Research ArticleImmunology

SMAC mimetics promote NIK-dependent inhibition of CD4+ TH17 cell differentiation

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Science Signaling  27 Aug 2019:
Vol. 12, Issue 596, eaaw3469
DOI: 10.1126/scisignal.aaw3469
  • Fig. 1 Expression of IL-17 in mouse and human TH17 cells after SM treatment.

    (A) Flow cytometry analysis of the viability of mouse TH17 cells differentiated with DMSO control or SM AT-406 for the indicated times. Data are means ± SD pooled from three experiments. (B) Flow cytometry analysis of IL-17A and IFN-γ production by mouse TH17 cells differentiated with DMSO or SM and treated for 48 hours. Dot plots (left) are representative of and quantified data with medians (right) are pooled from seven biological replicates. (C and D) Quantitative real-time PCR (qRT-PCR) analysis of Il17a and Il17f mRNA expression in mouse TH17 cells differentiated with DMSO or SM for 48 hours. Data with medians are pooled from five biological replicates. (E) Flow cytometry analysis of IL-17A and IFN-γ production by live human CD45RO+RORγt+ TH17 cells differentiated with DMSO or SM for 72 hours. Dot plots (left) are representative of and quantified data with medians (right) are pooled from four biological replicates. *P < 0.05, **P < 0.01 by Mann-Whitney test.

  • Fig. 2 SM induced transcriptional changes in TH17 cells.

    (A to C) Microarray analysis of mouse TH17 cells differentiated with DMSO or SM for 48 hours. Volcano plot of all differentially expressed genes after SM treatment (A) and network analysis of the differentially expressed genes annotated as regulators of downstream TH17 targets (B), and regulatory interaction nodes (C) are from the analysis of five biological repeats. Positive (solid lines) and negative (dotted lines) regulation and differential expression after SM treatment are indicated. (D and E) Comparison of model predicting whether the gene expression changes (B and C) correspond to measured gene expression (D) and ROC curve testing the qualitative performance of the prediction model (E). FC, fold change. In (C) circled symbols indicate inconsistent target change.

  • Fig. 3 SM induced proteomic changes in TH17 cells.

    (A to E) Mass spectrometry analysis of mouse TH17 cells differentiated with DMSO or SM for the indicated times. All twofold change protein changes across comparisons and 100 differentially expressed genes analyzed for functional similarity by tSNE analysis of STRING database annotations (A) are from two biological repeats. Proteins whose abundance changed from control by twofold or more in both replicates of SM-treated cells at 24 hours (B) or 48 hours (C) are colored by average fold change. Volcano plots indicate proteins with differential abundance in both biological replicates 24 hours after SM treatment within the TCR and NF-κB signaling cluster (D) or the cell cycle and mitochondrial activity clusters (E).

  • Fig. 4 SM drives NIK-dependent changes in TH17 cells.

    (A and B) Western blot for NIK and p100/p52 (A) or p52 and RelB subcellular location (B) in lysates of mouse TH17 cells treated with DMSO or SM for the indicated times. Blots (left) are representative of four experiments. Quantified band intensity values (right) are mean ± range pooled from all experiments. AU, arbitrary units. (C) Flow cytometry analysis of IL-17A and IFN-γ production by mouse CD4CRE-NIKF/F or control (Cre) TH17 cells treated with DMSO or SM for 48 hours. Dot plots (left) are representative of and quantified frequency data with medians (right) are pooled from five biological replicates. (D) qRT-PCR analysis of Il17a, Maf, Il10, and Il22 mRNA expression in mouse CD4CRE-NIKF/F or control (Cre) TH17 cells treated with DMSO or SM for 48 hours. Data with medians are from four experiments. *P < 0.05, **P < 0.01, ****P < 0.0001 by Fisher’s LSD ANOVA (A and B) or Mann-Whitney test (C and D).

  • Fig. 5 Both RelB and p52 contribute to SM-associated changes in TH17 cell gene expression.

    (A) Flow cytometry analysis of IL-17A and IFN-γ production by live, CD69+GFP+ mouse TH17 cells transduced with the indicated retrovirus. Dot plots (left) are representative of and quantified data (right) are pooled from five independent experiments. (B to G) qRT-PCR analysis of Il17a (B), Il22 (C), Maf (D), Il10 (E), Il17f (F), and Nrp1 (G) mRNA expression in FACS-sorted mouse GFP+ TH17 cells transduced with the indicated retrovirus. Data are pooled from five independent experiments. *P < 0.05, **P < 0.01 by paired t test.

  • Fig. 6 SM augments AhR-dependent production of IL-22.

    (A) Flow cytometry analysis of cMAF abundance in TH17 cells differentiated with DMSO or SM for 48 hours. Histograms (left) are representative of and quantified mean fluorescence intensity (MFI) data (right) are pooled from four biological replicates. (B) qRT-PCR analysis of Il22 mRNA expression in FACS-sorted mouse GFP+ TH17 cells transduced with the indicated retrovirus. Data are pooled from five biological replicates. (C) Flow cytometry analysis of IL-22 and IFN-γ production by TH17 cells differentiated with DMSO or SM and treated with AhR inhibitor (AhR-i) CH-223191 or FICZ, as indicated. Data with medians are from five biological replicates. *P < 0.05, **P < 0.01 by paired t test (A) or Mann-Whitney test (B and C).

  • Fig. 7 SM reduces IL-17A production and limits EAE disease progression.

    (A and B) Flow cytometry analysis of IL-17A and IFN-γ production by splenic CD44+CD4+ T (A) and γδ T (B) cells from MOG-immunized mice treated with DMSO or SM after 7 days. Contour plots (left) are representative of four experiments. Quantified data with medians (right) are from all experiments. (C) Clinical scores from MOG-immunized mice treated as indicated. Data are means ± SEM of 27 mice from four experiments. *P < 0.05, **P < 0.01, ****P < 0.0001 by Mann-Whitney test (A and B) or ANOVA with Bonferroni’s multiple comparisons test (C).

  • Fig. 8 SM skews the differentiation potential of TH cell subsets.

    (A) Flow cytometry analysis of cytokine production by splenic CD4+ T cells differentiated under the indicated TH conditions in the presence of DMSO or SM for 48 hours. Dot plots (left) are representative of and quantified data with medians (right) are pooled from at least three biological replicates. (B) Flow cytometry analysis of cytokine production by splenic CD4+ T cells were cultured under competitive conditions with IL-1β, IL-2, IL-4, IL-6, TGFβ1, and anti–IFN-γ for 48 hours. Dot plots (top left) are representative of four biological replicates. Quantified frequency data (right) are medians ± range pooled from all experiments. *P < 0.05, **P < 0.01 by Mann-Whitney test.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/596/eaaw3469/DC1

    Fig. S1. SMs do not prevent activation and proliferation of TH17 cells.

    Fig. S2. SMs promote NIK-dependent production of TNF in TH17 cells.

    Fig. S3. SMs do not alter RORγt, IRF4, or BATF protein amounts in TH17 cells.

    Fig. S4. Single cIAP deficiency cannot recapitulate SM-induced inhibition of TH17 cell differentiation.

    Fig. S5. Proteomic changes in TH17 cells compared to undifferentiated CD4+ T cells.

    Fig. S6. SMs induce global transcriptional and proteomic changes in TH17 cells.

    Fig. S7. Reduced abundance of metabolic proteins in TH17 compared to undifferentiated CD4+ T cells.

    Fig. S8. SM increases the abundance of noncanonical NF-κB and cell adhesion proteins in TH17 cells.

    Fig. S9. RelB induces the expression of Nfkb2 and reduces the expression of Il17a independently of p52.

    Fig. S10. SM treatment hinders RORγt binding to Il17a regulatory regions and increases the expression of immune checkpoint receptors in TH17 cells.

    Fig. S11. SM depletion of cIAP1 but not xIAP activates the noncanonical NF-κB and does not alter the nuclear translocation of TH17-associated TFs.

    Data file S1. Differentially expressed genes in DMSO- versus SM-treated TH17 cells.

    Data file S2. Differentially expressed proteins in DMSO- versus SM-treated TH17 cells.

    Data file S3. Proteins and pathways differentially regulated in TH17 compared to undifferentiated cells.

    Data file S4. Proteins and pathways differentially regulated in DMSO- and SM-treated TH17 cells.

  • The PDF file includes:

    • Fig. S1. SMs do not prevent activation and proliferation of TH17 cells.
    • Fig. S2. SMs promote NIK-dependent production of TNF in TH17 cells.
    • Fig. S3. SMs do not alter RORγt, IRF4, or BATF protein amounts in TH17 cells.
    • Fig. S4. Single cIAP deficiency cannot recapitulate SM-induced inhibition of TH17 cell differentiation.
    • Fig. S5. Proteomic changes in TH17 cells compared to undifferentiated CD4+ T cells.
    • Fig. S6. SMs induce global transcriptional and proteomic changes in TH17 cells.
    • Fig. S7. Reduced abundance of metabolic proteins in TH17 compared to undifferentiated CD4+ T cells.
    • Fig. S8. SM increases the abundance of noncanonical NF-κB and cell adhesion proteins in TH17 cells.
    • Fig. S9. RelB induces the expression of Nfkb2 and reduces the expression of Il17a independently of p52.
    • Fig. S10. SM treatment hinders RORγt binding to Il17a regulatory regions and increases the expression of immune checkpoint receptors in TH17 cells.
    • Fig. S11. SM depletion of cIAP1 but not xIAP activates the noncanonical NF-κB and does not alter the nuclear translocation of TH17-associated TFs.
    • Legends for data files S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Differentially expressed genes in DMSO- versus SM-treated TH17 cells.
    • Data file S2 (Microsoft Excel format). Differentially expressed proteins in DMSO- versus SM-treated TH17 cells.
    • Data file S3 (Microsoft Excel format). Proteins and pathways differentially regulated in TH17 compared to undifferentiated cells.
    • Data file S4 (Microsoft Excel format). Proteins and pathways differentially regulated in DMSO- and SM-treated TH17 cells.

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