The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines

Abstract

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor that plays a pivotal role in cellular defense against oxidative injury. Nrf2 signaling is involved in attenuating autoimmune disorders such as rheumatoid arthritis (RA). B cells play several roles in the pathogenesis of RA, such as in autoantibody production, antigen presentation, and T-cell activation. We investigated the anti-arthritic mechanisms of sulforaphane, an activator of Nrf2, in terms of its effect on B cells. To investigate the effect of sulforaphane on collagen-induced arthritis (CIA), sulforaphane was administered intraperitoneally after CIA induction. Hematoxylin and eosin-stained sections were scored for inflammation, pannus invasion, and bone and cartilage damage. We assessed the expression levels of inflammation-related factors by real-time PCR and the levels of various IgG subclasses by enzyme-linked immunosorbent assay. Sulforaphane treatment reduced the arthritis score and the severity of histologic inflammation in CIA mice. The joints from sulforaphane-treated CIA mice showed decreased expression of interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, receptor activator of NF-κB ligand, and tartrate-resistant acid phosphatase. Sulforaphane-treated mice showed lower circulating levels of type-II-collagen-specific IgG, IgG1, and IgG2a. In vitro, sulforaphane treatment significantly reduced the differentiation of lipopolysaccharide-stimulated murine splenocytes into plasma B cells and germinal-center B cells. Finally, sulforaphane significantly inhibited the production of IL-6, TNF-α, and IL-17 by human peripheral blood mononuclear cells stimulated with an anti-CD3 monoclonal antibody in a dose-dependent manner. Inhibition of differentiation into plasma B and Germinal Center B cells may be the mechanism underlying the anti-arthritic effect of sulforaphane.

Fig 1. Sulforaphane ameliorates collagen-induced arthritis (CIA) in mice.

(A) Arthritis score. Three weeks after CIA induction, CIA mice were intraperitoneally injected with sulforaphane three times per week (n = 5 per group); *P < 0.001. (B) Histological examination of the joints of CIA mice treated with sulforaphane. Mice were sacrificed on day 46 after CIA induction and tissue sections from the joints were stained with hematoxylin and eosin, and Safranin O. The histological scores of inflammations, cartilage damage, and bone damage are shown. ***P < 0.001, **P < 0.01.

Fig 2. Sulforaphane suppresses the expression of inflammatory cytokines in the joints of CIA mice.

(A) Immunohistochemical staining for interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, receptor activator of NF-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) in the synovium of CIA mice. Data are means ± standard deviation (SD) of three independent experiments. (B) Splenocytes from C57BL/6 mice were cultured with sulforaphane (1, 5, 10, or 50 μM) for 72 h, and the IL-6, IL-17, and TNF-α levels in the culture supernatant were measured by ELISA. (C) Cell viability by MTT analysis. Data are means ± SD. ***P < 0.001, **P < 0.01, *P < 0.05 (bars represent means).

Fig 3. Sulforaphane alters B cell differentiation.

(A) Splenocytes isolated from mice were analyzed by flow cytometry. The cells were stained with antibodies against CD138+B220low B cells for plasma B cells and GL7+B220+ B cells for germinal-center B cells. (B) Splenocytes were cultured with sulforaphane (5 or 10 μM) in the presence of 100 ng/mL lipopolysaccharide (LPS) for 3 days. The population of plasma B cells and germinal-center B cells were analyzed by flow cytometry. (B) Serum level of type II collagen (CII)-specific IgG 45 days after the first immunization. ***P < 0.001, **P < 0.01, *P < 0.05.

Fig 4. Sulforaphane decreases IL-6, TNF-α, IL-17, and total IgG levels.

(A) Peripheral blood mononuclear cells from healthy controls were cultured in medium without or with sulforaphane (1, 5, 10 μM) for 72 h in the presence of 0.5 μg/mL anti-CD3. The levels of IL-6, TNF-α, and IL-17 in the culture supernatant were assayed. (B) Synovial fluid mononuclear cells from RA patients were cultured with sulforaphane (1, 5, 10 μM) for 72 h in the presence of 100 ng/mL LPS and the level of total IgG in the supernatant was determined. ***P < 0.001, **P < 0.01.