Intranuclear Delivery of HIF-1α-TMD Alleviates EAE via Functional Conversion of TH17 Cells

Abstract

T helper 17 (TH17) cells are involved in several autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). In addition to retinoic acid receptor-related orphan nuclear receptor gamma t (ROR-γt), hypoxia-inducible factor-1α (HIF-1α) is essential for the differentiation and inflammatory function of TH17 cells. To investigate the roles of HIF-1α in the functional regulation of TH17 cells under the normal physiological condition without genetic modification, the nucleus-transducible form of transcription modulation domain (TMD) of HIF-1α (ntHIF-1α-TMD) was generated by conjugating HIF-1α-TMD to Hph-1 protein transduction domain (PTD). ntHIF-1α-TMD was effectively delivered into the nucleus of T cells without cellular cytotoxicity. ntHIF-1α-TMD significantly blocked the differentiation of naïve T cells into TH17 cells in a dose-dependent manner via IL-17A and ROR-γt expression inhibition. However, T-cell activation events such as induction of CD69, CD25, and IL-2 and the differentiation potential of naïve T cells into TH1, TH2, or Treg cells were not affected by ntHIF-1α-TMD. Interestingly, TH17 cells differentiated from naïve T cells in the presence of ntHIF-1α-TMD showed a substantial level of suppressive activity toward the activated T cells, and the increase of Foxp3 and IL-10 expression was detected in these TH17 cells. When mRNA expression pattern was compared between TH17 cells and ntHIF-1α-TMD-treated TH17 cells, the expression of the genes involved in the differentiation and functions of TH17 cells was downregulated, and that of the genes necessary for immune-suppressive functions of Treg cells was upregulated. When the mice with experimental autoimmune encephalomyelitis (EAE) were treated with ntHIF-1α-TMD with anti-IL-17A mAb as a positive control, the therapeutic efficacy of ntHIF-1α-TMD in vivo was comparable with that of anti-IL-17A mAb, and ntHIF-1α-TMD-mediated therapeutic effect was contributed by the functional conversion of TH17 cells into immune-suppressive T cells. The results in this study demonstrate that ntHIF-1α-TMD can be a new therapeutic reagent for the treatment of various autoimmune diseases in which TH17 cells are dominant and pathogenic T cells.

Keywords: Th17 cells; experimental autoimmune encephalomyelitis; functional conversion; hypoxia-inducible factor-1; protein transduction domain.

Figure 1

Examination of the functional influence of ntHIF-1α-TMD on CD4+ T-cell subsets. (A) The induced expression of the surface proteins associated with T-cell activation was analyzed by flow cytometry on CD4+ T cells activated by anti-CD3ϵ and anti-CD28 with or without ntHIF-1α-TMD treatment. Hph-1-PTD-fused enhanced green fluorescent protein (ntEGFP) was used as a negative control. (B) The secreted IL-2 in the supernatant from the cells in (A) was measured by ELISA. (C–F) The functional influence of ntHIF-1α-TMD on TH1, TH2, iTreg, or TH17 cells was evaluated by flow cytometry after 72 h incubation in the T-cell subset-skewing condition with ntHIF-1α-TMD. All experiments shown here were repeated three times. Data are represented as mean ± SEM (n ≥ 3), and the statistical analysis was examined using Student’s t-test. ns, not significant, *P < 0.05, **P < 0.01.

Figure 2

Inhibitory effect of ntHIF-1α-TMD on differentiation and function of TH17 cells. (A) Jurkat T cells were transfected with the plasmid containing the luciferase reporter gene whose expression is driven by the IL-17 promoter or the indicated gene. Luciferase activity was measured after ntHIF-1α-TMD treatment. (B, C) After incubation of naïve CD4+ T cells for 3 days in TH17-skewing condition, the level of secreted IL-17A in the supernatant was measured by ELISA. qRT-PCR was performed to evaluate the effect of ntHIF-1α-TMD on Rorc transcription. Experiments were performed in triplicated wells and repeated three times. Data are represented as mean ± SEM (n ≥ 4). Statistical analysis was performed using Student’s t-test. ns, not significant, **P < 0.01.

Figure 3

The functional conversion of TH17 cells into T cells with the immune-suppressive phenotype. (A) In the TH17-skewing condition, the change of IL-17A or Foxp3 expression in TH17 cells treated with ntHIF-1α-TMD were analyzed by flow cytometry. (B) The amount of IL-10 in the supernatant from T cells in (A) was measured by ELISA. (C, D) The immune-suppressive function of TH17 cells treated with ntHIF-1α-TMD was evaluated by suppression assay. (E) Expression heatmap of helper T cell-related genes. Z-score was calculated from log2(TPM+1). (F) GSEA-based enrichment plots of the Foxp3 targets cluster or TGF-β signaling pathway. Experiments in (A–D) were performed three times. Data are represented as mean ± SEM (n ≥ 3), and Student’s t-test was performed for statistical analysis. ns, not significant, *P < 0.05, **P < 0.01.

Figure 4

Therapeutic potential of ntHIF-1α-TMD in EAE disease models. (A) The treatment scheme of EAE-induced mice with phosphate-buffered saline (EAE induced), ntHIF-1α-TMD (100 μg/mouse), or anti-IL17A (40 μg/mouse) is represented. (B) A clinical score was measured until 21 days after EAE induction. (C) EAE-induced mice (w/or w/o treatment) were sacrificed 16 days after EAE induction. The spinal cords were harvested, and the histological analysis was performed by Luxol Fast Blue (C, upper panel) and H&E (C, lower panel) staining. (D) CD4+ T cells infiltrated into the spinal cord in (C) were counted, and the graph is represented as mean ± SEM (n ≥ 4). (E) CD4+ T cells in the lymph nodes harvested from the mice at day 16 were analyzed for the expression of IL-17A or Fopx3 by flow cytometry. (F) The level of IL-17A or IL-10 in the serum of mice at day 16 was measured by ELISA. The graphs are represented as mean ± SEM (n ≥ 3). The experiments in EAE disease model were independently performed three times. The group differences were analyzed by Student’s t-test as a statistical analysis. ns, not significant, *P < 0.05, **P < 0.01.