Skip to main content
PMC home page
PLOS ONE logoLink to PLOS ONE
. 2021 Feb 16;16(2):e0245986. doi: 10.1371/journal.pone.0245986

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

Su-Jin Moon 1,#, Jooyeon Jhun 2,#, Jaeyoon Ryu 2, Ji ye Kwon 2, Se-Young Kim 2, KyoungAh Jung 3, Mi-La Cho 2,3,4,‡,*, Jun-Ki Min 5,‡,*
Editor: David Douglass Brand6
PMCID: PMC7886167  PMID: 33592002

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.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by infiltration of immune cells (such as T and B cells) into the hyperplastic synovium, which manifests as pannus formation. The disease eventually leads to progressive joint destruction and reduces quality of life. The pathogenesis of RA involves various immune cells and inflammatory cytokines. B cells are phylogenetically the most recent evolutionary development in the immune system. The importance of B cells and plasma cells in the development and progression of RA was suggested [1] by detection of autoantibodies such as anti-cyclic citrullinated peptide (anti-CCP) and anti-rheumatoid factor (anti-RF) in patients with RA [2, 3]. In addition to its diagnostic utility, the presence of autoantibodies suggests a poor prognosis of RA, such as rapid joint destruction [4]. The fact that autoantibodies are detected in the majority of RA patients up to 14 years before disease onset implies that B cells are essential for its pathogenesis [5]. Furthermore, synovial B cells and plasma cells of RA patients show evidence of antigen-driven maturation and autoantibody production [6]. As autoantibody production leads to immune complex formation and cytokine release [7], plasma cell hyperreactivity is viewed as a key component of the development and perpetuation of RA [8, 9]. Therefore, B-cell depletion is used to treat RA [10, 11].

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cellular protective processes and modulates the expression of several antioxidant and anti-inflammatory genes. Nrf2 activation may regulate the production of factors implicated in the pathophysiology of rheumatic diseases, including RA [12], lupus nephritis [13], and osteoarthritis [14]. There has been little research on the effect of Nrf2 activity on B cells. One study examined the effect of Nrf2 activity on B cells in an animal model of chronic lung inflammation [15].

In this study, plasma-cell infiltration was significantly greater in Nrf2-/- compared with wild-type mice [15]. Here, we determined whether the anti-arthritic effect of sulforaphane in an animal model of RA is mediated by modulation of B-cell differentiation and activity.

Material and methods

Mice

Seven-week-old male DBA/1J mice (Orient Bio, Gyeonggi-do, Korea) were maintained under specific pathogen-free conditions and fed standard laboratory mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. The animals were housed five per cage in a room maintained under controlled temperature (21–22°C) and lighting (12 h light/dark cycle) conditions. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the School of Medicine and the Animal Research Ethics Committee of the Catholic University of Korea and were conducted in accordance with the Laboratory Animals Welfare Act according to the Guide for the Care and Use of Laboratory Animals. All experimental procedures were evaluated and conducted in accordance with the protocols approved by the Animal Research Ethics Committee at the Catholic University of Korea (Permit Number: CUMC 2016-0086-01). All procedures performed in this study followed the ethical guidelines for animal use.

Clinical scoring of CIA

CIA was induced in DBA1/J mice (n = 5). The experiment was performed three times. Mice were observed twice weekly for the onset, duration, and severity of joint inflammation for 7 weeks after the primary immunization. The mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Knee-joint inflammation was scored visually after dissection on a scale from 0 to 4 (0, normal; 1, mild swelling; 2, moderately severe arthritis involving toes and ankle; 3, severe arthritis involving an entire paw; and 4, severe arthritis). Scoring was performed by two independent observers.

Preparation of type II collagen Ag and immunization

CIA was induced in DBA1/J mice (n = 5). The experiment was performed three times. Male DBA/1J mice were immunized intradermally at the base of the tail with 100 μg of bovine type II collagen (CII) emulsified in complete Freund’s adjuvant (CFA; Arthrogen-CIA, Redmond, WA, USA) (1:1, w/v). Two weeks later, the mice were boosted by intradermal injection with 100 μg of bovine CII in incomplete Freund’s adjuvant (IFA; DIFCO, Detroit, MI, USA) (1:1, v/v). Three weeks after the primary immunization, collagen-induced arthritis (CIA) mice were injected intraperitoneally with 1.5 mM sulforaphane (200 μL of sulforaphane at 12.8 mg/mL/kg) every other day for 7 weeks. Control mice received vehicle (phosphate buffered saline) alone.

Histological assessment of arthritis

The hematoxylin and eosin-stained sections were scored for inflammation and cartilage damage. Inflammation was scored as follows: score 0, no inflammation; score 1, slight thickening of the lining layer or some infiltrating cells in the sublining layer; score 2, slight thickening of the lining layer plus some infiltrating cells in the sublining layer; score 3, thickening of the lining layer, influx of cells in the sublining layer, and presence of cells in the synovial space; and score 4, synovium highly infiltrated with many inflammatory cells. Scoring of cartilage erosion was performed as follows: score 0, no destruction; score 1, minimal erosion limited to single spots; score 2, slight-to-moderate erosion in a limited area; score 3, extended erosions; and score 4, general destruction. Neutrophils were enumerated in three adjacent sections.

Immunohistochemistry

Immunohistochemistry was performed using the Vectastatin ABCkit (Vector Laboratories, Burlingame, CA, USA). Joint tissue of sulforaphane- and vehicle-treated mice was incubated with primary antibodies to interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, receptor activator of NF-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. The sections were counterstained with hematoxylin and photographed using an Olympus photomicroscope (Tokyo, Japan). Mouse joint tissue was fixed in 4% paraformaldehyde, decalcified in ethylenediaminetetraacetic acid bone decalcifier, and embedded in paraffin. The resulting sections (7 μm) were stained with hematoxylin and eosin, Safranin O, and toluidine blue to detect proteoglycans. The number of positive cells was counted using Adobe Photoshop software (Adobe, USA) on high-power digital image (magnifcation: 400×). Positive cells were enumerated visually by three individuals, and the mean values were calculated.

Collagen-specific IgG assay

Serum was collected 7 weeks after the first immunization for determination of the collagen-specific total IgG, IgG1, and IgG2a levels. CII (40 μg/mL) in coating buffer (0.05 M sodium carbonate anhydrous in distilled water, pH 9.6) was used to coat 96-well flat-bottom plates at 4°C overnight. Serially diluted serum samples were incubated in the wells for 1 h at room temperature. The wells were washed with washing buffer (phosphate-buffered saline containing 50 mM Tris, 0.14 M NaCl, and 0.05% Tween 20), and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was added (Bethyl Laboratories, Montgomery, TX, USA). HRP activity was assayed by adding tetramethylbenzidine solution (eBioscience) and determining the absorbance at 450 nm.

Enzyme-linked immunosorbent assay

The supernatant was collected 3 days after sulforaphane treatment. The IL-6, IL-17, and TNF-α levels were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems). The absorbance at 405 nm was measured using an ELISA microplate reader (Molecular Devices).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrasolium bromide assay

Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the ability of mitochondria of viable cells to convert soluble MTT into an insoluble purple formazan reaction product. Cells were treated with MTT solution (5 mg/mL in Dulbecco’s modified Eagle’s medium without phenol red; Sigma-Aldrich) for 2 h. The MTT solution was aspirated and replaced with 200 mL/well dimethyl sulfoxide (DMSO). The absorbance at 540 nm of 100 mL of the reaction mixture was read at 540 nm. The results of two independent experiments performed in duplicate were pooled.

Isolation and stimulation of splenocytes

Splenocytes were prepared from the spleens of normal C57BL/6 mice. Splenocytes were maintained in RPMI1640 medium supplemented with 5% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) before being stimulated with lipopolysaccharide (LPS; 100 ng/mL, Sigma-Aldrich) for 3 days and subjected to flow cytometry.

Flow cytometry

Splenocytes were immunostained with various combinations of the following fluorescence-conjugated antibodies: APC-B220 (eBioscience, San Diego, CA, USA), PE-CD138, and FITC-GL-7 (BD Bioscience, San Diego, CA, USA). Events were collected on a FACSCalibur instrument (BD Bioscience). Data were analyzed using Flow Jo software (ver. 7.6; Treestar, Ashland, OR, USA).

Isolation of peripheral blood mononuclear cells and synovial fluid mononuclear cells

We prepared peripheral blood mononuclear cells (PBMCs) from heparinized blood and synovial fluid mononuclear cells (SFMCs) from heparinized synovial fluids by standard density gradient centrifugation over Ficoll–Paque (GE Healthcare Biosciences, Uppsala, Sweden). Cells were cultured in RPMI-1640 medium (Gibco BRL, Carlsbad, CA, USA) containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% FBS (Gibco BRL) that had been inactivated by heating to 55°C for 30 min. Suspensions of both cell types were dispensed into 48-well plates (Nunc, Rosklide, Denmark). Our study was approved by the institutional review board of Bucheon St. Mary’s Hospital and was performed in accordance with the Helsinki II Declaration. All patients were informed and gave their written consent.

Statistical analyses

Statistical analyses were conducted using the nonparametric Mann–Whitney U-test for comparisons of two groups and one-way analysis of variance with Bonferroni’s post hoc test for comparisons of multiple groups. GraphPad Prism (ver. 5.01; GraphPad Software Inc., San Diego, CA, USA) was used for all analyses. P < 0.05 was the threshold for statistical significance. The data are presented as means ± standard deviation (SD).

Results

Sulforaphane attenuated RA

First, we investigated the effect of sulforaphane on inflammation and joint destruction in CIA mice, which is dependent on autoantibody production [16]. Sulforaphane treatment of CIA mice ameliorated arthritis compared with vehicle-treated mice (Figs 1A and S1). Histological sections of hind-paw joints showed that sulforaphane attenuated inflammation, cartilage damage, and bone erosion (Fig 1B).

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

Fig 1

Open in a new tab

(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.

Anti-inflammatory effect of sulforaphane is associated with reduced expression of inflammatory cytokines in the joints of CIA mice

Next, we investigated the expression of inflammatory cytokines in vehicle- and sulforaphane-treated CIA mice. IL-6, IL-17, and TNF-α are proinflammatory cytokines that participate in the inflammatory process in the RA synovium and have systemic effects [17, 18]. Compared with vehicle-treated CIA mice, the joints from sulforaphane-treated CIA mice showed significantly fewer IL-6-, IL-17-, and TNF-α-expressing cells (Fig 2A). The receptor activator of NF-κB (RANK)/RANKL pathway plays a critical role in mediating articular bone erosion in RA. Focal bone loss in RA joints is mediated by osteoclasts, which express TRAP. Thus, we investigated the effect of sulforaphane on RANKL and TRAP expression in the joints of CIA mice. The reduced numbers of RANKL- and TRAP-positive cells in the joints of CIA mice treated with sulforaphane indicated suppression of osteoclastogenic activity (Fig 2A).

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

Fig 2

Open in a new tab

(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).

Next, we investigated the effect of sulforaphane on the production of proinflammatory cytokines. Splenocytes were isolated from C57BL/6 mice and cultured in the presence of an anti-CD3 antibody with or without sulforaphane (1 to 50 μM) for 72 h. The IL-6, IL-17, and TNF-α levels in the culture supernatant were decreased by sulforaphane in a dose-dependent manner (Fig 2B). By MTT assay, up to 50 μM sulforaphane was not cytotoxic to murine splenocytes (Fig 2C).

Effect of sulforaphane on B-cell differentiation in vitro and Ig production

Sulforaphane suppressed differentiation into CD138+B220low plasma cells and GL7+B220+ germinal-center B cells compared with vehicle (Fig 3A). The differentiation of B cells into antibody-secreting plasma cells is an antigen-driven and cytokine-dependent process. This suppression of differentiation into plasma cells by sulforaphane was due to decreased production of proinflammatory cytokines (Fig 2B). Therefore, we investigated the effect of sulforaphane on autoantibody production in vivo; sulforaphane-treated CIA mice had lower circulating levels of CII-specific IgG, IgG1, and IgG2a (Fig 3B).

Fig 3. Sulforaphane alters B cell differentiation.

Fig 3

Open in a new tab

(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.

Sulforaphane attenuated the production of IL-6, TNF-α, IL-17, and IgG in human PBMCs

Next, we confirmed that the anti-inflammatory and B cell-inhibitory effects of sulforaphane occur in human cells. Human PBMCs were cultured in the presence of absence of sulforaphane (dose, 1 to 10 μM) for 72 h in the presence of anti-CD3. Sulforaphane significantly inhibited the production of IL-6, TNF-α, and IL-17 by human PBMCs in a dose-dependent manner (Fig 4A). The production of IgG by RA SFMCs was inhibited by sulforaphane in a dose-dependent manner (Fig 4B). Therefore, sulforaphane attenuated RA by inhibiting the production of pathologic Igs.

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

Fig 4

Open in a new tab

(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.

Discussion

In this study, sulforaphane reduced the clinical and histologic scores of CIA mice. The anti-arthritic and anti-inflammatory effects of sulforaphane were due to suppression of the differentiation of naïve cells into plasma cells and GC B cells. This is the first report that sulforaphane exerts an anti-arthritic effect by regulating B-cell differentiation. The efficacy of sulforaphane in inflammatory arthritis including RA and gout has been shown in several in vitro and in vivo studies [19–21]. However, these studies did not postulate the mechanisms underlying the anti-arthritic effect [20, 22, 23]. Several studies that have investigated the efficacy of sulforaphane on T cells have been published in the past. Kong JS et al previously demonstrated the anti-inflammatory property of sulforaphane in RA T cells, regarding T cell proliferation as well as inflammatory cytokines production such as IL-17 and TNFα [20]. Liang J et al recently reported T cell-specific effects of sulforaphane especially regarding Th17 cell differentiation (including the expression of transcriptional factor RORγt and their related cytokines such as IL-17 and IL-22) [24]. To our knowledge, no study has investigated the effect of sulforaphane on B-cell differentiation. Because plasma cells are not affected by conventional immunosuppressive drugs such as steroids, cyclophosphamide, and B-cell-depleting agents, our finding that sulforaphane suppresses their differentiation into plasma cells is encouraging and suggests that plasma cell-targeted treatment strategies for RA may be effective.

The CIA mice exhibited T cell- as well as B cell-mediated immune responses against CII. CII-specific antibodies are important pathogenic factors in CIA mice, because they induce the destruction of cartilage and activation of synovial cells [25, 26]. The presence of GCs indicates a pathologic RA synovium. GCs generate plasma cells as well as memory B cells. Thus, a GC-targeting strategy may be effective against RA. Here, we showed that sulforaphane treatment of murine splenocytes suppressed their differentiation into GL7+B220+ GC B cells as well as CD138+ plasma cells. Similarly, Dahada et al. reported that GC B cells play a pivotal role in CIA [27].

In addition to autoantibody production, B cells, like T cells and macrophages, produce proinflammatory cytokines [28, 29]. B cells induce immune-cell trafficking by producing chemokines, to which they respond via cell-surface chemokine receptors. Furthermore, B cells act as antigen-presenting cells during the development of T cell-mediated autoimmune diseases [30]. Classically, B-cell activation is dependent on T cells. Affinity maturation takes place in germinal centers after B cells encounter antigen and receive T-cell help via the CD40/CD154 interaction. However, B cells can also be activated in the absence of direct T-cell help via FcγRIIb and a Toll-like receptor [31, 32].

Our findings suggest suppression of B cells, irrespective of T-cell activity suppression, to be a rational anti-inflammatory strategy for RA. However, few B cell-directed therapies for RA are available; one example is rituximab, a CD20 monoclonal antibody. However, rituximab is typically used as a second-line therapy rather than as a primary therapeutic agent. Thus, a new B cell-targeting therapeutic agent is needed.

In conclusion, systemic administration of sulforaphane attenuated arthritis and histological inflammation in a murine model of RA. Sulforaphane significantly reduced the expression of proinflammatory cytokines in inflamed joints. This anti-inflammatory effect of sulforaphane was due to inhibition of B-cell differentiation into CD138+B22low plasma cells and GL7+B220+ GC B cells. This is the first report that the anti-inflammatory effect of sulforaphane is mediated by inhibition of the differentiation of B cells into GC B cells and plasma cells, which are implicated in the pathogenesis of RA.

Supporting information

S1 Fig. Sulforaphane-mediated inhibition of the development of CIA (A,B).

A,B. Reduction in arthritis score in CIA mice treated with Sulforaphane. CIA mice were injected intraperitoneal injected with sulforaphane (1.5mM, 12.8mg/mL/kg) every other day 7 weeks.

(DOCX)

Click here for additional data file. (131.3KB, docx)

Abbreviations

RA

rheumatoid arthritis

CIA

collagen induced arthritis

Nrf2

Nuclear factor erythroid 2-related factor 2

TNF-α

tumor necrosis factor-α

IL

interleukin

CCP

citrullinated peptide

RF

rheumatoid factor

Data Availability

All relevant data are within the manuscript.

Funding Statement

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health & Welfare, Republic of Korea(HI14C1851). This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. NRF-2018R1C1B6005854). This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. NRF-2018R1A2B6007648).

References

  • 1.Steiner G, Smolen J. Autoantibodies in rheumatoid arthritis and their clinical significance. Arthritis Res. 2002;4 Suppl 2:S1–5. 10.1186/ar551 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Burmester GR, Feist E, Dorner T. Emerging cell and cytokine targets in rheumatoid arthritis. Nature reviews Rheumatology. 2014;10(2):77–88. Epub 2013/11/13. 10.1038/nrrheum.2013.168 . [DOI] [PubMed] [Google Scholar]
  • 3.Verheul MK, Fearon U, Trouw LA, Veale DJ. Biomarkers for rheumatoid and psoriatic arthritis. Clinical immunology (Orlando, Fla). 2015;161(1):2–10. Epub 2015/05/03. 10.1016/j.clim.2015.04.005 . [DOI] [PubMed] [Google Scholar]
  • 4.Syversen SW, Gaarder PI, Goll GL, Odegard S, Haavardsholm EA, Mowinckel P, et al. High anti-cyclic citrullinated peptide levels and an algorithm of four variables predict radiographic progression in patients with rheumatoid arthritis: results from a 10-year longitudinal study. Annals of the rheumatic diseases. 2008;67(2):212–7. Epub 2007/05/29. 10.1136/ard.2006.068247 . [DOI] [PubMed] [Google Scholar]
  • 5.Nielen MM, van Schaardenburg D, Reesink HW, van de Stadt RJ, van der Horst-Bruinsma IE, de Koning MH, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis and rheumatism. 2004;50(2):380–6. Epub 2004/02/12. 10.1002/art.20018 . [DOI] [PubMed] [Google Scholar]
  • 6.Tran CN, Lundy SK, Fox DA. Synovial biology and T cells in rheumatoid arthritis. Pathophysiology: the official journal of the International Society for Pathophysiology. 2005;12(3):183–9. Epub 2005/08/23. 10.1016/j.pathophys.2005.07.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Martinez-Gamboa L, Brezinschek HP, Burmester GR, Dorner T. Immunopathologic role of B lymphocytes in rheumatoid arthritis: rationale of B cell-directed therapy. Autoimmunity reviews. 2006;5(7):437–42. Epub 2006/08/22. 10.1016/j.autrev.2006.02.004 . [DOI] [PubMed] [Google Scholar]
  • 8.Tak PP, Doorenspleet ME, de Hair MJH, Klarenbeek PL, van Beers-Tas MH, van Kampen AHC, et al. Dominant B cell receptor clones in peripheral blood predict onset of arthritis in individuals at risk for rheumatoid arthritis. Annals of the rheumatic diseases. 2017;76(11):1924–30. Epub 2017/08/10. 10.1136/annrheumdis-2017-211351 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 9.De Clerck LS. B lymphocytes and humoral immune responses in rheumatoid arthritis. Clinical rheumatology. 1995;14 Suppl 2:14–8. Epub 1995/09/01. 10.1007/BF02215852 . [DOI] [PubMed] [Google Scholar]
  • 10.Leandro MJ, Edwards JC, Cambridge G. Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion. Annals of the rheumatic diseases. 2002;61(10):883–8. Epub 2002/09/14. 10.1136/ard.61.10.883 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close DR, et al. Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. The New England journal of medicine. 2004;350(25):2572–81. Epub 2004/06/18. 10.1056/NEJMoa032534 . [DOI] [PubMed] [Google Scholar]
  • 12.Wu WJ, Jia WW, Liu XH, Pan LL, Zhang QY, Yang D, et al. S-propargyl-cysteine attenuates inflammatory response in rheumatoid arthritis by modulating the Nrf2-ARE signaling pathway. Redox biology. 2016;10:157–67. Epub 2016/10/17. 10.1016/j.redox.2016.08.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Cordova EJ, Velazquez-Cruz R, Centeno F, Baca V, Orozco L. The NRF2 gene variant, -653G/A, is associated with nephritis in childhood-onset systemic lupus erythematosus. Lupus. 2010;19(10):1237–42. Epub 2010/05/29. 10.1177/0961203310367917 . [DOI] [PubMed] [Google Scholar]
  • 14.Li B, Jiang T, Liu H, Miao Z, Fang D, Zheng L, et al. Andrographolide protects chondrocytes from oxidative stress injury by activation of the Keap1-Nrf2-Are signaling pathway. Journal of cellular physiology. 2018;234(1):561–71. Epub 2018/08/03. 10.1002/jcp.26769 . [DOI] [PubMed] [Google Scholar]
  • 15.Lugade AA, Vethanayagam RR, Nasirikenari M, Bogner PN, Segal BH, Thanavala Y. Nrf2 regulates chronic lung inflammation and B-cell responses to nontypeable Haemophilus influenzae. American journal of respiratory cell and molecular biology. 2011;45(3):557–65. Epub 2011/01/11. 10.1165/rcmb.2010-0321OC [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wang H, Marsters SA, Baker T, Chan B, Lee WP, Fu L, et al. TACI-ligand interactions are required for T cell activation and collagen-induced arthritis in mice. Nature immunology. 2001;2(7):632–7. Epub 2001/06/29. 10.1038/89782 . [DOI] [PubMed] [Google Scholar]
  • 17.McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nature reviews Immunology. 2007;7(6):429–42. Epub 2007/05/26. 10.1038/nri2094 . [DOI] [PubMed] [Google Scholar]
  • 18.Veldhoen M. Interleukin 17 is a chief orchestrator of immunity. Nature immunology. 2017;18(6):612–21. Epub 2017/05/19. 10.1038/ni.3742 . [DOI] [PubMed] [Google Scholar]
  • 19.Silva Rodrigues JF, Silva ESC, Franca Muniz T, de Aquino AF, Neuza da Silva Nina L, Fialho Sousa NC, et al. Sulforaphane Modulates Joint Inflammation in a Murine Model of Complete Freund’s Adjuvant-Induced Mono-Arthritis. Molecules (Basel, Switzerland). 2018;23(5). Epub 2018/04/27. 10.3390/molecules23050988 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kong JS, Yoo SA, Kim HS, Kim HA, Yea K, Ryu SH, et al. Inhibition of synovial hyperplasia, rheumatoid T cell activation, and experimental arthritis in mice by sulforaphane, a naturally occurring isothiocyanate. Arthritis and rheumatism. 2010;62(1):159–70. Epub 2009/12/30. 10.1002/art.25017 . [DOI] [PubMed] [Google Scholar]
  • 21.Fragoulis A, Laufs J, Muller S, Soppa U, Siegl S, Reiss LK, et al. Sulforaphane has opposing effects on TNF-alpha stimulated and unstimulated synoviocytes. Arthritis research & therapy. 2012;14(5):R220 Epub 2012/10/18. 10.1186/ar4059 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Javaheri B, Poulet B, Aljazzar A, de Souza R, Piles M, Hopkinson M, et al. Stable sulforaphane protects against gait anomalies and modifies bone microarchitecture in the spontaneous STR/Ort model of osteoarthritis. Bone. 2017;103:308–17. Epub 2017/08/06. 10.1016/j.bone.2017.07.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yang G, Yeon SH, Lee HE, Kang HC, Cho YY, Lee HS, et al. Suppression of NLRP3 inflammasome by oral treatment with sulforaphane alleviates acute gouty inflammation. Rheumatology (Oxford, England). 2018;57(4):727–36. Epub 2018/01/18. 10.1093/rheumatology/kex499 . [DOI] [PubMed] [Google Scholar]
  • 24.Liang J, Jahraus B, Balta E, Ziegler JD, Hubner K, Blank N, et al. Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione. Frontiers in immunology. 2018;9:2584 Epub 2018/11/30. 10.3389/fimmu.2018.02584 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Nandakumar KS, Holmdahl R. Antibody-induced arthritis: disease mechanisms and genes involved at the effector phase of arthritis. Arthritis research & therapy. 2006;8(6):223 Epub 2007/01/27. 10.1186/ar2089 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Burkhardt H, Koller T, Engstrom A, Nandakumar KS, Turnay J, Kraetsch HG, et al. Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse. Arthritis and rheumatism. 2002;46(9):2339–48. Epub 2002/10/02. 10.1002/art.10472 . [DOI] [PubMed] [Google Scholar]
  • 27.Dahdah A, Habir K, Nandakumar KS, Saxena A, Xu B, Holmdahl R, et al. Germinal Center B Cells Are Essential for Collagen-Induced Arthritis. Arthritis & rheumatology (Hoboken, NJ). 2018;70(2):193–203. Epub 2017/10/19. 10.1002/art.40354 . [DOI] [PubMed] [Google Scholar]
  • 28.Schultze JL, Michalak S, Lowne J, Wong A, Gilleece MH, Gribben JG, et al. Human non-germinal center B cell interleukin (IL)-12 production is primarily regulated by T cell signals CD40 ligand, interferon gamma, and IL-10: role of B cells in the maintenance of T cell responses. The Journal of experimental medicine. 1999;189(1):1–12. Epub 1999/01/05. 10.1084/jem.189.1.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Pistoia V. Production of cytokines by human B cells in health and disease. Immunology today. 1997;18(7):343–50. Epub 1997/07/01. 10.1016/s0167-5699(97)01080-3 . [DOI] [PubMed] [Google Scholar]
  • 30.Serreze DV, Silveira PA. The role of B lymphocytes as key antigen-presenting cells in the development of T cell-mediated autoimmune type 1 diabetes. Current directions in autoimmunity. 2003;6:212–27. Epub 2002/11/01. 10.1159/000066863 . [DOI] [PubMed] [Google Scholar]
  • 31.Boackle SA. Complement and autoimmunity. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2003;57(7):269–73. Epub 2003/09/23. 10.1016/s0753-3322(03)00084-2 . [DOI] [PubMed] [Google Scholar]
  • 32.Carroll MC. The complement system in regulation of adaptive immunity. Nature immunology. 2004;5(10):981–6. Epub 2004/09/30. 10.1038/ni1113 . [DOI] [PubMed] [Google Scholar]

Decision Letter 0

David Douglass Brand

10 Nov 2020

PONE-D-20-25189

The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation.

PLOS ONE

Dear Dr. Cho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers have issues with the details concerning the numbers of mice used in each experiment and reviewer 2 wants specific information about how mice were handled.  How do the second and third arthritis induction experiments compare to that listed in 1A?

Both reviewers share concerns about the specificity of suppressive effects on B cells, suggesting that the effects may either be broad (covering multiple cell types) or worse, non-specific in nature.  These points will need to be addressed before this manuscript can be considered for publication. 

Reviewer 1's comment that "There is really no experiments in the manuscript documenting that sulforaphane is actually an activator of Nrf2" could be handled either through experimentation or simply through highlighting evidence from the literature, but it should be acknowledged somehow.

I would point out that the flow cytometry data needs attention as well.  It is difficult to determine exactly how the regions outlining specific populations were chosen. I have attached a copy of figure 3 on which annotations have been made to point out specifics.

The first issue is that of gating.  How are the dot plots shown in Figure 3A gated?  The only antibodies listed were those for B220, CD138 and GL-7.  Sometimes it is helpful to include a plot of ex-vivo splenocytes as a comparison to demonstrate that your antibodies have identified the populations that you intended.  

The box used to define B220low/CD138+ plasma cells does not really seem to specify any definable population even in the vehicle group.  It appears to cover B220 negative cells.   If one wants to define B220lo and B220hi populations, then arguably the best division may be the orange line I have indicated on the annotation, which would then make the plasma cells fall into the magenta colored rectangle.  This might change the conclusions.

At any rate, a more complete description of the strategies used to define specific populations should be provided.

--

Please submit your revised manuscript by Dec 25 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

David Douglass Brand

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS ONE requires experimental methods to be described in enough detail to allow suitably skilled investigators to fully replicate and evaluate your study. See https://journals.plos.org/plosone/s/submission-guidelines#loc-materials-and-methods for more information.

To comply with PLOS ONE submission guidelines, in your Methods section, please provide a more detailed description of your immunohistochemistry, ELISA, and flow cytometry methodology. In addition, please ensure that you describe the sources, catalog numbers, and dilutions of all primary and secondary antibodies used in your study.

3. Thank you for stating the following in the Competing Interests section:

'The authors declare that there is no conflict of interest regarding the publication of this article.' 

We note that one or more of the authors are employed by a commercial company: "Impact Biotech".

1.     Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

* Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Additional Editor Comments (if provided):

I would personally like to apologize for the length of time it took to get this review completed. Due to current circumstances, finding appropriately qualified reviewers for this work was particularly challenging but those agreeing to do so are indeed highly qualified for this subject area. Please look over their responses carefully as you decide how to proceed with this important work.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation

The manuscript investigates the effect of sulforaphane on collagen-induced arthritis. Mice are treated with sulforaphane intraperitoneally and the investigators demonstrate that there is a decrease in severity of arthritis. This is associated with decreases in histologic inflammation, decreases in cytokines IL-6, IL-17, TNF-a, and TRAP. The investigators also demonstrate that human PBMs can have inhibition in production of IL-6, TNF-a, and IL-17 when cultures with sulforaphane. This is an important finding and may have therapeutic implications.

However, there are some problems with the manuscript.

The title is misleading. The effects of sulforaphane appear to be broad and cause inhibition of both T cell cytokines, as well as antibody production and inhibition of differentiation into plasma cells. Therefore, the focus on B cells is misleading.

There is really no experiments in the manuscript documenting that sulforaphane is actually an activator of Nrf2.

There was no clear indication as to how many mice were used in the experiments described in figure 1A. It is important to know the number of mice in order to evaluate the validity of the results.

For the flow experiments, it is also important to know how many mice were evaluated and what are the means of the populations evaluated.

Reviewer #2: This paper shows that sulforophan, known as an inhibitor of Nrf2, suppress collagen induced arthritis. It is claimed it do so based on its effects on B cells. Major points:

1) All data are not shown. Its not acceptable to base a study on an arthritis experiment with n=5. Apparently the experiment has ben run three times so this is a selected experiment. If the same experiment has been done 3 times it should be pooled and calculated together. The pooled data can be shown in the paper and the single experiments int he suppl information.

2) It should be clearly stated that the experiment was done blindly and distributed in the cages randomly, especially as it is well known there is a strong cage effect in DBA/1 mice. It should also be stated that all animal experiments follow the ARRIVE guideline. Of course only if this was the case.

3) The treatment has a profound effect on the inflammatory response. It is likely to ha ve very broad effects and it will be difficult to say exactly what is the specific effect. Basically all readouts a re secondary effects to something that this high dose of sulforophan is dong, whatever that is. Thus, it is not possible to claim that the effect on arthritis is due to effects on B cells as there is no evidence for this. The treatment is given 3 weeks after priming which means that the B cells have been primed and a full antibody response been developed. What will happen is that if these mice, due to this unknown "toxic" effect of the treatment does not develop arthritis it will secondarily, dur to less exposure of inflamed cartilage as well as a less powerful immune system give lower antibody titres. It can be predicted whatever is given to a mice leading to such a suppression of arthritis development.

In conclusion. If the arthritis data holds its a valuable report. But the authors need to make it very clear that they cannot say anything about the specific effects about sulforophan action as all evidenced data are secondary to the arthritis effect per se. Regarding human cells it seems to have profound inhibitory effect on cytokine production and I am afraid that these cells are not very happy.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Figure 3 (Comments).pdf

Click here for additional data file. (1.1MB, pdf)
PLoS One. 2021 Feb 16;16(2):e0245986. doi: 10.1371/journal.pone.0245986.r002

Author response to Decision Letter 0

1 Dec 2020

Revised submission of manuscript PONE-D-20-25189

Dear David Douglass Brand

Editor-in-PLOS ONE

We thank the reviewers for their constructive and helpful comments concerning the manuscript. We have addressed the reviewers' concerns by either performing additional statistical analysis and revising the manuscript or explaining respectfully our rebuttal. The point-by-point replies are given in this letter. We hope that we have addressed satisfactorily all concerns raised by the reviewers, and that this manuscript is now suitable for publication.

Thank you again for the comments.

Sincerely yours,

Mi-La Cho, PhD

Department of Medical Lifescience, College of Medicine, The Catholic University of Korea, 222, Banpo-daero, Seocho-gu, Seoul, 06591, Republic of Korea

(Tel: 82 2 2258 7467, Fax: 82 2 2258 7473, E-mail address: iammila@catholic.ac.kr)

Comments from the editors and reviewers:

Reviewer #1: The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation

The manuscript investigates the effect of sulforaphane on collagen-induced arthritis. Mice are treated with sulforaphane intraperitoneally and the investigators demonstrate that there is a decrease in severity of arthritis. This is associated with decreases in histologic inflammation, decreases in cytokines IL-6, IL-17, TNF-a, and TRAP. The investigators also demonstrate that human PBMs can have inhibition in production of IL-6, TNF-a, and IL-17 when cultures with sulforaphane. This is an important finding and may have therapeutic implications.

However, there are some problems with the manuscript.

The title is misleading. The effects of sulforaphane appear to be broad and cause inhibition of both T cell cytokines, as well as antibody production and inhibition of differentiation into plasma cells. Therefore, the focus on B cells is misleading.

Answer: Thanks for valuable comments. The authors agree with your comments and revised as you required. The title was revised as follows. “The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation and inflammation cytokine.

There are really no experiments in the manuscript documenting that sulforaphane is actually an activator of Nrf2.

Answer: Thanks for your comments. We have no experimtents in the manuscript. But, many

studies have shown that Sulforaphane is a potent activator of the endogenous anti-oxidant

transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2).

Reference

1. Eri Kubo, Bhavana Chhunchha, Prerna Singh, Hiroshi Sasaki & Dhirendra P. Singh. Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress. Scientific Reports, 7:14130 (2017)

2. Albena T. Dinkova-Kostova, Jed W. Fahey and Thomas W. Kensler. KEAP1 and done? Targeting the NRF2 pathway with sulforaphane. Trends in Food Science & Technology, 69:257-269(2017)

3. Christine A. Houghton, Robert G. Fassett, and Jeff S. Coombes. Sulforaphane and Other Nutrigenomic Nrf2 Activators: Can the Clinician’s Expectation Be Matched by the Reality? Oxidative Medicine and Cellular Longevity, 7857186 (2016)

There was no clear indication as to how many mice were used in the experiments described in figure 1A. It is important to know the number of mice in order to evaluate the validity of the results. For the flow experiments, it is also important to know how many mice were evaluated and what are the means of the populations evaluated.

Answer: The authors agree with your comments and revised as you required. We added the method section. (marked by red color, Page_5_, line 16_). We analyzed the experiments 3 times and showed the most representative data.

Reviewer #2: This paper shows that sulforophan, known as an inhibitor of Nrf2, suppress collagen induced arthritis. It is claimed it do so based on its effects on B cells.

Major points:

1) All data are not shown. Its not acceptable to base a study on an arthritis experiment with n=5. Apparently the experiment has ben run three times so this is a selected experiment. If the same experiment has been done 3 times it should be pooled and calculated together. The pooled data can be shown in the paper and the single experiments in the suppl information.

Answer: Thanks for valuable comment which can clarify the method of present study. The experiment was performed three times. We analyzed the experiments 3 times and showed the most representative data. As the trend of the experiment was consistent, it was shown as a representative picture.

2) It should be clearly stated that the experiment was done blindly and distributed in the cages randomly, especially as it is well known there is a strong cage effect in DBA/1 mice. It should also be stated that all animal experiments follow the ARRIVE guideline. Of course only if this was the case.

Answer: Thanks for comments. We performed grouping prior to Sulforaphane administration. To proceed with group separation, arthritis score and serum CII specific IgG2a measurement were analyzed. Then, group divide was conducted by ranking the results of two analyzes of arthritis score and CII specific IgG2a.

All experimental procedures were evaluated and conducted in accordance with the protocols approved by the Animal Research Ethics Committee at the Catholic University of Korea (Permit Number: CUMC 2016-0086-01). All procedures performed in this study followed the ethical guidelines for animal use.

3) The treatment has a profound effect on the inflammatory response. It is likely to have very broad effects and it will be difficult to say exactly what is the specific effect. Basically all readouts are secondary effects to something that this high dose of sulforophan is dong, whatever that is. Thus, it is not possible to claim that the effect on arthritis is due to effects on B cells as there is no evidence for this. The treatment is given 3 weeks after priming which means that the B cells have been primed and a full antibody response been developed. What will happen is that if these mice, due to this unknown "toxic" effect of the treatment does not develop arthritis it will secondarily, dur to less exposure of inflamed cartilage as well as a less powerful immune system give lower antibody titres. It can be predicted whatever is given to a mice leading to such a suppression of arthritis development.

In conclusion. If the arthritis data holds its a valuable report. But the authors need to make it very clear that they cannot say anything about the specific effects about sulforophan action as all evidenced data are secondary to the arthritis effect perse. Regarding human cells it seems to have profound inhibitory effect on cytokine production and I am afraid that these cells are not very happy.

Answer: Thanks for comments. We agree with the referee’s concern. We also had a lot of thinking. Because high ROS concentrations may lead, through oxidative damage of cellular constituents, to various disorders. It was thought that it was important to find a concentration that could sustain homeostasis without toxic effects. We could expand the research in future study. We will expand our research in future studies.

Attachment

Submitted filename: Revised submission of manuscript PONE.docx

Click here for additional data file. (25.1KB, docx)

Decision Letter 1

David Douglass Brand

8 Dec 2020

PONE-D-20-25189R1

The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation and inflammation cytokine.

PLOS ONE

Dear Dr. Cho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

• Address the subject of the under-powered nature of the CIA study (both reviewers) either through the pooling of data or through new CIA experiments.  Your response did not properly address these previously.

• Provide the missing flow cytometric data in Figure three showing the nature of the splenocytes as taken from each group (sulforaphane treated and untreated) of CIA mice. 

==============================

Please submit your revised manuscript by Jan 22 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

David Douglass Brand

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Both reviewers showed concerns regarding the numbers of mice used in the CIA experiment. The second reviewer suggested pooling data and supplying individual experiments as supplementary data. These concerns are shared by this editor in that under no circumstances will five mice per group properly power a CIA experiment. A minimum of twice that number is required, with even larger numbers necessary in experiments where the detection of subtle differences are required. However, your response to the reviewers did not address their concerns. I am afraid that providing more information than that or new data using larger numbers per group will be required before these answers can be met.

There is an additional problem in that it appears that some data are inadvertently missing from this publication. Figure 3 suggests that there are flow data available from cells analyzed ex vivo from sulforaphane treated mice. This is very confusing because the section is entitled "Effect of sulforaphane on B-cell differentiation *in vitro* and Ig production". The second and third sentences say "The results showed that the population of CD138+B220low plasma cells was significantly decreased by sulforaphane treatment in CIA mice, whereas the population of GL7+B220+ germinal-center B cells did not differ between the two groups (Fig. 3a). Then, in vitro experiments were performed to determine the effects of sulforaphane on B cell differentiation. " This suggests that the first set of data was a flow cytometric analysis of splenocytes from each group of mice demonstrating a reduction in CD138+B220low plasma cells but no reduction in GL7+B220+ germinal-center B cells. Figure 3a, which is referred to twice in this section appears to only show the in vitro data. The labeling demonstrates the concentrations (5 and 10 µM sulforaphane) and clearly shows in vitro data due to the LPS stimulated absence of T cells (very few double negative cells) under any condition. What happened to the flow data demonstrating a reduction in CD138+B220low plasma cells but no reduction in GL7+B220+ germinal-center B cells taken from CIA mouse spleens?

Other points are

The title that you suggested including the words "and inflammation cytokine" is not proper English usage. Perhaps "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"

Strike the word "etiological" from the last sentence of the abstract or provide more information about what your were trying to say.

I am aware that this decision is a very difficult one to resolve, but since this editor agrees with the concerns of the two reviewers regarding properly powering the study, it cannot be ignored as you have done in your response.

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 16;16(2):e0245986. doi: 10.1371/journal.pone.0245986.r004

Author response to Decision Letter 1

2 Jan 2021

Comments from the editors and reviewers:

Reviewer #1: The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation

The manuscript investigates the effect of sulforaphane on collagen-induced arthritis. Mice are treated with sulforaphane intraperitoneally and the investigators demonstrate that there is a decrease in severity of arthritis. This is associated with decreases in histologic inflammation, decreases in cytokines IL-6, IL-17, TNF-a, and TRAP. The investigators also demonstrate that human PBMs can have inhibition in production of IL-6, TNF-a, and IL-17 when cultures with sulforaphane. This is an important finding and may have therapeutic implications.

Answer: We do appreciate the valuable comments from the reviewers about the manuscript. We have gone through the suggestive comments from all the reviewers and incorporated in the final revised version.

However, there are some problems with the manuscript.

The title is misleading. The effects of sulforaphane appear to be broad and cause inhibition of both T cell cytokines, as well as antibody production and inhibition of differentiation into plasma cells. Therefore, the focus on B cells is misleading.

Answer: Thanks for valuable comments. The authors agree with your comments and revised as you required. The title was revised as follows. “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.”

There are really no experiments in the manuscript documenting that sulforaphane is actually an activator of Nrf2.

Answer: Thanks for your comments. We have no experiments in the manuscript. But, many

studies have shown that Sulforaphane is a potent activator of the endogenous anti-oxidant

transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2).

Reference

1. Eri Kubo, Bhavana Chhunchha, Prerna Singh, Hiroshi Sasaki & Dhirendra P. Singh. Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress. Scientific Reports, 7:14130 (2017)

2. Albena T. Dinkova-Kostova, Jed W. Fahey and Thomas W. Kensler. KEAP1 and done? Targeting the NRF2 pathway with sulforaphane. Trends in Food Science & Technology, 69:257-269(2017)

3. Christine A. Houghton, Robert G. Fassett, and Jeff S. Coombes. Sulforaphane and Other Nutrigenomic Nrf2 Activators: Can the Clinician’s Expectation Be Matched by the Reality? Oxidative Medicine and Cellular Longevity, 7857186 (2016)

There was no clear indication as to how many mice were used in the experiments described in figure 1A. It is important to know the number of mice in order to evaluate the validity of the results. For the flow experiments, it is also important to know how many mice were evaluated and what are the means of the populations evaluated.

Answer: The authors agree with your comments and revised as you required. We added the method section. (marked by red color, Page_5_, line 17_). We analyzed the experiments 3 times and showed the most representative data. We provided all the arthritis score graphs. These graphs are shown below.

Reviewer #2: This paper shows that sulforophan, known as an inhibitor of Nrf2, suppress collagen induced arthritis. It is claimed it do so based on its effects on B cells.

Answer: We really appreciate your time and effort to edit our manuscript. In this revised manuscript, we have resolved most of the issues raised by the reviewers as you can see in our response to their comments below.

Major points:

1) All data are not shown. Its not acceptable to base a study on an arthritis experiment with n=5. Apparently the experiment has ben run three times so this is a selected experiment. If the same experiment has been done 3 times it should be pooled and calculated together. The pooled data can be shown in the paper and the single experiments in the suppl information.

Answer: Thanks for valuable comment which can clarify the method of present study. The experiment was performed three times. We analyzed the experiments 3 times and showed the most representative data. As the trend of the experiment was consistent, it was shown as a representative picture.

2) It should be clearly stated that the experiment was done blindly and distributed in the cages randomly, especially as it is well known there is a strong cage effect in DBA/1 mice. It should also be stated that all animal experiments follow the ARRIVE guideline. Of course only if this was the case.

Answer: Thanks for comments. We performed grouping prior to Sulforaphane administration. To proceed with group separation, arthritis score and serum CII specific IgG2a measurement were analyzed. Then, group divide was conducted by ranking the results of two analyzes of arthritis score and CII specific IgG2a.

All experimental procedures were evaluated and conducted in accordance with the protocols approved by the Animal Research Ethics Committee at the Catholic University of Korea (Permit Number: CUMC 2016-0086-01). All procedures performed in this study followed the ethical guidelines for animal use.

3) The treatment has a profound effect on the inflammatory response. It is likely to have very broad effects and it will be difficult to say exactly what is the specific effect. Basically all readouts are secondary effects to something that this high dose of sulforophan is dong, whatever that is. Thus, it is not possible to claim that the effect on arthritis is due to effects on B cells as there is no evidence for this. The treatment is given 3 weeks after priming which means that the B cells have been primed and a full antibody response been developed. What will happen is that if these mice, due to this unknown "toxic" effect of the treatment does not develop arthritis it will secondarily, dur to less exposure of inflamed cartilage as well as a less powerful immune system give lower antibody titres. It can be predicted whatever is given to a mice leading to such a suppression of arthritis development.

In conclusion. If the arthritis data holds its a valuable report. But the authors need to make it very clear that they cannot say anything about the specific effects about sulforophan action as all evidenced data are secondary to the arthritis effect perse. Regarding human cells it seems to have profound inhibitory effect on cytokine production and I am afraid that these cells are not very happy.

Answer: Thanks for comments. We agree with the referee’s concern. As pointed out by the reviewers, there is insufficient direct evidence for inducing the immunomodulatory effect of B cells in the animal model of rheumatoid arthritis as a direct response to the drug. Antioxidants such as sulforafan can appear when excessive inhibition of oxidative stress is accompanied by an inflammatory response. Therefore, when using antioxidant drugs, dosages that do not have cellular toxic effects and inflammatory responses should be used. However, there seems to be a difficulty because the dosage used for the drug is not proportional even between in vitro and in vivo experiments. As a reviewer's opinion, it seems difficult to identify directly in vivo that the therapeutic effect in an animal model is a direct regulatory response to B cells from our results. In the future, we will take this part into account and try to confirm it in subsequent studies. Thank you very much for your review comment.

Attachment

Submitted filename: Response to peer-reviewers_PLoS ONE.docx

Click here for additional data file. (178.9KB, docx)

Decision Letter 2

David Douglass Brand

12 Jan 2021

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.

PONE-D-20-25189R2

Dear Dr. Cho,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

David Douglass Brand

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

So long as the two additional severity scatter plots/Arthritis score graphs which were provided in your second response (Revision 2) are provided to the readers in the supplemental data section, the manuscript is acceptable.

However,

The wording is still confusing on page 11 would suggest removing lines 2 through 5 entirely.

Instead, start the section with line 6 but remove the word "Then"

Reviewers' comments:

Attachment

Submitted filename: Screen Shot 2021-01-11 at 4.07.43 PM.png

Click here for additional data file. (161.5KB, png)

Acceptance letter

David Douglass Brand

3 Feb 2021

PONE-D-20-25189R2

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.

Dear Dr. Cho:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. David Douglass Brand

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Sulforaphane-mediated inhibition of the development of CIA (A,B).

    A,B. Reduction in arthritis score in CIA mice treated with Sulforaphane. CIA mice were injected intraperitoneal injected with sulforaphane (1.5mM, 12.8mg/mL/kg) every other day 7 weeks.

    (DOCX)

    Click here for additional data file. (131.3KB, docx)
    Attachment

    Submitted filename: Figure 3 (Comments).pdf

    Click here for additional data file. (1.1MB, pdf)
    Attachment

    Submitted filename: Revised submission of manuscript PONE.docx

    Click here for additional data file. (25.1KB, docx)
    Attachment

    Submitted filename: Response to peer-reviewers_PLoS ONE.docx

    Click here for additional data file. (178.9KB, docx)
    Attachment

    Submitted filename: Screen Shot 2021-01-11 at 4.07.43 PM.png

    Click here for additional data file. (161.5KB, png)

    Data Availability Statement

    All relevant data are within the manuscript.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES