Monovalent glycoconjugates of sulforaphane prevent inflammation induced by lipopolysaccharide in human dendritic cells by inhibiting NF-ĸB signalling pathway

RESEARCH ARTICLE Monovalent glycoconjugates of sulforaphane prevent inflammation induced by lipopolysaccharide in human dendritic cells by inhibiting NF-ĸB signalling pathway Camila Leiva-Castro 1 | Ana Maria Múnera-Rodríguez 1 | Macarena Martínez-Bailén 2 | Ana Teresa Carmona 2 | Soledad López-Enríquez 1,3 | Francisca Palomares 1,3 1 Departamento de Bioquímica Médica y Biología Molecular e Inmunología, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain 2 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Sevilla, Spain 3 Institute of Biomedicine of Seville (IBiS)/ Virgen del Rocío University Hospital/Virgen Macarena University Hospital/University of Seville/CSIC, Sevilla, Spain Correspondence Soledad López-Enríquez and Francisca Palomares, Department of Medical Biochemistry and Molecular Biology, and Immunology School of Medicine, Universidad de Sevilla, Av. Sanchez Pizjuan s/n, 41009 Seville, Spain. Email: [email protected] and [email protected] Funding information Ministerio de Ciencia e Innovación, Grant/Award Numbers: PID2020-116460RB100, PID2023-147334OB-I00, RYC2021-031256-I; University of Seville, Grant/Award Numbers: 202200000421, 2023/00000482; Asociación Universitaria Iberoamericana de Postgrado Background and Purpose: Sulforaphane (SFN) has notable health benefits but faces challenges due to its poor solubility and delivery. This study investigates SFN-glycoconjugates effects on lipopolysaccharide (LPS)-induced inflammation in dendritic cells (DCs). With the aiming to enhance their therapeutic potential against inflammatory diseases. Novel monovalent SFN-glycoconjugates with mannose (Man) and fucose (0Fuc) were developed and tested for their anti-inflammatory and immune-modulatory properties in DCs from healthy donors under chronic LPS exposure. Experimental Approach: By leveraging therapeutic strategies, SFN-glycoconjugates significantly improved the solubility and bioavailability of SFN, thereby overcoming the limitations of traditional delivery methods. Monocyte-derived DCs were treated with SFN-glycoconjugates and subsequently exposed to a chronic inflammatory environment induced by LPS. Key Results: Our results showed that SFN-glycoconjugates enhance effectiveness in suppressing inflammation by targeting the p65 NF-κB pathway, without affecting MAPK signalling. SFN-glycoconjugates induce a tolerogenic immune response, characterized by increased IL-10 production and enhanced regulatory Tand B-cell proliferation. These effects surpass those of p65 NF-κB inhibition alone, highlighting a distinct and potent regulatory mechanism independent of MAPK pathways. Conclusion and Implications: The integration of food therapeutic strategies not only enhances the stability and delivery of bioactive compounds but also broadens their potential applications in functional foods and therapeutic approaches. In particular, SFN-glycoconjugates represent a promising option as biologically active compounds for inflammatory diseases, offering enhanced anti-inflammatory and immunomodulatory effects through optimized delivery systems and the activation of specific molecular pathways. Abbreviations: CFSE, 5,6-carboxyfluorescein diacetate N-succinimidyl ester; DCs, dendritic cells; HLA-DR, human leukocyte antigen-DR isotype; L, lipopolysaccharide; SFN, sulforaphane; SFNFuc, SFN-conjugate with fucose; SFNMan, SFN-conjugate with mannose; Treg, regulatory T-cells. Camila Leiva-Castro and Ana Maria Múnera-Rodríguez share first authorship. Soledad López-Enríquez and Francisca Palomares share last authorship. Received: 27 December 2024 Revised: 19 May 2025 Accepted: 26 May 2025 DOI: 10.1111/bph.70115 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2025 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. Br J Pharmacol. 2025;182:5157–5172. wileyonlinelibrary.com/journal/bph 5157 KEYWORDS dendritic cells, glycoconjugates, immunomodulation and regulatory Tand B-cells, inflammation, NF-κB pathway, sulforaphane 1|INTRODUCTION Inflammation is a complex physiological process that plays a crucial role in the organism immune response to various stimuli, ranging from the presence of pathogens to tissue damage and exposure to toxins (L. Chen et al., 2018). Sulforaphane (SFN), a bioactive compound found in cruciferous vegetables such as broccoli, has emerged as a promising candidate for modulating inflammation and its pathological consequences (Fernandez-Prades et al., 2023). SFN has been observed to exert anti-inflammatory effects by influencing key cellular signalling pathways, including the nuclear factor kappa B (NF-κB) pathways, which triggers cellular defence mechanisms, and mitogen-activated protein kinases (MAPKs), suggesting its therapeutic potential in a variety of inflammatory disorders (Deng et al., 2017; Mahn & Castillo, 2021). Our group has recently described that SFN pretreatment may induce a regulatory response by inhibiting NF-κBand MAPK signalling pathways in an inflammatory environment, along with inducing a regulatory pattern in human dendritic cells (DCs) (Fernandez-Prades et al., 2023; MuneraRodriguez et al., 2024). NF-κB is a central regulator of inflammation, controlling the expression of genes involved in immune responses. NF-κB remains inactive in the cytoplasm, sequestered by inhibitory proteins called IκBs (inhibitor of NF-κB(L. Chen et al., 2018; Lawrence, 2009). SFN has been observed to inhibit the release of inflammatory mediators such as thymus and activation-regulated chemokine, eotaxin-1 (CCL11) and vascular cell adhesion molecule-1 in cytokine-stimulated human corneal fibroblasts by blocking MAPK, NF-κB and signal transducer and activator of transcription 6 (STAT6) pathways (Yang et al., 2022). MAPKs represent a group of proteins that regulate cellular responses to diverse triggers, encompassing osmotic stress, growth factors and inflammatory agents like interleukin (IL-1β), tumour necrosis factor α(TNF-α) and IL-6 (Burkhard & Shapiro, 2010; L. Chen et al., 2018). Dysregulation of MAPK pathways is implicated in diseases such as Alzheimer's, Parkinson's and cancer (Kim & Choi, 2010). SFN modulation of MAPK signalling pathways may extend beyond inflammation to influence other cellular processes involved in disease pathogenesis (Saha et al., 2021; Zhang et al., 2022). Despite the promising anti-inflammatory properties caused by SFN (Mahn & Castillo, 2021), its translation into clinical application has encountered challenges due to its inherent limitations in bioavailability and stability. Although SFN demonstrates relatively higher bioavailability compared with some other nutraceuticals (Caponio et al., 2022), its precise mechanism of absorption and immune cell penetration remain inadequately understood. This limited understanding hampers effective administration of SFN and its targeted delivery to specific sites of action within the body (Wang et al., 2020). Consequently, there exists a critical imperative to investigate and develop alternative strategies aimed at augmenting the delivery mechanisms and therapeutic efficacy of SFN. In response to the challenges posed by SFN low bioavailability and stability, glycoconjugates (Kjaerup et al., 2014; Ramos-Soriano & Rojo, 2021) have emerged as a promising strategy to enhance its clinical efficacy in treating inflammatory and related diseases. Glycoconjugates are being actively investigated for their potential applications in vaccine development due to their ability to influence the immune response, specifically through the regulation of allergenspecific effects cells, which involves the activation of DCs (Sirvent et al., 2016). We propose that SFN-glycoconjugates, functionalized with carbohydrates such as mannose or fucose (SFN-conjugate with mannose [SFNMan] and/or SFN-conjugate with fucose [SFNFuc]), could significantly enhance and finely tune the interaction of SFN with immune cells, including DCs, thereby augmenting its therapeutic efficacy (Kjaerup et al., 2014; van Liempt et al., 2006). These What is already known •Sulforaphane (SFN) has anti-inflammatory properties but limited by poor solubility and inefficient delivery methods. •Chronic inflammation, often induced by lipopolysaccharides, in dendritic cells is involved in inflammatory diseases. What does this study add •Development of SFN-glycoconjugates conjugated are more effective than SFN by targeting the p65 NF-κB pathway alone. •However, these effects surpass those of p65 NF-κB inhibition, indicating mechanism independent of MAPK pathways. What is the clinical significance •SFN-glycoconjugates present a breakthrough in overcoming limitations involved in SFN delivery. •Their ability to target specific molecular pathways enhances their potential for treating chronic inflammatory diseases. 5158 LEIVA-CASTRO ET AL. 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License novel biologically active compounds not only aim to optimize the beneficial effects of SFN but also hold considerable promise in mitigating the effects of various inflammation-related diseases, including allergic pathologies (Rodriguez et al., 2019). By enhancing SFN's stability and solubility through conjugation with specific carbohydrates, SFNMan/SFNFuc-glycoconjugates offer a multifaceted approach to combatting inflammatory diseases, potentially leading to improved clinical outcomes and patient well-being, as observed with other glycoconjugates (Gringhuis et al., 2009; Palomares et al., 2022). Compared with alternative administration methods, such as nanoparticles (Zambrano et al., 2019), SFN-glycoconjugates present a range of advantages. Firstly, glycoconjugates exhibit greater stability compared with the free form of the compound, facilitating more effective interactions with immune cells (Palomares et al., 2022). Additionally, glycoconjugates can be designed to enhance the selectivity and specificity of SFN towards specific target cells, thereby reducing systemic exposure and minimizing unwanted side effects (Ribeiro-Viana et al., 2012). These advantages are particularly relevant in the context of chronic inflammatory diseases, where prolonged and targeted drug administration is essential to achieve optimal therapeutic outcomes (Palomares et al., 2019; Rodriguez et al., 2019). In this study, we will explore the potential of SFNglycoconjugates as a promising molecular therapy to improve the clinical efficacy of SFN in the treatment of inflammatory disorders and other related diseases. By gaining a deeper understanding of the molecular mechanisms underlying SFN-glycoconjugates and their ability to modulate inflammatory response, this research aims to develop more effective and targeted molecular therapeutic approaches to address a wide range of inflammatory and autoimmune diseases. 2|METHODS 2.1 |Study subjects Biological samples of peripheral blood were sourced from healthy individuals aged 18 years or older who provided informed consent. Study participants had no recent history of immunoregulatory drug use or immunological disorders. Sample collection was approved by the Research Ethics Committee of Hospitales Universitarios Virgen Macarena, Virgen del Rocío, under code: ID-SOL2022-21799. 2.2 |Sample collection and storing Sample collection involved venipuncture to procure peripheral blood, from which peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll gradient. The peripheral blood mononuclear cells were subsequently cryopreserved in liquid nitrogen. All procedures followed standard protocols established by the HUVM-IBiS Biobank, which operates under the Andalusian Public Health System Biobank framework. 2.3 |Monocyte-derived DCs (moDCs) moDCs were cultured in RMPI 1640 (Invitrogen, Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine,5mgml 1 gentamicin and 50 ngml 1 streptomycin. Differentiation was achieved for 6 days by exposing the cells to 200 ngml 1 GM-CSF and 100 ngml 1 IL-4; the cultured was maintained at 5% CO 2 and 37C (Benito-Villalvilla et al., 2022). 2.4 |MoDCs treatment with bioactive molecules Before treatment, moDCs were incubated in 96-well plates (Thermo Fisher Scientific, Waltham, MA) at 37C and 5% CO 2 for 10 min to allow for basal stabilization. Following this, moDCs were treated under different experimental conditions. Experimental conditions included SFNMan and SFNFuc both at 25 μM (with solubility of 60% PBS and 40% DMSO for both), lipopolysaccharide (LPS) from Escherichia coli 0127: B8 100 ngml 1 as a positive control to induce chronic inflammation (Sigma-Aldrich, St. Louis, MO, USA), SFN 10 μM (soluble in 100% DMSO) and unstimulated moDCs serving as the negative control. The protective properties and the immunological changes induced by the SFN-glycoconjugates were examined by preincubating moDCs with SFN-glycoconjugates for 1 h prior to LPS addition, thereby inducing a strong chronic inflammatory response. To compare their outcomes, the SFN was also incubated for 1 h with LPS to generate chronic inflammation. 2.5 |Signalling assay The Immuno-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018). Monocyte-derived DCs (MoDCs; 7.5 10 5 ) were grown in 96-well plates at 37C and 5% CO 2 for 10 min. For effective intracellular staining of p65 NF-κB and MAPKs, the moDCs were fixed with 1% paraformaldehyde and permeabilized using BD Phosflow Perm Buffer III (Becton, Dickinson, [BD], Franklin Lakes, NJ). Specific monoclonal antibodies (moAbs), detected using different lasers, were used to identify changes in the different signalling pathways: MAPK (pT180/pY182) (BD Biosciences Cat# 560313, RRID:AB_1645295), p65 NF-KB (BD Biosciences Cat# 558423, RRID:AB_647222) and JNK (BD Biosciences Cat# 562481, RRID:AB_11153116) (Table S1). Flow cytometry was employed to assess changes in signalling pathways, analysing a total of 10,000 events per experimental condition, measured using a Miltenyi MACSQuantVYB cytometer (Miltenyi LEIVA-CASTRO ET AL.5159 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Biotec, North Rhine-Westphalia, Germany) and analysed in FlowJo software (BD). Results were presented as percentages of marker expression. 2.6 |Phenotype changes in monocyte-derived DCs (moDCs) MoDCs (10 10 5 ) were cultivated in 96-well plates at 37C and 5% CO 2 after 48 h. As previously stated, various experimental conditions were tested. To evaluate the effects of SFN-glycoconjugates, moDCs were preincubated with the glycoconjugates for 1 h before the addition of LPS. The cells were stained with fluorochrome-labelled moAbs: CD80 (BD Biosciences Cat# 555683, RRID:AB_396035), CD83 (BD Biosciences Cat# 551073, RRID:AB_398488), CD86 (BioLegend Cat# 305423, RRID:AB_2260067), HLA-DR (EXBIO Praha Cat# PO690-T100, RRID:AB_3696487) and CD274 (PD-L1) (BD Biosciences Cat# 568080, RRID:AB_3684021) (Table S2) to evaluate their maturation. Flow cytometry was used to analyse the expression levels of maturation markers, including CD80, CD83, CD86, HLA-DR and PDL1. In fact, elevated expression of these markers indicates a mature state, while their low expression and/or the absence of these phenotypic changes suggest an immature state, as described in other studies (Jin et al., 2010; Wu et al., 2017). Cell vitality was assessed using the LIVE/DEAD viability kit (Thermo Fisher Scientific). Flow cytometry data were collected with a Miltenyi MACSQuantVYB cytometer (Miltenyi Biotec) and analysed using FlowJo software (BD). Results were expressed as the frequency of expression for each surface marker on the moDCs. 2.7 |Cytokine determination Cytokine production (IFN-γ, IL-17, IL-5, TNF-αand IL-10) was quantified using a human ProcartaPlex Multiplex (Thermo Fisher Scientific). After 48 h, supernatants from moDC cultures were collected. Samples (80 μl) and standards were incubated with a magnetic bead mix overnight at 4C. Subsequently, biotinylated antibodies were added and incubated for 300 min at room temperature (RT). Streptavidin was then added and incubated for another 300 min at RT. Following each incubation, the plate was washed and prepared for detection using Bio-Plex if 200 (Bio-Rad, Laboratories, Inc, Hercules, CA, USA) with a reading buffer. Data analysis was performed using Bio-Plex Data Analysis Software (Bio-Rad) and the LOD (Limit of Detection) for each cytokine was established according to the standards supplied with the kit of ProcartaPlex Multiplex (Table S4). The results were expressed as the concentration (pgml 1 ) of each cytokine. 2.8 |Specific proliferative response As previously described, moDCs (1.5 10 3 ) were seeded in 96-well plates and prestimulated with SFNMan, SFNFuc (both at 25 μM) and SFN (10 μM) in the presence or absence of LPS (100 ngml 1 ) for 48 h. After this prestimulation, the moDCs were cocultured with autologous peripheral blood mononuclear cells (1.5 10 5 ), previously labelled with 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Thermo Fisher Scientific), at a 10:1 ratio in a final volume of 250 μl of complete medium for 6 days at 37C and 5% CO 2 . This probe, CFSE, was used to evaluate proliferative responses of Tand B-cells, in addition to determining the lymphocyte subsets (CD3 + T-, CD3  CD19 + Band CD3 + CD4 + FOXP3 + T-cells, as Treg-cells) (Palomares et al., 2018). The fluorescent moAbs employed were CD3 (BioLegend Cat# 980004, RRID:AB_2632620), CD4 (Miltenyi Biotec Cat# 130-113-230, RRID:AB_2726041), FoxP3 (BD Biosciences Cat# 560889, RRID:AB_10584329) and CD19 (BD Biosciences Cat# 557835, RRID:AB_396893) (Table S3). The cytokine production capacity was confirmed by measuring IL-10 (PE antihuman IL-10, BioLegend Cat# 501404, RRID:AB_ 315170) and IFN-γ(VioGreen antihuman IFN-γ, Miltenyi Biotec Cat# 130-130-447, RRID:AB_2922300) levels. Brefeldin A (5 mgml 1 ) was added at a concentration of 5 μgml 1 (1/1000) for 3 h, followed by fixation with the Cytofix/CytoPerm Fixation/Permeabilization Solution Kit (BD). To identify Tregs, the Human FOXP3 Buffer Set (BD) was used for intracellular staining. Results were expressed as the percentage of CFSE low cells within each cell subpopulation under different experimental conditions, as well as the percentage of IL-10 and IFN-γproduction in proliferating T-cells, Tregs and B-cells. 2.9 |Blocking assay To investigate the role of the p65 NF-κB and MAPK signalling pathways in the immune response induced by SFN (10 μM) and SFNglycoconjugates (25 μM) in moDCs, blocking assays were conducted. Monocyte-derived DCs (MoDCs) were preincubated with selective blockers for 1 h at 37C. These blockers included MG-132 (1 μM, a potent inhibitor of p65 NF-κB activation (Tanimura et al., 2021); SB203580 (10 μM, known for its ability to inhibit p38 MAPK (Lin et al., 2015; and SP600125 (10 μM), functioning as a JNK inhibitor, preventing the activation of inflammatory genes (Lin et al., 2015), regardless of the absence or presence of LPS. Changes in the signalling pathways (p65 NF-κB and MAPK) were assessed at 10 min, while alterations in the maturation state were measured at 48 h as previously described. Results were expressed as the frequency of expression for each surface marker on the moDCs. Additionally, alterations in the proliferative response of various Tand B-cell subgroups were studied. Autologous moDCs were preincubated with the described blockers, followed by specific assays for the proliferative response, expressed as the percentage of CFSE low for each cell subgroup under varying experimental conditions, along with the percentage of IL-10 and IFN-γpresent in proliferating T-, Tregand B-cells. 5160 LEIVA-CASTRO ET AL. 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2.10 |Statistical analysis The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2025). For variables that did not display a significant normal distribution, nonparametric tests were applied. Comparisons of related samples were conducted using the Friedman test to assess differences among experimental conditions in repeated measures. Post hoc tests were only performed when the P-value reached P< 0.05. When significant differences were identified via the Friedman test, subsequent pairwise comparisons of related samples were carried out using the Wilcoxon signed-rank test. Significant differences were reported as Pvalues. The FIGURE 1 Modulation of p65 NF-κB and MAP kinase signalling pathway expression. (a) The bars and symbols represent the mean (SD) of the percentages of p65 NF-κB expression and (b) of different MAPK expression, for each experimental condition (n=12). imDC, immature dendritic cells; L, lipopolysaccharide (LPS);MG132 (MG) inhibitor of p65 NF-κB+L incubated for 1 h followed by LPS stimulation; SFN +L, SFN incubated for 1 h followed by LPS stimulation; SB +L and SP +L, the different blockers incubated for 1 h followed by LPS stimulation; SB, SB203580, blocker of p38 MAPK; SFN, sulforaphane; SFNMan and SFNFuc, SFN-glycoconjugates with mannose or fucose; SFNMan +L and SFNFuc +L, SFN-glycoconjugates incubated for 1 h followed by LPS stimulation; SP, SP600125 as a JNK inhibitor. The Friedman test was used to detect differences in related samples across multiple comparisons, indicating significant Pvalues. The Wilcoxon test was used for pairwise comparisons of related samples, indicating the exact p values. LEIVA-CASTRO ET AL.5161 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 2 Changes in the expression of DCs surface molecule. The bars and symbols represent the mean (SD) of the percentages of expression of CD80, CD83, CD86, PD-L1 and HLA-DR on moDCs under different experimental conditions (n=12). imDC, immature dendritic cells; L, lipopolysaccharide (LPS); SFN +L, SFN incubated for 1 h followed by LPS stimulation; SFN, sulforaphane; SFNMan and SFNFuc, SFNglycoconjugates with mannose or fucose; SFNMan +L and SFNFuc +L, SFN-glycoconjugates incubated for 1 h followed by LPS stimulation. The Friedman test was used to detect differences in related samples across multiple comparisons, indicating significant Pvalues. The Wilcoxon test was used for pairwise comparisons of related samples, indicating the exact Pvalues. 5162 LEIVA-CASTRO ET AL. 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License sample size included in the statistical analysis was at least 8 data points per group (n =8), where n represents the number of independent values. Statistical analyses were conducted using GraphPad Prism 7. Blinding was included in the statistical analysis to minimize bias. 2.11 |Materials The synthesis of sulforaphane (SFN)-glycoconjugates was performed through coupling reactions between an amino-derivative of SFN and a FIGURE 3 Changes in the cytokine production on DCs. The bars and symbols represent the mean (SD) of the concentration of each cytokine (pgml 1 )(n=8) on moDCs under different experimental conditions. imDC, immature dendritic cells; L, lipopolysaccharide (LPS); SFN +L, SFN incubated for 1 h followed by LPS stimulation; SFN, sulforaphane; SFNMan and SFNFuc, SFN-glycoconjugates with mannose or fucose; SFNMan +L and SFNFuc +L, SFN-glycoconjugates incubated for 1 h followed by LPS stimulation. The Friedman test was used to detect differences in related samples across multiple comparisons, indicating significant Pvalues. The Wilcoxon test was used for pairwise comparisons of related samples, indicating the exact Pvalues. LEIVA-CASTRO ET AL.5163 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 4 Legend on next page. 5164 LEIVA-CASTRO ET AL. 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License mannoor fuco-configured carbohydrate containing an isothiocyanate moiety (see the Supporting Information for further experimental details, including Figures S1–S4). Sulforaphane, brefeldin A, DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA), while gentamicin and streptomycin were purchased from Norton (Madrid, Spain), GM-CSF and IL-4 from R&D Systems Inc. (Minneapolis, MN, U.S.A). In addition, glutamine was purchased from Whittaker (Pittsburgh, PA, U.S.A). Details of other materials, suppliers, and tools are provided in the specific sections and in Table S5. 2.12 |Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander et al., 2023). 3|RESULTS 3.1 |Sulforaphane (SFN)-glycoconjugates did not produce cellular cytotoxicity SFN-glycoconjugates were engineered to enhance certain physicochemical properties of SFN (Figure S5A), such as stability and bioavailability, without increasing cellular toxicity. To assess this, moDCs were incubated for 48 h with various concentrations of SFN and its glycoconjugates. Our results indicate that glycosylation of SFN increased its usable concentration in in vitro studies compared with SFN alone. No cytotoxic effects were observed when moDCs were stimulated with 10 μM SFN, however higher concentrations of SFN compromised cell viability. Similarly, no cytotoxic effects were observed with the SFNManand SFNFuc-glycoconjugates at any of the concentrations tested (Figure S5B). 3.2 |SFN-glycoconjugates modulate the p65 NFκB but not MAPK signalling pathway The protective effects of SFN-glycoconjugates were investigated to elucidate their molecular mechanisms in mitigating LPS-induced inflammation, focusing on the p65 NF-κB and MAPK signalling pathways, which play pivotal roles in inflammatory responses. The SFN-glycoconjugates did not induce changes in p65 NF-κB levels compared with immature DCs. However, our results revealed that LPS instigates p65 NF-κB activation, a process significantly attenuated by SFN, SFN-glycoconjugates and the selective p65 NF-κB blocker, MG-132. Pretreatment with SFN, SFN-glycoconjugates and MG-132 for 1 h prior to LPS exposure significantly reduced LPS-induced p65 NF-κB expression compared with LPS-pretreated moDCs (Figure 1a). Previous studies by our group demonstrated that SFN pretreatment in moDCs involved the p38 MAPK and JNK signalling pathways in modulating immune responses towards a regulatory pattern (Munera-Rodriguez et al., 2024). Based on these findings, we aimed to analyse whether SFN-glycoconjugates could regulate these signalling pathways. Results showed that LPS stimulation induced the phosphorylation of p38 MAPK (pp38 MAPK) and JNK (pJNK), with pronounced up-regulation compared with immature DCs, SFN and their respective blockers (SB203580 and SP600125). However, SFNglycoconjugates significantly reduced pJNK levels compared with LPS-treated moDCs (Figure 1b). Additionally, moDCs treated with SFN for 1 h followed by LPS (L) stimulation (SFN +L) demonstrated significantly reduced activation of the MAPK and JNK signalling pathways compared with LPStreated moDCs, resembling results obtained with SB203580 +L and SP600125 +L (Figure 1b). Conversely, no significant differences were noted in the inhibition of the p38 MAPK and JNK pathways in SFNglycoconjugate-pretreated moDCs challenged with LPS. These findings suggest that the chemical nature of the SFN-glycoconjugates lacks the capacity to regulate the MAPK pathways. 3.3 |SFN-glycoconjugates induce changes in the moDC maturational status To investigate the immunomodulatory impact of SFN-glycoconjugates on moDCs, we assessed their influence on the expression of key surface regulatory molecules, specifically, CD80, CD83, CD86, PD-L1 and HLA-DR. Initially, moDCs were treated with SFN and SFNglycoconjugates. Neither SFN nor its glycoconjugates induced changes in the expression of any markers compared with the immature state. However, a significant increase in the percentage of HLADR was observed in the presence of SFNFuc. In contrast, stimulation with LPS up-regulated the expression of all maturation markers compared with immature DCs (Figure 2). Furthermore, to explore whether SFN and SFN-glycoconjugates could influence the expression of moDC regulatory molecules in the presence of LPS (to induce an inflammatory microenvironment), FIGURE 4 Proliferative T and B response induced by the SFN-glycoconjugate pretreatment. The bars and symbols represent the mean and standard deviation of the different percentages of CFSE low for T-, Band Treg-cells and the percentages of IFN-γand IL-10 levels in proliferating T-, Tregand B-cells under different experimental conditions (n=8–11). CFSE, 5,6-carboxyfluorescein diacetate N-succinimidyl ester; L, lipopolysaccharide (LPS); SFN +L, SFN incubated for 1 h followed by LPS stimulation; SFN, sulforaphane; SFNMan and SFNFuc, SFNglycoconjugates with mannose or fucose; SFNMan +L and SFNFuc +L, SFN-glycoconjugates incubated for 1 h followed by LPS stimulation; Unstim, unstimulated cells. The Friedman test was used to detect differences in related samples across multiple comparisons, indicating significant Pvalues. The Wilcoxon test was used for pairwise comparisons of related samples, indicating the exact Pvalues. LEIVA-CASTRO ET AL.5165 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. 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British Journal of Pharmacology, 182(21), 5157–5172. https://doi.org/10.1111/bph.70115 5172 LEIVA-CASTRO ET AL. 14765381, 2025, 21, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70115 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [23/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License