Yuyng Sun, Jiqing Tng, Cui Li, Jun Liu,b,*, Hijie Liu,*

a College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

b Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China

Keywords:

BMDMs

Intestinal inflammation

IL-10

Macrophage phenotype

STAT3

Sulforaphane

A B S T R A C T

Innate immunity, particularly macrophages, is critical for intestinal homeostasis.Sulforaphane, a dietary isothiocyanate from cruciferous vegetables, has been reported to protect against intestinal inflammation.However, the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet.In this study, sulforaphane effectively attenuated dextran sodium sulphate (DSS) induced intestinal inflammation in murine model.Of note, sulforaphane skewed the switching from classically (M1) to alternatively (M2) activated phenotype both in intestinal and bone marrow-derived macrophages (BMDMs).The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide (LPS) plus interferon gamma-γ (IFN-γ) were suppressed by sulforaphane while M2 marker gene expression levels were improved.This resulted in alteration of inflammatory mediators,particularly interleukin-10 (IL-10), both in colon tissues and culture medium of BMDMs.Subsequently,IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling.This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections.Moreover, anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation.Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.

1.Introduction

Inflammatory bowel diseases (IBD), mainly consisting of Crohn’s disease (CD) and ulcerative colitis (UC), are relapsing diseases along with severe damage to the gastrointestinal and colonic mucosa [1].Although the etiology of IBD has not been fully elucidated, a currently accepted interpretation is that environment factors, especially the diet,lead to abnormal immune responses a nd gut microbiota dysbiosis in IBD patients [2].Over the past two decades, most studies on intestinal immunity have stressed that adaptive immunity drives the progression of IBD.For instance, both CD and UC are driven by T helper cells(Thcells) responses [3].Recently, researches have suggested that innate immune responses play a vital role in the development of intestinal inflammation as well [4].

As a major component of innate immunity, macrophages can help to maintain intestinal homeostasis through the modulation of immune responses.They are known to display excellent plasticity and adopt polarized phenotypes with specific functional characteristics [5].Traditionally, macrophages are subdivided into two populations: classically activated macrophages (M1/CAM)and alternatively activated macrophages (M2/AAM) [6].During intestinal inflammation, the monocytes are recruited to lamina propria and subsequently differentiated into specific immature macrophages activated by pathogen-associated molecular patterns(PAMPs).Then the macrophages secret pro-inflammatory cytokines to aggravate inflammatory response and barrier damage.Elimination of inflammation and barrier reconstruction are driven by macrophages shifting from M1 to M2 phenotype, which triggered by efferocytosis [7].In several experimental models of IBD, infiltration of activated macrophages into the lamina propria facilitates the development of intestinal inflammation.A disequilibrium of M1/M2 macrophages contributes to colitis development, while the severity can be reduced with the increased percentage of M2 macrophages [8,9].Therefore,macrophages phonotype alternation might be a novel therapeutic approach in IBD.

Drug therapy is the common method for treating IBD.Nowadays, several medical methods are available for IBD patients,including 5-aminosalicylates, corticosteroids, antibiotics, and immunomodulators [10].Classic drugs such as 5-aminosalicylates and corticosteroids directly suppress intestinal inflammatory macrophages by blocking nuclear factor κB (NF-κB) signaling pathways [7].Other immunomodulators such as infliximab block inflammatory factorsmediated inflammatory responses and promote M2 macrophages to attenuate IBD [7].However, severe side effects have been reported to accompany with long-term administration of above medicines, such as endocrine disorders, hypertension, and lymphoma [11].In contrast,natural phytochemicals usually have low toxicity and high efficiency for IBD treatment, such as terpeniods, polyphenols, flavonoids, and alkaloids [12].These phytochemicals work by modulating microbiota,enhancing antioxidant capacity, and alternating adaptive immune responses [13,14].However, few researches have explored the effects of those natural phytochemicals on IBD in the perspective of macrophages-mediated innate immunity.

Sulforaphane (1-isothiocyanato-4-(methyl-sulfinyl)) is a dietary isothiocyanate from cruciferous vegetables of broccoli, cabbage, kale,and arugula with anti-inflammatory activities [15].In broccoli or broccoli sprouts, sulforaphane can be converted from glucoraphanin,a glucosinolate [16].Several clinical studies support that sulforaphane exhibits several health-promoting effects including antioxidant,antimicrobial, and anticancer activities [17].Specially, sulforaphane owns excellent anti-inflammatory bioactivity in digestive system,respiratory tract, and urinary system.For example, sulforaphane enables to decrease inflammatory biomarkers serum pepsinogens I and II and to alleviate gastric inflammation caused byHelicobacter pyloricolonization [18].In addition, sulforaphane can reduce the inflammatory response in asthmatics patientsviaNrf2 mediated pathways [19].In vitro, sulforaphane suppresses human bronchial epithelial cells inflammation caused by particulate matterviaERK/JNK signaling pathway [20].Moreover, sulforaphane can alleviate inflammation through preventing nuclear translocation of NF-κB, which can activate the transcription of inflammatory genes [21].In animal model, sulforaphane suppresses NF-κB activation and tumor necrosis factor (TNF-α)level to inhibit inflammation in nephropathy induced by cisplatin [22].Having observed that strong anti-inflammatory capacity of sulforaphane, we expect it to exert the same effects in IBD.

Sulforaphane has been reported to regulate the phenotype of macrophages which can benefit the treatment of several diseases.For example, the tubular epithelial inflammation in nephrocalcinosis was relieved by sulforaphane through facilitating M2 macrophage differentiationviaNrf2-miR-93-TLR4/IRF1 signaling pathway [23].Sulforaphane promoted microglia differentiation from M1 to M2 and improved neuromotor dysfunction caused by hyperammonemia [24].For PAM-induced THP1-derived macrophages, the phenotype was altered by sulforaphane from M1 to M2 with upregulated IL-10 and CD206 expression [25].In this study, we investigate whether sulforaphane exert anti-inflammatory effect in IBD by regulating macrophage polarization.

In this study, the effects of sulforaphane on experimental intestinal inflammation were investigated using a murine model of dextran sodium sulphate (DSS)-induced colitis.The molecular mechanisms involved in sulforaphane induced modulation of macrophage functional phenotypes and the contribution to the alternation of intestinal inflammation were studied bothin vivoandin vitro.

2.Materials and methods

2.1 Materials and reagents

Sulforaphane (purity >95%) was obtained from TRC Inc.(Toronto, Canada).Antibodies against phospho-pSTAT3 (Y705)(ab76315), F4/80 (ab6640), STAT3 (ab68153), Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 594 goat anti-rat IgG were purchased from Abcam PLC.(Cambridge, UK).Dulbecco’s modified Eagle’s medium (DMEM/F12), fetal bovine serum (FBS), andL-glutamine were provided by Corning Inc.(Corning, NY, USA).M-CSF was obtained from Peprotech Inc.(NJ, USA).Penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA).WST-1 cell proliferation cytotoxicity assay kit was purchased from Roche Pharmaceutical Ltd.(Mannheim, Germany).LPS was obtained from Sigma (St.Louis, MO, USA).ELISA kits of IL-1β, IL-6,IL-10, tumor necrosis factor TNF-α, and interferon gamma IFN-γ were purchased from Biolegend (San Diego, CA, USA), as well as Brilliant Violet 510 conjugated anti-mouse CD86 antibody, APC conjugated anti-mouse CD68 antibody, PE conjugated anti-mouse CD206 antibody, PC-Cy7 conjugated anti-mouse F4/80 antibody, and anti-mouse CD16/32 antibody.ECL chemiluminescence detection kit was obtained from Bio-Rad (Hercules, CA, USA).Anti-IL-10 neutralizing antibody (αIL-10; clone JES5-16E3) was received from Bioscience (San Diego, CA, USA).GoScript? Reverse transcriptase kit was provided by Promega (Madison, WI, USA).TB Green Premix Ex Taq II was purchased from Takara Bio Inc.(Kyoto, Japan).

2.2 Animals and experimental design

Six- to eight-week-old male C57BL/6JNifdc mice obtained from Vital River Laboratory Animal Technology Co., Ltd.(Beijing,China) were kept in polypropylene cages.All mice were raised under standard conditions of 12-h light/dark cycle at (25 ± 2) °C with a relative humidity of (55 ± 5)%.Mice had free access to basal diet(Vital River Laboratory Animal Technology Co., Ltd., Beijing, China)and water.After one-week adaption, mice were randomly divided into 5 groups (n= 5 in each group): CON group (water and phosphatebuffered saline (PBS)), DSS group (2.5% DSS and PBS), H-SF/DSS group (40 mg/kg·d sulforaphane and 2.5% DSS) , M-SF/DSS group(20 mg/kg·d sulforaphane and 2.5% DSS), L-SF/DSS group(10 mg/kg·d sulforaphane and 2.5% DSS).All groups were treated for 14 days and DSS was gavaged from day 8 to day 14 (7 days) to induce the acute colitis.All experimental procedures were approved by Institutional Animal Ethics Committee of China Agricultural University (Approval number: 20185001-3).

2.3 Assessment of colitis

During the experiment, the body weight, stool consistency, and rectal bleeding of mice in different treatment groups were recorded daily and the disease activity index was calculated accordingly [26].At day 14, all mice were sacrificed through cervical dislocation.The colon was collected and the length was measured after flushing with ice-cold PBS.One third of the length to the midpoint of the colon(1–2 cm) was formalin-fixed and aparaffin-embedded.After trimming for longitudinal sectioning, histological analysis on the hematoxylin and eosin (H&E) stained colon section was conducted and scored as epithelium (E) and infiltration (I), respectively [27].The remaining colon tissue was scraped for mucosa collection and then subjected for subsequent enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR).

2.4 Immunofluorescent staining

For immunofluorescent labeling, the paraffin-embedded colon tissues were cut into 3 mm-thick sections.Then, the sections were deparaffinized through xylene incubation twice (each for 10 min)and rehydrated with decreasing percentage of ethanol.After antigen retrieval by hot citrate buffer (pH = 6, 95 °C, and 30 min), the colon sections were blocked with nonfat milk (5% in PBS) for 1 h.Subsequently, primary antibodies of rabbit monoclonal anti-STAT3 and rat anti-F4/80 were incubated with the blocked colon sections at 4 °C overnight.The colon sections were then incubated with secondary antibodies of Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-rat IgG for 2 h at room temperature (RT,22-23 °C).After washing three times with PBS, nuclear staining was conducted using DAPI and all samples were analyzed by an inverted confocal microscope system (MaiTai-FV1000, Olympus Corporation, Japan).

2.5 Preparation and treatment of bone marrow-derived macrophages

Bone marrow cells were flushed out from the marrow of the femurs and tibias of uninjured adult WT male BALB/c mice with Dulbecco’s phosphate-buffered saline (DPBS) [28].The cells were collected into a sterile falcon tube and centrifuged at 600 ×gfor 5 min.The cell pellets were resuspended in DMEM/F12 medium supplemented with heat-inactivated fetal bovine serum (FBS; 10%),penicillin/streptomycin (1%), andL-glutamine.Cells were then seeded in sterile petri dishes and cultured for 6 days at 37 °C under a humidified atmosphere with 5% CO2.M-CSF (50 ng/mL) was used for inducing the cell differentiation and the purity of derived bone marrow-derived macrophages (BMDMs) was confirmed by flow cytometer to be > 95%.BMDMs were then treated as follows: (1)Ctrl group: cell culture medium; (2) M1 group: LPS (10 ng/mL) +IFN-γ (20 ng/mL); (3) M1+H-SF, M1+M-SF, and M1+L-SF group:LPS (10 ng/mL) + IFN-γ (20 ng/mL) + sulforaphane at 10.0, 1.0, and 0.1 μmol/L, respectively.To investigate the mediation effects of IL-10 in activation of anti-inflammation, BMDMs were blocked with the neutralizing antibody to IL-10 at 5 μg/mL.

2.6 qRT-PCR analysis

Total RNA extraction from tissue samples and BMDMs were conducted following the standard protocol using Trizol reagent.RNA was quantified using a NanoDrop spectrophotometer and RNA purity was determined by calculating 260/280 nm ratios.Then, cDNA synthesis was performed using a GoScript? Reverse Transcriptase kit in a thermocycler (Bio-Rad Laboratories, CA, USA).PCR reaction was initiated with TB Green Premix Ex Taq II (TliRNaseH Plus) in CFX Connect Real-Time System following the program: firstly an initial denaturation step at 95 °C for 30 s, then 40 cycles consisting of 5 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, after which the gene fragments were completely melted at appropriate temperature.The primer sequences for targeted genes were listed in Table 1 and GAPDH was used as internal control for normalization.

Table 1Primer sequences used for qRT-PCR.

2.7 Cytokine assay

ELISA was performed for analyzing the release of cytokines in the colon tissues and BMDMs after specific treatments.The homogenate of colon tissue samples and the supernatant of BMDMs culture medium were collected and the levels of TNF-α, IL-1β,IL-6, and IL-10 were measured using ELISA kits following the manufacturer’s instructions.

2.8 Cell surface staining and flow cytometry analysis

Colonic lamina propria cells were isolated according to the method described previously [29].Cells were stained by fluorochromelabeled mouse antibodies of Brilliant Violet 510 labeled anti-mouse CD86, PE-Cy7 labeled anti-mouse F4/80, APC labeled anti-mouse CD68, and PE labeled anti-mouse CD206.The cytometry analysis was performed on a FACS Aria II Cell Sorter (Becton, Dickinson, NJ,USA) and the data were processed with FlowJo Software V.10.1 (Tree Star, Ashland, OR, USA).

2.9 Western blotting

Proteins expressed in BMDMs were harvested using RIPA buffer with protease inhibitor cocktail (200:1) and quantified through BCA assay [30].Aliquots (20 μg) of protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes.After blocking with nonfat milk (5% in TBS with 0.1% Tween 20), the membranes were incubated with primary antibodies of rabbit anti-STAT3 (1:1 000; Abcam), rabbit anti-pSTAT3 (1:1 000), and mouse anti-β-actin (1:1 000) under gently shaken at 4 °C for overnight.Finally, the membranes were incubated (RT for 1 h) with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin IgG and visualized by ChemiDoc XRS system (Bio-Rad; CA, USA).The densitometry analysis on each blot was performed with Image J software (NIH; MD, USA).

2.10 Statistical analysis

Data were presented as mean ± SD and analyzed by GraphPad Prism version 7 software (GraphPad Software, Inc.; La Jolla, CA,USA).Statistical significance of difference was determined using one-way analysis of variance, followed by multiple comparisons with Dunnett’s test.Significance level was set asP< 0.05.

3.Results

3.1 Sulforaphane attenuated DSS-induced intestinal inflammation in mice

Mice that received sulforaphane for 7 days prior to DSS treatment developed less severe symptoms of intestinal inflammation as compared with the normal control group.Weight loss induced by DSS was partially mitigated by sulforaphane (P< 0.05, Fig.1A).Sulforaphane effectively attenuated the colonic symptoms of stool formation and fecal bleeding with lowered disease activity index (DAI) score (P< 0.05, Fig.1A) comparing with the DSS treatment group.The shortening effects of DSS on colon length were diminished by 33.3%, 29.1%, and 40.1%, respectively, after oral exposure to sulforaphane (10, 20, and 40 mg/kg·d) (Fig.1D).Moreover, sulforaphane administration significantly increased the gene expression levels ofZO-1andOccludinin DSS-treated mice which were associated with the intestinal barrier integrity (P< 0.05,Figs.1B and 1C).Histology analysis showed that colitis mice treated with sulforaphane had noticeably lower levels of monocytes infiltration into mucosa with less edema (P< 0.05, Figs.1E and 1F).Sulforaphane (10, 20, and 40 mg/kg·d) administration reduced the histology scores by 17.1%, 25.7% and 54.2%, respectively.Those changes suggested that sulforaphane mitigated DSS-induced colonic damage with improved epithelial integrity.

Fig.1 Effects of sulforaphane on DSS induced intestinal inflammation in mice.Body weight change, diarrhoea status, rectal bleeding, and DAI of DSS treated mice with or without sulforaphane intervention (A).Expression of ZO-1 (B) and Occludin (C) in the colon of mice; Changes in colon length (D), histologic analysison the colonic section (× 200, bar = 400 μm), and accordingly the histological score of DSS-induced colitic mice with or without sulforaphane treatment(E, F).F1.Ctrl, F2.DSS, F3.DSS/L-SF, F4.DSS/M-SF, F5.DSS/H-SF.Data are expressed as means ± SD (ns, not significant; *P < 0.05; **P < 0.01 vs control group; #P < 0.05; ##P < 0.01 vs DSS group).

Fig.1(Continued)

3.2 Sulforaphane reduced inflammatory leukocytes accumulation and altered macrophage phenotyping

As shown in Fig.2A, DSS treatment significantly increased the release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in colon tissues (by 1.1, 1.3 and 2.4 folds, respectively)when compared with the normal control group.Mice fed with sulforaphane (20 and 40 mg/kg·d) significantly inhibited the DSS induced accumulation of pro-inflammatory cytokines (P< 0.05).On the contrary, the levels of anti-inflammatory cytokine IL-10 in groups by oral exposure to sulforaphane (20 and 40 mg/kg·d)were 1.09- and 1.43-fold higher as compared with DSS treatment group, respectively (P< 0.01).Moreover, DSS treatment significantly increased the percentage of F4/80+CD68+colonic macrophages while the percentage of F4/80+CD206+was not significantly affected when compared with the normal control group.The oral exposure to sulforaphane decreased the percentage of F4/80+CD68+macrophages with boosted F4/80+CD206+macrophages.Consistently, the expression levels of macrophages polarization marker genesIL-1β,iNOS,ARG-1,IL-10, andPTX3were significantly increased by DSS treatment (Fig.2B).Notably,sulforaphane administration lowered the gene expression levels ofIL-1βandiNOSthat associated with M1 polarization dose-dependently(P< 0.05).The expression of genes related to M2 polarization,includingARG-1,MR,CD163,IL-10, andPTX3, was significantly upregulated by sulforaphane treatment.Data here suggested that sulforaphane promoted macrophage M2 phenotype with altered release of inflammatory leukocytes in colonic lamina propria.

Fig.2 Effects of sulforaphane on the phenotype of intestinal macrophages and the release of inflammatory leukocytes.The levels of inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-10 in the colon of DSS-induced colitic mice (A).The expression of marker genes that associated with macropahge polarization in the colonic tissues (B).Flow cytometry analysis on the macrophages in the colon of DSS-induced colitic mice with or without sulforaphane treatment (C)and accordingly the percentage of M1 and M2 macrophages based on the surface markers CD68 and CD206 (D).Data are expressed as means ± SD (ns, not significant; #P < 0.05; ##P < 0.01 vs DSS group).

Fig.2(Continued)

Fig.2(Continued)

3.3 Sulforaphane skewed M2 phenotype switching of BMDMs

The effects of sulforaphane on phenotypes of BMDMs were analyzedin vitro.Upon the treatment by LPS plus IFN-γ, the gene expression levels ofIL-1βandiNOSthat associated with M1 phenotype were significantly increased by 67 and 107 times as compared with the control (Fig.3A).Sulforaphane suppressed the gene expression levels ofIL-1βandiNOSinduced by LPS plus IFN-γ in a dose dependent manner (P< 0.01; Fig.3A).The gene expression levels ofARG-1,MR, andCD163in BMDMs treated by LPS plus IFN-γ were not significantly changed with or without sulforaphane(Fig.3B).However, sulforaphane recovered the LPS plus IFN-γ induced suppression on gene expression levels ofIL-10andPTX3that associated with M2 macrophage phenotype (P< 0.01, Fig.3C).The cytokines release in the culture medium of BMDMs were measured by ELISA.Treatment of LPS plus IFN-γ significantly increased the release of IL-6, TNF-α, and IL-1βas compared with those of the control group.Interestingly, the level of anti-inflammatory cytokine IL-10 in the supernatant of BMDMs culture medium was also increased by LPS plus IFN-γ.Sulforaphane (10 μmol/L)significantly decreased the production of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 (by 26.6%, 56.3%, and 90.6%, respectively),while IL-10 release was further increased by 2.5 folds (P< 0.01;Fig.3D).Flow cytometry analysis revealed that LPS plus IFN-γ significantly increased the expression of surface marker CD86 (Fig.3E).Sulforaphane exposure suppressed CD86 expression on BMDMs that induced by LPS plus IFN-γ treatment.Thus, sulforaphane could switch the LPS plus IFN-γ induced M1 phenotype of BMDMs into M2 phenotype with altered inflammation status.

Fig.3 Effects of sulforaphane on phenotype switching and cytokine release in BMDMs.The expression of M1 phenotype related marker genes IL-1β and iNOS in BMDMs (A).The expression of M2 phenotype related marker genes ARG-1, MR, CD163, IL-10, PTX3, and MERTK in BMDMs (B and C).Release of cytokines IL-6, TNF-α, IL-1β, and IL-10 in BMDMs with or without sulforaphane treatment as measured by ELISA (D).Expression of surface marker CD86 in BMDMs with or without sulforaphane treatment (E).Data are expressed as means ± SD (ns, not significant; #P < 0.05; ##P < 0.01 vs M1 group).

Fig.3(Continued)

3.4 IL-10 mediated anti-inflammatory response of sulforaphane

Having observed phenotype switching of BMDMs induced by sulforaphane, we set out to investigate the involved molecular mechanisms.Notably, sulforaphane further promoted STAT3 activation in BMDMs induced by LPS plus IFN-γ treatment (1.35 folds, Figs.4A and 4B).In the presence of neutralizing αIL-10,pSTAT3/STAT3 was significantly reduced by 31% by sulforaphane in LPS plus IFN-γ treated BMDMs.Coincidentally, αIL-10 neutralization significantly up-regulated the gene expression levels ofIL-1βandiNOSthat associated with M1 phenotype in LPS plus IFN-γ treated BMDMs with or without sulforaphane (Fig.4C,P< 0.01).Conversely, M2 phenotype associated gene expression levels ofCD163,IL-10, andPTX3in LPS plus IFN-γ treated BMDMs were significantly suppressed in the presence of αIL-10 with or without sulforaphane (Fig.4C,P< 0.05).Interestingly,ARG-1andMRexpression levels in LPS plus IFN-γ treated BMDMs were increased after αIL-10 neutralization (Fig.4C,P< 0.01).On the other hand, sulforaphane significantly suppressed the LPS plus IFN-γ inducedARG-1andMRexpression levels in the presence of αIL-10.Consistently, the production of pro-inflammatory cytokines of IL-6,TNF-α, and IL-1β in LPS plus IFN-γ treated BMDMs were significantly increased after αIL-10 neutralization while IL-10 production was decreased independently on sulforaphane (Fig.4D,P< 0.05).Moreover, αIL-10 neutralization significantly increased the expression of cell surface marker of CD86 on LPS plus IFN-γ treated BMDMs.On the other hand, CD86 expression on LPS plus IFN-γ treated BMDMs was significantly reduced in the presence of sulforaphane (Fig.4E).Data suggested that the activation of IL-10/STAT3 cascade might be involved in sulforaphane induced phenotype switching of BMDMs.

Fig.4 The role of IL-10/STAT3 signaling in mediation of sulforaphane induced phenotype switching in BMDMs.(A) Western blotting of STAT3 expression in BMDMs induced by sulforaphane with or without αIL-10 neutralization, and (B) accordingly the level of phosphorylation of STAT3.(C) The expression of marker genes associated with macrophage polarization in BMDMs before and after αIL-10 neutralization.(D) The effects of αIL-10 neutralization on the release cytokines in BMDMs.(E) Expression of surface marker CD86 in BMDMs before and after αIL-10 neutralization.Data are expressed as means ± SD (ns, not significant;**P < 0.01 vs control group; #P < 0.05; ##P < 0.01 vs M1 group; +P < 0.05; ++P < 0.01, ANTI-IL10 M1 group vs WT M1 group; ^P < 0.05; ^^P < 0.01, ANTI-IL10 M1+H-SF group vs WT M1+H-SF group).

Fig.4(Continued)

3.5 Sulforaphane attenuated DSS induced intestinal inflammation through STAT3

Having demonstrated that IL-10/STAT3 signaling mediated the sulforaphane induced genotype switching in BMDMs, we further analyzed whether STAT3 was involved in sulforaphane regulated intestinal inflammation by immunofluorescence.Macrophages were identified by the expression of F4/80 [31].As shown in Fig.5,immunofluorescence analysis indicated merely a small number of p-STAT3-positive cells in the colon sections from DSS-treated mice,but sulforaphane treatments significantly increased the abundance of p-STAT3-positive cells.Those further confirmed that sulforaphane attenuated DSS induced intestinal inflammation through IL-10/STAT3 signaling mediated macrophage phenotype switching.

Fig.5 Immunofluorescence analysis on STAT3 activation in the colon sections of DSS-treated mice.pSTAT3 positive macrophages were indicated by white arrows.Data are expressed as means ± SD (ns, not significant; ##P < 0.01 vs DSS group).

4.Discussion

In the present study, sulforaphane was found to mediate the DSS induced intestinal inflammation with attenuated colonic damage and improved epithelial integrity.The molecular mechanisms involved were further studied and macrophage phenotype switching contributed to the observed protective effects of sulforaphane.IL-10/STAT3 signaling cascade was involved in the phenotype switching of macrophages bothin vitroandin vivo.

IBD has become a worldwide disease and it is associated with damage to intestinal structure, abnormality of epithelial barrier,and infiltration of inflammatory cells [7].Due to the serious side effects and complications of drugs, it is of particular interests to find new strategies for IBD treatment.Dietary intervention with nutrients and phytochemicals from fruits and vegetables, such as fibers, polysaccharides, polyphenols, and flavonoids, have great potential for IBD treatment [12].As a cruciferous vegetable derived dietary isothiocyanate, sulforaphane possesses various health beneficial effects with high bioavailability and low toxicity [32,33].Sulforaphane also showed anti-inflammatory activity and protected against DSS-induced colitis with reduced expression of inflammatory markers and increased expression of Nrf2-dependent genes [34].Moreover, sulforaphane reversed DSS induced gut microbiota dysbiosis and shifted the balance toButyricicoccuson[35].Besides,sulforaphanehas also been reported to attenuate chemotherapy drug 5-fluorouracil induced intestinal mucositis.Gene expression related to antioxidant defense (Nrf2andHO-1) and tight junction (claudin-1)was significantly improved by sulforaphane with no significant effect on the short-chain fatty acids (SCFAs) concentration [36].In this study, sulforaphane treatment significantly attenuated DSS induced weight loss, colon length shortening, and fecal bleeding.Moreover,mucosa monocytes infiltration and edema were mitigated by sulforaphane with improved intestinal integrity.This was proved by the increasedZO-1andoccludinexpression in DSS-treated mice after sulforaphane administration.

Previously, adaptive immune responses were thought to drive the pathogenesis of IBD and Th17/Treg was treated as an attractive therapeutic target.However, recent studies have found that innate immunity also occupies main stage in the development of IBD.Macrophages are an important contributor to innate immunity and possess capacity in immunoregulation and tissue reparation [5].Evidence exists that sulforaphane suppresses M1 macrophages and results in the switch to M2 polarization [37,38].A study by Ali et al.[37]implied that sulforaphane was repressive on gene expression levels includingIL-6,IL-1βandTNF-αand boosted M2 gene markerMRC1relevant to mannose receptor CD206 in LPS-induced THP-1-derived macrophages.Similarly, it was found that sulforaphane prevented RAW264.7 murine macrophages from LPS-induced inflammation by reduction of proinflammatory mediators along with activation of Nrf2/HO-1 signal [38].In intestinal inflammation,miR-155/QKI pathway might be a key regulatory approach to control macrophages polarization and beneficial to the inflammation reduction in sulforaphane pretreated colitis mice [39].These results indicated that sulforaphane exerted anti-inflammatory effect usually accompanied by switching macrophages states.Furthermore, other phytochemicals such as flavonoid could alleviate DSS-induced colitis by reducing the ratio of M1/M2 macrophages [40,41].Here,sulforaphane prevented macrophages M1 polarization and promoted macrophage M2 phenotype as evidenced by the expression of marker genes related to macrophage polarization both in colonic lamina propria and BMDMs.

IL-10 was a crucial cytokine which exerts strong antiinflammatory activity by regulating excessive immune responses to maintain intestinal homeostasis.Several transcription factors such as c-Maf, GATA3, and NF-κB p50 were able to activate IL-10 transcription in various immune cells including Tregs,macrophages, and dendritic cells [42].Mutually, IL-10 could also adjust diverse immune cells to exhibit anti-inflammatory and protective effects on IBD by means of binding to IL-10Rα and IL-10Rβ (IL-10 receptors) expressed on the surface of these immune cells and triggering the expression of anti-inflammatory downstream genes [42].Macrophages expressed highest level of IL-10 receptors so that moderate reaction of macrophages to IL-10 signaling was a key approach to manage intestinal inflammation [43].For instance, intestinal inflammatory macrophages were strongly inactivated after encountering IL-10 signal [44].Besides, IL-10 could also reduce the expression of characteristic surface biomarkers such as CD86 and MHC Class II and block the proinflammatory mediators secretion (IL-6 and TNF-α) to decrease tissue damage [42,45].Consistently, our study found that the accumulation of proinflammatory cytokines induced by DSS treatment was suppressed with improved release of anti-inflammatory cytokine IL-10, thus consequently with attenuation of intestinal inflammation.Moreover,several studies have demonstrated the correlation between IL-10 and IBD in both clinical and experimental models.For instance,individuals deficient in function of IL-10 or IL-10 receptors were more likely to develop serious intestinal inflammation [43].In animal models, IL-10-/-mice boosted spontaneous colitis and administration ofLactococcus lactissecreting IL-10 significantly suppressed DSS induced gut damage both in WT and IL-10-/-mice [46,47].The observations showed the irreplaceable impact of IL-10 on preventing intestinal inflammation.

For IL-10-regulated anti-inflammatory effects, STAT3 is considered as a key effector molecule which contributes to inflammation treatment and intestinal homeostasis.The receptor binding of IL-10 induced the activation of JAK1 and further triggered the phosphorylation of STAT3 [48].Then the phosphorylated STAT3 homodimers translocate to nucleus promptly and stimulate the target genes expression such asBcl3andEtv3which damaged NF-κB activity to suppress pro-inflammatory cytokines production [49].IL-10/STAT3 signaling was reported to mediate the M2 polarization of macrophages, thus facilitated phagocytosis effect and stimulated IL-10 and TGF-βproduction.However, the deficiency of IL-10 signaling lead to STAT3 inactivation [7].In this study, sulforaphane promoted IL-10 production in LPS plus IFN-γ treated BMDMs and skewed the phenotype switching from M1 to M2 with distinct STAT3 activation.On the other hand, in the presence of neutralizing αIL-10, sulforaphane induced M2 phenotype switching of BMDMs was suppressed with reduced STAT3 phosphorylation.This was further confirmedin vivoby immunofluorescence analysis that sulforaphane significantly increased p-STAT3-positive cells in the colon sections from DSS-treated mice.Wang et al.[50]found that Cinobufacini (a Chinese medicine) improved STAT3 phosphorylation and the ratio of M2/M1 in RAW264.7 macrophages and protected against DSS-induced colitis.STAT3 activation contributed to the C.EDA(a compound preparation consisted of an antioxidant and a terpenoid)induced M2 differentiation of intestinal macrophages with reduced release of M1-associated cytokines and repair of colon barrier [51].These consequences were parallel with our findings.

5.Conclusions

Sulforaphane mitigated the DSS induced experimental intestinal inflammation with attenuated weight loss, colonic symptoms of stool formation and fecal bleeding but improved intestinal barrier integrity and histological pattern.Sulforaphane mediated macrophage phenotype switching from M1 to M2 with altered release of inflammatory leukocytes contributed to the relief of the inflammatory status bothin vitroandin vivo.The activation of IL-10/STAT3 signaling was involved insulforaphane mediated macrophage phenotype switching (Fig.6).Thus, sulforaphane might be a potential food material for the development of new dietary therapy for IBD.

Fig.6 Diagram of sulforaphane attenuate DSS induced experimental colitis.DSS treatment induced M1 phenotype switching of intestinal macrophages and consequently increased release of pro-inflammatory cytokines which further surged intocolon tissues of mice.Sulforaphane protected DSS induced colitis via IL-10/STAT3 signaling mediated M2 phenotype switching of macrophages, which led to increased IL-10 production and up-regulation of related genes expression.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We gratefully acknowledge the financial support provided by“Modern Food Processing, Food Storage, Transportation Technology,and Equipment” State Key Research and Development Plan(2017YFD0400204) and the National Science Foundation of China(31972091).