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

## TL;DR

This study shows how the m6A demethylase FTO helps regulate TNF-α expression in human macrophages infected with Toxoplasma gondii, revealing a new epigenetic mechanism.

## Contribution

The study uncovers a novel post-transcriptional regulatory mechanism of TNF-α expression in human macrophages during T. gondii infection via m6A modification.

## Key findings

- T. gondii infection increases FTO levels and decreases m6A modification in TNF-α mRNA.
- Knocking down FTO dampens the immune response and allows uncontrolled parasite proliferation.
- YTHDF2 binding to TNF-α mRNA is reduced, promoting TNF-α expression by inhibiting mRNA degradation.

## 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.

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.