Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis

T helper 17 (Th17) cells are a subset of CD4+ T helper cells involved in the inflammatory response in autoimmunity. Th17 cells secrete Th17 specific cytokines, such as IL-17A and IL17-F, which are governed by the master transcription factor RoRγt. However, the epigenetic mechanism regulating Th17 cell function is still not fully understood. Here, we reveal that deletion of RNA 5-methylcytosine (m5C) methyltransferase Nsun2 in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates Th17 cell-induced colitis pathogenesis. Mechanistically, RoRγt can recruit Nsun2 to chromatin regions of their targets, including Il17a and Il17f, leading to the transcription-coupled m5C formation and consequently enhanced mRNA stability. Our study demonstrates a m5C mediated cell intrinsic function in Th17 cells and suggests Nsun2 as a potential therapeutic target for autoimmune disease.

| The expression of Nsun2 immune organs and validation of Nsun2 knockout mice. a, The mRNA level of Nsun2 in mouse organs were detected by RT-qPCR (n = 3 per group). b, Strategies for construction of Nsun2 knockout mice. c, d, The knockout efficiency of Nsun2 was detected. Both RNA and protein were extracted from the spleen, lymph node, and thymus of wild-type and Nsun2 -/mice respectively, and then carried out RT-qPCR (n = 3 per group) (c) and western blot (d) to detect Nsun2 expression. Data are representative of two independent experiments. P values are calculated by using two-tailed unpaired Student's t-test. Error bars represent mean ± s.e.m (a, c). Source data are provided as a Source Data file.

Supplementary Fig. 4 | Nsun2 is critical for Th17 cells differentiation in vitro.
a. Gating strategy related to Fig. 1e-f and Supplementary Fig. 4b-c. b, c, The in vitro differentiation frequencies of Th1, Th2, Treg, Th17 and CD8 + T cells in wild-type and Nsun2 -/mice were analyzed by FACS. Summary of the frequency of each cells are shown in c (n = 3 per group). d, e, The mRNA level of Il17a and Il17f in littermate wild-type and Nsun2 -/-T subsets were detected by RT-qPCR (n = 3 per group). All data are at least two independent experiments. P values are calculated by using two-tailed unpaired Student's t-test. Error bars represent mean ± s.e.m (c-e). Source data are provided as a Source Data file. Fig. 5 | Strategies for construction of Nsun2 cKO mice. a, Schema for gene targeting to generate floxed Nsun2 mice, the KO first mice (tm1a) were mated with FLPeR mice to delete the Neo cassette to generate Nsun2-floxed mice (tm1c), Nsun2 conditional KO mice should be generated by crossing with cre mice (tm1d). b, Genotyping for generating Nsun2 floxed mice. The heterozygous floxed mice (#64) was typed by two pairs of primers: loxp1(f1: 391 bp, WT: 251 bp); loxp2(f2: 182 bp, WT: 128 bp) versus positive control (PC) and negative control (NC). c, Strategy for construction of Nsun2 f/f ; Cd4-Cre +/mice. d, Determine conditional knockout of Nsun2 in T cells sorting from lymph nodes in wild-type or Nsun2 cKO mice by western blot, and the Nsun2 protein expression in liver, brain and kidney as the control.

Supplementary Fig. 6 | T cell-specific Nsun2 deletion impairs Th17 generation. a, b,
The mRNA expression of Il17a (a) and Il17f (b) in wild-type and Nsun2 cKO Th17 cells were detected by RT-qPCR (n = 6 for wild-type; n = 9 for Nsun2 cKO ). c, d, The secretions of IL-17A (c) and IL-17F (d) was detected by ELISA. The supernatants were collected from wildtype and Nsun2 cKO Th17-inducing media as indicated time points (0-4 days) (n = 3 per group). e, The mRNA expression of Rorc in Th17 cells was detected by RT-qPCR (n = 9 per group). All data are at least two independent experiments. P values are calculated by using two-tailed unpaired Student's t-test. Error bars represent mean ± s.e.m (a-e). Source data are provided as a Source Data file. a, Footprints of nucleosome around TSSs using ATAC-seq of wild-type and Nsun2 cKO Th17 cells. b, Venn plots showing the overlap of ATAC-seq peaks between two replicates of wildtype and Nsun2 cKO Th17 cells, as well as the overlap of ATAC-seq peaks detected in wild-type Th17 cells for this study and GSE113721. c, Scatter plots showing the repeatability of the ATAC-seq enrichment between two replicates of wild-type and Nsun2 cKO Th17 cells. P values are calculated by using two-sided student's t-test. d, Genome-wide distribution of peaks for wild-type and Nsun2 cKO Th17 cells identified by MACS2. e, Box plots showing promoter enrichment of H3K4me3 (left; GSE40918) and gene expression (right; mRNA-seq) in wildtype Th17 cell for genes with different promoter enrichment of ATAC-seq. Only the top 15K genes with highest promoter enrichment of ATAC-seq were used and then were divided into three equal groups in this analysis. P values were determined by two-way Mann-Whitney U test and only value between Q1-1.5*IQR and Q3+1.5*IQR has been shown in boxplot. f, IGV tracks showing the ATAC-seq, H3K4me3, and mRNA-seq signal in Th17 cells. g, ATAC-seq summit-centered heatmap of ATAC-seq signal in wild-type and Nsun2 cKO , as well as RORγt ChIP-seq signal (GSE40918)  (left) and m 5 C peaks (right). c, Box plots displaying the variation of GC ratio containing in all random (in Black), Nsun2 (in Red) and m 5 C peaks (in Green). P values were determined by two-way Mann-Whitney U test and only value between Q1-1.5*IQR and Q3+1.5*IQR has been shown in boxplot. d, The distribution of Nsun2 (in Red) and m 5 C peaks (in Green) along mRNA 5'UTR, CDS and 3'UTR. e, Venn plot showing the overlay between Nsun2 targets and m 5 C genes. f, Venn diagram displaying the overlay between down-regulated genes and Nsun2 targets with m 5 C. g, Pie plot showing the ratio of RORγt targets among the genes which were Nsun2 targets with m 5 C and were also down-regulated upon Nsun2 depletion. h, IGV tracks displaying ATAC-seq, RoRγt ChIP-seq (GSE40918), mRNA-seq, Nsun2 RIP-seq (IP and input) and m 5 C MeRIP-seq (IP and input) enrichment near Il17f. All data are representative of two independent experiments. Supplementary Fig. 11 | Nsun2 controls Th17 associated mRNA stability through marking with m 5 C modification. a, The mRNA level of Il17f in Th17 cells of rescuing Nsun2-WT, Nsun2-C321A, and Vector in were detected by RT-qPCR (n = 3 per group). b, The half-lives of Il17f in wild-type and Nsun2 cKO were detected by RT-qPCR (n = 3 per group). Sorted naïve CD4 + T cells from wildtype and Nsun2 cKO mice respectively were induced under Th17-inducing condition for 4 days, and inhibiting RNA transcription in Th17 cells by treating with Actinomycin D, and harvesting the cells as indicated times (0, 20, and 40 min). The residual RNAs were normalized to 0 min. c, m 5 C MeRIP-qPCR analysis results showing the 424 cytosine site of Il17a marks m 5 C modification (n = 3 per group). d, The RNA abundance of the Il17a-m 5 C (in Red) and Il17a-C (in Black) reporter in Nsun2 cKO Th17 cells were detected by RT-qPCR and normalized to the value at 0 min (n = 3 per group). Gapdh served as an internal RNA control. All data are two independent experiments. P values are calculated by using two-tailed unpaired Student's t-test (a-d). Error bars represent mean ± s.e.m. Source data are provided as a Source Data file. Supplementary Fig. 12 | Nsun2 deficiency ameliorates inflammations. a-e, The phenotype of wild-type and Nsun2 -/mice under DSS-induced. Littermate wild-type and Nsun2 -/mice were treated with 3% DSS in drinking water. The body weight changes (a), representative macrograph of colons (b), summary of colon length (n = 6 per group) (c), hematoxylin-eosin staining indicated the severity of colons damage and inflammatory infiltration (Scale bar, 100 μm) (d), and colitis score (n = 18 images for WT and n = 15 images Nsun2 -/-) (e). f, g, The representative macrograph of colons compared between wild-type and Nsun2 cKO mice before or after DSS treatment, and summary of colon length (n = 6 per group) (g). All data are at least two independent experiments. P values are calculated by two-tailed unpaired Student's t-test (a, c, e and g). Error bars represent mean ± s.e.m. Source data are provided as a Source Data file.

Supplementary Fig. 13 | Nsun2 deficiency ameliorates DSS-induced colitis.
Gating strategies related to Fig. 4d-g. The flow cytometry analysis of T subsets in mesenteric lymph nodes and spleen.

Supplementary Fig. 14 | Nsun2 cKO naïve T cells does not promote disease in CD45RB hi
adoptive transfer colitis mouse model. a, The body weight changes of Rag1 -/host mice transferred with wild-type and Nsun2 cKO naïve CD45RB hi T cell, followed by the treatment (two times per week) with neutralizing antibody of IL-17A alone or IL-17A plus IL-17F. b-h, The phenotype of wild-type (n = 5) and Nsun2 cKO (n = 5) naïve T cell adoptively transferring into Rag1 -/recipient mice. The representative macrograph of mice (b), representative image of spleens (c) and colons (d), colon length (n = 5 per group) (e), colitis score (n = 5 per group) (f). g. The representative FACS profiles of IFNγ + CD4 + T, IL-4 + CD4 + T, IL-17A + CD4 + T and Foxp3 + CD4 + T cells frequency in colonic lamina propria CD4 + T cells from recipient mice transferring CD4 + CD25 − CD45Rb hi naïve T cells of wild-type or Nsun2 cKO mice (Gating strategy related to Fig. 4k). PBS vehicle injection was used as control. n = number of biological replicates. P values are calculated by using two-tailed unpaired Student's t-test (a, e and f). Error bars represent mean ± s.e.m. Source data are provided as a Source Data file. Supplementary Fig. 15 | Blockage of IL-17A or both IL-17A and IL-17F signaling eliminates colitis development in CD45RB hi adoptive transfer colitis. a-c, The phenotype of Rag1 -/host mice transferred with wild-type and Nsun2 cKO naïve CD45RB hi T cell, followed by the treatment (two times per week) with neutralizing antibody of IL-17A alone or IL-17A plus IL-17F. The representative macrograph of colons (a), colon length (b), and hematoxylin-eosin staining indicated the severity of colons damage and inflammatory infiltration (Scale bar, 100 μm) (c). The control group of each experiment shown in Supplementary Fig. 13 d, e, and g. n = number of biological replicates. P values are calculated by using two-tailed unpaired Student's t test (b). Error bars represent mean ± s.e.m. Source data are provided as a Source Data file. Supplementary Fig. 16 | Neutralization of IL-17A and IL-17F alleviates the colitis development in DSS-induced mice. a-f, The wild-type (n = 5) and Nsun2 cKO (n = 5) mice were treated with 3% DSS in drinking water after blockage of IL-17A (250 μg/mouse) or IL-17F (250 μg/mouse) alone, or both IL-17A (250 μg/mouse) and IL-17F (250 μg/mouse) signaling via antibodies. Five mice were used in each group. Body weight changes (a), representative macrograph of spleens (b) and colons (c), the colon length (n = 5 mice per group) (d), hematoxylin-eosin staining indicated the severity of colons damage and inflammatory infiltration (Scale bar, 100 μm) (e), and colitis score (n = 5 mice per group) (f). n = number of biological replicates. P values are calculated by using two-tailed unpaired Student's t-test (a, d and f). Error bars represent mean ± s.e.m. Source data are provided as a Source Data file.  Gold) and Nsun2 cKO +DSS treatments. The aberrantly changed cell types during colitis were marked with black asterisk (left). In colitis, the rescued increased cell types upon Nsun2 deletion were marked with red asterisk, the rescued decreased cell types were marked with green asterisk (right).