Apigenin, a non-mutagenic dietary flavonoid, suppresses lupus by inhibiting autoantigen presentation for expansion of autoreactive Th1 and Th17 cells

Abstract

Introduction: Lupus patients need alternatives to steroids and cytotoxic drugs. We recently found that apigenin, a non-mutagenic dietary flavonoid, can sensitize recurrently activated, normal human T cells to apoptosis by inhibiting nuclear factor-kappa-B (NF-kappaB)-regulated Bcl-xL, cyclooxygenase 2 (COX-2), and cellular FLICE-like inhibitory protein (c-FLIP) expression. Because sustained immune activation and hyperexpression of COX-2 and c-FLIP contribute to lupus, we treated SNF1 mice that spontaneously develop human lupus-like disease with apigenin.

Methods: SNF1 mice with established lupus-like disease were injected with 20 mg/kg of apigenin daily and then monitored for development of severe nephritis. Histopathologic changes in kidneys, IgG autoantibodies to nuclear autoantigens in serum and in cultures of splenocytes, along with nucleosome-specific T helper 1 (Th1) and Th17 responses, COX-2 expression, and apoptosis of lupus immune cells were analyzed after apigenin treatment.

Results: Apigenin in culture suppressed responses of Th1 and Th17 cells to major lupus autoantigen (nucleosomes) up to 98% and 92%, respectively, and inhibited the ability of lupus B cells to produce IgG class-switched anti-nuclear autoantibodies helped by these Th cells in presence of nucleosomes by up to 82%. Apigenin therapy of SNF1 mice with established lupus suppressed serum levels of pathogenic autoantibodies to nuclear antigens up to 97% and markedly delayed development of severe glomerulonephritis. Apigenin downregulated COX-2 expression in lupus T cells, B cells, and antigen-presenting cells (APCs) and caused their apoptosis. Autoantigen presentation and Th17-inducing cytokine production by dendritic cells were more sensitive to the inhibitory effect of apigenin in culture, as evident at 0.3 to 3 muM, compared with concentrations (10 to 100 microM) required for inducing apoptosis.

Conclusions: Apigenin inhibits autoantigen-presenting and stimulatory functions of APCs necessary for the activation and expansion of autoreactive Th1 and Th17 cells and B cells in lupus. Apigenin also causes apoptosis of hyperactive lupus APCs and T and B cells, probably by inhibiting expression of NF-kappaB-regulated anti-apoptotic molecules, especially COX-2 and c-FLIP, which are persistently hyperexpressed by lupus immune cells. Increasing the bioavailability of dietary plant-derived COX-2 and NF-kappaB inhibitors, such as apigenin, could be valuable for suppressing inflammation in lupus and other Th17-mediated diseases like rheumatoid arthritis, Crohn disease, and psoriasis and in prevention of inflammation-based tumors overexpressing COX-2 (colon, breast).

Figure 1

Apigenin suppressed nucleosome-specific interferon-gamma (IFN-γ) response and IgG-autoantibody production. Splenocytes from 5- to 6-month-old unmanipulated SNF1 mice were stimulated with nucleosomes in the presence of various amounts of apigenin or vehicle (dimethyl sulfoxide-phosphate-buffered saline). (a) Apigenin markedly suppressed IFN-γ responses by nucleosome-specific T cells in enzyme-linked immunosorbent spot assay. (b) Apigenin significantly reduced the level of IgG class autoantibodies in nucleosome-stimulated lupus Th cell-B cell coculture assays. *P < 0.001, **P < 0.01, and ^xP < 0.02. dsDNA, double-stranded DNA; SNF1, (SWR × NZB)F1; ssDNA, single-stranded DNA.

Figure 2

Dose response for in vivo treatment with apigenin for suppressing interferon-gamma (IFN-γ) response to nucleosomes. Three-month-old unmanipulated SNF1 mice were treated daily with apigenin at 3 mg/kg (13.89 μM), 6 mg/kg (27.8 μM), and 20 mg/kg (0.93 mM). Treatment with 20 mg/kg apigenin for 2 weeks markedly suppressed IFN-γ response to nuclesosomes ex vivo. Values are mean ± standard error of the mean. *P < 0.001, **P < 0.01, ^xP < 0.02, and ⁺P < 0.05. SNF1, (SWR × NZB)F1.

Figure 3

In vivo treatment with apigenin reduced nucleosome-specific Th1, Th17, and IgG autoantibody production. In vivo treatment with apigenin (20 mg/kg) for 2 months markedly reduced nucleosome-specific Th1 and Th17 responses and IgG autoantibody production ex vivo as compared with vehicle-treated SNF1 mice. (a) Splenocytes from apigenin- or vehicle-treated SNF1 mice were stimulated with nucleosomes and analyzed for Th1(left panel) and Th17 (right panel) responses by enzyme-linked immunosorbent spot assay. 'CD3' indicates results upon stimulation with optimal amount of anti-CD3 antibody (0.2 μg/mL). (b) IgG autoantibody levels of anti-dsDNA, anti-ssDNA, anti-nucleosome, and anti-histone in culture supernatants of lupus Th cell-B cell-nucleosome cocultures were analyzed by enzyme-linked immunosorbent assay. *P < 0.001, **P < 0.01, ^xP < 0.02, and ⁺P < 0.05. dsDNA, double-stranded DNA; IL-17, interleukin-17; SNF1, (SWR × NZB)F1; ssDNA, single-stranded DNA; Th, T helper.

Figure 4

Apigenin treatment in vivo suppresses IgG anti-nuclear autoantibodies and lupus nephritis. (a) Treatment for 1 month and 2 months resulted in significant reduction of IgG autoantibody levels in serum of SNF1 mice as compared with vehicle treatment. (b) Another group of mice was treated with apigenin or vehicle and monitored for the incidence of severe nephritis. Apigenin treatment markedly delayed incidence of nephritis (log-rank test, ⁺⁺P = 0.00313). (c) With treatment regimens identical to those in (b), renal histopathologic features of lupus nephritis were evaluated. Apigenin treatment significantly lowered the histopathology score of nephritis. (d) Representative histopathology figures of kidneys with treatment regimens identical to those in (b); hematoxylin and eosin stain (× 200). (e) Total IgG levels in serum of apigenin- or vehicle-treated mice were measured by enzyme-linked immunosorbent assay. *P < 0.001, **P < 0.01, and ^xP < 0.02. Ag, antigen; AutoAb, autoantibody; DMSO-PBS, dimethyl sulfoxide-phosphate-buffered saline; dsDNA, double-stranded DNA; Nuc, nucleosome; SNF1, (SWR × NZB)F1; ssDNA, single-stranded DNA.

Figure 5

Effect of apigenin on nucleosome-induced Th1 and Th17 responses and antigen presentation function of antigen-presenting cells (APCs). T cells and APCs from 3-month-old unmanipulated SNF1 mice were pulsed with various amounts of apigenin or vehicle for 1 hour, and crisscross cocultures were done in the presence of nucleososome (10 μg/mL). Apigenin pre-exposure suppressed autoantigen-presenting ability of APCs and resulted in inhibition of Th1 (a) and Th17 (b) responses more markedly than pre-exposure of the responding T cells to apigenin. *P < 0.001, **P < 0.01, and ^xP < 0.02. Api, apigenin; IFN-γ, interferon-gamma; IL-17, interleukin-17; SNF1, (SWR × NZB)F1; Th, T helper.

Figure 6

Effect of apigenin on cyclooxygenase 2 (COX-2) expression and apoptosis. Intracellular COX-2 expression followed treatment with apigenin or vehicle for 3 months. (a) COX-2 expression in representative histograms of spleen cell subsets. (b) Representative dot plot of gated CD4 T cells (percentage shown in right upper quadrant). (c) Compiled results from three experiments. Treatment with apigenin markedly suppressed COX-2 expression in gated CD4⁺ T cells, B cells, dendritic cells (DCs), and macrophages, but there was no difference in total CD11b⁺ cells or CD8⁺ T cells. (d) In vitro treatment with apigenin induced apoptosis of lupus T cells, B cells, DCs, and macrophages from SNF1 mice after 24-hour incubation. Culture with 30 μM apigenin resulted in a twofold increase in percentage of specific apoptosis in DCs and macrophages than in T and B cells. Apoptotic cells were analyzed in gated cell subsets to calculate percentage of specific apoptosis, as described in Materials and methods (n = 5 per stain). *P < 0.001, **P < 0.01, ^xP < 0.02, and ⁺P < 0.05 for (c) and (d). SNF1, (SWR × NZB)F1.

Figure 7

Apigenin markedly decreased interleukin-6 (IL-6) production by lupus dendritic cells (DCs). DCs from unmanipulated 3-month-old SNF1 mice were stimulated with nucleosomes (30 μg/mL), CpG (2.5 μg/mL), and R837 (1 μg/mL) in the presence of various amounts of apigenin. IL-6 in culture supernatant was measured by enzyme-linked immunosorbent assay. *P < 0.001, **P < 0.01, and ⁺P < 0.05. Nuc, nucleosome; SNF1, (SWR × NZB)F1.