AhR-ROR-γt complex is a therapeutic target for MAP4K3/GLKhighIL-17Ahigh subpopulation of systemic lupus erythematosus

Abstract

The cytokine IL-17A plays critical roles in the pathogenesis of autoimmune diseases. The frequencies of MAP kinase kinase kinase kinase 3 [also named germinal center kinase-like kinase (GLK)]-overexpressing T cells are correlated with disease severity of systemic lupus erythematosus (SLE). T-cell-specific GLK-transgenic mice develop spontaneous autoimmune responses through IL-17A. GLK signaling selectively stimulates IL-17A production in murine T cells through inducing aryl hydrocarbon receptor (AhR)-retinoic acid receptor-related orphan nuclear receptor-γt (ROR-γt) complex formation. Here, we investigated whether GLK-induced AhR-ROR-γt complex in T cells is a therapeutic target for human SLE. The population of GLK⁺IL-17A⁺ T cells was enhanced in the peripheral blood from patients with SLE compared with that of healthy controls using flow cytometry. The receiver operating characteristic curve analysis showed that increased GLK⁺IL-17A⁺ T-cell population in peripheral blood reflected an active stage of SLE. In addition, peripheral blood T cells from patients with SLE displayed induction of ROR-γt phosphorylation and the AhR-ROR-γt (and AhR-phosphorylated ROR-γt) complex. Moreover, we identified a small-molecule inhibitor, verteporfin, that inhibited GLK kinase activity and AhR-ROR-γt interaction. The small-molecule inhibitor verteporfin suppressed the disease severity in autoimmune mouse models and IL-17A production in T cells from patients with SLE. Collectively, the GLK-induced AhR-ROR-γt (and AhR-phosphorylated ROR-γt) complex is a therapeutic target for the GLK^highIL-17A^high subpopulation of human patients with SLE.-Chuang, H.-C., Chen, Y.-M., Chen, M.-H., Hung, W.-T., Yang, H.-Y., Tseng, Y.-H., Tan, T.-H. AhR-ROR-γt complex is a therapeutic target for MAP4K3/GLK^highIL-17A^high subpopulation of systemic lupus erythematosus.

Keywords: SLE; autoimmune disease; verteporfin.

Figure 1

The frequencies of GLK⁺ T_h17 cells are increased in patients with SLE. A–D) Flow cytometry analyses of GLK⁺ and IL-17⁺ T cells [CD3-gated, CD3 plus CD4–gated, CD3 plus CD8–gated, or CD3 plus CD4⁻CD8⁻ (DN)–gated] from the PBLs of 6 healthy controls and 18 patients with SLE; the results from a representative healthy control (HC) and a representative patient with SLE (SLEDAI = 12) are shown. E) Statistical analyses of GLK⁺IL-17⁺ T cells in the healthy control group and the patient with SLE group are shown. F) Positive correlation and significant regression between SLEDAI and the frequencies of GLK⁺IL-17⁺ T cells in the CD4⁺ T subset from all patients with SLE (in red) and healthy controls (in blue). (Pearson correlation coefficient: r = 0.729, P = 0.000053). G) ROC curves of the frequencies of GLK⁺IL-17⁺ cells in T-cell subsets (CD3⁺, CD4⁺, CD8⁺, or CD4⁻CD8⁻ DN subset) for detection of active SLE (SLEDAI ≧ 12). The area under the curve values of individual GLK⁺IL-17⁺ subpopulations: 0.935, P = 0.002 (CD3⁺ T cells); 0.944, P = 0.001 (CD4⁺ T subset); 0.792, P = 0.036 (CD8⁺ T subset); and 0.759, P = 0.062 (CD4⁻CD8⁻ DN T subset).

Figure 2

Phosphorylated ROR-γt interacts with AhR in human autoimmune T cells. A) Immunoblotting analyses of phosphorylated ROR-γt (Ser-489) and ROR-γt in peripheral blood T cells freshly isolated from 3 patients with SLE, 1 patient with RA, and 3 healthy controls (HCs). B) Confocal microscopy analyses of PLA for the interaction between AhR and ROR-γt in peripheral blood T cells, which were freshly isolated from 27 patients with SLE and 23 healthy controls (3 representative healthy controls are shown; 20 additional healthy controls and 3 patients with RA are shown in Supplemental Fig. S2). C) Confocal microscopy analyses of PLA for the interaction between endogenous AhR and phosphorylated ROR-γt proteins in peripheral blood T cells (3 representative patients with SLE and 1 healthy control are shown; 22 additional patients with SLE, 20 additional healthy controls, and 2 patients with RA are shown in Supplemental Fig. S3). For PLA, red dots represent direct interaction signals. T-cell nucleus was stained with DAPI (blue color). Original magnification, ×630. Scale bar, 10 μm.

Figure 3

Verteporfin (C1) blocks the AhR–ROR-γt complex in human autoimmune T cells. A) Luciferase activity of NF-κB–driven reporter assays in stable GLK-transfected CHO-K1 cells that were treated with or without C1 under the indicated concentration. The fold changes are presented relative to the value of vector control. B) Recombinant proteins of GLK kinase domain plus recombinant proteins of GST-tagged kinase-dead PKC-θ (K409W) were subjected to in vitro kinase assays in the presence or absence of C1. The fold changes are presented relative to the value of the substrate alone as a control. Means ± sd of are shown. C) The AhR–ROR-γt complex was inhibited by C1. Confocal microscopy analyses of PLA for the interaction between AhR and either ROR-γt or phosphorylated ROR-γt (S489) in peripheral blood T cells treated with C1 (5 μM) for 30 min. The T cells were freshly isolated from 4 patients with SLE and 1 patient with RA. Original magnification, ×630. D) Quantification of the PLA signals shown in panel C. *P < 0.05, ***P < 0.001 (2-tailed Student’s t test).

Figure 4

Verteporfin (C1) suppresses IL-17 production and autoimmune responses in mice. A–C) Effect of C1 administration (0.4 μg/g body weight every 3 d for a period of 30 d) on serum IL-17A and autoantibodies in 20-wk-old Lck-GLK Tg mice. B) ELISA of serum IL-17A, IL-6, TNF-α, IFN-γ, and IL-17F in Lck-GLK Tg mice treated with C1 or PBS for 16 d. C) ELISA of serum autoantibodies in Lck-GLK Tg mice treated with C1 or PBS for 30 d. Means ± sem are shown; n = 4/group. D) EAE induction in wild-type C57BL/6J mice treated with PBS or C1 (0.4 μg/g body weight every 3 d). Clinical scores are presented on a scale of 1–5. PBS group, n = 10; C1 group, n = 7. E) ELISA of IL-17A in the sera from MOG-immunized mice at day 16. F) Induction of CIA in wild-type C57BL/6J mice treated with C1 (0.4 μg/g body weight every 3 d) or PBS. Clinical scores are presented on a scale of 1–16; n = 4/group. G) ELISA of serum IL-17A in collagen-immunized mice at d 28. Data shown are representatives of 3 independent experiments. α-dsDNA, anti-dsDNA antibody; ANA, anti-nuclear antibody; N.S., not significant; RF, rheumatoid factor. Means ± sem are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed Student’s t test).

Figure 5

Verteporfin (C1) blocks IL-17 production of human T cells. A) ELISA of various cytokines in supernatants of murine primary T cells stimulated with anti-CD3 or CD28 and cotreated with C1 (5 or 10 μM) for 3 d. Means ± sd are shown; n = 3/group. B) ELISA of various cytokines in supernatants of human T cells. T cells were stimulated with anti-CD3 or CD28 and cotreated with C1 (1 or 5 μM) for 3 d. HC, healthy control; N.S., not significant. Means ± sd are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed Student’s t test).

Figure 6

GLK-deficient mice display an increased lifespan. A) Survival curves of wild-type or GLK-deficient mice were calculated by the life-table method. Shown are survival plots representing pooled data from 10 wild-type mice and 15 GLK-deficient mice. GLK-deficient mice exhibited a significant extension of lifespan relative to wild-type mice (Wilcoxon test, P = 0.001). B) 16-mo-old wild-type mice displayed gray hair, whereas 38-mo-old GLK-deficient mice still displayed healthy hair. C) ELISA of various cytokines in the sera of 4.5-mo-old wild-type, 20-mo-old wild-type, or 20-mo-old GLK-deficient mice. Means ± sem are shown; n = 4/group. WT, wild-type mice; GLK KO, GLK-deficient mice; N.S., not significant. *P < 0.05, **P < 0.01 (2-tailed Student’s t test).