Immunotoxicity Evaluation of Trihalophenolic Disinfection By-Products in Mouse and Human Mononuclear Macrophage Systems: The Role of RNA Epitranscriptomic Modification in Mammalian Immunity

Abstract

Background:

2,4,6-Trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are three widely detected trihalophenolic disinfection by-products (DBPs). Previous studies have mainly focused on the carcinogenic risk and developmental toxicity of 2,4,6-trihalophenols. Very little is known about their immunotoxicity in mammals.

Objectives:

We investigated the effects of 2,4,6-trihalophenols on mammalian immunity using a mouse macrophage model infected with bacteria or intracellular parasites and aimed to elucidate the underlying mechanisms from an epitranscriptomic perspective. The identified mechanisms were further validated in human peripheral blood mononuclear cells (PBMCs).

Methods:

The mouse macrophage cell line RAW264.7 and primary mouse peritoneal macrophages were exposed to different concentrations of TCP, TBP, and TIP. The pro-inflammatory marker Ly6C, the survival of the bacterium Escherichia coli (E. coli), and the parasite burden of Toxoplasma gondii (T. gondii) were assessed. Furthermore, the global gene expression profiling of macrophages following exposure to 2,4,6-trihalophenols was obtained through RNA-sequencing (RNA-seq). The effects of 2,4,6-trihalophenols on RNA $N^6\text{-methyladenosine}$ (${\mathrm{m}}^6\mathrm{A}$) methyltransferases and total RNA ${\mathrm{m}}^6\mathrm{A}$ levels were evaluated using Western blotting and dot blot, respectively. Transcriptome-wide ${\mathrm{m}}^6\mathrm{A}$ methylome was analyzed by ${\mathrm{m}}^6\mathrm{A}\text{-seq}$. In addition, expression of ${\mathrm{m}}^6\mathrm{A}$ regulators and total RNA ${\mathrm{m}}^6\mathrm{A}$ levels in human PBMCs exposed to 2,4,6-trihalophenols were detected using quantitative reverse transcriptase polymerase chain reaction and dot blot, respectively.

Results:

Mouse macrophages exposed to TCP, TBP, or TIP had lower expression of the pro-inflammatory marker Ly6C, with a greater difference from control observed for TIP-exposed cells. Consistently, macrophages exposed to such DBPs, especially TIP, were susceptible to infection with the bacterium E. coli and the intracellular parasite T. gondii, indicating a compromised ability of macrophages to defend against pathogens. Intriguingly, macrophages exposed to TIP had significantly greater ${\mathrm{m}}^6\mathrm{A}$ levels, which correlated with the greater expression levels of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases. Macrophages exposed to each of the three 2,4,6-trihalophenols exhibited transcriptome-wide redistribution of ${\mathrm{m}}^6\mathrm{A}$. In particular, the ${\mathrm{m}}^6\mathrm{A}$ peaks in genes associated with immune-related pathways were altered after exposure. In addition, differences in ${\mathrm{m}}^6\mathrm{A}$ were also observed in human PBMCs after exposure to 2,4,6-trihalophenols.

Discussion:

These findings suggest that 2,4,6-trihalophenol exposure impaired the ability of macrophages to defend against pathogens. This response might be associated with notable differences in ${\mathrm{m}}^6\mathrm{A}$ after exposure. To the best of our knowledge, this study presents the first ${\mathrm{m}}^6\mathrm{A}$ landscape across the transcriptome of immune cells exposed to pollutants. However, significant challenges remain in elucidating the mechanisms by which ${\mathrm{m}}^6\mathrm{A}$ mediates immune dysregulation in infected macrophages after 2,4,6-trihalophenol exposure. https://doi.org/10.1289/EHP11329

Introduction

Disinfection is indispensable in water treatment to inactivate harmful pathogens and protect public health. However, during the disinfection process, disinfectants, such as chlorine and monochloramine, can react with natural organic matter and halides to form numerous halogenated disinfection by-products (DBPs).^1–3 Ever since the first identification of 2,4,6-trihalophenols, including 2,4,6-trichlorophenol (TCP), 2,4,6-tribromophenol (TBP), and 2,4,6-triiodophenol (TIP), with a powerful precursor ion scan method in chlorinated drinking water,^1,4,5 studies have shown that aromatic DBPs are the non-negligible contributors to total organic halogen and in vivo (in marine polychaete Platynereis dumerilii embryos and zebrafish embryos and zebrafish embryos) toxicity of chlorinated drinking water.^6–8 The concentrations of TCP, TBP, and TIP in drinking water treatment plants from the Yangtze River Delta Region of China and the simulated drinking water samples were up to 215, 57, and $33\text{\hspace{0.17em}ng}/\mathrm{L}$, respectively.^2,3 2,4,6-Trihalophenols can enter the human body through water intake and dermal contact^9,10 and have been detected in human urine¹¹ and serum.¹² TCP, TBP, and TIP have been associated with a number of adverse effects, such as carcinogenicity (using the general population and mouse and rat models),¹³ genotoxicity [using in vitro V79 Chinese hamster cells¹⁴ and human peripheral blood mononuclear cells (PBMCs)¹⁵], and developmental toxicity (using in vitro human extended pluripotent cells¹⁶ and the marine polychaete Platynereis dumerilii¹⁷).¹⁸ Therein, TCP and other polychlorophenols (combined exposures) were classified as possible human carcinogens (Group 2B) by the World Health Organization’s International Agency for Research on Cancer in 1999.¹³ TBP was reported to cause DNA damage in human PBMCs using an in vitro assay.¹⁵ Recent studies using the marine polychaete worm¹⁷ and human¹⁶ and mouse¹⁹ cell line models have indicated that TIP is more developmentally toxic and cytotoxic compared with its chlorinated and brominated analogs. However, previous studies mainly focused on the carcinogenic, developmental, and mutagenic toxicity of 2,4,6-trihalophenols. Very little is known about their immunotoxicity in mammals.

The macrophage is a type of vital immune cell that plays essential roles in maintaining immune balance and organizing defense against infection.²⁰ Sensing pathogen attack, macrophages can directly eliminate invading pathogens and induce a series of immune reactions.²¹ Our previous study found that treatment with $200\mathrm{\mu }\mathrm{M}$ TIP or TBP remarkably induced M2-dominant polarization of macrophages, whereas treatment with TCP triggered an M1-dominant polarization of macrophages.¹⁹ A growing body of evidence has suggested that macrophage dysfunction is associated with various diseases.²⁰ For example, the unexpected M2 polarization of macrophages might contribute to uncontrolled infection as explored in mice.²² Therefore, evaluating the effects of three 2,4,6-trihalophenols on macrophage-mediated resistance to infection could facilitate our understanding of the complex host–pathogen–environment interactions.

In recent years, emerging in vitro evidence has suggested that RNA $N^6\text{-methyladenosine}$ (${\mathrm{m}}^6\mathrm{A}$) modification is involved in anti-infectious immunity and could regulate the function of macrophages.^23–27 Methylation of adenosine at the $N^6$ position (${\mathrm{m}}^6\mathrm{A}$) is the most abundant chemical modification on eukaryotic mRNA.^28,29 In eukaryotic species, ${\mathrm{m}}^6\mathrm{A}$ modification is regulated by three types of functional proteins, including methyltransferases, demethylases, and ${\mathrm{m}}^6\mathrm{A}$ binding proteins.³⁰ ${\mathrm{m}}^6\mathrm{A}$ modification is catalyzed by a multicomponent methyltransferase complex containing methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor 1 associated protein (WTAP).³¹ Demethylases, including fat mass and obesity associated (FTO) and alkylation repair homolog 5 (ALKBH5), are responsible for removing ${\mathrm{m}}^6\mathrm{A}$ from mRNA.^32,33 In addition, YT521-B homology domain family 1 (YTHDF1), YTHDF2, YTHDF3, YT521-B homology domain containing 1 (YTHDC1), and YTHDC2 are ${\mathrm{m}}^6\mathrm{A}$ binding proteins, which directly recognize this mark and modulate the splicing, translation, and stability of mRNA.³⁴ ${\mathrm{m}}^6\mathrm{A}$ has been shown to be responsive to cellular stress conditions, such as heat in bovine mammary epithelial cells³⁵ and nutrition deprivation in neuroblastoma cells,³⁶ and to play a functional role in the stress response.³⁷ Studies in recent years have shown exposure to environmental stressors, such as air pollution³⁸ and cigarette smoke,³⁹ to be associated with global ${\mathrm{m}}^6\mathrm{A}$ changes in lung epithelial cells and peripheral blood, respectively, from participants in the Beijing Truck Diver Air Pollution Study. Although research on ${\mathrm{m}}^6\mathrm{A}$ modification is in its infancy in the field of environmental health, lacking studies with transcript-level ${\mathrm{m}}^6\mathrm{A}$, its importance to macrophage polarization²³ and immune response against infection^24,26,27 is indisputable. In light of these findings, we predict that ${\mathrm{m}}^6\mathrm{A}$ modification might be associated with the immunotoxic effects of 2,4,6-trihalophenols on macrophages.

In the present study, we evaluated the immunotoxicity and immunomodulatory effects of three 2,4,6-trihalophenols on macrophages. Escherichia coli (E. coli) and Toxoplasma gondii (T. gondii) infection models were employed to investigate macrophage-mediated resistance to infection after TCP, TBP, and TIP treatment. In addition, we mapped and compared the ${\mathrm{m}}^6\mathrm{A}$ methylomes of macrophages in response to TCP, TBP, and TIP exposure. The underlying mechanism of 2,4,6-trihalophenol-induced immunotoxicity was explored from an epitranscriptomic perspective. Finally, the toxicity-related alteration of ${\mathrm{m}}^6\mathrm{A}$ was validated in human PBMCs.

Methods

Study Design

The prespecified objective of the present study was to investigate the immunotoxicity and immunomodulatory effects of three 2,4,6-trihalophenols (TCP, TBP, and TIP) on mammalian mononuclear macrophage systems and to reveal the underlying mechanism. For ethical reasons, the mouse macrophage cell line RAW264.7 was mainly employed to investigate the immunotoxicity and immunomodulatory effects of 2,4,6-trihalophenols (Figure S1). Using the RAW264.7 cell line, the cell viability and expression of the macrophage pro-inflammatory marker Ly6C was assessed after 2,4,6-trihalophenol exposure. RAW264.7 cell-mediated resistance to infection after 2,4,6-trihalophenol exposure was evaluated using the bacterium E. coli, as well as the parasite T. gondii, infection models. The transcriptomes and ${\mathrm{m}}^6\mathrm{A}$ methylomes of RAW264.7 cells in response to TCP, TBP, and TIP exposure were obtained by RNA- uencing (RNA-seq) and ${\mathrm{m}}^6\mathrm{A}\text{-seq}$. Effects of 2,4,6-trihalophenols on ${\mathrm{m}}^6\mathrm{A}$ regulators and total RNA ${\mathrm{m}}^6\mathrm{A}$ levels in RAW264.7 cells were evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blotting, and dot blot. We also analyzed the ${\mathrm{m}}^6\mathrm{A}$ modification of immune-related genes using ${\mathrm{m}}^6\mathrm{A}$ immunoprecipitation qPCR (${\mathrm{m}}^6\mathrm{A}\text{-IP-qPCR}$) on RAW264.7 cells that were challenged with parasite T. gondii infection. To further verify the effects of 2,4,6-trihalophenols on ${\mathrm{m}}^6\mathrm{A}$ methyltransferases, mouse peritoneal macrophages were employed. Furthermore, we evaluated the effects of 2,4,6-trihalophenols on human PBMCs. After exposure to 2,4,6-trihalophenols, expression of ${\mathrm{m}}^6\mathrm{A}$ regulators and total RNA ${\mathrm{m}}^6\mathrm{A}$ levels in human PBMCs were respectively detected using qRT-PCR and dot blot. According to the results of the Cell Counting Kit 8 (CCK8; Cat. #A311-01, Vazyme) assay, the concentrations used in this study were 50, 100, and $200\mathrm{\mu }\mathrm{M}$, which are higher than the environmental concentrations of 2,4,6-trihalophenols. We intentionally used these higher concentrations for better observation of the acute effects of 2,4,6-trihalophenols.

Chemicals

The TCP (Cat. #T0390), TBP (Cat. #T0349), and TIP (Cat. #T0452) used in this study were purchased from TCI Shanghai. Purity of TCP, TBP, and TIP was $>97.0\%$, $>98.0\%$, and $>98.0\%$, respectively (Table S1). TCP, TBP, and TIP were dissolved in dimethyl sulfoxide (DMSO; Cat. #D2650, Sigma-Aldrich, purity $\ge 99.7\%$) to prepare a $400\text{\hspace{0.17em}mM}$ stock solution for in vitro study. All stock solutions were stored at $-20°\mathrm{C}$. Because the final concentration of DMSO in the cell culture medium was $<0.1\%$ (vol/vol), $0\mathrm{\mu }\mathrm{M}$ was used as a negative control for exposure experiments.

Animal Experiments and Isolation of Mouse Peritoneal Macrophages

C57BL/6 mice (6–8 wk old and weighing $18–20\mathrm{g}$) were purchased from the Animal Core Facility of Nanjing Medical University (Nanjing, China). This study was approved by the Animal Ethics Committee of Nanjing Medical University, and mouse experiments were performed in accordance with the approved guidelines (permit no.: NJMU/IACUC-2007024). Forty mice were euthanized by carbon dioxide (${\mathrm{CO}}_2$) inhalation. Each mouse was injected with $7\mathrm{mL}$ of phosphate buffered saline (PBS) with 1% fetal bovine serum (FBS; Cat. #S711-001S, Lonsera) into the peritoneal cavity after euthanasia. After gently massaging the mouse’s abdomen, as much as possible of the injected PBS was collected using a $5\text{-mL}$ syringe. Then, the cells were combined and centrifuged at $500g$ for 5 min at 4°C. The cells were counted by hemocytometer and cultured in 6-well culture plates ($2\times {10}^6$ cells/well) in Dulbecco’s modified Eagle medium (DMEM; Cat. #C11995500BT, Gibco) containing 10% FBS (Cat. #S711-001S, Lonsera), 1% $100\times \text{penicillin}$ and streptomycin (Cat. #15140122, Gibco). After adhering for 1 h, the peritoneal macrophages attached to the culture plates and nonadherent cells were washed off with PBS. Then, the mouse peritoneal macrophages were treated with $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h (3 wells per treatment, $n=3$ technical replicates).

Isolation of Human PBMCs

This study was approved by the ethics review board of Nanjing Medical University [permit no.: NJMU/(2021) 668]. Three healthy volunteers were male, 20–30 y of age, had never smoked or used illicit drugs. Blood samples were processed immediately after collection. First, $30\mathrm{mL}$ of venous blood samples from the healthy volunteers were mixed with PBS in a 1:1 ratio. Then, Ficoll (Cat. #17144002, Cytiva) was added to the centrifuge tube. The diluted blood sample was carefully layered onto the Ficoll (4:3 vol ratio of diluted blood sample to Ficoll) and centrifuged at room temperature for 30 min at $400g$ with the brake off. The separated mononuclear cells were washed twice with at least three volumes of PBS and centrifuged at $300g$ for 10 min at room temperature. After that, isolated PBMCs were cultured in 6-well culture plates ($1\times {10}^6$ cells/well) in DMEM (Cat. #C11995500BT, Gibco) containing 10% FBS (Cat. #S711-001S, Lonsera), 1% $100\times \text{penicillin}$ and streptomycin (Cat. #15140122, Gibco). After 24 h cultivation, the isolated mononuclear cells were treated with $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h. PBMCs from the three donors served as three biological replicates ($n=3$ biological replicates).

Cell Culture of RAW264.7 Cells and 2,4,6-Trihalophenol Exposure

Mouse macrophage RAW264.7 cells were the kind gift of Z. Xu, Nanjing Medical University. The RAW264.7 cells were cultured in DMEM (Cat. #C11995500BT, Gibco) containing 10% FBS (Cat. #S711-001S, Lonsera), 1% $100\times \text{penicillin}$ and streptomycin (Cat. #15140122, Gibco). Serial subculture dilution was performed when the cell confluence of RAW264.7 cells reached 70%–80%. The RAW264.7 cells were then cultured in 96-well culture plates ($1\times {10}^4$ cells/well), 24-well culture plates ($1\times {10}^5$ cells/well), or 6-well culture plates ($5\times {10}^5$ cells/well). After adhering overnight, the RAW264.7 cells were treated for 24 h with different concentrations of TCP, TBP, and TIP ranging from 0 to $800\mathrm{\mu }\mathrm{M}$ ($n=3$ wells per treatment for CCK8 assay and flow cytometry).

CCK8 Assay

CCK8 cell counting kit (Cat. #A311-01, Vazyme) was used to detect the cell viability after TCP, TBP, or TIP exposure, following the manufacturer’s instructions. The RAW264.7 cells ($1\times {10}^4$ cells/well) were seeded into 96-well culture plates and allowed to adhere overnight. Then different concentrations [0 (control), 50, 100, 200, 400, or $800\mathrm{\mu }\mathrm{M}$] of TCP, TBP, and TIP were added into the 96-well culture plates and incubated with the RAW264.7 cells for 24 h ($n=3$ wells per treatment). Subsequently, the cell culture medium was replaced with fresh DMEM containing 10% CCK8 solution. After 1 h incubation, the absorbance at $450\mathrm{\hspace{0.17em}nm}$ was measured by a Synergy HT Microplate Reader (BioTek). The absorbance at $450\mathrm{\hspace{0.17em}nm}$ from each well represented the number of cells per well. The relative absorbance (relative cell viability) was calculated as follows: relative $\text{absorbance}=\left[\left(\text{experimental\hspace{0.17em}}\mathrm{absorbance}-\text{background\hspace{0.17em}}\mathrm{absorbance}\right)/\left(\text{untreated\hspace{0.17em}control\hspace{0.17em}}\mathrm{absorbance}-\text{background\hspace{0.17em}}\mathrm{absorbance}\right)\right]$. The concentration $0\mathrm{\mu }\mathrm{M}$ was employed as the untreated control. CCK8 assay was repeated twice with independent sample preparations.

Flow Cytometry

To evaluate the differences in the proportion of lymphocyte antigen 6 complex locus C positive [lymphocyte antigen 6 complex, locus C (${\text{Ly}6\mathrm{C}}^{+}$)] macrophages, RAW264.7 cells were stimulated with TCP, TBP, or TIP [0 (control), 50, 100, or $200\mathrm{\mu }\mathrm{M}$] for 24 h ($n=3$ wells per treatment). Then, the cells were collected and incubated with anti-mouse CD16/32 antibody (1:200 dilution; Cat. #156604, BioLegend) at 4°C for 15 min. Next, the cells were incubated with fluorescein isothiocyanate (FITC) antimouse F4/80 antibody (1:500 dilution; Cat. #123108, BioLegend), allophycocyanin (APC) antimouse CD11b antibody (1:100 dilution; Cat. #101212, BioLegend), and peridinin–chlorophyll–protein complex (PerCP)/Cyanine 5.5 antimouse Ly-6C antibody (1:150 dilution; Cat. #128012, BioLegend) in the dark at 4°C for 30 min. Then, the samples’ labels were detected by FACSVerse flow cytometer (BD Biosciences) and analyzed by FlowJo software (version 10.0.7; Tree Star).

Bacteria Culture

E. coli 8099 (CGMCC 1.3373) was purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. The preparation of competent cells for transformation was performed by adopting the calcium chloride (${\text{CaCl}}_2$) method. One hundred nanograms of pEF 3-YFP-2 plasmids (Cat. #20106, Addgene), which contain the gene of enhanced yellow fluorescent protein (EYFP) and kanamycin-resistance gene (kanR), were added into $100\mathrm{\mu }\mathrm{L}$ of competent E. coli cells. The mixture was gently rotated and incubated on ice for 30 min. Then the mixture was bathed in water at 42°C for 90 s and quickly placed on ice for 1–2 min. Six hundred microliters of Luria-Bertani (LB) broth was added before $200\text{\hspace{0.17em}rpm}$ shaking at 37°C for 1 h. Then $100\mathrm{\mu }\mathrm{L}$ of the cultures were spread on an LB agar plate (Cat. #HB0129, Hopebio) supplemented with $50\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ kanamycin (Cat. #K1030, Solarbio) and cultured overnight at 37°C. A single bacterial colony picked from the agar plate was inoculated to LB broth supplemented with $50\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ kanamycin. After overnight cultivation, the E. coli/pEF 3-YFP-2 cultures were centrifuged ($\mathrm{3,000}g$, 10 min at room temperature), suspended in 20% glycerol, and then stored at $-20°\mathrm{C}$. The stored E. coli/pEF 3-YFP-2 cells were added into $5\mathrm{mL}$ of fresh LB broth supplemented with $50\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ kanamycin at a ratio of 1:100 (vol/vol), and was cultivated until ${\text{OD}}_{600}$ reached 0.6–0.8 with shaking ($200\text{\hspace{0.17em}rpm}$) at 37°C. To calculate bacterial concentration via the number of colony forming units (CFUs) per milliliter, the cultures were serially diluted 10-fold by sequential transfer of $100\mathrm{\mu }\mathrm{L}$ into $900\mathrm{\mu }\mathrm{L}$ of ultrapure sterile water. The last dilution of $1\mathrm{mL}$ was separately spread out on three LB agar plates supplemented with $50\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ of kanamycin and cultured at 37°C overnight. Then three plates with colonies for each sample were taken for calculating CFUs. The same method was used in subsequent experiments to determine the number of E. coli/pEF 3-YFP-2.

Preparation of T. gondii Tachyzoites

Both T. gondii RH tachyzoites and green fluorescent protein (GFP)–expressing RH tachyzoites were the kind gifts of J. Shen, Anhui Medical University. They were preserved in liquid nitrogen. Human foreskin fibroblasts (HFFs) were also the kind gift of J. Shen and used to culture T. gondii tachyzoites. HFF cells were cultured in DMEM (Cat. #C11995500BT, Gibco) containing 10% FBS (Cat. #S711-001S, Lonsera), 1% $100\times \text{penicillin}$ and streptomycin (Cat. #15140122, Gibco). When the confluence of HFF cells reached $\sim 80\%$ in a $25{\text{-cm}}^2$ culture flask, $2.5\times {10}^5$ T. gondii RH tachyzoites or GFP-RH tachyzoites were added to the cell cultures and cultivated for 3–4 d at 37°C in 5% ${\mathrm{CO}}_2$. After observing the release of a large number of tachyzoites, the cells were scraped off from the culture flask. All cultures with tachyzoites were filtered through a $3\text{-}\mathrm{\mu }\mathrm{m}$ pore-sized track-etched membrane (Cat. #111112, Whatman). Then the filtrate containing the tachyzoites was saved for subsequent infection assay. Tachyzoites were counted with a hemocytometer under a Primovert microscope (Zeiss).

Macrophage Infection Assay

In the bacterium infection model, RAW264.7 cells were cultured in 24-well culture plates ($1\times {10}^5$ cells/well) and allowed to adhere overnight. The RAW264.7 cells were incubated with TCP, TBP, and TIP at different concentrations (0, 50, or $100\mathrm{\mu }\mathrm{M}$) for 24 h. After incubation, a well of untreated cells was detached with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Cat. #25200072, Gibco) to count the number of cells per well by hemocytometer. The number of E. coli/pEF 3-YFP-2 was calculated as aforementioned. Then, the cell cultures were rinsed with PBS three times and the medium was replaced with fresh DMEM containing 10% FBS, $50\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ kanamycin, and E. coli/pEF 3-YFP-2 at a multiplicity of infection (MOI) of 100 bacteria per cell (MOI of 100).⁴⁰ After incubation in the incubator (37°C, 5% ${\mathrm{CO}}_2$) for 1.5 h, infected macrophage cultures were directly centrifuged at $\mathrm{3,000}g$ for 10 min at room temperature to collect the total bacteria ($n=3$ biological replicates). To collect intracellular bacteria only ($n=3$ biological replicates), infected RAW264.7 cells, in culture plates, were washed with PBS three times before centrifugation. After centrifugation ($\mathrm{3,000}g$, 10 min at room temperature), equivalent ultrapure sterile water was added for resuspension. Then, the bacterial suspensions were serially diluted and spread on kanamycin-resistant agar plates as mentioned above for quantifying surviving bacteria (in colony forming units per milliliter).

In the parasite infection model, RAW264.7 cells were pretreated with 50 or $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h ($n=3$ biological replicates, each with two technical replicates). The concentration $0\mathrm{\mu }\mathrm{M}$ was employed as the control ($n=3$ biological replicates, each with two technical replicates). Then the cells were infected with prepared T. gondii tachyzoites at an MOI of 1 in fresh medium for 24 h. The parasite burden of T. gondii in macrophages was calculated as the ratio of the mRNA levels of T. gondii internal transcribed spacer 1 (ITS1) gene to normalized RAW264.7 cell number, which was adjusted by the cell viability measured by CCK8 assay. The method to detect the mRNA levels of ITS1 is described below.

qRT-PCR

The mRNA levels of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases, ${\mathrm{m}}^6\mathrm{A}$ demethylases, ${\mathrm{m}}^6\mathrm{A}$ binding proteins, T. gondii ITS1 gene, mouse tumor necrosis factor-alpha ($Tnf\text{-}\mathrm{\alpha }$), and eukaryotic translation initiation factor 2-alpha kinase 2 (Eif2ak2) genes were detected by qRT-PCR. Total RNA of RAW264.7 cells (with or without T. gondii infection), mouse peritoneal macrophages, and human PBMCs was extracted via TRIzol reagent (Cat. #15596018, Invitrogen). Total RNA was quantified using NanoDrop 2000 (Thermo Fisher Scientific). RNA purity was confirmed by determining the optical density 260/280 (OD260/OD280) absorption ratio. The ratio of OD260/OD280 of the extracted RNA ranged from 1.8 to 2.0. One microgram of total RNA for each sample was reverse-transcribed using HiScript III RT Supermix (Cat. #R323-01, Vazyme) in Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific). Semiquantitative RT-PCR was carried out in a LightCycler 96 Instrument (Roche) containing complementary DNA (cDNA; $50\text{\hspace{0.17em}ng}$ cDNA per $20\mathrm{\mu }\mathrm{L}$ of reaction), $10\mathrm{\mu }\mathrm{M}$ primer pairs, and SYBR qPCR Master Mix (Cat. #Q411-02, Vazyme). The reference gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. The cycling conditions were as follows. The first step was pre-denaturation (1 cycle, 95°C for 30 s). The second step was two-step amplification (40 cycles, 95°C for 10 s and 60°C for 30 s). The third step was for the melting curve (1 cycle, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s). The primers were designed using the National Center for Biotechnology Information (NCBI) Primer–Basic Local Alignment Search Tool (BLAST) (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The sequences of the gene-specific primers used in this study are listed in Table S2.

Dot Blot

The ${\mathrm{m}}^6\mathrm{A}$ dot blot assay is a classic and relatively straightforward method to detect global ${\mathrm{m}}^6\mathrm{A}$ differences in total RNA.⁴¹ Total RNA from RAW264.7 cells and human PBMCs was extracted via TRIzol (Cat. #15596018, Invitrogen). Then the total RNA was diluted to 1,000, 800, 500, 400, 250, and $200\text{\hspace{0.17em}ng}/\mathrm{\mu }\mathrm{L}$, serially diluted, and spotted on a nylon membrane (Cat. #FFN13, Beyotime Biotechnology). The membrane was cross-linked at ultraviolet (UV) $254\mathrm{\hspace{0.17em}nm}$, $0.12{\mathrm{J}/\mathrm{cm}}^2$. After UV cross-linking, the blotted membrane was blocked in PBS containing 0.1% Tween-20 (PBST; Cat. #30189328, Sinopharm Chemical Reagent Company) and 5% nonfat milk (Cat. #FD0080, FDbio Science) for 1 h at room temperature. The membrane was then incubated with ${\text{anti-m}}^6\mathrm{A}$ antibody (1:1,000 dilution; Cat. #ab284130, Abcam) at 4°C overnight. The membrane was incubated with horseradish peroxidase (HRP)–conjugated antirabbit immunoglobulin G (IgG; 1:5,000 dilution; Cat. #7074, Cell Signaling Technology) at room temperature for 1 h and visualized by FDbio-Dura ECL Kit (Cat. #FD8020, FDbio Science). Dot blot analysis of RNA from RAW264.7 cells was performed three independent times ($n=3$ biological replicates).

Western Blotting

RAW264.7 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Cat. #20-188, Millipore) supplemented with protease and phosphatase inhibitor cocktail (Cat. #P1045, Beyotime Biotechnology), and phenylmethylsulfonyl fluoride (PMSF; Cat. #ST506, Beyotime Biotechnology). Lysates were centrifuged at $\mathrm{12,000}g$ for 15 min at 4°C. The supernatant was then collected in a new Eppendorf tube. Protein concentrations were measured via bicinchoninic acid (BCA) assay (Cat. #PA115, TIANGEN) and equalized with PBS and sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Cat. #P0015L, Beyotime Biotechnology). After boiling for 10 min, an equal amount ($20\mathrm{\mu }\mathrm{g}$) of protein extracts was subjected to gels and separated by SDS-PAGE. Subsequently, the proteins were transferred onto a fitted polyvinylidene fluoride (PVDF) membrane (Cat. #ISEQ00010, Millipore), blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST; Cat. #30189328, Sinopharm Chemical Reagent Company) for 2 h at room temperature and incubated with 1:1,000 diluted primary antibodies in TBST at 4°C overnight. For Western blotting, $N^6\text{-mA}$ Methyltransferase Antibody Sampler Kit (1:1,000 dilution for METTL3, METTL14, and WTAP; Cat. #69159) and GAPDH Rabbit mAb (1:1,000 dilution; Cat. #2118) were bought from Cell Signaling Technology. After incubation with secondary antibody HRP-conjugated antirabbit IgG (1:5,000 dilution; Cat. #7074, Cell Signaling Technology) at room temperature for 1 h, the membrane was visualized by FDbio-Dura ECL Kit (Cat. #FD8020, FDbio Science). Images were taken by ChemiDoc Touch Imaging System (Bio-Rad). Western blotting bands were quantified with Image Lab software (version 6.0; Bio-Rad). All the Western blotting experiments were performed three independent times ($n=3$ biological replicates).

RNA-Seq and $m^{6}A\text{-}Seq$

RAW264.7 cells were treated with $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h ($n=2$ biological replicates). Total RNA was extracted using TRIzol (Cat. #15596018, Invitrogen). Poly(A) RNA was purified from no less than $50\mathrm{\mu }\mathrm{g}$ of total RNA using Dynabeads Oligo(dT)²⁵ (Cat. #61005, Thermo Fisher Scientific) by two rounds of purification. Poly(A) RNA was fragmented at 94°C for 5 min in fragmentation buffer [$10\text{\hspace{0.17em}mM}$ Tris-hydrochloride (Tris-HCl), pH 7.0, $10\text{\hspace{0.17em}mM}$ zinc chloride (${\text{ZnCl}}_2$)]. The reaction was stopped with $0.5\mathrm{M}$ EDTA. RNA was precipitated using $3\mathrm{M}$ sodium acetate, glycogen, and ethanol as referred to in previously published methods.⁴² The RNA was pelleted, washed, and resuspended in RNase-free water. A portion of RNA fragments served as the input control in RNA-seq. For ${\mathrm{m}}^6\mathrm{A}\text{-seq}$, ${\mathrm{m}}^6\mathrm{A}$-specific antibody (Cat. #202003, Synaptic Systems) was incubated with RNA fragments in $1\times \text{IP}$ buffer [$10\text{\hspace{0.17em}mM}$ Tris-HCl, pH 7.4, $150\text{\hspace{0.17em}mM}$ sodium chloride (NaCl), 0.1% Igepal CA-630] on a rotator for 2 h at 4°C. While the samples were incubated, Protein A/G beads (Cat. #20421, Thermo Fisher Scientific) were washed and blocked in IP buffer supplemented with bovine serum albumin (BSA) ($0.5\mathrm{mg}/\mathrm{mL}$) for 2 h. The RNA mixtures were then incubated with the previously blocked Protein A/G beads for another 2 h at 4°C. After that, the supernatant was discarded and the beads that were bonded with ${\mathrm{m}}^6\mathrm{A}\text{-methylated}$ RNA fragments were washed with IP buffer three times and then subjected to elution. In brief, the beads were eluted twice in $100\mathrm{\mu }\mathrm{L}$ of elution buffer [$1\times \text{IP}$ buffer containing 6.7 mM ${\mathrm{m}}^6\mathrm{A}$ 5′-monophosphate sodium salt (Cat. #sc-215524) bought from Santa Cruz Biotechnology] with head-over-tail rotation for 1 h at 4°C. Then the beads were spun down and carefully removed, retaining the supernatant. The desired ${\mathrm{m}}^6\mathrm{A}\text{-enriched}$ RNA fragments were retained in the supernatant. All the supernatants were combined, and RNA was concentrated by ethanol precipitation. The desired ${\mathrm{m}}^6\mathrm{A}\text{-enriched}$ RNA was used to construct a cDNA library in parallel with input control. The ${\mathrm{m}}^6\mathrm{A}\text{-enriched}$ RNA was reverse-transcribed into cDNA using SuperScript II reverse transcriptase (Cat. #18064071, Invitrogen). Next, E. coli DNA polymerase I (Cat. #M0209, NEB), RNase H (Cat. #M0297, NEB) and 2′-deoxyuridine 5′-triphosphate (dUTP) solution (Cat. #R0133, Thermo Fisher Scientific) were used to synthesize U-labeled second-stranded DNAs. Each strand’s blunt ends received an A-base addition so as to be ready for ligation to the indexed adapters. The fragments were ligated with dual-index adapters, and size selection was conducted with AMPure XP beads (Cat. #A63880; insert size: 180–220 bp). The U-labeled second-stranded DNAs were treated with the heat-labile uracil–DNA glycosylase (UDG) enzyme (Cat. #M0280, NEB), and the ligated products were amplified with PCR to create a sequencing library. The PCR conditions were as follows: initial denaturation at 95°C for 3 min; 8 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s; and then final extension at 72°C for 5 min. The library quality was assessed using an Agilent Bioanalyzer 2100 system. Last, we performed paired-end sequencing (PE150) on an Illumina Novaseq 6000 platform [sequencing primers: VAHTS i5 PCR Primer: 5′-AATGATACGGCGACCACCGAGATCTACAC(i5)ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3′; VAHTS i7 PCR Primer: 5′-CAAGCAGAAGACGGCATACGAGAT(i7)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3′]. All the sequencing experiments were performed with two biological replicates ($n=2$).

Quality control of ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ data was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc; version 0.11.9). Low-quality bases and adapter contamination were deleted by Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore; version 0.6.5) with Q20 and Q30 as sequencing quality scores. Mouse rRNA sequences from NCBI (NR_046233.2) were downloaded. The sequence reads were aligned to the rRNA reference using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml; version 1.0.0) and the rRNA alignments were discarded. Unmapped reads were aligned to the GRCm38 reference genome using HISAT2 (http://daehwankimlab.github.io/hisat2) with default parameters. Peaks were identified using R software (version 3.4.4; R Development Core Team) by R package exomePeak (https://www.bioconductor.org/packages/3.3/bioc/html/exomePeak.html) and annotated by ANNOVAR (http://www.openbioinformatics.org/annovar/). The distribution of ${\mathrm{m}}^6\mathrm{A}$ was plotted by R package Guitar (version 2.6.0).⁴³ Motif finding on ${\mathrm{m}}^6\mathrm{A}$ was done using HOMER (http://homer.ucsd.edu/homer; version 4.11) findMotifs.pl program.

Reads mapping and processing of RNA-seq data were performed as described for ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ data above. StringTie (https://ccb.jhu.edu/software/stringtie) was used to calculate the number of reads mapped to each gene. R package EdgeR (version 3.3.2)⁴⁴ was used for differential expression analysis. Genes with $|{\log }_2\text{Fold}$ difference $|\ge 1$ and false discovery rate (FDR) $<0.05$ were defined as differentially expressed genes (DEGs). The raw RNA-seq and ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE223045. Bioinformatic analyses were performed using the OmicStudio tools available at https://www.omicstudio.cn/tool.

$m^{6}A\text{-IP-qPCR}$ on RNA Extracted from T. gondii-Infected RAW264.7 Cells

RAW264.7 cells were cultured in $10\text{-cm}$ dishes ($3\times {10}^6$ cells/dish). After adhering overnight, the RAW264.7 cells were pretreated with $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h. Then, the treated cells were rinsed, replenished with normal culture medium, and infected with T. gondii at an MOI of 1 for another 24 h. The supernatant was discarded and the cells were collected for RNA extraction with TRIzol (Cat. #15596018, Invitrogen). RNA from different batches of cells was pooled to perform the ${\mathrm{m}}^6\mathrm{A}\text{-IP-qPCR}$ assay ($n=2$ biological replicates). Total RNA was fragmented and immunoprecipitated with ${\mathrm{m}}^6\mathrm{A}$ antibody as described above in ${\mathrm{m}}^6\mathrm{A}\text{-seq}$.⁴² The untreated fragmented RNA served as input control. Then, the ${\mathrm{m}}^6\mathrm{A}\text{-enriched}$ RNA fragments and input control were used to synthesize the first strand cDNA for qRT-PCR analysis as mentioned above. Primers targeting ${\mathrm{m}}^6\mathrm{A}\text{-enriched}$ regions of $Tnf\text{-}\mathrm{\alpha }$ and Eif2ak2 were designed based on up- and downstream sequences of predicted ${\mathrm{m}}^6\mathrm{A}$ sites (Table S2).

Statistical Analyses

Statistical analyses were mainly done using SPSS software (version 17.0; IBM) and GraphPad Prism (version 8.0.1; GraphPad). Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by the Tukey post hoc test. Unpaired Student’s $t$-test was conducted for two-group comparison. Statistical significance was set at $p<0.05$. The corresponding numeric data for all figures are provided in the Supplemental Excel Files.

Results

Cell Viability of Mouse Macrophages after 2,4,6-Trihalophenol Exposure

The CCK8 assay was employed to measure the cell viability of mouse macrophage RAW264.7 cells after exposure to TCP, TBP, and TIP. The RAW264.7 cells were treated with TCP, TBP, and TIP over a range of concentrations [0 (control), 50, 100, 200, 400, or $800\mathrm{\mu }\mathrm{M}$] for 24 h. From the OD values of the CCK8 assay, there was a downward trend of cell survival rate with increasing concentration of 2,4,6-trihalophenols, especially at high concentrations (Figure S2). For TCP, the cell survival rate was significantly lower at concentrations of 400 and $800\mathrm{\mu }\mathrm{M}$ ($p<0.001$). For TBP, the cell survival rate was significantly lower at concentrations of 200, 400, and $800\mathrm{\mu }\mathrm{M}$ ($p<0.001$). For TIP, statistically significant differences in cell survival rate were observed at concentrations of $50\mathrm{\mu }\mathrm{M}$ ($p<0.05$), $100\mathrm{\mu }\mathrm{M}$ ($p<0.01$), and 200, 400, and $800\mathrm{\mu }\mathrm{M}$ ($p<0.001$).

Expression of Ly6C on Macrophages

We stimulated RAW264.7 cells with different concentrations [0 (control), 50, 100, or $200\mathrm{\mu }\mathrm{M}$] of all three 2,4,6-trihalophenols. The expression of Ly6C on macrophages was measured by flow cytometry. The percentages of ${\text{Ly}6\mathrm{C}}^{+}$ cells in the TBP and TIP groups were gradually less with increasing concentrations ($p<0.01$ for the 50 and $100\mathrm{\mu }\mathrm{M}$ TBP groups, $p<0.001$ for the $200\mathrm{\mu }\mathrm{M}$ TBP group and all the TIP groups; Figure 1A,B). The percentages of ${\text{Ly}6\mathrm{C}}^{+}$ cells were also lower after 100 and $200\mathrm{\mu }\mathrm{M}$ TCP exposure ($p<0.05$ for $100\mathrm{\mu }\mathrm{M}$, $p<0.001$ for $200\mathrm{\mu }\mathrm{M}$). The difference in Ly6C median fluorescence intensity (MFI) was generally consistent with that of ${\text{Ly}6\mathrm{C}}^{+}$ cell percentage. Ly6C MFI was inversely proportional to the concentration of TCP, TBP, and TIP (Figure 1C). The rank order of potency for inhibiting Ly6C expression was $\text{TIP}>\text{TBP}>\text{TCP}$.

Figure 1.

The expression of Ly6C on mouse macrophages after exposure to TCP, TBP, and TIP. (A) The expression of Ly6C on RAW264.7 macrophages (${\mathrm{F}4/80}^{+}{\text{CD}11\mathrm{b}}^{+}$ gated cells) was evaluated by flow cytometry. One representative of three independent experiments is shown. (B) The percentage of ${\text{Ly}6\mathrm{C}}^{+}$ cells in ${\mathrm{F}4/80}^{+}{\text{CD}11\mathrm{b}}^{+}$ gated RAW264.7 cells. Data were analyzed using FlowJo software and are shown as $\text{mean}\pm \text{SD}$ ($n=3$ technical replicates). Statistical significance was determined using one-way ANOVA with the Tukey post hoc test. *$p<0.05$, **$p<0.01$, ***$p<0.001$. The corresponding data are presented in Excel Table S1. (C) MFI of Ly6C expression on RAW264.7 macrophages. Data were analyzed using FlowJo software and are shown as $\text{mean}\pm \text{SD}$ ($n=3$ technical replicates). Statistical significance was determined using one-way ANOVA with the Tukey post hoc test. *$p<0.05$, ***$p<0.001$. The corresponding data are presented in Excel Table S2. Note: ANOVA, analysis of variance; MFI, median fluorescence intensity; SD, standard deviation; SSC, side scatter; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

Mouse Macrophage-Mediated Resistance to Infection

E. coli/pEF 3-YFP-2 strain, which expressed YFP and was kanamycin-resistant, was used to construct the bacterial infection model. The RAW264.7 cells were pretreated with different concentrations of TCP, TBP, and TIP. Then the RAW264.7 cells were washed and infected with E. coli/pEF 3-YFP-2 at an MOI of 100. Compared with the control group, the number of surviving E. coli inside the infected macrophages was significantly greater in the 50 and $100\mathrm{\mu }\mathrm{M}$ TIP-pretreated groups ($p<0.01$; Figure 2A). Meanwhile, E. coli CFU was not significantly different between the control group and the TCP- or TBP-pretreated groups at the concentration of $100\mathrm{\mu }\mathrm{M}$ ($p=0.295$ for TCP, $p=0.165$ for TBP), or even lower in the $50\mathrm{\mu }\mathrm{M}$ TCP-pretreated group ($p=0.009$). It is known that in addition to phagocytosis, macrophages can also kill bacteria by secreting inflammation cytokines. Therefore, we further analyzed the total surviving bacteria in the cell culture, both inside and outside RAW264.7 cells. The number of total surviving E. coli was significantly greater after exposure to 50 and $100\mathrm{\mu }\mathrm{M}$ TBP and TIP, whereas TCP had no statistically significant effect on the number of total surviving bacteria (Figure 2B).

Figure 2.

Effects of 2,4,6-trihalophenols on phagocytosis and pathogen clearance of mouse macrophages. (A) Surviving bacteria inside TCP-, TBP-, or TIP-pretreated RAW264.7 cells. RAW264.7 cells were pretreated with 0, 50 or $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenol (TCP, TBP, or TIP) for 24 h. Then RAW264.7 cells were washed with PBS and infected with E. coli/pEF 3-YFP-2 at an MOI of 100 for 1.5 h. The number of surviving bacteria inside RAW264.7 cells was counted as CFU/mL. Data are represented as $\text{mean}\pm \text{SD}$ ($n=3$ biological replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. **$P<0.01$. The corresponding data are presented in Excel Table S3. (B) Total surviving bacteria in RAW264.7 cultures pretreated with TCP, TBP, or TIP. RAW264.7 cells were pretreated with 0, 50 or $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenol (TCP, TBP, or TIP) for 24 h. Then RAW264.7 cells were washed with PBS and infected with E. coli/pEF 3-YFP-2 at an MOI of 100 for 1.5 h. The number of total surviving bacteria was counted as CFU/mL. Data are represented as $\text{mean}\pm \text{SD}$ ($n=3$ biological replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. *$p<0.05$, **$p<0.01$, ***$p<0.001$. The corresponding data are presented in Excel Table S4. (C,D) Parasite burden of intracellular parasite T. gondii in macrophages after exposure to TCP, TBP, or TIP. RAW264.7 cells were seeded at the same cell number and pretreated with $50\mathrm{\mu }\mathrm{M}$ (C) or $100\mathrm{\mu }\mathrm{M}$ (D) 2,4,6-trihalophenol (TCP, TBP, or TIP) for 24 h. Then RAW264.7 cells were infected with T. gondii in fresh medium for another 24 h. The parasite burden of T. gondii in macrophages was calculated as the ratio of the mRNA levels of the T. gondii ITS1 gene to normalized RAW264.7 cell number, which was adjusted by cell viability measured by CCK8 (Figure S2). Results are shown as fold differences compared with the control group ($0\mathrm{\mu }\mathrm{M}$). $1=\text{no\hspace{0.17em}difference}$. Data are represented as $\text{mean}\pm \text{SD}$ ($n=3$ biological replicates, each with two technical replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. *$p<0.05$, ***$p<0.001$. The corresponding data are summarized in Excel Table S5. Note: ANOVA, analysis of variance; CCK8, Cell Counting Kit 8; CFU, colony forming units; MOI, multiplicity of infection; PBS, phosphate buffer saline; SD, standard deviation; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

Moreover, the intracellular parasite T. gondii was used to construct the parasite infection model. RAW264.7 cells were pretreated with 50 and $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h. No significant difference in the parasite burden of T. gondii was observed between the $50\mathrm{\mu }\mathrm{M}$ TCP-exposure group and the control group (Figure 2C; Figure S3). However, macrophages exposed to $50\mathrm{\mu }\mathrm{M}$ TBP and TIP had about 2- and 3-fold greater parasite burden, respectively ($p<0.05$ for TBP, $p<0.001$ for TIP). Perhaps owing to the toxicity of 2,4,6-trihalophenols to T. gondii, macrophages exposed to $100\mathrm{\mu }\mathrm{M}$ TIP had only 1.7-fold greater parasite burden ($p<0.05$), whereas there were no statistically significant differences in the parasite burden in the $100\mathrm{\mu }\mathrm{M}$ TCP- or TBP-pretreated groups (Figure 2D).

Global Gene Expression Profiling of Macrophages

To thoroughly evaluate the effects of 2,4,6-trihalophenols on macrophages, we performed global gene expression profiling by RNA-seq (Tables S3 and S4, Figure S4). We identified DEGs (${|\log }_2\text{Fold}$ difference $|\ge 1$ and FDR $<0.05$) in the exposure groups. After TCP, TBP, and TIP exposure, a total of 3,069, 608, and 3,894 genes were respectively up-regulated. Meanwhile, TCP, TBP, and TIP exposure also respectively down-regulated 547, 4,447, and 929 genes of macrophages (Figure 3A). For TCP and TIP, the number of up-regulated DEGs was greater than that of down-regulated DEGs. Interestingly, TIP exposure was associated with more up-regulated DEGs than its chlorinated and brominated analogs.

Figure 3.

Significantly altered genes and pathways of mouse macrophages after exposure to $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenols. (A) Venn diagram representing up- and down-regulated DEGs in RAW264.7 cells after exposure to TCP, TBP, or TIP. DEGs were defined as ${|\log }_2\text{Fold}$ difference $|\ge 1$ and FDR $<0.05$. (B) SAPs in the TCP-, TBP-, and TIP-exposure groups. The up-regulated or down-regulated DEGs after TCP, TBP, or TIP exposure were used for GO enrichment analysis. The SAPs inside the gray box have at least two DEGs and FDR $<0.05$. The dot color and size are proportional to the FDR value and DEG number, respectively. The corresponding data are presented in Excel Table S6. RNA-seq was performed with two biological replicates ($n=2$). Note: DEGs, differentially expressed genes; FDR, false discovery rate; GO, Gene Ontology; MHC, major histocompatibility complex; $\text{NF-}\mathrm{\kappa }\mathrm{B}$, nuclear factor kappa-light-chain-enhancer of activated B cells; SAPs, significantly altered pathways; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

To further interpret the transcriptome data, DEGs in the 2,4,6-trihalophenol–treated groups were selected for Gene Ontology (GO) enrichment analysis. The GO enrichment results indicated that genes among immune-related pathways differed after exposure to 2,4,6-trihalophenols compared with control (Figure 3B). However, the pathways associated with differentially expressed genes [significantly altered pathways (SAPs), DEGs $\ge 2$, and FDR $<0.05$] were quite different in different exposure groups. TCP exposure was associated with differences in genes from pathways associated with the interaction between macrophages and other lymphocytes, especially T cells and natural killer cells. Specifically, TCP exposure was associated with the differential expression of genes involved in lymphocyte homeostasis, T cell proliferation, T cell costimulation, natural killer cell lectin-like receptor binding, innate immune response, and type I interferon production pathways. For the TBP group, genes associated with microglial cell activation and cytokine-mediated signaling pathways were differentially expressed. For the TIP group, genes from pathways related to antigen presentation and signal transduction were differentially expressed, including the inhibitory major histocompatibility complex (MHC) class I receptor activity pathway, the negative regulation of nuclear factor kappa-light-chain-enhancer of activated B cells ($\text{NF-}\mathrm{\kappa }\mathrm{B}$) transcription factor activity pathway, the negative regulation of cytokine-mediated signaling pathway, and the lymphocyte apoptotic process pathway. Meanwhile, genes associated with pathways regulating the killing of cells of other organisms, viral entry into host cell, and viral genome replication were all differentially expressed.

Expression of $m^{6}A$ Regulators in Macrophages

We first determined the transcriptional expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases (METTL3, METTL14, and WTAP) using qRT-PCR. Compared with the control group, the mRNA levels of Mettl3 were $>4.9$-fold higher in the $200\mathrm{\mu }\mathrm{M}$ TBP- and TIP-exposure groups ($p<0.01$ for TBP, $p<0.001$ for TIP; Figure 4A). Consistent with this, compared with the control group, transcriptional expression of Mettl14 was significantly higher after exposure to $200\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP by $\sim 2$-fold ($p<0.01$ for TCP, $p<0.05$ for TBP, $p<0.01$ for TIP; Figure 4B). For Wtap, the $200\mathrm{\mu }\mathrm{M}$ TIP-exposure group exhibited a statistically significantly greater mRNA abundance ($p<0.01$; Figure 4C). Then, we evaluated the transcriptional expression of the ${\mathrm{m}}^6\mathrm{A}$ demethylases (FTO and ALKBH5). Exposure to $200\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP was associated with greater transcriptional expression of Fto ($p<0.001$ for TCP, $p<0.01$ for TBP, $p<0.05$ for TIP; Figure 4D). The mRNA levels of Alkbh5 were greater after the administration of $200\mathrm{\mu }\mathrm{M}$ TBP or TIP ($p<0.05$ for TBP, $p<0.001$ for TIP; Figure 4E). There were no statistically significant differences in the mRNA levels of the ${\mathrm{m}}^6\mathrm{A}$ binding proteins in all exposure groups except Ythdf2 ($p<0.05$ for the $200\mathrm{\mu }\mathrm{M}$ TCP group, $p<0.05$ for the $200\mathrm{\mu }\mathrm{M}$ TBP group, $p<0.001$ for the $200\mathrm{\mu }\mathrm{M}$ TIP group; Figure 4F–J).

Figure 4.

The effects of TCP, TBP, and TIP on the expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases, demethylases, and ${\mathrm{m}}^6\mathrm{A}$ binding proteins in RAW264.7 cells. (A–J) Transcriptional expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases, demethylases, and ${\mathrm{m}}^6\mathrm{A}$ binding proteins in RAW264.7 cells. After exposure to different concentrations (0, 50, 100, $200\mathrm{\mu }\mathrm{M}$) of TCP, TBP, or TIP for 24 h, the mRNA levels of Mettl3, Mettl14, Wtap, Fto, Alkbh5, Ythdf1, Ythdf2, Ythdf3, Ythdc1, and Ythdc2 were quantitatively measured via qRT-PCR. Results are shown as fold differences compared with the control group ($0\mathrm{\mu }\mathrm{M}$). $1=\text{no\hspace{0.17em}difference}$. Data are expressed as $\text{mean}\pm \text{SD}$ ($n=3$ biological replicates, each with two technical replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. *$p<0.05$, **$p<0.01$, ***$p<0.001$. The corresponding data are presented in Excel Table S7. (K,L) Protein abundance of METTL3, METTL14, and WTAP in RAW264.7 cells. After exposure to different concentrations (0, 50, 100, $200\mathrm{\mu }\mathrm{M}$) of TCP, TBP, or TIP for 24 h, protein abundance of METTL3, METTL14, WTAP, and the loading control (GAPDH) was analyzed using Western blotting. Western blotting was repeated with independent sample preparations three times ($n=3$ biological replicates; Figure S5). (K) One representative figure for each ${\mathrm{m}}^6\mathrm{A}$ methyltransferase was shown. (L) Western blotting results were quantified with Image Lab. $1=\text{no\hspace{0.17em}difference}$. Data are expressed as $\text{mean}\pm \text{SD}$ ($n=3$ biological replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. *$p<0.05$, **$p<0.01$, ***$p<0.001$. The corresponding data are summarized in Excel Table S8. Note: ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ${\mathrm{m}}^6\mathrm{A}$, $N^6\text{-methyladenosine}$; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SD, standard deviation; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

The mRNA levels of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases were significantly higher in exposed cells, especially Mettl3. Therefore, it was necessary to evaluate the protein abundance of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases in macrophages. When the exposure concentration increased to 100 or $200\mathrm{\mu }\mathrm{M}$, the protein abundance of METTL3 was significantly greater in three exposure groups compared with the control group (Figure 4K,L; Figure S5). For METTL14, cells exposed to each of 2,4,6-trihalophenols did not exhibit any statistically significant differences in protein expression. For WTAP, the protein abundance was significantly higher after the administration of $200\mathrm{\mu }\mathrm{M}$ TCP, $200\mathrm{\mu }\mathrm{M}$ TBP, and 100 and $200\mathrm{\mu }\mathrm{M}$ TIP.

Because there was no significant difference in the transcriptional levels of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases in RAW264.7 cells after exposure to $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenols, the transcriptional expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases was further evaluated in mouse peritoneal macrophages after exposure to $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, and TIP (Figure S6). Compared with the control group, mouse peritoneal macrophages exposed to TCP or TBP exhibited significantly greater transcriptional expression of Wtap ($p<0.01$ for the TCP- and TBP-exposure groups); however, the mRNA levels of Mettl3 in the TCP- and TBP-exposure groups were lower $p<0.05$ (for the TCP- and TBP-exposure groups). Meanwhile, RNA-seq results of RAW264.7 cells showed that there was greater Mettl14 expression in the TIP group and lower Mettl14 expression in the TBP group after exposure to $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenols (Table S5).

RNA $m^{6}A$ Modification Status in Macrophages

To determine whether the ${\mathrm{m}}^6\mathrm{A}$ levels differed in the TCP-, TBP-, and TIP-exposure groups, we measured the global ${\mathrm{m}}^6\mathrm{A}$ levels of total RNA from 2,4,6-trihalophenol–treated RAW264.7 cells using an ${\mathrm{m}}^6\mathrm{A}$ dot blot. Methylene blue staining was used as a loading control after visualizing in white light. After exposure to 50, 100, or $200\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenols, we detected differences in ${\mathrm{m}}^6\mathrm{A}$ levels in the 2,4,6-trihalophenol exposure groups. TIP-exposed cells exhibited greater modification levels (Figure 5; Figure S7).

Figure 5.

The ${\mathrm{m}}^6\mathrm{A}$ levels in RAW264.7 cells after exposure to TCP, TBP, or TIP at concentrations of (A) $50\mathrm{\mu }\mathrm{M}$, (B) $100\mathrm{\mu }\mathrm{M}$, or (C) $200\mathrm{\mu }\mathrm{M}$. The ${\mathrm{m}}^6\mathrm{A}$ levels of total RNA were measured by dot blot (upper panel). Methylene blue staining was used as a loading control (lower panel). Dot blot analysis was repeated with independent sample preparations three times ($n=3$ biological replicates; Figure S7). Note: ${\mathrm{m}}^6\mathrm{A}$, $N^6\text{-methyladenosine}$; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

Macrophage Transcriptome-Wide $m^{6}A$ Profile ($m^{6}A\text{-}Seq$)

We used ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ to profile ${\mathrm{m}}^6\mathrm{A}$ levels across the transcriptome of RAW264.7 cells with or without 2,4,6-trihalophenol exposure. In both the control and 2,4,6-trihalophenol–treated groups, the ${\mathrm{m}}^6\mathrm{A}$ peaks were mainly distributed in the 3′ untranslated (UTR) and exon regions, with only a small number of peaks harboring in the 5′UTR, intron, and intergenic regions (Figure 6A). The TIP group had a higher number of transcripts with multisite ${\mathrm{m}}^6\mathrm{A}$ modifications (${\mathrm{m}}^6\mathrm{A}$ peak number per transcript $\ge 3$) than other exposure groups and the control group (Figure S8). Meanwhile, the TIP group had the lowest number of transcripts with a single-site ${\mathrm{m}}^6\mathrm{A}$ modification (${\mathrm{m}}^6\mathrm{A}$ peak number per $\text{transcript}=1$), and the control group had the highest.

Figure 6.

${\mathrm{m}}^6\mathrm{A}\text{-seq}$ analysis of mouse macrophages after exposure to $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP. (A) Distribution of ${\mathrm{m}}^6\mathrm{A}$ peaks across the transcriptome of RAW264.7 cells with or without 2,4,6-trihalophenol exposure. (B) Diagram representing the overlap of ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ genes with or without TCP, TBP, or TIP exposure. (C) Venn diagrams representing ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ genes in the 2,4,6-trihalophenol exposure groups and the control group. (D) GO pathways of RAW264.7 cells enriched among uniquely ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ genes after 2,4,6-trihalophenol exposure. The uniquely ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ genes after TCP, TBP, or TIP exposure were used for GO enrichment analysis. The red, blue, and green boxes indicate the TCP-, TBP-, and TIP-exposure groups, respectively. GO enrichment analysis showed a significant enrichment in immune-related, infection-associated and cancer-associated signaling pathways (FDR $<0.05$). The ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ was performed with two biological replicates ($n=2$). Note: ESCRT, endosomal sorting complex required for transport; FDR, false discovery rate; GO, Gene Ontology; ${\mathrm{m}}^6\mathrm{A}$, $N^6\text{-methyladenosine}$; MHC, major histocompatibility complex; $\text{NF-}\mathrm{\kappa }\mathrm{B}$, nuclear factor kappa-light-chain-enhancer of activated B cells; NiK, nuclear factor kappa $\mathrm{\beta }$-inducing kinase; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol; UTR, untranslated region.

The ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ data demonstrated significant differences in ${\mathrm{m}}^6\mathrm{A}$ levels in all exposure groups. In total, 7,338 genes were commonly modified in all groups (Figure 6B). Six hundred thirty-four, 190, and 1,006 genes were uniquely modified with ${\mathrm{m}}^6\mathrm{A}$ after TCP, TBP, and TIP exposure, respectively (Figure 6C). TIP exposure not only up-regulated the expression of most genes (Figure 3A), but it also exhibited the biggest influence on the ${\mathrm{m}}^6\mathrm{A}$ levels of macrophages compared with TCP and TBP.

Furthermore, GO enrichment analysis of uniquely ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ genes after TCP, TBP, or TIP exposure were performed. Significantly enriched pathways were closely associated with inflammation and pathogen resistance (Figure 6D). TCP-exposed cells had different ${\mathrm{m}}^6\mathrm{A}$ peaks in genes related to immune-related pathways, including cellular immunity and inflammatory response. Specifically, cells exposed to TCP had higher levels of ${\mathrm{m}}^6\mathrm{A}$ in genes related to the T cell activation pathway, the regulatory T cell apoptotic process pathway, the chronic inflammatory response pathway, and the cytokine-mediated signaling pathways (tumor necrosis factor- and interferon-gamma–mediated signaling pathways). Cells exposed to TBP exhibited greater ${\mathrm{m}}^6\mathrm{A}$ levels in genes related to several immune-related pathways, especially pathogen defense pathways, such as the induced systemic resistance pathway, the leukocyte activation pathway, the neutrophil-mediated killing of bacterium pathway, and the regulation of the viral budding pathway. Similar to the TBP group, the ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ genes unique to the TIP group were associated with several pathways related to immune response and pathogen resistance. Notably, cells exposed to TIP had higher levels of ${\mathrm{m}}^6\mathrm{A}$ in genes related to the antibacterial humoral response pathway, as well as a defense response to gram-positive and gram-negative bacterium pathways.

Joint analysis of RNA-seq and ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ data was used for enriching DEGs with significantly altered ${\mathrm{m}}^6\mathrm{A}$ modifications after TCP, TBP, or TIP exposure. After 2,4,6-trihalophenol exposure, genes associated with several notably different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways had significantly different levels of ${\mathrm{m}}^6\mathrm{A}$ modifications (Figure 7A). Therein, the ${\mathrm{m}}^6\mathrm{A}$ levels of genes associated with pattern recognition receptors [retinoic acid-inducible gene-I (RIG-I)-like receptors and nucleotide-binding and oligomerization domain (NOD)-like receptors], the inflammatory mediator regulation of transient receptor potential (TRP) channels pathway, and the $\text{NF-}\mathrm{\kappa }\mathrm{B}$ signaling pathway were significantly different after exposure compared with control. Consistent with these immune-related pathways, several ${\mathrm{m}}^6\mathrm{A}\text{-altered}$ KEGG pathways were associated with pathogen infection (Figure 7A). These pathways were related to virus, bacterium, and parasite infection, including the herpes simplex virus 1 infection pathway, the Salmonella infection pathway, and the African trypanosomiasis pathway. In addition, in the TIP group, genes associated with the endometrial cancer and thyroid cancer pathways were remarkably different and had significantly different ${\mathrm{m}}^6\mathrm{A}$ peaks.

Figure 7.

The association of ${\mathrm{m}}^6\mathrm{A}$ with altered pathways and immune-related genes in RAW264.7 cells after 2,4,6-trihalophenol exposure. (A) KEGG pathways enriched among DEGs together with significantly altered ${\mathrm{m}}^6\mathrm{A}$ in RAW264.7 cells after $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP exposure. The red, blue, and green boxes represent the TCP-, TBP-, and TIP-exposure groups, respectively. The SAPs have at least three DEGs and FDR $<0.05$. The corresponding data are presented in Excel Table S9. (B) The effects of 2,4,6-trihalophenol pretreatment on ${\mathrm{m}}^6\mathrm{A}$ levels of $Tnf\text{-}\mathrm{\alpha }$ and Eif2ak2 in RAW264.7 cells challenged with T. gondii. RAW264.7 cells were pretreated with $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h. Then, the cells were rinsed, replenished with normal culture medium, and infected with T. gondii for another 24 h. The ${\mathrm{m}}^6\mathrm{A}$ levels were analyzed using ${\mathrm{m}}^6\mathrm{A}\text{-IP-qPCR}$. $1=\text{no\hspace{0.17em}difference}$. Data are expressed as $\text{mean}\pm \text{SD}$ ($n=2$ biological replicates). Unpaired Student’s $t$-test was conducted for two-group comparison. *$p<0.05$, **$p<0.01$. The corresponding data are summarized in Excel Table S10. Note: DEGs, differentially expressed genes; Eif2ak2, eukaryotic translation initiation factor 2-alpha kinase 2; FDR, false discovery rate; IP, immunoprecipitation; KEGG, Kyoto Encyclopedia of Genes and Genomes; ${\mathrm{m}}^6\mathrm{A}$, $N^6\text{-methyladenosine}$; mTOR, mammalian target of rapamycin; $\text{NF-}\mathrm{\kappa }\mathrm{B}$, nuclear factor kappa-light-chain-enhancer of activated B cells; NOD, nucleotide-binding and oligomerization domain; qPCR, quantitative polymerase chain reaction; RIG, retinoic acid-inducible gene; SAPs, significantly altered pathways; SD, standard deviation; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol; TNF, tumor necrosis factor; TRP, transient receptor potential; UTR, untranslated region.

Furthermore, we analyzed the ${\mathrm{m}}^6\mathrm{A}$ modifications of immune-related genes using ${\mathrm{m}}^6\mathrm{A}\text{-IP-qPCR}$ on RAW264.7 cells that were challenged with parasite T. gondii infection. RAW264.7 cells were pretreated with $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP for 24 h. Then, the treated cells were rinsed, replenished with normal culture medium, and infected with T. gondii for another 24 h. The ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ results showed that ${\mathrm{m}}^6\mathrm{A}$ peaks in the 5′UTR or 3′UTR of the inflammatory cytokine $Tnf\text{-}\mathrm{\alpha }$ were up-regulated after 24 h-exposure to TBP or TIP (Table S6). After T. gondii infection, ${\mathrm{m}}^6\mathrm{A}$ peaks in the $3\mathrm{\prime }\text{UTR}$ of $Tnf\text{-}\mathrm{\alpha }$ remained up-regulated in both the TBP and TIP groups when compared with the control group ($p<0.01$ for TBP, $p<0.05$ for TIP). In addition, ${\mathrm{m}}^6\mathrm{A}$ peaks in the 5′UTR of $Tnf\text{-}\mathrm{\alpha }$ were also up-regulated in both the TBP and TIP groups ($p<0.05$; Figure 7B). The ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ results showed that ${\mathrm{m}}^6\mathrm{A}$ peaks in the 3′UTR of protein kinase R Eif2ak2 were up-regulated after TCP or TIP exposure (Table S6). After T. gondii infection, ${\mathrm{m}}^6\mathrm{A}$ peaks in the 3′UTR of Eif2ak2 were still up-regulated in the TCP and TIP groups ($p<0.05$; Figure 7B).

Effects of 2,4,6-Trihalophenols on Human PBMCs

Finally, the effects of TCP, TBP, and TIP on ${\mathrm{m}}^6\mathrm{A}$ modifications of human immune cells were evaluated. Human PBMCs were exposed to $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, and TIP. The transcriptional expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases, demethylases, and ${\mathrm{m}}^6\mathrm{A}$ binding proteins were measured. Compared with the control group, the mRNA levels of WTAP were significantly higher in the TBP group ($p<0.05$), whereas the mRNA levels of METTL3 were higher in the TCP group ($p<0.05$; Figure 8A–C). Meanwhile, exposure to TBP was associated with significantly greater transcriptional expression of demethylase FTO ($p<0.001$) and ${\mathrm{m}}^6\mathrm{A}$ binding protein YTHDF2 ($p<0.001$), YTHDF3 ($p<0.001$), and YTHDC1 ($p<0.01$; Figure 8D–J).

Figure 8.

The effects of TCP, TBP, and TIP on the expression of ${\mathrm{m}}^6\mathrm{A}$ regulators and global ${\mathrm{m}}^6\mathrm{A}$ levels in human PBMCs. (A–J) Transcriptional expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases, demethylases, and ${\mathrm{m}}^6\mathrm{A}$ binding proteins in human PBMCs after 2,4,6-trihalophenol exposure. After exposure to $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, and TIP for 24 h, mRNA levels of METTL3, METTL14, WTAP, FTO, ALKBH5, YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 were quantitatively measured via qRT-PCR. Results are shown as fold differences compared with the control group ($0\mathrm{\mu }\mathrm{M}$). $1=\text{no\hspace{0.17em}difference}$. Data are expressed as $\text{mean}\pm \text{SD}$ ($n=3$ biological replicates). Data were analyzed by one-way ANOVA with the Tukey post hoc test. *$p<0.05$, **$p<0.01$, ***$p<0.001$. The corresponding data are summarized in Excel Table S11. (K) Global ${\mathrm{m}}^6\mathrm{A}$ levels in human PBMCs after exposure to $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenols. The ${\mathrm{m}}^6\mathrm{A}$ levels of total RNA were measured by dot blot analysis (upper panel). Methylene blue staining was used as a loading control (lower panel). Note: ANOVA, analysis of variance; ${\mathrm{m}}^6\mathrm{A}$, $N^6\text{-methyladenosine}$; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SD, standard deviation; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol; TIP, 2,4,6-triiodophenol.

Furthermore, we detected the global ${\mathrm{m}}^6\mathrm{A}$ levels of total RNA from 2,4,6-trihalophenol–treated human PBMCs. After $100\mathrm{\mu }\mathrm{M}$ 2,4,6-trihalophenol exposure, the ${\mathrm{m}}^6\mathrm{A}$ levels were all greater in the TCP-, TBP-, and TIP-exposure groups compared with the control group (Figure 8K).

Discussion

In this study, the results of the CCK8 assay demonstrated that TCP, TBP, and TIP could impair the cell viability of mouse macrophage RAW264.7 cells at high concentrations. Ly6C is an important pro-inflammatory marker of macrophages. The recruitment of ${\text{Ly}6\mathrm{C}}^{+}$ macrophages was closely related to a variety of host inflammatory and anti-infectious responses in mice.^45,46 The lower expression of Ly6C in cells exposed to 2,4,6-trihalophenols suggested that trihalophenolic DBPs might affect macrophage-mediated resistance to infection (Figure 1). Considering the differences among pathogens, we used both bacteria and intracellular parasites to investigate the effects of 2,4,6-trihalophenols on phagocytosis and the pathogen clearance ability of macrophages. Cells exposed to TBP or TIP demonstrated greater numbers of E. coli after infection, suggesting that exposure might affect the cells’ response to or elimination of bacterial infection (Figure 2B). Interestingly, bacteria were identified intracellularly, but they were apparently not killed by the cells exposed to TIP, suggesting that TIP may have inhibited the bactericidal ability of macrophages but not the ability to phagocytize them (Figure 2A). T. gondii is a protozoan parasite capable of infecting humans and many warm-blooded animals.⁴⁷ T. gondii infection displays obvious opportunistic pathogenic characteristics in human immunodeficiency virus (HIV)-positive people,⁴⁸ solid-organ transplant recipients,⁴⁹ and pregnant women.^50,51 Under normal conditions, macrophages inhibit the overproliferation of T. gondii.^52,53 However, in immunocompromised hosts, T. gondii proliferates rapidly in macrophages.⁵⁴ Therefore, we used the parasite burden of T. gondii to evaluate macrophage-mediated resistance to parasite infection. In this study, mouse macrophages exposed to TBP or TIP appeared less able to kill T. gondii, and TIP had a stronger effect than its brominated analog (Figure 2C,D). RNA-seq results showed differences in genes associated with antigen presentation and signal transduction in TIP-exposed cells (Figure 3) that was commensurate with the impaired ability of RAW264.7 cells to control parasite infection after exposure to TIP. In sum, the present study demonstrated that exposure to 2,4,6-trihalophenols, especially TIP, induced remarkable immune suppression of mouse macrophages.

According to previous reports, the concentrations of 2,4,6-trihalophenols in drinking water samples in China reached $0.46–215\text{\hspace{0.17em}ng}/\mathrm{L}$,^2,3 and the total organic halogen (TOX) in drinking water samples of 23 cities in the United States and Canada were $\sim 22–273\mathrm{\mu }\mathrm{g}/\mathrm{L}$.⁵⁵ Although the environmental concentrations of 2,4,6-trihalophenols are much lower than the concentrations used in this study, it was reported that 2,4,6-trihalophenols could remain in the body for a relatively long time.^11,12,56,57 In addition, in special occupational settings, the ubiquity of exposure to 2,4,6-trihalophenols leads to abundant enrichment of such pollutants in particular individuals. For example, TCP was detected in the urine of sawmill workers (where chlorophenols were used to prevent fungal growth in lumber after sawing) at concentrations ranging from 1 to $11.8\mathrm{\mu }\mathrm{M}$,⁵⁶ which were up to $\sim \mathrm{4,000}$ times higher than the average concentration of TCP detected in human urine ($0.59\mathrm{\mu }\mathrm{g}/\mathrm{L}$) in the German Environmental Survey.¹¹ Therefore, our results can inform the immunotoxicity risk of long-term exposure or occupational exposure to 2,4,6-trihalophenols.

It was reported that the toxicity of an electrophilic DBP to a nucleophile site and that of a nucleophilic DBP to an electrophile site might increase with decreasing energy of the lowest unoccupied molecular orbital (${\mathrm{E}}_{\text{LUMO}}$; related to molecular reactions with nucleophiles) and increasing energy of the highest occupied molecular orbital (${\mathrm{E}}_{\text{HOMO}}$_; related to molecular reactions with electrophiles).⁸ There was an obvious decrease of the three 2,4,6-trihalophenols in ${\mathrm{E}}_{\text{LUMO}}$ and an increasing trend in ${\mathrm{E}}_{\text{HOMO}}$ from TCP to TIP.^17,8 Thus, the toxicity of 2,4,6-trihalophenols commonly followed the rank order of iodinated $\text{DBP}>\text{brominated}$ $\text{analog}>\text{chlorinated}$ analog.^17,58,59

Different from DNA and protein modifications, epitranscriptomic methylation modulates gene expressions and functionalities more rapidly and flexibly following exposure to environmental toxicants.^38,39,60 There are $>100$ kinds of chemical modifications located on RNA, among which ${\mathrm{m}}^6\mathrm{A}$ was the most prevalent internal modification.⁶¹ Studies in recent years have shown that exposure to environmental stressors could induce global ${\mathrm{m}}^6\mathrm{A}$ modification changes. Specifically, exposure to carcinogens [particulate matter (PM) and sodium arsenite] and endocrine disruptors [bisphenol A (BPA) and vinclozolin], had obvious effects on the global ${\mathrm{m}}^6\mathrm{A}$ modifications.³⁸ Participants exposed to high PM $\le 2.5\mathrm{\mu }\mathrm{m}$ in aerodynamic diameter (${\mathrm{PM}}_{2.5}$) exhibited higher expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases (METTL3 and WTAP), demethylases (FTO and ALKBH5), and binding protein Heterogeneous Nuclear Ribonucleoprotein C (HNRPC) compared with those in the low-exposure control group.³⁸ Compared with nonsmokers, the total ${\mathrm{m}}^6\mathrm{A}$ levels in the peripheral blood of smokers were lower by 10.7%.³⁹ In addition, ${\mathrm{m}}^6\mathrm{A}$ modification was also involved in the intergenerational toxic mechanism of benzo[a]pyrene (BaP), a widespread pollutant in aquatic environments.⁶⁰

In this study, exposure to 2,4,6-trihalophenols was associated with differences in ${\mathrm{m}}^6\mathrm{A}$ levels in mouse macrophages, accompanied by differences in the abundance of methyltransferases (Figures 4 and 5). Therein, cells exposed to TIP exhibited the most obvious differences in global ${\mathrm{m}}^6\mathrm{A}$ levels and expression of ${\mathrm{m}}^6\mathrm{A}$ methyltransferases. Based on these results, we speculated that after 2,4,6-trihalophenol exposure, the observed immune suppression of macrophages might be associated with remarkable differences in RNA ${\mathrm{m}}^6\mathrm{A}$ modifications.

In addition, we also detected the effects of 2,4,6-trihalophenols on ${\mathrm{m}}^6\mathrm{A}$ modifications of human PBMCs (Figure 8). Global ${\mathrm{m}}^6\mathrm{A}$ levels of human PBMCs were greater in all three 2,4,6-trihalophenol exposure groups than in the control group. The results suggested that 2,4,6-trihalophenol–induced ${\mathrm{m}}^6\mathrm{A}$ disturbance was not unique to mouse macrophages. Differences in ${\mathrm{m}}^6\mathrm{A}$ regulators differed between RAW264.7 cells and human PBMCs. In RAW264.7 cells, the transcriptional expressions of ${\mathrm{m}}^6\mathrm{A}$ regulators did not exhibit any significant differences after exposure to $100\mathrm{\mu }\mathrm{M}$ TCP, TBP, or TIP. However, the mRNA levels of partial ${\mathrm{m}}^6\mathrm{A}$ regulators in PBMCs were significantly different after the same treatment. This is, perhaps, because PBMCs are a mixture of immune cell types. The immune cells interact to regulate the functions of each other. In addition, primary cells might be more susceptible than immortalized cell lines.^62–64

Although recent environmental toxicology studies have gradually paid attention to RNA ${\mathrm{m}}^6\mathrm{A}$ modification, the specific genes or pathways modulated by ${\mathrm{m}}^6\mathrm{A}$ following environmental pollutant exposure remain unclear. There is still a lack of in-depth research on pollutant-induced variations of ${\mathrm{m}}^6\mathrm{A}$ modification. In this study, the dot blot results exhibited the global differences in ${\mathrm{m}}^6\mathrm{A}$ modifications in response to 2,4,6-trihalophenol exposure; however, it was a nonquantitative result. A more advanced and precise study was required for the ${\mathrm{m}}^6\mathrm{A}$ profile. Thus, we performed the high-throughput ${\mathrm{m}}^6\mathrm{A}\text{-seq}$. Transcriptome-wide ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ revealed the distribution patterns of ${\mathrm{m}}^6\mathrm{A}$ across mouse macrophage transcriptome after exposure to 2,4,6-trihalophenols (Figure 6). Exposure to 2,4,6-trihalophenols affected the ${\mathrm{m}}^6\mathrm{A}$ levels of several genes associated with immune-related pathways. Joint analysis of ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ and RNA-seq data confirmed the potential association of abnormal ${\mathrm{m}}^6\mathrm{A}$ levels with altered immune-related pathways (Figure 7A), suggesting ${\mathrm{m}}^6\mathrm{A}$ as a potential biomarker for the immunotoxicity of environmental pollutants. To the best of our knowledge, this was the first time that ${\mathrm{m}}^6\mathrm{A}\text{-seq}$ has been used to map the ${\mathrm{m}}^6\mathrm{A}$ modification in macrophages in the area of environmental health.

The immune system is critical to the survival of individuals when dealing with internal and external pathogens. Thus, 2,4,6-trihalophenol-induced ${\mathrm{m}}^6\mathrm{A}$ variation and immune dysfunction of macrophages deserve special attention. However, how is ${\mathrm{m}}^6\mathrm{A}$ related to the immune dysfunction in DBP-pretreated cells that are also challenged with infection? To probe this process, we performed the ${\mathrm{m}}^6\mathrm{A}\text{-IP-qPCR}$ on RAW264.7 cells that were also challenged with parasite T. gondii. $\text{TNF-}\mathrm{\alpha }$ is an inflammatory cytokine produced by macrophages and plays a vital role in cell resistance against T. gondii.⁶⁵ EIF2AK2 is known as protein kinase R and selectively regulates the gene expression in the immune response.⁶⁶ After T. gondii infection, the ${\mathrm{m}}^6\mathrm{A}$ levels of $Tnf\text{-}\mathrm{\alpha }$ and Eif2ak2 remained significantly up-regulated in the 2,4,6-trihalophenol–pretreated groups compared with the control group (Figure 7B). The ${\mathrm{m}}^6\mathrm{A}\text{-modified}$ RNAs can be degraded by ${\mathrm{m}}^6\mathrm{A}$ binding proteins,⁶⁷ and then the expressions of corresponding genes are down-regulated. These findings were commensurate with the dampened ability of macrophages to control infection after 2,4,6-trihalophenol exposure. In the future, further studies are needed to get a full and in-depth understanding of the underlying mechanism.