m(6)A methyltransferase METTL3 programs CD4(+) T-cell activation and effector T-cell differentiation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder in which excessive CD4+ T-cell activation and imbalanced effector T-cell differentiation play critical roles. Recent studies have implied a potential association between posttranscriptional N6-methyladenosine (m6A) modification and CD4+ T-cell-mediated humoral immunity. However, how this biological process contributes to lupus is not well understood. In this work, we investigated the role of the m6A methyltransferase like 3 (METTL3) in CD4+ T-cell activation, differentiation, and SLE pathogenesis both in vitro and in vivo. The expression of METTL3 was knocked down and METTL3 enzyme activity was inhibited using siRNA and catalytic inhibitor, respectively. In vivo evaluation of METTL3 inhibition on CD4+ T-cell activation, effector T-cell differentiation, and SLE pathogenesis was achieved using a sheep red blood cell (SRBC)-immunized mouse model and a chronic graft versus host disease (cGVHD) mouse model. RNA-seq was performed to identify pathways and gene signatures targeted by METTL3. m6A RNA-immunoprecipitation qPCR was applied to confirm the m6A modification of METTL3 targets. METTL3 was defective in the CD4+ T cells of SLE patients. METTL3 expression varied following CD4+ T-cell activation and effector T-cell differentiation in vitro. Pharmacological inhibition of METTL3 promoted the activation of CD4+ T cells and influenced the differentiation of effector T cells, predominantly Treg cells, in vivo. Moreover, METTL3 inhibition increased antibody production and aggravated the lupus-like phenotype in cGVHD mice. Further investigation revealed that catalytic inhibition of METTL3 reduced Foxp3 expression by enhancing Foxp3 mRNA decay in a m6A-dependent manner, hence suppressing Treg cell differentiation. In summary, our findings demonstrated that METTL3 was required for stabilizing Foxp3 mRNA via m6A modification to maintain the Treg differentiation program. METTL3 inhibition contributed to the pathogenesis of SLE by participating in the activation of CD4+ T cells and imbalance of effector T-cell differentiation, which could serve as a potential target for therapeutic intervention in SLE.

Lu S ，Wei X ，Zhu H ，Hu Z ，Zheng M ，Wu J ，Zhao C ，Yang S ，Feng D ，Jia S ，Zhao H ，Zhao M ... -  《-》

METTL3-Mediated N6-Methyladenosine Methylation Modifies Foxp3 mRNA Levels and Affects the Treg Cells Proportion in Peripheral Blood of Patients with Asthma.

To investigate the regulatory effect and mechanism of methyltransferase-like protein 3 (METTL3)-mediated N6-methyladenosine methylation (m6A) on forkhead box protein 3 (Foxp3) levels and the proportion of regulatory T (Treg) cells in the peripheral blood of patients with asthma. Flow cytometry and ELISA were used to detect the differences in the proportions of Treg cells and serum interleukins (ILs) 4 and 7, respectively, in the peripheral blood between healthy individuals and patients with different asthma conditions. Reverse transcription-quantitative PCR (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels, respectively, of METTL3 and Foxp3 in CD4+ T cells in the peripheral blood samples of different groups. M6A blot and m6A coimmunoprecipitation-PCR were used to detect the global and Foxp3 mRNA m6A levels, respectively, in the peripheral blood CD4+ T cells. CD4+ T cells collected from the peripheral blood of patients with asthma were subjected to in vitro transfection to knockdown the METTL3 levels and observe changes in the Foxp3 mRNA, protein, m6A levels, and RNA stability. Flow cytometry and ELISA were used to detect the changes in the Treg cell proportion and IL-4 and IL-17 levels in the cell culture supernatant. Compared with the healthy individuals, the ratio of Treg cells to peripheral blood CD4+ T cells was significantly decreased and the Foxp3 mRNA and protein expression was downregulated in patients with asthma with disease progression. The Foxp3 mRNA and protein expression levels were positively correlated with the Treg cell proportion and negatively correlated with IL-17 expression. The global and Foxp3 mRNA m6A levels were increased in the peripheral blood CD4+ T cells of patients with asthma. METTL3 expression was significantly higher in the peripheral blood CD4+ T cells of patients with asthma compared with healthy individuals. After METTL3 knockdown, the Foxp3 mRNA m6A level was reduced, and the stability of Foxp3 mRNA and protein expression was increased. YTHDF2 could bind to the m6A site in 3'UTR of Foxp3 mRNA. Knockdown of YTHDF2 regulated the level and stability of Foxp3 mRNA. METTL3 knockdown reduced the ratio of Treg cells to CD4+ T cells and the IL-4 and IL-17 secretion levels from CD4+ T cells in the peripheral blood of patients with asthma. High METTL3 expression in the peripheral blood CD4+ T cells of patients with asthma increased the m6A level and reduced the stability of Foxp3 mRNA in a YTHDF2-dependent way, thereby reducing the expression of Foxp3 and the proportion of Treg cells.

Fan L ，Wu J ，Wang H ，Chen Q ，He X ，Wang Q ，Yang Z ... -  《-》

Foxp3+ Helios+ regulatory T cells are expanded in active systemic lupus erythematosus.

Alexander T ，Sattler A ，Templin L ，Kohler S ，Groß C ，Meisel A ，Sawitzki B ，Burmester GR ，Arnold R ，Radbruch A ，Thiel A ，Hiepe F ... -  《-》

METTL3-dependent m(6)A modification programs T follicular helper cell differentiation.

Yao Y ，Yang Y ，Guo W ，Xu L ，You M ，Zhang YC ，Sun Z ，Cui X ，Yu G ，Qi Z ，Liu J ，Wang F ，Liu J ，Zhao T ，Ye L ，Yang YG ，Yu S ... -  《Nature Communications》

Downregulation of circular RNA ETS1 promotes SLE activity and inhibits Treg cell differentiation through miR-1205/FoxP3 molecular axis.

Zou H ，Ma S ，Li L ，Xia X ，Zhou Y ，Zhang R ... -  《-》