reported in association with increases in pro-inflammatory cytokines, such as TNFα, including recurrent pregnancy loss, pre-eclampsia, and recurrent implantation failure ( Alijotas-Reig et al. 2017 , Romanowska-Prochnicka et al. 2021 ). While pro-inflammatory cytokines have important roles in early placental functions and embryonic development, dysfunction in this complex coordination between pro-inflammatory and anti-inflammatory responses can be a risk factor for pregnancy loss and adverse outcomes ( Romanowska-Prochnicka et al. 2021 ). Moreover, immunosuppressive effects of chemical exposures are associated with increase susceptibility to pathogenic organisms, as demonstrated by a number of drug therapies that are associated with increased risk for bacterial and viral infections ( Chastain and Stover 2023 ). The extent to which environmental exposures cause similar effects in exposed populations is not well understood, despite compelling findings indicated that this may be the case ( Lee et al. 2019 , Stephens et al. 2023 , Ebel et al. 2025 ). Overall, the appropriateness of inflammatory responses during pregnancy is highly complex and requires a carefully coordinated balance of immune system responses. Thus, DCVC-induced suppression of pro-inflammatory cytokine release could disrupt this highly intricate balance of immune system regulation at various stages throughout gestation. Immunosuppression could also increase susceptibility to intrauterine infections and complications arising from common infections, while also posing problems for developmental stages that require pro-inflammatory signaling, such as implantation and parturition ( Dropulic and Lederman 2016 ). For TNFα release, benchmark doses were 0.21 μM, 0.20 μM, and 0.22 μM DCVC across LPS, LTA, and zymosan, respectively. For IL-1β release, benchmark doses were 0.13 μM, 0.35 μM, and 0.42 μM DCVC across LPS, LTA, and zymosan, respectively ( Table 1 ). These are the concentrations that have a meaningful biological impact on macrophage function, based on suppression of TNFα and IL-1β release. The benchmark doses of DCVC that were calculated are 100-fold smaller than doses of the direct upstream metabolite, DCVG, that have been detected in the blood of female volunteers exposed to the occupational exposure limit of 100 ppm, with peak blood concentrations of 13.4 μM ( Lash et al. 1999 ). Additionally, our data show that suppression of TNFα and IL-1β release are more sensitive endpoints (lower benchmark dose) compared to DCVC-induced cytotoxicity, which had a benchmark dose of 13.3 μM. Transcription factors were significantly enriched for genes impacted by DCVC, both with LPS-simulation (CEBPD and VDR) as well as without LPS (EPAS1, MITF, NFATC2, RUNX2 and TP53), with EGRI and TRERF1 enriched for both treatments. These results suggest that DCVC induces changes in the macrophage transcriptomic regulatory network that could interfere with both baseline macrophage activity and infection- induced immune responses in macrophages. Importantly for the context of macrophage activity in gestational tissues like the placenta and decidualized uterus, several of these transcription factors play important roles during pregnancy. For example, TRERF1 is involved in the production of steroid hormones and there is evidence of its involvement in endometriosis, which increases risk of preterm birth, miscarriage, and low birth weight infants (Cao et al. 2018). Additionally, EGR1 has an important role in the early events of pregnancy establishment, including uterine receptivity and decidualization, and a study by Szwarc et al. ( Szwarc et al. 2019 ) showed that EGR1 levels are decreased in the endometrium of women with recurrent implantation failure. Lastly, CEBPD has been shown to play an important role in endocrine signaling and the stimulation of labor pathways at parturition ( Lu et al. 2021 ). To our knowledge, studies linking TCE exposure in women to endometriosis and/or implantation failure have not been conducted and present an important knowledge gap that requires further research. The transcription factor NF-κB functions to mediate inflammatory responses by inducing the expression of pro-inflammatory genes, including those encoding cytokines, and activating differentiation of the M1 pro-inflammatory macrophage phenotype ( Liu et al. 2017 ). As shown in Figures 6A and 6B , DCVC down-regulated target genes of NF-κB, including those coding the pro-inflammatory cytokines TNFα, IL-1β, IL-12β, IL-18, and IL-6. Given the crucial role of these cytokines in coordinating cell-mediated immune responses and summoning immune cells, such as macrophages, to the site of infection or inflammation, DCVC-induced suppression of their expression could hinder the capacity of the immune system to respond to infection. In the context of pregnancy, this could increase susceptibility to intrauterine infection and worsen disease outcomes ( Yang et al. 2019 , Mezouar et al. 2021 ). Lastly, VDR is an important transcription factor for the regulation of hormone secretions, placental immune functions, as well as cell proliferation and differentiation during pregnancy. Decreased VDR expression has been associated with pre-eclampsia, preterm birth, and fetal growth restriction ( Knabl et al. 2017 ). Thus, DCVC-induced suppression of VDR expression could result in adverse pregnancy outcomes. Vitamin D supplementation has been shown to significantly increase VDR expression ( Medeiros et al. 2020 ). During pregnancy, Vitamin D supplementation is associated with increased birth weight and birth length ( Perez-Lopez et al. 2015 ). In addition, VDR plays a crucial role in the induction of antimicrobial peptides, which are essential for host defense mechanisms in mucosal tissues ( Wang et al. 2004 ). Thus, while not previously studied, Vitamin D supplementation during pregnancy among women with known exposure to TCE could be a potential therapeutic intervention to aid in fetal development and achieve a healthy birthweight. The impact of volatile organic compound metabolites like DCVC in dysregulating transcriptional networks in macrophages in the gestational compartment and potential impacts on pregnancy warrants further study. DCVC also down-regulated expression of genes involved in the major steps of phagocytosis in LPS- stimulated macrophages. Given the key role that phagocytosis plays in fighting microbial infections, these impacts have significant implications for DCVC impacts on macrophage functions in gestational tissue and the ability to mount an immune defense against intrauterine pathogens. Prolonged exposure to intrauterine infection is harmful to both the pregnant person and the infant ( Krohn et al. 1997 , Goldenberg et al. 2000 , Kwon et al. 2022 , Megli and Coyne 2022 , Fowler and Simon 2024 ). Complications of chronic intrauterine infection in the neonate include neurologic abnormalities, respiratory distress, cerebral palsy, neonatal sepsis, and neonatal death ( Anthony et al. 1978 , Goldenberg et al. 2000 , Farr et al. 2015 ). Maternal complications include severe pelvic infections, sepsis, and postpartum hemorrhage ( Fowler and Simon 2024 ). Thus, TCE exposure could lead to the formation of immunosuppressive metabolites that could contribute to severe adverse pregnancy outcomes.

This study suggests that in macrophages, DCVC impacts genes associated with transcriptional regulation of immune processes, potentially increasing maternal susceptibility to intrauterine infection, and disrupting immune homeostasis during pregnancy. Additionally, DCVC acts in a dose-dependent fashion to suppress the release of important cytokine under inflammatory conditions across three distinct stimulators of inflammation that are associated with common intrauterine infection. Lastly, this study utilized lower doses than previously utilized in our studies of DCVC immunosuppression and still showed impacts on macrophage function and cytokine release in a THP-1 macrophage model. Future studies should investigate functional impacts of DCVC on macrophage phagocytotic capacity. In addition, further investigation of TCE metabolite impacts on anti-inflammatory macrophage functions should be conducted, as macrophages in the gestational compartment play an important role in promoting immune tolerance of the semi-allogenic fetus, e.g., through the release of anti-inflammatory cytokines ( Parasar et al. 2021 ). Therefore, to adequately assess the effects of TCE exposure during pregnancy, whether TCE metabolites affect both pro- and anti-inflammatory processes in macrophages during pregnancy will need to be determined in future studies. Because cell culture studies cannot replicate the complex interactions between multiple immune cell types and tissues that occur through pregnancy, animal models will likely be required to determine if the effects observed in this study have relevance for in vivo systems. The effect of DCVC and other TCE metabolites on additional immune cell types that play important roles during intrauterine infections should also be assessed. For example, neutrophils and monocytes are the most abundant leukocytes in the amniotic fluid of women with intra-amniotic infections. These cells have critical functions like trapping and killing pathogens (neutrophils) and the release of pro-inflammatory cytokines (neutrophils and monocytes). The extent to which DCVC and other TCE metabolites also inhibit the functions of these cells should be assessed to more fully understand the impact of TCE exposures on the immune system, particularly during unique life events like pregnancy ( Gomez-Lopez et al. 2019 ).

Environmental toxicants can alter immune responses in human and animal models, demonstrating that toxicants can both enhance or suppress immune responses depending on the biological context ( Hartung and Corsini 2013 , Martins Costa Gomes et al. 2021 ). Toxicant-induced suppression could increase susceptibility to infectious diseases in exposed populations, worsening disease outcomes ( Winans et al. 2011 ). The immunotoxic effects of most environmental contaminants are poorly understood, and many environmental toxicants have not been evaluated for their adverse impact on the immune system. Trichloroethylene (TCE) is a volatile synthetic compound that is mainly used in the manufacturing of refrigerants and as a degreasing solvent for metal equipment ( Waters et al. 1977 , Elkin et al. 2020 ). Due to its widespread use, large amounts of TCE are released into the environment, with an estimated two million pounds of TCE released in the United States in 2017 ( De Miranda and Greenamyre 2020 ). Human exposure to TCE can occur through inhalation via vapor intrusion into homes in contaminated areas, ingestion via drinking water, and dermal absorption via showering or in occupational settings. After exposure, TCE is rapidly absorbed into the lungs or gastrointestinal tract and distributed to target organs via systemic circulation. TCE is metabolized via two major pathways, i.e., (1) cytochrome-dependent oxidation, and (2) conjugation with glutathione. The oxidation pathway generates trichloroacetic acid (TCA), while glutathione conjugation generates S -(1,2-dichlorovinyl)-glutathione (DCVG), which is then further metabolized into the reactive intermediate S -(1,2)-dichlorovinyl-L-cysteine (DCVC) ( Lash et al. 2000 , Elkin et al. 2020 ). TCE has been shown to have immunotoxic effects, including both activation and suppression of the immune/inflammation responses ( Iavicoli et al. 2005 , Selgrade and Gilmour 2010 , Lee et al. 2019 ). TCE exposure has been shown to lead to activation of inflammatory responses in macrophages in the organs like the liver in vivo ( Gao et al. 2024 , Zhang et al. 2024 ). Disruption of anti-microbial functions in immune cells like macrophages could be a significant threat to public health via increased susceptibility to bacterial or viral infections. Selgrade and Gilmore demonstrated that mice exposed to 200 ppm of TCE had higher bacteria counts in their lungs compared to air only controls. In addition, they showed that lung macrophages from TCE exposed mice were less capable of phagocytosis ( Selgrade and Gilmour 2010 ). Pregnancy leads to a shift in the maternal body’s complex immune responses to simultaneously tolerate and protect the growing fetus ( Abu-Raya et al. 2020 ). Infection at the maternal-fetal interface can lead to adverse birth and neonatal outcomes ( Goldenberg et al. 2005 , Agrawal and Hirsch 2012 ). Resident macrophages in the gestational compartment play an important role in preventing and eliminating infections ( Fakonti et al. 2021 ). Despite the importance of understanding toxicant-pathogen interactions at the maternal-fetal interface, few studies investigating immune responses have been conducted. Previous studies have demonstrated that toxicants can modify immune function in both placental macrophages and in the THP-1 macrophage cell line in a similar manner, suggesting that immunotoxicity responses observed in cell lines may be relevant for toxicant impacts on resident macrophages in the gestational compartment ( Tetz et al. 2015 , Li et al. 2021 , Stephens et al. 2023 ). TCE and its metabolites can cross the placenta, as demonstrated by early studies when TCE was used as an anesthetic gas, in which TCE was detected in the umbilical vein and artery ( Beppu 1968 , Laham 1970 ). Direct metabolism of TCE may also occur directly in gestational tissues. For example, glutathione S-transferases, the enzymes that catalyze reactions in the glutathione conjugation pathway of TCE metabolism, are expressed in the placenta, in addition to γ-glutamyltransferase, the enzyme that converts DCVG to DCVC ( Elkin et al. 2020 ). Mouse studies also suggest that TCE metabolites like trichloroacetic acid (TCA) are produced in the feto-placental unit independent of maternal metabolism ( Ghantous et al. 1986 ). DCVC induces oxidative stress, including lipid peroxidation in the rat placenta, as well as apoptosis and mitochondrial membrane depolarization in the HTR-8 first trimester trophoblast cell line, suggesting that DCVC impacts in gestational tissues could contribute to adverse birth outcomes ( Elkin et al. 2018 , Elkin et al. 2019 , Loch-Caruso et al. 2019 , Elkin et al. 2022 ). Additionally, DCVC, not TCA, has been shown to suppress the release of pro-inflammatory cytokines in response to a common pathogen (group B streptococcus ) in a fetal membrane explant model ( Boldenow et al. 2015 ). The fetal membranes represent an early line of defense against intrauterine infection, as both layers of the human fetal membranes (chorion and amnion) are known to express anti-microbial peptides ( Ramuta et al. 2021 ). Thus, these findings suggest that gestational tissues are an important target of DCVC toxicity. However, DCVC impacts on specific immune cell types, including macrophages, in gestational tissues remain poorly understood. It was previously shown that DCVC suppresses lipopolysaccharide (LPS)-stimulated cytokine release and transcriptomic immune pathways in a macrophage cell model ( Harris et al. 2022 ). However, important questions remain about DCVC immunotoxicity in the context of pregnancy, including which transcription factors mediate transcriptomic responses to DCVC. In addition, while LPS is a prominent pathogen- associated molecular pattern molecule (PAMP) associated with gram-negative bacteria ( Farhana and Khan 2024 ), pregnant women are exposed to a variety of bacterial and fungal pathogens that elicit host immune/inflammation responses via multiple, distinct PAMPs ( Lopez and Aterman 1968 , McDonald and Chambers 2000 , Stock et al. 2017 ) and it is unknown if DCVC immunosuppression occurs for all PAMPs or is limited to specific PAMPs like LPS. Finally, to improve the ability to interpret DCVC effects, it is important to understand the potency of immunosuppressive effects of DCVC in macrophages, relative to other toxicity endpoints like cytotoxicity. The overall goals of this study were to assess the dose-response of DCVC inhibition of pro-inflammatory cytokine release for three different PAMPs (LPS, LTA, and zymosan) relevant to intrauterine pathogenic infections and to identify DCVC impacts on the expression of genes that regulate a key anti-microbial function of macrophages (phagocytosis) ( Ashburner et al. 2000 , Gene Ontology et al. 2023 ). We hypothesized that DCVC treatment causes a dose-dependent suppression of pro-inflammatory cytokine response to LPS, LTA, and zymosan and that DCVC would down-regulate genes involved in immune processes including phagocytosis. DCVC concentrations for this study were selected to be similar to or lower than blood levels of the TCE metabolite directly upstream of DCVC in the glutathione metabolism pathway ( S -(1,2- dichlorovinyl)-glutathione) detected in female volunteers after a one-time inhalation exposure to TCE at the occupational exposure limit (100 ppm), i.e., ~13μM (with blood levels returning to baseline after ~12 hours) ( Lash et al. 1999 ).