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

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 macrophages against T. gondii, this study utilized T. gondii tachyzoites from the RH strain to infect human THP-1 macrophages. qPCR and ELISA results showed that T. gondii infection mounted the expression of TNF-a. RNA-seq profiling showed that T. gondii infection was associated with difference in genes from pathway associated with TNF signaling. Expression of m6A regulators were evaluated using qPCR and Western blotting. T. gondii infection increased the abundance of m6A methyltransferase WTAP and demethylase FTO. Joint analysis of RNA-seq and m6A-seq data was utilized for enriching differentially expressed genes with significantly altered m6A modifications. After T. gondii infection, the m6A levels of genes associated with TNF signaling were significantly altered. In this study, we found that m6A methylation involved in T. gondii infection induced TNF-a expression. Overall design: In this study, T. gondii RH strain tachyzoites were utilized to infect THP-1 macrophages, forming the infected group, while untreated macrophages served as the control group. Following the extraction of RNA from both groups of cells, a portion was retained as the input sample. The IP assay was then employed to isolate the m6A fragment from both the control and infected groups. These RNA fractions containing m6A fragments were then employed as IP samples for the control and infected groups.