Halofuginone inhibits TH17 cell differentiation by activating the amino acid starvation response

Abstract

A central challenge for improving autoimmune therapy is preventing inflammatory pathology without inducing generalized immunosuppression. T helper 17 (TH17) cells, characterized by their production of interleukin-17, have emerged as important and broad mediators of autoimmunity. Here we show that the small molecule halofuginone (HF) selectively inhibits mouse and human TH17 differentiation by activating a cytoprotective signaling pathway, the amino acid starvation response (AAR). Inhibition of TH17 differentiation by HF is rescued by the addition of excess amino acids and is mimicked by AAR activation after selective amino acid depletion. HF also induces the AAR in vivo and protects mice from TH17-associated experimental autoimmune encephalomyelitis. These results indicate that the AAR pathway is a potent and selective regulator of inflammatory T cell differentiation in vivo.

Fig. 1

Selective inhibition of T_H17 differentiation by HF. (A) Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled T cells were activated in the presence of dimethyl sulfoxide (DMSO), 40 nM MAZ1310, or titrating concentrations of HF. CFSE dilution and the percentages of cells expressing CD25, IFN-γ⁺ IL-4⁻ (T_H1 cells), IL-4⁺ IFN-γ⁻ (T_H2 cells), or IL-17⁺ IFNγ⁻ (T_H17 cells) cells are displayed as mean values ± SD normalized to MAZ1310-treated cells. IC₅₀ values are listed next to corresponding lines ± SD. (B) Intracellular cytokine expression in primary human T cells treated with DMSO, HF, or MAZ1310. (C) Differentiating T_H17 cell cultures were incubated with HF at the indicated times after activation. Intracellular cytokine expression was determined on day four. The mean percentage of T_H17 cells is shown ± SD (error bars). *P < 0.005, relative to MAZ1310-treated T cells. (D) CFSE-labeled T cells were differentiated to T_H17 in the presence of HF or MAZ1310. After intracellular cytokine staining, CFSE peaks were gated and mean percentages of IL-17⁺ T cells within each peak are displayed ± SD (error bars). All data represent at least three similar experiments.

Fig. 2

HF activates the AAR pathway. (A) Histogram of microarray data from differentiating T_H17 cells treated with HF or MAZ1310 for 3 or 6 hours. Red dots indicate transcripts increased greater than twofold by HF-treatment at both 3 and 6 hours. Text and arrows denote several defined AAR genes (27). (B) Quantitative real-time PCR was performed on cDNA from unstimulated T cells or those activated for 4 hours in the presence of MAZ1310 or HF. Asns, Gpt2, or eIF4Ebp1 mRNA expression was normalized to Hprt levels and are shown as mean values ± SD. (C) Unstimulated or TCR-activated T cells were treated with DMSO, 40 nM MAZ1310, or HF and were lysed for Western blotting after 4 hours. (D) Activated T cells were lysed at the indicated times after treatment with MAZ1310 or HF. ATF4 protein is indicated by an arrow. NS, nonspecific band. Microarray data were generated from three biological replicates. Other data represent two to three experiments.

Fig. 3

Regulation of T_H17 differentiation by amino acids. (A) Lysates from TCR-activated T cells treated with MAZ1310 or HF for 4 hours were analyzed by Western blotting. (B) Unstimulated or activated T cells were cultured in complete medium, medium lacking Cys/Met (-Cys/Met), or complete medium containing tunicamycin, HF, or MAZ1310 and lysed after 4 hours for Western blot analysis. Xbp-1 splicing assay was performed on isolated cDNA (6). (C) Activated T cells were cultured without cytokines or polarized to T_H1, T_H2, iT_reg, or T_H17 cells. Titrating concentrations of Cys/Met are indicated. The percentages of T_H1, T_H2, and T_H17 cells, and those expressing CD25 or Foxp3, were determined as in Fig. 1A and are displayed as mean values ± SD, normalized to cells cultured in complete medium. (D) T cells differentiated as in (C) were treated with tunicamycin as indicated. T cell activation and differentiation was determined as in (C). (E) Activated T cells were treated with DMSO or HF. Some cultures were supplemented with 5× or 10× amino acids (6), and lysates were analyzed after 4 hours by Western blotting. (F) T cells activated in T_H17 polarizing conditions were treated with DMSO or HF plus amino acids, as indicated, and stained for intracellular cytokine expression. These data represent three experiments.

Fig. 4

HF inhibits T_H17-associated autoimmune inflammation in vivo. (A) Wild-type mice were immunized with phosphate-buffered saline or MOG33-55 emulsified in CFA and treated daily with DMSO or HF, and disease was monitored (6). (B) After PLP-specific T cell transfer, recipients were treated daily with DMSO or HF, and disease was monitored. Mean EAE scores are displayed. (C) (Left) Paraaortic lymph nodes from MOG-immunized mice treated with DMSO or HF were harvested on day 6. Mononuclear cells were cultured without (top) or with (bottom) PMA and ionomycin and stained for intracellular cytokines. (Right) Mononuclear cells isolated from CNS tissue of DMSO-treated (score = 2) or HF-treated (score = 0) mice were cultured and stained for intracellular cytokines as above. TCRβ⁺ CD4⁺ cells were gated on for analyses. (D) (Top) Splenocyte lysates were prepared from DMSO- or HF-treated mice and analyzed by Western blotting. (Bottom) AAR-associated gene expression (Asns, Gpt2, eIF4Ebp1) was analyzed by QPCR on cDNA from spleens of mice treated with DMSO or HF. Transcript levels were normalized to Hprt and are displayed as mean expression ± SD (error bars) from triplicate samples. EAE data are cumulative from three independent experiments; other data represent two to three similar experiments.