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Research Article

The m6A demethylase FTO regulates TNF-α expression in human macrophages following Toxoplasma gondii infection

  • Min Qin,

    Roles Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft

    Affiliations Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China, Department of Pathology, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Nan Gao,

    Roles Formal analysis, Investigation, Validation, Visualization, Writing – original draft

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Jierui Sun,

    Roles Investigation, Methodology

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Shuqing Lin,

    Roles Investigation, Methodology

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Tingting Hu,

    Roles Investigation, Methodology

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Xinjian Liu,

    Roles Supervision

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Rong Zhang,

    Roles Supervision

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Yong Wang ,

    Roles Supervision, Writing – review & editing

    * E-mail: qiujingfan@njmu.edu.cn (JQ); yongwsh@njmu.edu.cn (YW)

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯
  • Jingfan Qiu

    Roles Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing

    * E-mail: qiujingfan@njmu.edu.cn (JQ); yongwsh@njmu.edu.cn (YW)

    Affiliation Key Laboratory for Pathogen Infection and Control of Jiangsu Province, Department of Pathogen Biology and Immunology, Nanjing Medical University, Nanjing, Jiangsu, China

    ⨯

Abstract

Toxoplasma gondii (T. gondii) is an opportunistic parasite. After infection, macrophages finely regulate the immune response to restrict parasite proliferation. It is well-known that N6-methyladenosine (m6A) plays a critical role in fine-tuning gene expression. To investigate whether m6A modification is involved in regulating the anti-infection immune response in human macrophages against T. gondii, this study utilized T. gondii tachyzoites from the RH strain to infect human THP-1 macrophages. qPCR and ELISA results show that T. gondii infection mounted the expression of TNF-α, IL-1β, and IL-6. Transcriptomic data suggest that the infection of T. gondii induced differential gene expression in pathways associated with TNF signaling and cytokine-cytokine receptor interaction. Meanwhile, expression of m6A regulators were evaluated using qPCR and Western blotting. T. gondii infection increased the abundance of m6A demethylase FTO and methyltransferase WTAP. Joint analysis of RNA-seq and m6A-seq data was utilized for enriching differentially expressed genes (DEGs) with significantly altered m6A modifications. Intriguingly, following T. gondii infection, the m6A levels of DEGs associated with toxoplasmosis, TNF signaling pathway, and NF-κB signaling pathway were significantly different. The m6A-IP-qPCR assay further confirmed that T. gondii infection led to the decrease in the levels of m6A modification in the 3’UTR and 5’UTR regions of TNF-α mRNA. Knocking-down of FTO retarded the infection induced-decrease in the levels of m6A modification in TNF-α transcripts, accompanied by dampened immune response and uncontrolled T. gondii proliferation. Furthermore, the YTHDF2 RIP assay indicates that T. gondii infection remarkably weakened the binding of YTHDF2 to TNF-α mRNA, which could mount TNF-α expression by inhibiting the degradation of TNF-α mRNA. Overall, these findings suggest that m6A plays a role in the T. gondii infection-induced immune response in human macrophages, uncovering a new molecular mechanism for the regulation of TNF-α expression from an epitranscriptomic aspect.

Author summary

One intriguing phenomenon in T. gondii infection and immunity is that the immune response induced by T. gondii is diverse in various hosts. Our previous research shows that in mouse macrophages, T. gondii microneme protein 3 (MIC3) activates TNF-α transcription via the TLR11/MyD88/NF-κB pathway. T. gondii infection can also induce TNF-α production in humans. However, human cells lack TLR11, leading us to predict that the mechanisms by which T. gondii regulates TNF-α expression in human macrophages differ from those in mouse macrophages. In this study, we found that T. gondii infection increased the abundance of m6A demethylase FTO. Joint analysis of RNA-seq and m6A-seq data indicates significant differences in the m6A levels of differentially expressed genes related to the TNF signaling pathway after T. gondii infection. Results of m6A-IP-qPCR assay further confirmed that T. gondii infection decreased the m6A modification levels in the 3’UTR and 5’UTR regions of TNF-α mRNA. Knocking-down FTO hindered the infection-induced decrease in m6A modification levels of TNF-α transcripts, accompanied by a dampened immune response and uncontrolled T. gondii proliferation. Additionally, T. gondii infection significantly weakened the binding of YTHDF2 to TNF-α mRNA, thereby promoting TNF-α expression by inhibiting TNF-α mRNA degradation. In sum, these findings explain why human macrophages lacking TLR11 can still express TNF-α after T. gondii infection and uncover a novel post-transcriptional regulation mechanism for TNF-α.

Citation: Qin M, Gao N, Sun J, Lin S, Hu T, Liu X, et al. (2025) The m6A demethylase FTO regulates TNF-α expression in human macrophages following Toxoplasma gondii infection. PLoS Negl Trop Dis 19(7): e0013289. https://doi.org/10.1371/journal.pntd.0013289

Editor: Leonardo Augusto, UNMC: University of Nebraska Medical Center, UNITED STATES OF AMERICA

Received: February 16, 2025; Accepted: June 25, 2025; Published: July 15, 2025

Copyright: © 2025 Qin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files. The raw sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE288205.

Funding: This work was supported by National Natural Science Foundation of China (Grant numbers 81802030 and 22476096 to JQ; https://www.nsfc.gov.cn/) and the Natural Science Foundation of Jiangsu Province, China (Grant number BK20200088 to JQ; https://kxjst.jiangsu.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Toxoplasma gondii (T. gondii) is an important opportunistic parasite. In the healthy population, T. gondii infection does not cause obvious clinical symptoms [1,2]. However, infection occurs in the elderly and the immunocompromised individuals (such as AIDS patients, cancer patients and organ transplant recipients) could develop into acute toxoplasmosis, causing encephalitis, and even death [1,2]. Thus, the immune status of the host plays an important role in the development and outcome of T. gondii infection.

Macrophages play an indispensable role in resisting T. gondii infection and regulating immune homeostasis [3]. Among the inflammatory cytokines secreted by macrophages, tumor necrosis factor-α (TNF-α) has a highly pleiotropic effect in diverse cellular processes [4]. First of all, TNF-α is involved in controlling T. gondii infection. Blocking TNF-α may cause the latent infection of T. gondii to be reactivated, increasing the risk of acute toxoplasmosis [5]. Additionally, TNF-α could drive the conversion of tachyzoites to bradyzoites, resulting in the transition of toxoplasmosis from acute to chronic phase [6]. Furthermore, in spite of the protective role of TNF-α, excessive production of TNF-α can exacerbate inflammation and lead to immunopathology, contributing to the symptoms of acute toxoplasmosis, such as encephalitis [7]. Thus, it is of paramount importance to investigate the regulatory mechanisms of TNF-α after T. gondii infection.

In previous study, our research group focused on elucidating the transcriptional regulation mechanism of TNF-α. We demonstrated that MIC3 (microneme protein 3) activates TNF-α transcription via the TLR11/MyD88/NF-κB pathway in mouse macrophages [8]. In humans, T. gondii infection could also induce TNF-α production and human cells discriminate between viable and killed T. gondii tachyzoites [9–11]. However, human cells lack TLR11. Thus, we predicted that the mechanisms by which T. gondii regulates TNF-α expression in human macrophages are distinctly different from those in mouse macrophages.

It is well-known that post-transcriptional regulation helps cells respond more quickly to external stimuli than transcriptional regulation. Does post-transcriptional regulation play a role in modulating TNF-α expression in T. gondii-infected human macrophages? To answer this question, we focused on the most abundant internal mRNA modification, N6-methyladenosine (m6A), which has been reported as an important mechanism of post-transcriptional regulation [12]. m6A is an essential epitranscriptomic mark. Deposition of m6A is installed by a large protein complex called methyltransferases, consisting of METTL3, METTL14 and WTAP [13]. Demethylases, mainly FTO and ALKBH5, could remove existing m6A modification from mRNA [14,15]. The combination of methyltransferases and demethylases makes m6A modification a dynamic and reversible process. Finally, m6A modifications are directly recognized and bound by YTH-domain family proteins, including YTHDF1, YTHDF2, YTHDF3, YTHDC1 and YTHDC2 [16–18]. YTH-domain family proteins activate the downstream post-transcriptional regulation pathways, including promoting the degradation of mRNA.

In this study, we found that T. gondii infection significantly altered mRNA m6A modifications in human macrophages and increased the abundance of m6A demethylase FTO. Interestingly, there are m6A modification sites on human TNF-α mRNA. T. gondii could govern the expression of FTO to down-regulate m6A modification in the 3’UTR and 5’UTR regions of TNF-α mRNA, thereby mounting the TNF-α production in human macrophages. We believe that what we have learned about m6A and T. gondii-induced TNF-α production will provide important insights into the pathogenesis of toxoplasmosis and the T. gondii host-pathogen relationship.

Materials and methods

Ethics statement

This study was approved by the Ethics Review Board of Nanjing Medical University [permit no.: NJMU/ (2018) 498].

Study design

This study aimed to investigate the role of m6A modification in regulating the anti-infection immune response of human macrophages against T. gondii. Human THP-1 macrophages were infected with tachyzoites of T. gondii RH strain to establish an in vitro infection model. Uninfected cells served as controls. qPCR was performed to measure the mRNA abundance of inflammation-related genes. The secretion of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were measured by ELISA. The expression of m6A regulators was detected at the mRNA and protein levels using qPCR and Western blotting. RNA-seq was conducted to identify differentially expressed genes in T. gondii-infected macrophages compared to controls. m6A-seq was used to profile m6A modifications in mRNA transcripts. The m6A-IP-qPCR assay was performed to validate m6A modification levels in TNF mRNA. FTO was knocked down in THP-1 macrophages using lentiviral shRNA. Scramble shRNA served as the negative control. Infected FTO-knockdown cells were analyzed for m6A modification levels in TNF transcripts, expression of inflammation-related genes, secretion of inflammatory cytokines, and T. gondii proliferation. To investigate the role of m6A readers, RIP assay was performed to detect whether YTHDF1 or YTHDF2 had a direct effect on TNF-α mRNA.

Cell culture

THP-1 cells (human monocytic cell line) and HFF cells (human foreskin fibroblast cell line) were cultured in 1640 medium (Gibco BLR, MD, USA) and Dulbecco’s modified Eagle medium (Gibco BLR, MD, USA), respectively. Both media were supplemented with 10% fetal bovine serum (Gemini, CA, USA) and 1% 100 ×ばつ penicillin and streptomycin (Gibco BLR, MD, USA). To induce differentiation into a macrophage phenotype, THP-1 cells were treated with 10 ng/ml phorbol myristate acetate (PMA, Sigma-Aldrich) for 72 h. For infection assay, THP-1 macrophages were infected with T. gondii RH tachyzoites and GFP-RH tachyzoites at a 1:1 ratio.

Preparation of T. gondii tachyzoites

T. gondii RH tachyzoites and GFP-expressing RH tachyzoites were serially subcultured in HFF cell lines. After observing 80% tachyzoite release, cell supernatants were filtered through a 3 μm pore size track-etched membrane (Shanghai Nengthink Filtration Technology Co. Ltd., Shanghai, China) to collect T. gondii RH tachyzoites and GFP-RH tachyzoites.

Real-time qRT-PCR

Total RNA was extracted from cell line samples and T. gondii tachyzoites using TRIZOL (Invitrogen, CA, USA). The quality and integrity of the extracted RNA for real-time qRT-PCR and m6A-IP-qPCR were evaluated using NanoDrop 2000 (NanoDrop, Wilmington, USA) and Bioanalyzer 2100 (Agilent, CA, USA). Samples with OD260/280 > 1.8, and RNA integrity number (RIN) > 7.0 were then reverse-transcribed to cDNA using HiScript III RT Supermix (Vazyme, Nanjing, China). Semi-quantitative RT-PCR, including cDNA, SYBR Green (Vazyme, Nanjing, China), and 10 μM primer pairs, was performed in a LightCycler 96 Instrument (Roche, Basel, Switzerland). Sequences of gene-specific primers used in this study are provided in S1 Table. Relative expression levels of these genes were calculated using the 2−(△しろさんかく△しろさんかくCt) method.

Enzyme-linked immunosorbent assay (ELISA)

After 24 h of T. gondii tachyzoites infection, THP-1 macrophage supernatants were harvested. The concentrations of TNF-α, IL-1β, and IL-6 in supernatants were detected using Human TNF-α, IL-1β, and IL-6 ELISA Kits (DaKeWe, Nanjing, China) respectively, following the manufacturer’s instructions.

Western blotting

For extracting total protein, THP-1 macrophages were lysed in RIPA lysis buffer (Millipore, MA, USA). After centrifugation at 12,000 g for 15 min at 4°C, the supernatants of lysates were collected. Protein concentrations were measured using a BCA assay (TIANGEN, Beijing, China). Protein samples were boiled with SDS-PAGE sample loading buffer (Beyotime, Shanghai, China) for 10 min. Appropriate amount of protein sample was resolved on a 10% SDS-PAGE gel, transferred to a 0.2 μm PVDF membrane (Millipore, MA, USA), and then blocked in TBST with 5% non-fat milk. Antibodies included N6-mA Methyltransferase (METTL3, METTL14, and WTAP) Antibody Sampler Kit (1:1000, Cell Signaling Technology), anti-FTO antibody (1:1000, Abcam), anti-YTHDF1 antibody (1:1000, Proteintech), anti-YTHDF2 antibody (1:1000, Proteintech), and HRP-linked anti-rabbit secondary antibody (1:2000, Cell Signaling Technology). Visualization was performed using ECL luminescence solution (FDbio Science, Hangzhou, China) on a ChemiDocTM imaging system (Bio-RAD, CA, USA).

RNA-seq and m6A-seq

T. gondii RH strain tachyzoites were utilized to infect THP-1 macrophages, forming the infected group, while untreated THP-1 macrophages served as the control group. Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA). The quality and integrity of the extracted RNA were evaluated by NanoDrop 2000 (NanoDrop) and Bioanalyzer 2100 (Agilent), and were confirmed by agarose gel electrophoresis, as previously reported [19]. Samples with OD260/280 > 1.8, RIN > 7.0, and total amount > 50 μg were processed further. Ribosomal RNA of total RNA was depleted using Epicentre Ribo-Zero Gold Kit (Illumina, CA, USA). Magnesium RNA Fragmentation Module (NEB, MA, USA) was used to fragment RNA into small pieces for 7 min at 86°C. The fragmented RNA was saved for RNA-seq as input control. For m6A-seq, the cleaved RNA fragments and m6A-specific antibody (Synaptic Systems, Germany) were incubated for 2 h at 4°C in IP buffer. 50 mM Tris-HCl, 750 mM NaCl, and 0.5% Igepal CA-630 were used as IP buffer. Subsequently, the IP RNA was reverse-transcribed to create the cDNA, which were next used to synthesize U-labeled second-stranded DNAs. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Then adapters are ligated to the fragments. After the heat-labile UDG enzyme (NEB) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation (1 cycle, 95°C for 3 min); amplification (8 cycles, 98°C for 15 s, 60°C for 15 s, and 72°C for 30 s); and then final extension (1 cycle, 72°C for 5 min). Finally, paired-end sequencing (PE150) was performed on an Illumina NovaseqTM 6000, and the data were analyzed by LC-Bio Technology CO., Ltd. (Hangzhou, China). All the sequencing experiments were performed with two biological replicates. The raw sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE288205. Raw reads obtained from the sequencing machine include reads containing adapters and low quality bases which will affect the following bioinformatics analysis. Thus, to get high quality clean reads, reads were further filtered by fastp (https://github.com/OpenGene/fastp, v0.19.4) [20]. For RNA-seq, genes with |log2Fold change| ≥ 1 and P < 0.05 were defined as differentially expressed genes (DEGs). DEGs were used to perform KEGG pathway enrichment analyses. For m6A-seq, DEGs with significantly altered m6A modification [false discovery rate (FDR) < 0.05] after T. gondii infection were used to perform KEGG pathway enrichment analyses.

m6A-IP qPCR

Firstly, total RNA was isolated using TRIZOL (Invitrogen, CA, USA) and its concentration was adjusted to 1 μg/μl. Subsequently, total RNA was fragmented for 5 min at 94°C in Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific, MA, USA). 1 μg fragmented RNA was reserved as input control. The immunoprecipitation (IP) mixture was incubated for 2 h at 4°C, including m6A-specific antibody (Abcam) or mouse IgG (Beyotime), RnaseOUT (Invitrogen), Ribonucleoside vanadyl complexes (RVC, Sigma), and fragmented RNA. Meanwhile, Protein A/G Mix Magnetic Beads (Millipore, MA, USA) was blocked with BSA (Beyotime, Shanghai, China). Finally, magnetic beads were added into the incubated IP mixture for another 2 h at 4°C. The RNA in the magnetic beads was eluted twice and precipitated for further qRT-PCR detection.

FTO shRNA knock-down assay

Lentiviral shRNA was used to knock down of FTO in THP-1 cells. shRNA targeting sequences are shown below. Human FTO (shFTO): 5’-GGATGACTCTCATCTCGAAGG-3’. Scramble control sequence (NC): 5’-TTCTCCGAACGTGTCACGTAA-3’. Lentiviral particles harboring shRNA targeting FTO (shFTO) and negative control shRNA (NC) were designed and constructed by HANBIO (Shanghai, China). THP-1 cells were cultured in 6-well culture plates using Opti-MEM medium. Then cells were incubated in lentivirus-containing medium with polybrene (Sigma-Aldrich), and selected with puromycin (Solarbio, Beijing, China). The multiplicity of infection (MOI) of lentivirus was 60. FTO knock-down efficiency was assessed via qRT-PCR and Western blotting.

RNA-binding protein immunoprecipitation (RIP) assay

Anti-YTHDF1 antibody (Proteintech) or anti-YTHDF2 antibody (Proteintech) and Protein A/G Mix Magnetic Beads (Millipore) were pre-incubated overnight at 4°C. 20% percent of cell extracts were taken out as input control. Remaining cell lysates were incubated with previous magnetic bead-antibody complex for 4 h. After elution, RNA was isolated from IP fractions and input control for further detection.

Statistical analysis

Data from ELISA and qPCR assay was analyzed using Graphpad Prism software (GraphPad). Student’s t-test was used for comparisons between two groups. The P value < 0.05 was considered statistically significant.

Results

T. gondii infection upregulated inflammatory cytokine expression in human macrophages

During T. gondii invasion, T. gondii antigens interact with host immune cells to trigger the immune responses [21]. Therefore, to analyze the impact of T. gondii infection on human macrophages, we used T. gondii tachyzoites from RH strain to infect THP-1 macrophages. Inflammatory-related gene expression and the secretion of inflammatory cytokines were measured at 6 h, 12 h, 18 h and 24 h after infection (Fig 1).

Fig 1. mRNA abundance of inflammatory-related genes and secretion of inflammatory cytokines in THP-1 macrophages infected with T. gondii tachyzoites.

A-F: Transcriptional expression of inflammatory-related genes in THP-1 macrophages after T. gondii infection. mRNA abundance of TNF-α (A), iNOS (B), Arg-1 (C), IL-1β (D), IL-10 (E), and IL-6 (F) were quantitatively measured via qRT-PCR. Results are shown as fold changes compared with the control group. Data are represented as the mean ± SD (n = 3). Statistical significance was determined using Student’s t-test. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. G-I: Secretion of inflammatory cytokines in THP-1 macrophages after T. gondii infection. The secretion of TNF-α (G), IL-1β (H) and IL-6 (I) were detected by ELISA. Results are shown as fold changes compared with the control group. Data are represented as the mean ± SD (n = 3). Comparisons between two groups were made by Student’s t-test. **, P < 0.01; ***, P < 0.001.

https://doi.org/10.1371/journal.pntd.0013289.g001

The mRNA abundance of TNF-α, IL-1β and IL-6 were significantly up-regulated from 6 h to 24 h post infection (Fig 1A, 1D, and 1F), while those of iNOS, Arg-1 and IL-10 did not change significantly from 18 h to 24 h post infection (Fig 1B, 1C, and 1E). The up-regulation amplitude was IL-6 > IL-1β > TNF-α, and the up-regulation amplitude increased with extended infection time within 18 h. Protein abundance of TNF-α, IL-1β, and IL-6 increased at 24 h after infection (Fig 1G, 1H, and 1I), consistent with mRNA abundance. Interestingly, unlike mRNA abundance, protein abundance of TNF-α exhibited the largest increase after T. gondii infection (Fig 1G). In sum, the results showed that T. gondii tachyzoites could induce the expression of inflammatory-related genes and promote the secretion of inflammatory cytokines, including TNF-α, IL-1β and IL-6.

T. gondii infection increased the expression of m6A methyltransferase WTAP and demethylase FTO

In the regulation of host immune response against pathogens, post-transcriptional regulation responds more rapidly than transcriptional regulation. RNA m6A modification plays an important role in post-transcriptional regulation in various pathogen infection models [22–25]. After T. gondii infection, the transcriptional expression of methyltransferases, demethylases and m6A-specific binding proteins (m6A reader proteins) were detected by qRT-PCR (Fig 2A to 2J). At different time points post-infection from 0 h to 24 h, WTAP was highly up-regulated at the transcriptional level, accompanied by a slight decrease in demethylase ALKBH5 (Fig 2C and 2E). Meanwhile, the m6A demethylase FTO also showed an upward trend from 18 h to 24 h after T. gondii infection (Fig 2D). The mRNA abundance of methyltransferase METTL3, and m6A-specific binding protein YTHDF2 and YTHDF3 significantly increased from 18 h to 24 h after infection (Fig 2A, 2G, and 2H).

Fig 2. mRNA and protein abundance of m6A methyltransferases, demethylases and m6A-specific binding proteins in THP-1 macrophages infected with T. gondii tachyzoites.

A-J: 6A methyltransferases, m6A demethylases and m6A-specific binding proteins in THP-1 macrophages after T. gondii infection. mRNA abundance of METTL3 (A), METTL14 (B), WTAP (C), FTO (D), ALKBH5 (E), YTHDF1 (F), YTHDF2 (G), YTHDF3 (H), YTHDC1 (I), and YTHDC2 (J) were quantitatively measured by qRT-PCR. Results are shown as fold changes compared with the control group. Data are represented as the mean ± SD (n = 3). Comparisons between two groups were made by Student’s t-test. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. K-P: Western blotting analysis for m6A methyltransferases, m6A demethylases, and m6A-specific binding proteins. Protein abundance of FTO (K), METTL3 (L), YTHDF1 (M), METTL14 (N), YTHDF2 (O), and WTAP (P) were measured by Western blotting.

https://doi.org/10.1371/journal.pntd.0013289.g002

Then, the protein abundance of m6A methyltransferases, demethylases and m6A-specific binding proteins were measured at 6 h, 12 h, 18 h, and 24 h after infection. The results showed a significant increase in WTAP and FTO compared to the control group (Fig 2K and 2P). As for METTL3, METTL14, YTHDF1, and YTHDF2, T. gondii infection causes slight or no significant changes in protein abundance (Fig 2L-2O).

T. gondii infection affected TNF signaling in human macrophages

To thoroughly evaluate the effects of T. gondii infection on THP-1 macrophages, we performed global gene expression profiling using RNA-seq. |log2Fold change| ≥ 1 and P < 0.05 were used as criteria to identify DEGs (differentially expressed genes) between the infected group and the control group. After 24 h-infection of T. gondii in THP-1 macrophages, a total of 4887 DEGs were screened, including 1992 up-regulated genes and 2895 down-regulated genes (Fig 3A). Clustering analysis indicated a significant upregulation of TNF-α expression level after T. gondii infection (Fig 3B). Additionally, the expression of several members of the TNF super-family, including TRAP1, TNFAIP3, and TNFAIP8, were notably altered. KEGG pathway enrichment analysis showed that DEGs were enriched in TNF signaling pathway, toxoplasmosis pathway, cytokine-cytokine receptor interaction pathway, NF-κB signaling pathway, and so on (Fig 3C). These results suggest that T. gondii infection could induce an immune response in human macrophages. Proinflammatory cytokine TNF-α significantly changed after infection.

Fig 3. Significantly altered genes and pathways of macrophages after T. gondii infection.

A: Volcano plot of differentially expressed genes. The red dots indicate up-regulated DEGs after T. gondii infection. The blue dots indicate down-regulated DEGs after T. gondii infection. The gray dots indicate genes without any significant change. Genes with |log2Fold change| ≥ 1 and P < 0.05 are defined as DEGs. B: Heatmap of DEGs related to TNF pathway after T. gondii infection. The blue color indicates down-regulated genes. The red color indicates up-regulated genes. C: KEGG pathway enrichment analysis of DEGs. The size of the dot is proportional to the number of enriched DEGs, and the rich factor indicates the degree of enrichment.

https://doi.org/10.1371/journal.pntd.0013289.g003

T. gondii infection influenced RNA m6A modification in human macrophages

To systematically analyze changes in RNA m6A modification in T. gondii-infected THP-1 macrophages, we performed m6A-seq. After 24 h-infection of T. gondii in THP-1 macrophages, m6A peaks were mainly distributed in the 3’ UTR and the 5’ UTR regions in both control and infected groups (Fig 4A). T. gondii infection increased methylation in the 3’ UTR region, while methylation in the 5’ UTR region decreased. Differentially methylated m6A peaks (|log2Fold change| ≥ 1 and P < 0.05) were identified, with 1440 total (647 up-regulated and 793 down-regulated) in the infected group compared to the control group (Fig 4B). KEGG pathway enrichment analysis of DEGs with significantly altered m6A modification (FDR < 0.05) after T. gondii infection revealed that such genes were enriched in toxoplasmosis pathway, TNF signaling pathway, NF-κB signaling pathway, and so on (Fig 4C).

Fig 4. Transcriptome-wide m6A methylation profiling of THP-1 macrophages by m6A-seq after T. gondii infection.

A: Distribution of m6A peaks across the transcriptome of THP-1 macrophages. B: Volcano plot of differentially methylated m6A peaks. The red dots indicate significantly up-regulated m6A methylation after T. gondii infection (log2Fold change ≥ 1 and P < 0.05). The blue dots indicate significantly down-regulated m6A methylation after T. gondii infection (log2Fold change ≤ -1 and P < 0.05). The gray dots indicate m6A methylation without any significant changes. C: KEGG pathway enrichment analysis of DEGs with significantly altered m6A modification (FDR < 0.05) after T. gondii infection. The size of the dot is proportional to the number of enriched DEGs, and the rich factor indicates the degree of enrichment.

https://doi.org/10.1371/journal.pntd.0013289.g004

T. gondii infection downregulated the m6A modification in the 5’UTR and 3’UTR region of TNF-α mRNA in human macrophages

The m6A modification is not uniformly distributed in different mRNAs. Given the importance of TNF-α in responding to T. gondii infection, we probed the m6A modification sites on TNF-α mRNA. Firstly, the online prediction tool SRAMP was used to predict the potential m6A modification sites on human TNF-α mRNA. The results showed that there were 9 potential m6A modification sites in the 5’UTR and 3’UTR regions of TNF-α mRNA (S2 Table). The m6A-seq results suggested that m6A modifications in the 5’UTR and 3’UTR region of TNF-α mRNA decreased after T. gondii infection. Furthermore, we used m6A-IP-qPCR to verify the above findings. The m6A modification levels decreased at sites both in the 5’UTR region and in the 3’UTR region after T. gondii infection (Fig 5).

Fig 5. Detection of TNF-α mRNA m6 A sites after T. gondii infection.

The m6A modification sites on TNF-α mRNA were detected by m6A-IP-qPCR. Fold change = 1 indicates no change (n = 3).

https://doi.org/10.1371/journal.pntd.0013289.g005

Knock-down of FTO attenuated TNF-α expression and led to uncontrolled proliferation of T. gondii in human macrophages

FTO, a key m6A demethylase, was upregulated in THP-1 macrophages after T. gondii infection. To investigate the function of FTO in T. gondii-induced immune response in macrophages, we knocked down FTO in THP-1 macrophages by shRNA (S1 Fig). In FTO knock-down cell lines, m6A modification levels at sites in the 5’UTR and 3’UTR regions of TNF-α mRNA did not decrease significantly after T. gondii infection (Fig 6A), while T. gondii infection down-regulated the m6A modification levels at sites in the 5’UTR and 3’UTR regions of TNF-α mRNA in wildtype cells (Fig 5). These results indicate that FTO plays a role in T. gondii-induced down-regulation of m6A modifications in TNF-α mRNA.

Fig 6. Effects of FTO knock-down on THP-1 macrophage immune response induced by T. gondii.

A: Changes of m6A modification on TNF-α mRNA in FTO knock-down cell lines after T. gondii infection. Results are expressed as fold change relative to the control group. Fold change = 1 indicates no change (n = 3). B-G: mRNA abundance of inflammatory-related genes, including TNF-α (B), iNOS (C), Arg-1 (D), IL-1β (E), IL-10 (F), and IL-6 (G), were quantitatively measured via qRT-PCR. Results were shown as fold changes compared with the control group. Data were expressed as the mean ± SD (n = 3). Comparisons between two groups were made by Student’s t-test. ns, P > 0.05; *, P < 0.05; ***, P < 0.001. H-J: Secretion of TNF-α (H), IL-1β (I), and IL-6 (J) were detected by ELISA. Results were shown as fold changes compared with the control group. Data were expressed as the mean ± SD (n = 3). Comparisons between two groups were made by Student’s t-test. ns, P > 0.05; ***, P < 0.001. K: The mRNA abundance of TOXO ITS1 was measured by qRT-PCR. L: The proliferation of T. gondii GFP-expressing tachyzoites was observed by fluorescence microscope.

https://doi.org/10.1371/journal.pntd.0013289.g006

In the shFTO group, mRNA abundance of TNF-α, IL-1β, and IL-6 was significantly lower than that in the control group, while the transcriptional levels of iNOS, Arg-1, and IL-10 remained unchanged (Fig 6B-6G). The levels of secreted cytokines after T. gondii infection decreased significantly for TNF-α and IL-1β in the shFTO group compared to the control group, and the secretion of IL-6 in the shFTO group showed a decreasing trend but was not statistically significant (Fig 6H-6J). FTO knock-down weakened the immune response induced by T. gondii, as evidenced by decreased expression of inflammation-related genes and reduced secretion of inflammatory cytokines.

Additionally, after T. gondii infection, the mRNA levels of TOXO ITS1, which indicate parasite burden of T. gondii, increased significantly in the FTO knock-down group compared with the control group (Fig 6K). Meanwhile, enhanced proliferation of T. gondii green fluorescent protein (GFP)-expressing tachyzoites was also observed by the fluorescence microscope in the FTO knock-down group (Fig 6L). These results further demonstrated that FTO knock-down weakened the anti-infection immune response against T. gondii.

T. gondii infection inhibited YTHDF2 binding to TNF-α mRNA

YTHDF proteins are known to bind m6A sites and could promote the decay of m6A-modified mRNA [26]. An RNA-binding protein immunoprecipitation (RIP) assay was performed to detect whether YTHDF1 or YTHDF2 had a direct effect on TNF-α mRNA. After T. gondii infection, decreased binding to TNF-α mRNA was observed for YTHDF1 (about 2-fold, P < 0.05, Fig 7A). Notably, the YTHDF2 RIP assay showed that after T. gondii infection, the binding of YTHDF2 to TNF-α mRNA significantly down-regulated (about 11-fold, P < 0.001), indicating T. gondii infection remarkably weakened the binding of YTHDF2 to TNF-α mRNA (Fig 7B). This could be attributed to the reduced m6A modifications on TNF-α mRNA after T. gondii infection. These findings suggest that T. gondii infection may mount TNF-α expression by inhibiting the degradation of TNF-α mRNA.

Fig 7. YTHDF1 RIP and YTHDF2 RIP.

A: qRT-PCR detection of the changes of TNF-α enriched by YTHDF1 and IgG. B: qRT-PCR detection of the changes of TNF-α enriched by YTHDF2 and IgG. Results were shown as fold changes compared with the control group. Data were expressed as the mean ± SD (n = 3). Comparisons between two groups were made by Student’s t-test. *, P < 0.05; ***, P < 0.001.

https://doi.org/10.1371/journal.pntd.0013289.g007

Discussion

This study focuses on the post-transcriptional regulation mediated by m6A modification in human macrophages after T. gondii infection, especially the role of m6A in post-transcriptional regulation of TNF-α and the effect of T. gondii infection on it. In this study, m6A-seq and RNA-seq were conducted to systematically observe the changes of RNA m6A modification after T. gondii infection. Our transcriptome analysis showed that the expression level of TNF-α was significantly up-regulated in T. gondii-treated THP-1 macrophages. Further KEGG pathway analysis indicated that DEGs were enriched in TNF signaling pathway and cytokine-cytokine receptor interaction pathway. Based on the m6A-seq data, the m6A peaks were mainly enriched in the 3’UTR and the 5’UTR regions. In the infected group, the m6A methylation in the 3’UTR region was increased, concomitant with decreased methylation in the 5’UTR region, suggesting that the mRNA splicing, stability and translation were affected. In addition, KEGG pathway enrichment analysis of DEGs with significantly altered m6A modification showed that DEGs in toxoplasmosis pathway, TNF signaling pathway, NF-κB signaling pathway were highly enriched. All these findings suggest that there is a close relationship between m6A modification alterations and T. gondii-induced immune responses, especially the pro-inflammatory cytokine TNF-α.

TNF-α biosynthesis is regulated by a variety of complex mechanisms, which mainly occur in gene transcription, mRNA transport, stability and translation, intracellular signal transduction and other stages [27–30]. At the transcriptional level, TNF-α expression is co-regulated by transcription factors, co-regulators, and chromatin modifications [27]. Our research group found that MIC3, which is a T. gondii excretory-secretory antigen, could induce the immune response of mouse macrophages through TLR11/MyD88/NF-κB signaling pathway, and initiate the TNF-α production against T. gondii. Post-transcriptional regulation is also essential in TNF-α synthesis. Different microRNAs (miRNAs) and AU-rich elements (AREs) mediate direct or indirect modulation of TNF-α, triggering mRNA degradation, translation inhibition, or translation activation of TNF-α mRNA [28–30]. Two recent studies have tried to explore the role of m6A in TNF-α production [31,32]. In human macrophages, the loss of m6A writer components eliminates m6A modification of TNF transcripts, thereby enhancing mRNA stability and TNF-α production [31]. Conversely, Tnf-α mRNA in murine macrophages was not the direct target of the m6A modification [32]. Unlike the commonly studied lipopolysaccharide (LPS)-induced inflammatory response, our study utilized T. gondii to infect THP-1 macrophages, and focused on the role of the m6A demethylase FTO. We found that after T. gondii infection, the upregulated expression of FTO attenuated the m6A modification of TNF transcripts, thereby enhancing the TNF mRNA stability and subsequently increasing TNF-α production. Specifically, we found that there were 9 potential m6A modification sites on human TNF-α mRNA. After T. gondii infection, the m6A modification levels at sites in the 5’UTR and 3’UTR regions of TNF-α mRNA decreased. Meanwhile, T. gondii infection significantly up-regulated the expression of the demethylase FTO in human macrophages. In FTO-knockdown cell lines, the m6A modification levels at sites in the 5’UTR region, especially in the 3’UTR region of TNF-α mRNA, did not significantly decrease after T. gondii infection. This suggests that the T. gondii infection-induced decrease in the m6A modification levels on TNF-α mRNA is associated with FTO. After T. gondii infection, the binding of TNF-α mRNA to the YTH domain family proteins decreased, suggesting that the degradation of TNF-α mRNA was reduced, which would result in increased TNF-α production. Our study elucidated, for the first time, the molecular mechanism of TNF-α expression in human macrophages induced by T. gondii infection from an m6A perspective.

In host-pathogen interactions, m6A modification exhibits a rapid onset of action and plays a crucial role in regulating downstream pathways [33,34]. On one hand, m6A modification affects pathogen gene expression by regulating the stability and translational efficiency of pathogen-derived mRNAs, thereby helping pathogens to escape host immune recognition [25,35,36]. On the other hand, m6A modification influences the expression of immune-related genes, and regulates the activation and differentiation of immune cells, thereby shaping host immune responses [22–24,32,37,38]. For example, after HIV-1 infection, the global m6A modification level of T cell transcriptome was significantly increased, and the immune response was subsequently changed [22]. In the case of T. gondii, while two recent studies reported the m6A methylation profiles of T. gondii tachyzoites and bradyzoites [19,39], our study, to the best of our knowledge, is the first to present the m6A landscape across the transcriptome of human macrophages after T. gondii infection.

Currently, research on m6A modification in the field of parasitology is limited to characterizing the m6A modification sites and modification levels of the parasites’ own RNA, identifying homologous m6A regulatory proteins, and focusing on the regulatory role of m6A modification in the parasite life cycle. For example, there are abundant m6A modifications on RNA of the bloodstream form and procyclic form of Trypanosoma brucei [40]. Subsequent studies reported that the m6A localized in the poly(A)-tailed mRNA of the variant surface glycoprotein (VSG) of Trypanosoma brucei inhibits RNA degradation by blocking deadenylation, thus ensuring the stability of VSG transcript [41,42]. PfMT-A70, pfYTH1 and pfYTH2 are m6A writer or reader proteins encoded by Plasmodium falciparum [43]. m6A marks are enriched near the 3’-boundary of T. gondii transcripts [39]. METTL3 and WTAP are essential in T. gondii viability and depletion of these two writer components prevents parasite replication [39]. Regarding m6A methylation differences among different T. gondii genotypes, pairwise comparisons of the RH strain (Type I), ME49 strain (Type II), and VEG strain (Type III) identified 735, 192, and 615 differentially methylated peaks (DMPs), as well as 172, 41, and 153 differentially methylated genes (DMGs) [19]. Enrichment analyses associated these DMPs and DMGs with cellular components (e.g., Golgi apparatus, plasma membrane) and signaling pathways (e.g., endocytosis, mTOR signaling), suggesting a role for m6A in the pathobiology of T. gondii.

It is well-known that T. gondii infection universally induces Th1 responses during the acute stage of the disease [44,45]. However, different strains of T. gondii induce varying types and levels of cytokines and signaling molecules, ultimately leading to distinct infection outcomes [44–46]. For example, after RH strain (type I) infection, macrophages exhibit only low levels of iNOS expression, resulting in negligible NO production; in contrast, following ME49 strain (type II) infection, macrophages express higher levels of iNOS and produce more NO [44]. Moreover, infection with different strains of T. gondii can induce macrophages to polarize into different subtypes [47]. It has been reported that Toxoplasma polymorphic effectors, such as ROP16 and GRA15, are involved in the regulation of these processes [47,48]. In this study, we investigated the regulatory role of m6A in the immune response of macrophages after infection with T. gondii RH strain. As different T. gondii strains trigger distinct immune responses in macrophages, besides Toxoplasma polymorphic effectors, whether m6A plays a role in the distinct host immune responses induced by different T. gondii strains is interesting and deserves further investigation.

In this study, we used T. gondii tachyzoites and human macrophages to investigate the role of m6A in innate immunity against infection. This model reflects the immune response of human macrophages in the acute stage of infection. From an m6A perspective, our findings reveal the impact of T. gondii, and potentially Apicomplexan protozoa, on public health. Notably, our findings are constrained to acute toxoplasmosis. In immunocompetent individuals, T. gondii infection is usually asymptomatic, existing in the form of bradyzoites. Therefore, the role of m6A in patients in the chronic stage or those with latent infection needs to be further investigated. Additionally, T. gondii infection in healthy populations has been linked to an increased risk of certain diseases, such as neuropsychiatric disorders, metabolic diseases, and autoimmune disorders [49–51]. An increasing number of studies have shown associations between m6A and these diseases [52–54]. However, whether a causal relationship exists among T. gondii infection, m6A alteration, and these diseases awaits further study.

In sum, this study provides new insights into the role of m6A modification in macrophage immune responses against T. gondii. Additionally, our findings uncover a new molecular mechanism for the immediate gene activation of TNF-α, through which m6A modification regulates TNF-α expression.

Supporting information

S2 Table. Prediction of m6A modification sites on human TNF-α mRNA.

https://doi.org/10.1371/journal.pntd.0013289.s002

(DOCX)

S1 Fig. The efficacy of FTO knock-down.

A: qRT-PCR analysis of FTO knock-down efficacy. The mRNA abundance of FTO was measured by qRT-PCR. Data are shown as mean ± SD (n = 3). Statistical significance was determined using Student’s t-test. ***, P < 0.001. B: Western blotting analysis of FTO knock-down efficacy.

https://doi.org/10.1371/journal.pntd.0013289.s003

(TIF)

S1 Text. Original images for Western blotting.

https://doi.org/10.1371/journal.pntd.0013289.s004

(DOCX)

S1 Data. Original values used to build graphs.

https://doi.org/10.1371/journal.pntd.0013289.s005

(XLSX)

Acknowledgments

We thank Prof. Peng Huang from the Department of Epidemiology at Nanjing Medical University for helpful discussion about statistical methods of this manuscript.

References

  1. 1. Lewis JM, Clifford S, Nsutebu E. Toxoplasmosis in immunosuppressed patients. Rheumatology (Oxford). 2015;54(11):1939–40. pmid:25969518
  2. 2. Teimouri A, Mohtasebi S, Kazemirad E, Keshavarz H. Role of Toxoplasma gondii IgG Avidity Testing in Discriminating between Acute and Chronic Toxoplasmosis in Pregnancy. J Clin Microbiol. 2020;58(9):e00505-20. pmid:32321784
  3. 3. Wang Q, Cao Y, Ye S, Ding M, Ge W, Liang Y, et al. Trem2/Syk/PI3K axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via modulating decidual macrophages. PLoS Pathog. 2024;20(9):e1012543. pmid:39250507
  4. 4. Tian T, Wang M, Ma D. TNF-α, a good or bad factor in hematological diseases? Stem Cell Investig. 2014;1:12. pmid:27358858
  5. 5. El-Sayed NM, Ismail KA, Badawy AF, Elhasanein KF. In vivo effect of anti-TNF agent (etanercept) in reactivation of latent toxoplasmosis. J Parasit Dis. 2016;40(4):1459–65. pmid:27876967
  6. 6. Ricard J, Pelloux H, Pathak S, Pipy B, Ambroise-Thomas P. TNF-alpha enhances Toxoplasma gondii cyst formation in human fibroblasts through the sphingomyelinase pathway. Cell Signal. 1996;8(6):439–42. pmid:8958446
  7. 7. Zhang Y, Chen H, Chen Y, Wang L, Cai Y, Li M, et al. Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis. Parasit Vectors. 2014;7:372. pmid:25128410
  8. 8. Qiu J, Xie Y, Shao C, Shao T, Qin M, Zhang R, et al. Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway. PLoS Negl Trop Dis. 2023;17(2):e0011105. pmid:36730424
  9. 9. Subauste CS, Wessendarp M. Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma. J Immunol. 2000;165(3):1498–505. pmid:10903756
  10. 10. Derouich-Guergour D, Aldebert D, Vigan I, Jouvin-Marche E, Marche PN, Aubert D, et al. Toxoplasma gondii infection can regulate the expression of tumour necrosis factor-alpha receptors on human cells in vitro. Parasite Immunol. 2002;24(5):271–9. pmid:12060321
  11. 11. Tosh KW, Mittereder L, Bonne-Annee S, Hieny S, Nutman TB, Singer SM, et al. The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion. J Immunol. 2016;196(1):345–56. pmid:26597011
  12. 12. He L, Li H, Wu A, Peng Y, Shu G, Yin G. Functions of N6-methyladenosine and its role in cancer. Mol Cancer. 2019;18(1):176. pmid:31801551
  13. 13. Yang Y, Hsu PJ, Chen Y-S, Yang Y-G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res. 2018;28(6):616–24. pmid:29789545
  14. 14. Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7(12):885–7. pmid:22002720
  15. 15. Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang C-M, Li CJ, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013;49(1):18–29. pmid:23177736
  16. 16. Patil DP, Pickering BF, Jaffrey SR. Reading m6A in the Transcriptome: m6A-Binding Proteins. Trends Cell Biol. 2018;28(2):113–27. pmid:29103884
  17. 17. Shi H, Zhang X, Weng Y-L, Lu Z, Liu Y, Lu Z, et al. m6A facilitates hippocampus-dependent learning and memory through YTHDF1. Nature. 2018;563(7730):249–53. pmid:30401835
  18. 18. Du H, Zhao Y, He J, Zhang Y, Xi H, Liu M, et al. YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex. Nat Commun. 2016;7:12626. pmid:27558897
  19. 19. Wei C, Cao H, Li C, Song H, Liu Q, Zhu X, et al. Differences in N6-methyladenosine (m6A) methylation among the three major clonal lineages of Toxoplasma gondii tachyzoites. Journal of Integrative Agriculture. 2024.
  20. 20. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884–90. pmid:30423086
  21. 21. Sana M, Rashid M, Rashid I, Akbar H, Gomez-Marin JE, Dimier-Poisson I. Immune response against toxoplasmosis-some recent updates RH: Toxoplasma gondii immune response. Int J Immunopathol Pharmacol. 2022;36:3946320221078436. pmid:35227108
  22. 22. Lichinchi G, Gao S, Saletore Y, Gonzalez GM, Bansal V, Wang Y, et al. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells. Nat Microbiol. 2016;1:16011. pmid:27572442
  23. 23. Zheng Q, Hou J, Zhou Y, Li Z, Cao X. The RNA helicase DDX46 inhibits innate immunity by entrapping m6A-demethylated antiviral transcripts in the nucleus. Nat Immunol. 2017;18(10):1094–103. pmid:28846086
  24. 24. Lichinchi G, Zhao BS, Wu Y, Lu Z, Qin Y, He C, et al. Dynamics of Human and Viral RNA Methylation during Zika Virus Infection. Cell Host Microbe. 2016;20(5):666–73. pmid:27773536
  25. 25. Tan B, Liu H, Zhang S, da Silva SR, Zhang L, Meng J, et al. Viral and cellular N6-methyladenosine and N6,2’-O-dimethyladenosine epitranscriptomes in the KSHV life cycle. Nat Microbiol. 2018;3(1):108–20. pmid:29109479
  26. 26. Zaccara S, Jaffrey SR. A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA. Cell. 2020;181(7):1582-1595.e18. pmid:32492408
  27. 27. Shebzukhov IV, Kuprash DV. Transcriptional regulation of TNF/LT locus in immune cells. Mol Biol (Mosk). 2011;45(1):56–67. pmid:21485497
  28. 28. Huang H-C, Yu H-R, Huang L-T, Huang H-C, Chen R-F, Lin I-C, et al. miRNA-125b regulates TNF-α production in CD14+ neonatal monocytes via post-transcriptional regulation. J Leukoc Biol. 2012;92(1):171–82. pmid:22581933
  29. 29. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science. 2007;318(5858):1931–4. pmid:18048652
  30. 30. Salvi V, Gianello V, Tiberio L, Sozzani S, Bosisio D. Cytokine Targeting by miRNAs in Autoimmune Diseases. Front Immunol. 2019;10:15. pmid:30761124
  31. 31. Haag SM, Xie S, Eidenschenk C, Fortin J-P, Callow M, Costa M, et al. Systematic perturbation screens identify regulators of inflammatory macrophage states and a role for TNF mRNA m6A modification. Nat Genet. 2024;56(11):2493–505. pmid:39443811
  32. 32. Tong J, Wang X, Liu Y, Ren X, Wang A, Chen Z, et al. Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation. Sci Adv. 2021;7(18):eabd4742. pmid:33910903
  33. 33. Zhou J, Wan J, Shu XE, Mao Y, Liu X-M, Yuan X, et al. N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response. Mol Cell. 2018;69(4):636-647.e7. pmid:29429926
  34. 34. Leseva MN, Buttari B, Saso L, Dimitrova PA. Infection Meets Inflammation: N6-Methyladenosine, an Internal Messenger RNA Modification as a Tool for Pharmacological Regulation of Host-Pathogen Interactions. Biomolecules. 2023;13(7):1060. pmid:37509095
  35. 35. Li N, Hui H, Bray B, Gonzalez GM, Zeller M, Anderson KG, et al. METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection. Cell Rep. 2021;35(6):109091. pmid:33961823
  36. 36. Lu M, Zhang Z, Xue M, Zhao BS, Harder O, Li A, et al. N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I. Nat Microbiol. 2020;5(4):584–98. pmid:32015498
  37. 37. Liu Y, Liu Z, Tang H, Shen Y, Gong Z, Xie N, et al. The N6-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of STAT1 mRNA. Am J Physiol Cell Physiol. 2019;317(4):C762–75. pmid:31365297
  38. 38. Chen J, Song H-X, Hu J-H, Bai J-S, Li X-H, Sun R-C, et al. Classical swine fever virus non-structural protein 5B hijacks host METTL14-mediated m6A modification to counteract host antiviral immune response. PLoS Pathog. 2024;20(3):e1012130. pmid:38551978
  39. 39. Holmes MJ, Padgett LR, Bastos MS, Sullivan WJ Jr. m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii. PLoS Pathog. 2021;17(7):e1009335. pmid:34324585
  40. 40. Liu L, Zeng S, Jiang H, Zhang Y, Guo X, Wang Y. Differential m6A methylomes between two major life stages allows potential regulations in Trypanosoma brucei. Biochem Biophys Res Commun. 2019;508(4):1286–90. pmid:30573362
  41. 41. Maran SR, de Lemos Padilha Pitta JL, Dos Santos Vasconcelos CR, McDermott SM, Rezende AM, Silvio Moretti N. Epitranscriptome machinery in Trypanosomatids: New players on the table?. Mol Microbiol. 2021;115(5):942–58. pmid:33513291
  42. 42. Viegas IJ, de Macedo JP, Serra L, De Niz M, Temporão A, Silva Pereira S, et al. N6-methyladenosine in poly(A) tails stabilize VSG transcripts. Nature. 2022;604(7905):362–70. pmid:35355019
  43. 43. Baumgarten S, Bryant JM, Sinha A, Reyser T, Preiser PR, Dedon PC, et al. Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development. Nat Microbiol. 2019;4(12):2246–59. pmid:31384004
  44. 44. Gavrilescu LC, Denkers EY. IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection. J Immunol. 2001;167(2):902–9. pmid:11441097
  45. 45. Mordue DG, Monroy F, La Regina M, Dinarello CA, Sibley LD. Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines. J Immunol. 2001;167(8):4574–84. pmid:11591786
  46. 46. Boothroyd JC, Grigg ME. Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease?. Curr Opin Microbiol. 2002;5(4):438–42. pmid:12160866
  47. 47. Jensen KDC, Wang Y, Wojno EDT, Shastri AJ, Hu K, Cornel L, et al. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation. Cell Host Microbe. 2011;9(6):472–83. pmid:21669396
  48. 48. Zhu W, Li J, Pappoe F, Shen J, Yu L. Strategies Developed by Toxoplasma gondii to Survive in the Host. Front Microbiol. 2019;10:899. pmid:31080445
  49. 49. Yang L, Wang B, Yang Z, Zheng S, Xin Z, Wu S, et al. Toxoplasma gondii infection positively associated with schizophrenia: Evidences from UK Biobank cohort and case-controlled studies. J Psychiatr Res. 2024;175:243–50. pmid:38749298
  50. 50. Tian A-L, Gu Y-L, Zhou N, Cong W, Li G-X, Elsheikha HM, et al. Seroprevalence of Toxoplasma gondii infection in arthritis patients in eastern China. Infect Dis Poverty. 2017;6(1):153. pmid:29065914
  51. 51. Reeves GM, Mazaheri S, Snitker S, Langenberg P, Giegling I, Hartmann AM, et al. A Positive Association between T. gondii Seropositivity and Obesity. Front Public Health. 2013;1:73. pmid:24400300
  52. 52. Liufu C, Luo L, Pang T, Zheng H, Yang L, Lu L, et al. Integration of multi-omics summary data reveals the role of N6-methyladenosine in neuropsychiatric disorders. Mol Psychiatry. 2024;29(10):3141–50. pmid:38684796
  53. 53. Huang Y, Xue Q, Chang J, Wang Y, Cheng C, Xu S, et al. M6A methylation modification in autoimmune diseases, a promising treatment strategy based on epigenetics. Arthritis Res Ther. 2023;25(1):189. pmid:37784134
  54. 54. Hu H, Li Z, Xie X, Liao Q, Hu Y, Gong C, et al. Insights into the role of RNA m6A modification in the metabolic process and related diseases. Genes Dis. 2023;11(4):101011. pmid:38560499

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