BET bromodomain inhibition suppresses TH17-mediated pathology

Abstract

Interleukin (IL) 17-producing T helper (T(H)17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine T(H)17 differentiation from naive CD4(+) T cells, as well as in the activation of previously differentiated T(H)17 cells. We provide evidence that BET controls T(H)17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector T(H)17-associated cytokines, including IL17, IL21, and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in T(H)17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in T(H)17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of autoimmunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and T(H)17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions.

Figures

Figure 1.

BET bromodomain inhibition selectively blocks human TH17 differentiation. (A) Human CD4+CD45RA+ naive T cells were purified from peripheral blood, cultured for 6 d under TH17 polarizing conditions with DMSO or 150 nM JQ1, and analyzed by flow cytometry. Data are representative of three to five independent experiments. Statistical significance was determined by Student t test, and the corresponding p-values are indicated. Percentage of IL-17–, IL-22–, and IL-10–positive cells are indicated in red (DMSO) and blue (JQ1). (B) Human naive T cells were cultured under TH1, TH2, and T reg polarizing conditions with DMSO or 150 nM JQ1, and analyzed by flow cytometry. Data are representative of two independent experiments. (C–F) RNA was obtained from the indicated T cell subsets and analyzed by real-time qPCR for the indicated genes at 48 h (C and E). Total cell numbers from the corresponding experiments are shown in panels D and F. Data are representative of three to five independent experiments. Error bars represent standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05).

Figure 2.

BET bromodomain inhibition blocks human TH17 differentiation by controlling multiple TH17-associated genes. (A) Human naive T cells were cultured under TH17 conditions in the presence of 150 nM JQ1 or DMSO for 48 h and the expression of the indicated genes was investigated by qPCR. Error bars represent standard deviation. Data are representative of two to three independent experiments. Statistical significance was determined by Student’s t test (**, P < 0.01). (B) Human T cells were lentivirally transduced with hairpins targeting BRD2 (red bars) or BRD4 (blue bars) and cultured under TH17-polarizing conditions for 6 d. Two individual hairpins per gene were used (sh1 and sh2). Expression of BRD2, BRD4, and RORC was measured by qPCR; NTC, nontargeting control. IL-17A, IL-17F, and IL-21 protein was quantitated by Luminex. Data are representative of two independent experiments. Error bars represent standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01). (C) Human naive T cells were stimulated with anti-CD3/CD28 for 24 or 48 h in the presence or absence of exogenous human recombinant IL-21 and JQ1 as indicated, and IL21 expression was measured by qPCR. Data are representative of two independent experiments and two independent donors. Error bars represent standard deviation. (D) Human naive T cells were cultured under TH17-differentiating conditions for the indicated times in the presence or absence of exogenous human recombinant IL-21 and JQ1 as indicated, and phospho-Stat3 was measured by flow cytometry. Data are representative of two independent experiments and two independent donors. (E and F) Samples from D (5 d, bottom) were analyzed by qPCR for the indicated transcripts. Data are representative of two independent experiments and two independent donors. Error bars represent standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01); NS: not significant. (G and H) Human naive T cells were cultured under TH17 conditions for 6 d. After that time, cells were washed and rested for 16 h prior restimulation with anti-CD3/anti-CD28 for 48 h in the presence of DMSO control or 150 nM JQ1. Cytokine levels (luminex) or transcript levels (qPCR) are shown. Data are representative of two independent experiments. Error bars represent standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05).

Figure 3.

BET bromodomains control mouse TH17 differentiation. (A) Mouse CD4+CD62Lhi naive T cells were cultured under TH17 conditions in the presence of 150 nM JQ1 or DMSO control, and their activation state was evaluated by CD69 staining by FACS after 24 h of culture. Data are representative of three independent experiments. (B) Mouse naive T cells were cultured under TH17 conditions in the presence of 150 nM JQ1 or DMSO control, and IL-17 induction was measured by intracellular staining (contour plots) and ELISA (bars); IL-22 and TNF were measured by ELISA (bars). Data are representative of three independent experiments. (C) JQ1 dose response inhibition was monitored by ELISA (IL-17A) and Cell Titer Glo (viability). Data are representative of three independent experiments. (D) Mouse naive T cells were cultured under TH17 conditions in the presence of 150 nM JQ1 or DMSO control for 48 h, and whole genome gene expression was evaluated on the Affymetrix exon array platform: heatmap display, log2 expression of each gene is mean-centered, and normalized to have a standard deviation of 1; GSEA plot was calculated from the same dataset. Data represent two independent biological replicates. (E) Mouse naive T cells were differentiated into TH17 cells for 4 d and reactivated for 18 h with anti-CD3/CD28 in the absence (filled bars) or presence (open bars) of increasing concentrations of JQ1, and IL-17A and TNF production were monitored by AlphaLISA. Data are representative of three independent experiments. (F) Brd2, Brd4, H3K4me3, and H4Ac4 ChIP was performed from naive T cells differentiated for 20 h under TH17, TH1, or TH2 conditions and treated with 150 nM JQ1 or DMSO control, and examined by quantitative PCR with specific primers for the CNS2 genomic region of the Il17 locus. IgG was used as a control. Data are representative of two to five independent experiments. (G) BET bromodomains control TH17 differentiation in vivo. Mice were injected with anti-CD3 at times 0 and 48 h, and the duodenal T cell population was analyzed by flow cytometry 4 h after the last anti-CD3 injection in animals treated with vehicle control or 30 mg/kg JQ1 twice per day. Plasma levels of IL-17 and IL-10 were measured by ELISA (H). Data are representative of three independent experiments (n = 4–6 mice per group). All panels, error bars represent standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01).

Figure 4.

Inhibition of BET bromodomains protects mice from autoimmunity. (A) CIA disease progression in DBA/1 mice treated with vehicle (blue line, n = 6) or 30 mg/kg JQ1 (red line, n = 14) for 14 d. Onset of dosing is presented as day 0 in the horizontal axis. (B) Histopathology at day 14 of one representative specimen from each group. Bars, 100 µm. Data are representative of two independent experiments. Error bars represent standard deviation. *, P < 0.05 by Student’s unpaired t test, two-tailed. (C) EAE disease progression in C57/B6 mice was monitored daily for changes in clinical score (left), body weight (middle), and disease incidence (right). Unimmunized animals (naive; n = 20) were followed in parallel. Treatment with DMSO (vehicle; n = 30), JQ1 (30 mg/kg; n = 25), or dexamethasone (Dex; n = 30) was started 2 wk after immunization and lasted for the remaining duration of the study. Data are representative of two independent experiments. Error bars represent standard deviation. *, P < 0.05 by Student’s unpaired t test, two-tailed. (D) Single cell suspensions prepared from cervical lymph nodes of mice with EAE were restimulated with 10 µg/ml MOG peptide for 72 h and culture supernatants were assayed for the production of IFN-γ (left) and IL-17 (right) by ELISA; **, P < 0.001 by Student’s t test. Error bars represent standard error of the mean. (E) Cells were isolated from the CNS of mice treated with DMSO, JQ1, or dexamethasone on day 17 after immunization with MOG peptide and stimulated ex vivo with PMA/ ionomycin, followed by intracellular cytokine staining and FACS analysis.