Research ArticleIMMUNOLOGY

Epigenetic initiation of the TH17 differentiation program is promoted by Cxxc finger protein 1

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Science Advances  09 Oct 2019:
Vol. 5, no. 10, eaax1608
DOI: 10.1126/sciadv.aax1608
  • Fig. 1 TH17 cell differentiation was severely impaired in Cxxc1-deficient mice.

    (A and B) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from dLckcreCxxc1fl/fl or WT mice were differentiated into TH17 cells with (A) IL-6 and TGF-β1 or (B) IL-1β, IL-6, and IL-23 for 96 hours and then restimulated for intracellular cytokine staining. One of five to seven experiments is shown. (C and D) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from ERT2creCxxc1fl/fl mice were differentiated into TH17 cells with (C) IL-6 and TGF-β1 or (D) IL-1β, IL-6, and IL-23 for 96 hours in the presence or absence of 4-OHT (4-Hydroxytamoxifen) and then restimulated for intracellular cytokine staining. One of five experiments is shown. (E and F) Naive CD4+ T cells from RORγtcreCxxc1fl/fl or WT mice were differentiated into TH17 cells with (E) IL-6 and TGF-β1 or (F) IL-1β, IL-6, and IL-23 for 96 hours and then restimulated for intracellular cytokine staining. One of seven experiments is shown. (G) Intracellular staining of IL-17A in lipoprotein lipase (LPL) CD4+ T cells in the small intestines of RORγtcreCxxc1fl/fl and WT mice. One of four experiments is shown. Error bars show the means ± SD. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 using the Student’s t test.

  • Fig. 2 Cxxc1 deficiency restricts T cell–mediated autoimmunity and increases sensitivity to C. rodentium infection.

    (A) Mean clinical scores for EAE in Rag1−/− recipients of RORγtcreCxxc1fl/fl (n = 13) or WT (n = 11) naive CD4+ T cells after being immunized with MOG35–55, complete Freund’s adjuvant (CFA), and pertussis toxin. Data are summed from three independent experiments. (B) Representative histology of the spinal cord of Rag1−/− mice after EAE induction (day 25). Hematoxylin and eosin (H&E) staining (left), Luxol fast blue (F&B) staining (right). (C) On day 20 after the induction of EAE in Rag1−/− hosts, CD4+ T cells were analyzed from leukocytes isolated from the CNS, draining lymph nodes (dLNs), and spleen and further analyzed for the frequency of IL-17A+ and IFN-γ+ T cells (left). Summary of CNS IL-17A+CD4+ and IFN-γ+CD4+ T cells in Rag1−/− hosts (right). One representative of three experiments is depicted. (D) The frequency of Foxp3+ cells from CNS-infiltrating lymphocytes in Rag1−/− EAE mice was determined at day 20 after immunization (top). Summary of CNS Foxp3+CD4+ cells in Rag1−/− hosts (bottom). One representative of three experiments is depicted. (E) Splenocytes were rechallenged with the MOG peptide (0, 5, and 25 μg/ml) for 3 days, and then, cytokine production was measured by enzyme-linked immunosorbent assay (ELISA). (F) Body weight changes of Rag1−/− recipients of naive CD4+ T cells from RORγtcreCxxc1fl/fl (n = 12) or WT (n = 11) mice after oral inoculation with C. rodentium at the indicated time points. Data are summed from three independent experiments. (G) Colon length for Rag1−/− recipients of naive CD4+ T cells from RORγtcreCxxc1fl/fl or WT mice after oral inoculation with C. rodentium at day 7. Summary of colon lengths in Rag1−/− hosts (right). (H) Histological analysis of representative colons from Rag1−/− hosts 7 days after inoculation. (Photo credit: Feng Lin, Institute of Immunology, Zhejiang University School of Medicine). (I) C. rodentium colony-forming units (CFUs) in the colon 7 days after inoculation. Data are summed from three independent experiments. (J) FACS analysis of IL-22 expression from isolated LPLs in Rag1−/− hosts at day 7 after inoculation. One of five experiments is shown. Error bars show the means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 using the Student’s t test.

  • Fig. 3 Cxxc1-deficient TH17 cells exhibit a Treg cell–like expression profile.

    (A) Naive CD4+ T cells (CD4+CD25CD62LhiCD44lo) from WT and RORγtcreCxxc1fl/fl mice were differentiated in the presence of TGF-β1 and IL-6 (TH17) for 72 hours, and the total RNA from the cells was analyzed by RNA-seq [STAR (structured transparent accessible reporting) method]. Scatter diagram showing changes in gene expression in WT and Cxxc1-deficient TH17 cells. Down-regulated genes are indicated in blue; up-regulated genes are indicated in red. (B) Heatmap of the fold change (log2) for differentially expressed genes (false discovery rate < 0.05 in TH17 cells is shown). (C) Pathway analysis of the down-regulated genes (left) and up-regulated genes (right). (D) The expression of the selected transcripts was quantified in TH17 cell samples differentiated from naive CD4+ T cells with TGF-β1 and IL-6 for 72 hours by real-time qPCR. One of five experiments is shown. Error bars show the means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 using the Student’s t test.

  • Fig. 4 Genome-wide maps of Cxxc1 binding and H3K4me3 modifications in TH17 cells differentiated from naive CD4+ T cells with TGF-β1 and IL-6 for 24 hours.

    (A) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from WT and RORγtcreCxxc1fl/fl mice were differentiated in the presence of TGF-β1 and IL-6 (TH17) for 24 hours, and ChIP-seq analysis was conducted to map genome-wide Cxxc1-binding sites in WT TH17 cells. Distribution of the genetic features across the whole mouse genome (mm10) (left) and the distribution of Cxxc1-binding peaks in TH17 cells (right). (B) Distribution of Cxxc1-binding peaks across extended gene bodies in TH17 cells. The tag density of Cxxc1 binding to gene bodies [between the transcription start site (TSS) and the transcription termination site (TTS)], as well as 3-kb upstream of the TSS and 3-kb downstream of the TTS regions of all RefSeq (mm10) genes, was calculated. (C) Enrichment of Cxxc1-binding peaks on CGIs. The tag density of Cxxc1 binding to CGIs and 5-kb flanking regions was calculated. (D) Overlapped regions between Cxxc1-binding sites, H3K4me3 sites, and RNA-seq down-regulated genes in WT and Cxxc1-deficient TH17 cells. (E) Integrative Genomics Viewer browser view of Cxxc1-binding peaks (red) in WT TH17 cells and H3K4me3 markers (blue) in WT and Cxxc1-deficient TH17 cells. (F) Naive WT CD4+ T cells were sorted and cultured under TH17 differentiation conditions (TGF-β and IL-6) for 24 hours, and ChIP-qPCR analysis of Cxxc1 binding at the indicated gene loci was performed. (G) Naive CD4+ T cells from WT and RORγtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-β1 and IL-6 for 24 hours, and H3K4me3 modifications at the indicated gene loci were detected by ChIP-qPCR. The statistical significance was determined by Student’s t test. Error bars show the means ± SD. *P ≤ 0.05, **P ≤ 0.01.

  • Fig. 5 The IL-6/STAT3 signaling pathway was defective after Cxxc1 deletion.

    (A and B) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from RORγtcreCxxc1fl/fl and WT mice were differentiated into TH17 cells with IL-6 and TGF-β1 (A) or IL-1β, IL-6, and IL-23 (B). The expression of IL-6Rα was measured by flow cytometry (left), and the mean fluorescence intensity (MFI) of IL-6Rα at different time points was measured (right). One of six experiments is shown. (C) Naive CD4+ T cells from RORγtcreCxxc1fl/fl and WT mice were differentiated into TH17 cells with IL-6 and TGF-β1, and the supernatants from cell cultures were collected at indicated time points. The amounts of IL-6Rα were then measured by ELISA. One of four experiments is shown. (D and E) Purified naive CD4+ T cells were stimulated for the indicated times with IL-6 (10 ng/ml). Phosphorylated and total STAT3 proteins were detected by Western blot assays (D) or flow cytometry (E). One of five experiments is shown. (F) Naive CD4+ T cells from WT and RORγtcreCxxc1fl/fl mice were polarized into TH17 cells in the presence of TGF-β and IL-6, and varying concentrations of IL-6Rα antibody were added. The expression levels of IL-17A and IL-17F were then analyzed by intracellular staining after 72 hours. One of six experiments is shown. (G and H) Naive CD4+ T cells from WT and RORγtcreCxxc1fl/fl mice were cultured in the presence of TGF-β1 and varying concentrations of IL-6 for 72 hours, and then, the expression levels of IL-17A, IL-17F, and Foxp3 were analyzed by intracellular staining after restimulation. One of seven experiments is shown. The statistical significance was determined by Student’s t test. Error bars show the means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 6 Cxxc1 mediates TH17 cell differentiation by mediating IL-6Rα expression.

    (A) Naive CD4+ T cells from WT and RORγtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-β1 and IL-6, and 20 to 24 hours, later the cells were transfected with the indicated retrovirus (Mock, Cxxc1, IL-6Rα, and IL-6ST). IL-17A and IL-17F levels were then measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours. One of six experiments is shown. (B) Sorted naive CD4+ T cells were differentiated into TH17 cells in the presence of TGF-β1 and IL-6, and 20 to 24 hours later, the cells were transfected with the indicated retrovirus. IL-6Rα levels were then measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours. One of five experiments is shown. (C) Naive CD4+ T cells from WT and RORγtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-β1 and IL-6, and 20 to 24 hours later, the cells were transfected with the indicated retrovirus [Mock, STAT3 (WT), STAT3 (A662C, N664C), and STAT3 (Y705A)]. IL-17A and IL-17F levels were then measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours. One of five experiments is shown. (D) Naive CD4+ T cells from WT and RORγtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-β1 and IL-6, and 20 to 24 hours later, the cells were transfected with the indicated retrovirus. CD4+GFP+ cells were then sorted after retrovirus infection for 72 hours and transferred into RAG1−/− hosts. Two days later, the recipient mice were immunized with MOG35–55 and FCA plus pertussis toxin to induce EAE. Clinical scores were recorded and calculated each day for the indicated times. Data are summed from three independent experiments. (E) IL-17A and IFN-γ production by CD4+ T cells isolated from CNS, draining lymph nodes, and spleens of Rag1−/− mice at the peak of disease. One representative of three experiments is depicted. Error bars show the means ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 using the Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax1608/DC1

    Supplementary Materials and Methods

    Fig. S1. Phenotype of Cxxc1 conditional KO mice.

    Fig. S2. Analysis of T cell differentiation in vitro from WT and Cxxc1-deficient mice.

    Fig. S3. Phenotype of RORγtcreCxxc1wt/wt and RORγtcreCxxc1fl/fl mice.

    Fig. S4. Cxxc1 deficiency restricts T cell–mediated autoimmunity.

    Fig. S5. Cxxc1 deficiency increased sensitivity to C. rodentium infection.

    Fig. S6. Cxxc1 regulates TH17 differentiation with its H3K4me3 function.

    Fig. S7. Genome-wide maps of Cxxc1 binding and H3K4me3 modifications in TH17 cells differentiated from naive CD4+ T cells with TGF-β1 and IL-6 for 24 hours.

    Fig. S8. The IL-6–Stat3 signaling pathway was defective after Cxxc1 deletion.

    Fig. S9. Cxxc1 mediates TH17 cell differentiation by mediating IL-6Rα expression.

    Table S1. Down regulated genes in Cfp1 deficient Th17 cells.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Phenotype of Cxxc1 conditional KO mice.
    • Fig. S2. Analysis of T cell differentiation in vitro from WT and Cxxc1-deficient mice.
    • Fig. S3. Phenotype of RORγtcreCxxc1wt/wt and RORγtcreCxxc1fl/fl mice.
    • Fig. S4. Cxxc1 deficiency restricts T cell–mediated autoimmunity.
    • Fig. S5. Cxxc1 deficiency increased sensitivity to C. rodentium infection.
    • Fig. S6. Cxxc1 regulates TH17 differentiation with its H3K4me3 function.
    • Fig. S7. Genome-wide maps of Cxxc1 binding and H3K4me3 modifications in TH17 cells differentiated from naive CD4+ T cells with TGF-β1 and IL-6 for 24 hours.
    • Fig. S8. The IL-6–Stat3 signaling pathway was defective after Cxxc1 deletion.
    • Fig. S9. Cxxc1 mediates TH17 cell differentiation by mediating IL-6Rα expression.
    • Table S1. Down regulated genes in Cfp1 deficient Th17 cells.

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