Gut barrier makes a huge research gap between in vivo and in vitro studies of orally bioactive polysaccharides: whether/how they contact the related cells in vivo. A hyperbranched heteroglycan RAP from Radix Astragali, exerting antitumor and immunomodulatory effects in vitro and in vivo, is right an example.
Carbohydrate Polymers 292 (2022) 119653 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol A lymphatic route for a hyperbranched heteroglycan from Radix Astragali to trigger immune responses after oral dosing Quanwei Zhang a, 1, Lifeng Li a, 1, Shuang Hao b, Man Liu a, Chuying Huo a, Jianjun Wu a, Hongbing Liu a, Wanrong Bao a, Hongming Zheng a, Zhipeng Li a, Huiyuan Cheng a, Hauyee Fung a, Tinlong Wong a, Pingchung Leung c, Shunchun Wang d, Ting Li e, Ge Zhang a, Min Li a, Zhongzhen Zhao a, Wei Jia a, Zhaoxiang Bian a, Timothy Mitchison f, Jingchao Zhang b, *, Aiping Lyu a, *, Quanbin Han a, *, Handong Sun g a School of Chinese Medicine, Hong Kong Baptist University, Hong Kong 999077, China The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450000, China c State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Hong Kong 999077, China d Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China e State Key Laboratory for Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau 999078, China f Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston 02115, United States g State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China b A R T I C L E I N F O A B S T R A C T Keywords: Radix Astragali Polysaccharide Intact Targeting route Antitumor immune responses Gut barrier makes a huge research gap between in vivo and in vitro studies of orally bioactive polysaccharides: whether/how they contact the related cells in vivo A hyperbranched heteroglycan RAP from Radix Astragali, exerting antitumor and immunomodulatory effects in vitro and in vivo, is right an example Here, we determined first that RAP's antitumor activity is immune-dependent Being undegraded and non-absorbing, RAP quickly entered Peyer's patches (PPs) in h where it directly targeted follicle dendritic cells and initiated antitumor immune responses RAP was further delivered to mesenteric lymph node, bone marrow, and tumor By contrast, the control Dendrobium officinale polysaccharide did not enter PPs These findings revealed a blood/microbiotaindependent and selective lymphatic route for orally administrated RAP to directly contact immune cells and trigger antitumor immune responses This route bridges the research gap between the in vitro and in vivo studies and might apply to many other bioactive polysaccharides Introduction The gut wall barrier to macromolecules remains an unsolved chal lenge for developing orally-delivered macromolecular therapeutics (Scaldaferri et al., 2012) Many natural polysaccharides, being safe and effective, show great potential to be medicines (Mohammed et al., 2021; Yu et al., 2018) However, few of them are developed to medications because their efficacy on the human body is often doubted due to their poor bioavailability Inspiration is found from some polysaccharides that could quickly affect the immune system after oral dosing (Jiang et al., 2010; Schepetkin & Quinn, 2006; Yu et al., 2018) It is hypothe sized that there might be a blood-independent route for these polysaccharides to work in body Understanding how polysaccharides access the immune system may open new doors in developing oral de livery of polysaccharide-based vaccines or drugs Here, we take Radix Astragali polysaccharide RAP as a case study to test the hypothesis Radix Astragali is the most frequently used ‘Qi’ tonic herb medicine in tumor therapy with Traditional Chinese Medicine In our previous study, a hyperbranched heteroglycan RAP (1334 kDa) was purified from this herb medicine (Yin et al., 2012) Its chemical structure was characterized by monosaccharide composition, partial acid hydro lysis, methylation analysis, GC-MS, NMR spectra, SEM and AFM mi croscopy The backbone of RAP consists of 1,2,4-linked Rhap, α-1,4linked Glcp, α-1,4-linked GalAp6Me, β-1,3,6-linked Galp, with branched * Corresponding authors E-mail addresses: zhangjingchao126@126.com (J Zhang), aipinglu@hkbu.edu.hk (A Lyu), simonhan@hkbu.edu.hk (Q Han) These authors contributed equally https://doi.org/10.1016/j.carbpol.2022.119653 Received 24 January 2022; Received in revised form May 2022; Accepted 22 May 2022 Available online 27 May 2022 0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 at O-4 of the 1,2,4-linked Rhap and O-3 or O-4 of β-1,3,6-linked Galp The side chains are mainly α-T-Araf and α-1,5-linked Araf with O-3 as branching points, having trace Glc and Gal The terminal residues are Tlinked Araf, T-linked Glcp, and T-linked Galp As a major component, it looks similar to the reported heteroglycans from the same herb medicine (Zheng et al., 2020) RAP is a typical example of the poor bioavailability concern Orally administrated RAP exhibited promising antitumor activities in vivo, particularly the synergism in combination with Taxol in increasing survival rate of tumor bearing mice, and protection of the bone marrow of cyclophosphamide-treated mice (Bao et al., 2019; Bao et al., 2021) The mechanism investigations in vitro have revealed its inducing effects on macrophages via TLR4 signaling pathway, inducing phenotype po larization to antitumor M1 via Notch signaling pathway, and protection on mice/human hematopoietic stem cells (HSCs) via regulation of FOS expression (Bao et al., 2021; Wei et al., 2016; Wei et al., 2019) How ever, there is a huge gap between these in vivo and in vitro activities: whether/how the poorly bioavailable polysaccharide contact immune cells after oral administration (Barclay et al., 2019) Interestingly, we found that orally administrated RAP might be able to enter the bone marrow to protect chemotherapy-induced myelosup pression (Bao et al., 2021) Our preliminary data further indicated that RAP could quickly induce immune responses in the Peyer's Patches (PPs) of the small intestine in h, consistent with what we observed on macrophages in vitro (Wei et al., 2016) Therefore, a reasonable hy pothesis is that intact RAP may enter PPs and directly contact immune cells to trigger immune responses As the immune sensors in the small intestine, PPs, playing an essential role in linking intestinal antigens and the host mucosal immune responses (Jung et al., 2010), may provide a possible route Actually, PPs have been mentioned as a gateway for glucans, but the detection solely relied on the fluorescence or radioactivity signals and was poorly validated (De Jesus et al., 2014; Sakai et al., 2019) There are still a few doubts, including 1) whether the observed signal was only the fluores cent/radioactivity flag rather than the polymer; 2) whether poly saccharides stayed intact carbohydrate polymer in the PPs; 3) what is the direct target of intact polysaccharide in PPs; 4) whether PPs is accessible to all polysaccharides (Hashimoto et al., 1991; Pedro et al., 2021; Rice et al., 2005; Smet et al., 2013; Vetvicka et al., 2007; Xie et al., 2016; Zheng et al., 2022) The gateway via PPs for intact poly saccharides needs to be verified In this study, we compared RAP's effects on tumor growth between normal and nude mice first and confirmed that its antitumor effects are immune system-dependent We further tracked orally dosed RAP in the gastrointestinal tract using a series of methods including carbohydrate testing, fluorescence imaging, HPLC-FLD, and flow cytometry The re sults indicated that RAP, as an undegraded carbohydrate polymer, quickly entered the PPs to specifically activate DCs and trigger immune responses Confocal micrographs further revealed that DCs might sub sequently transport RAP from PPs to mesenteric lymph nodes (MLN), bone marrow, and even tumor tissue This interesting delivery route for intact RAP entering PPs must be selective as another control poly saccharide DOP from Dendrobium officinale could not take the same route These findings provide convincing evidence of an efficient lymphatic route for intact RAP to directly trigger immune responses after oral administration This interesting route might be also applicable to many other immunomodulatory carbohydrate polymers Diego, CA, USA) Phenol‑sulfuric acid, 4′ ,6-diamidino-2-phenylindole DAPI, fluorescein isothiocyanate isomer I FITC, methyl sulphoxide, and other related chemical reagents were all purchased from Sigma-Aldrich Corp (St Louis, MO, USA) High glucose Dulbecco Modified Eagle Medium (DMEM), heated-inactivated fetal bovine serum (FBS), GMCSF, and IL-4 were bought from the Thermo Fisher Scientific (Cleve land, OH, USA) 2.2 Mice and cell BALB/C and nude mice were purchased from the Chinese University of Hong Kong Five- to eight-week-old mice were used in this study The animals were provided with a standard pellet diet and purified water and maintained under controlled temperature and humidity conditions, with 12 h light/dark cycles All animal experiments followed the Ani mals Ordinance guidelines, Department of Health, Hong Kong SAR ((1665) in DH/HA&P/8/2/6, (19-151) in DH/HT&A/8/2/6) Macrophage RAW264.7 cells were bought from American Type Culture Collection (ATCC) and cultured in high glucose DMEM with 10% FBS in a humidified incubator at 37 ◦ C under an atmosphere of 5% CO2 2.3 Preparation of polysaccharides RAP was prepared from the water extract of the dried roots of Astragalus membranaceus and stored at ◦ C (Yin et al., 2012) In our previous work, its structure was elucidated by monosaccharide composition, partial acid hydrolysis, and methylation analysis, and further confirmed by FT-IR, GC–MS, and 1H and 13C NMR spectra, SEM and AFM microscopy (Yin et al., 2012) Before using the polysaccharide RAP, the average molecular weight, purity, the 1H NMR spectrum, 13C NMR spectrum, and monosaccharides composition of RAP was detected RAP was labeled with fluorescein isothiocyanate isomer I (FITC) as re ported (Li et al., 2019) Briefly, RAP (1.0 g) was dissolved in methyl sulphoxide (8 mL) containing a few drops of pyridine FITC (80 mg) was added to the RAP solution, followed by dibutyltin dilaurate (16 μL) The mixture was heated for h at 95 ◦ C After precipitation in ethanol (90% v/v) to collect the precipitate and remove the free dye, the FITC-RAP was re-dissolved in water and purified by molecular sieve (3 kDa cutoff) Using a procedure reported previously, control polysaccharide DOP was prepared from Dendrobium officinale and similarly labeled with FITC (Li et al., 2019) 2.4 4T1 breast tumor mouse model and treatment First of all, we optimized the dosages (50, 100, and 200 mg/kg) by testing RAP-induced immune responses in PPs, and further evaluated the selected dosage for RAP's suppression against 4T1 breast tumor in BALB/c female mice, with cisplatin (4 mg/kg) as the positive control Five-week-old BALB/c female and nude female mice were used for the tumor model (10 mice/group) Before establishing the tumor model, mice were pre-treated with 100 mg/kg RAP for seven days In accor dance with a previous study (Pulaski & Ostrand-Rosenberg, 2000), the implantation of T1 cells was performed on the seventh day Briefly, 4T1 cells (2 × 104/mouse) were implanted with a 27-G needle RAP treatment continued until the last day of sacrificing mice When tumors begin to develop, a vernier caliper was used to measure tumor diameters and calculate tumor volume After three weeks of tumor growth, animals were sacrificed, and organ samples were harvested for investigation, including tumor weight were weighted, and immune cells and cytokines were detected in the tumor and immune system Materials and methods 2.1 Materials Flow cytometry antibodies were bought from BioLegend (San Diego, CA, USA) CD11c primary antibody and goat anti-rat IgG antibody conjugated with Alexa Fluor 568 were purchased from Abcam (Cam bridge, UK) All mouse ELISA kits were purchased from eBioscience (San 2.5 Carbohydrate detection of intestinal contents by phenol‑sulfuric acid method The intestinal contents (i.e., stomach, the small intestine, and the Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 large intestine) of BALB/C mice that had received oral doses of RAP (10 mg/mouse) were collected at 1, 2, 3, 4, h after oral administration (n = each group) Similarly, the intestinal contents of mice that had not received RAP were collected as control Carbohydrates in the samples were detected by the phenol‑sulfuric acid method (Masuko et al., 2005) In detail, samples were homogenized and centrifuged at 15,000 rpm for 10 50 μL of each supernatant was injected in a well of 96-well microplate to which 150 μL of concentrated sulfuric acid was added and mixed rapidly 30 μL of 5% phenol in water was then added into the mixed solution, and it was incubated for at 90 ◦ C in a static water bath The plate was then cooled to room temperature and wiped dry for detection at A490 nm by a microplate reader Glucose was used as a reference standard to establish standard curves The experiments were repeated three times then homogenized and centrifuged at 15,000 rpm for 10 to obtain the supernatant for cytokine production assessment using ELISA kits According to the manufacturer's instructions, cytokines interleukin (IL-6), IL-12, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), monocyte chemotactic protein-1 (MCP-1), macro phage colony-stimulating factor (M-CSF), and interferon-gamma (IFN-γ) were determined using ELISA kits 2.10 Immunofluorescence staining and confocal microscopy Frozen sections of PPs from the ligated loop assay were washed three times with PBS and blocked with 5% normal goat serum in PBS for h Sections were incubated with anti-mouse CD11c antibody overnight at ◦ C PPs sections were washed three times with PBS and then treated with Alexa Fluor 568 secondary goat anti-rat antibody for h at RT in dark, followed by three PBS washes The cell nuclei were stained with μg/mL 4′ ,6-diamidino-2-phenylindole (DAPI) for 15 Sections were washed three times with PBS and mounted with an anti-fade mounting medium Images were captured with a Leica TCS SP8 confocal laser scanning microscope 2.6 Dynamic distribution of FITC-RAP after oral administration FTIC-RAP (10 mg/mouse) was orally administrated to each mouse (n = 6) The mice were sacrificed at 0, 1, 2, 3, 4, and h later, and blood and major tissues, including liver, spleen, kidney, stomach, small in testine, mesenteric lymph nodes (MLN), Peyer's patches (PPs), caecum, and colon, were collected and imaged using an IVIS Lumina XR in vivo imaging system (PerkinElmer) immediately The molecular size of FITCRAP was monitored using high-performance gel-permeation chroma tography Similarly, the dynamic distribution of FITC-DOP control in PPs was investigated For assay by confocal microscopy, cell suspensions from tissues or bone marrow were collected and detected for RAP dis tribution in vivo 2.11 Cell culture and treatment Macrophage RAW264.7 cells were used to confirm effects of RAP on macrophage differentiation as reported in previous study (Wei et al., 2019) In brief, RAW264.7 cells were treated with RAP at different concentrations (0.001, 0.01, 0.1, 1, 10, 100 μg/mL) After 24 h incu bation, cell suspensions were collected for ELISA assay According to the results, we chose an optimal concentration for the following study To figure out the effects of RAP on macrophage differentiation, we treated RAW264.7 cells with RAP at the optimal concentration and detected the surficial markers of macrophages by flow cytometry, including F4/80, CD80, CD86, and CD206 Antibodies for flow cytometry are shown in Table To figure out the potential receptors of RAP on DCs, bone marrowderived dendritic cells (BMDCs) were isolated as reported in previous study (Granucci et al., 2012) In brief, femur bones were collected and transferred into dishes with 70% ethanol on ice for min/time, repeated three times BM cells were collected, washed, and resuspended with 1× RBS water lysis to remove the red blood cells 2× 106 cells/mL were prepared with culture medium (RPMI-1640 + 10% FBS + 20 mM penicillin/streptomycin +20 ng/mL rmGM-CSF) 10 mL cell suspension was added into each petridish and was incubated at 37 ◦ C, 5% CO2 for days After removing old medium, 10 mL of fresh culture medium with 20 ng/mL GM-CSF was added to each petridish, and the cells were incubated for another days BMDCs were treated with RAP for 24 h at the optimal concentration and then screened the expression of surficial toll-like receptors by flow cytometry, including TLR1, TLR2, TLR4, TLR5, and TLR6 Antibodies are shown in Table 2.7 High-performance gel permeation chromatography coupled with fluorescence detector (HPGPC-FLD) analysis The tissues collected as described above were homogenized using a 3-fold volume of 0.1 mol/L phosphate buffer (pH 7.4) and centrifuged at 15,000 rpm for 10 PP collected from the ligated loop assay model was homogenized using 200 μL PBS and centrifuged at 15,000 rpm for 10 The supernatant was collected and stored at − 20 ◦ C for chro matographic analysis The separation was achieved on a TSK GMPWXL column (300 × 7.8 mm i.d., 10 μm) system operated at 40 ◦ C using an Agilent-1100 HPLC system equipped with FLD Ammonium acetate aqueous solution (20 mM) was used as a mobile phase at a 0.6 mL/min flow rate The excitation wavelength and emission wavelength of FLD were 495 and 515 nm, respectively 2.8 Western blotting (WB) PPs from different groups were treated with RAP (100 mg/kg), collected at time points (0, 1, 2, 3, and h) after oral administration, and prepared for WB In brief, PPs collected at different time points were lysed with RIPA protein extraction reagent containing protease and phosphatase inhibitors for 30 Protein samples were separated by 10% SDS–PAGE and transferred to a PVDF membrane Membranes were blocked in 5% blocker milk (BioRad) at room temperature (RT) for h then incubated with primary antibodies at ◦ C overnight with shaking According to our previous study (Wei et al., 2016), the primary anti bodies were GAPDH, P38, p-P38, P65, p-P65, ERK, and p-ERK The membranes were washed three times with PBST (0.1% Tween 20) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for h Protein bands were visualized using enhanced chemiluminescence (ECL) detection reagent and medical X-ray film Gray value of each band was evaluated with ImageJ software Table Antibodies used for flow cytometry analysis 2.9 ELISA for quantitative analysis of cytokines For ELISA assay, PPs were collected 24 h after 100 mg/kg RAP or RAP at different concentrations (50, 100, and 200 mg/kg) treatment, Fluorophore Antibody Clone Vendor APC FITC PE PE/Cyanine7 PE PE PE/Cyanine7 PE PerCP PE PE PE/Cyanine Alexa Fluor647 PE Anti-CD11c Anti-MHCII Anti-CD4 Anti-CD8a Anti-CD19 Anti-CD80 Anti-F4/80 Anti-CD86 Anti-CD206 Anti-TLR1Anti-TLR2 Anti-TLR4 Anti-TLR5 Anti-TLR6 N418 M5/114.15.2 GK1.5 53–6.7 1D3/CD19 16-10A1 BM8 A17199A C068C2 TR23 CB225 MTS510 ACT5 418,601 BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend BioLegend TheremoFisher BioLengend BioLegend BioLegend R&D systems Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 2.12 Flow cytometry analysis Results For the detection of RAP-induced immune responses, tumor tissues and PPs were minced then ground with a syringe plug PPs collected from mice were ground with a syringe plug for the isolation and detection of immune cells Tissues and cells were collected and rinsed with ice-cold PBS at 400 g for min, then filtered through a 70 μm filter Single-cell suspensions were prepared for antibody staining Cells were incubated with antibodies or the matching isotypes for 25 at room temperature The stained cells were rinsed twice, resuspended in PBS, and analyzed by FACSAria III (BD Biosciences) Data were analyzed with FlowJo V10 software Antibodies for flow cytometry are shown in Table 3.1 RAP's antitumor activity is immune system-dependent As shown in Supplementary Fig 1A–D, the average molecular weight and purity of RAP remain unchanged, and the 1H NMR spectrum, 13C NMR spectrum, and monosaccharides composition further confirmed its stable chemistry The average molecular weight of RAP was 1334 kDa And RAP was composed of Rha, Ara, Glc, Gal and GalA in a molar ratio of 0.03:1.00:0.26:0.37:(0.28) The antitumor beneficial effects of polysaccharides isolated from Radix Astragali have been closely associated with its immunomodula tory effects (Li et al., 2020; Yang et al., 2013; Lijing Zhou et al., 2017) Here we first optimized RAP's dosages by testing RAP-induced immune responses in PPs, and the results showed that 100 mg/kg is the optimal to induce significant increase the IL-6 and TNF-α production in 24 h after oral administration (Supplementary Fig 2) The further evaluation, with cisplatin as the positive control, confirmed that this dosage significantly suppressed the growth of 4T1 breast tumor (Supplementary Fig 3) And it was much safer than the positive control cisplatin which caused half death Therefore, 100 mg/kg was selected for the subsequent tests To determine if RAP's antitumor effects are dependent on its immunomodulatory effects, we compared RAP's effects on the 4T1 2.13 Statistical analysis Each experiment was independently repeated three times Statistical analysis was performed by IBM SPSS Statistics 25 software As noted in figure legends, all data are shown as mean ± SD Statistical differences between each experimental group were analyzed by Student's t-test or one-way ANOVA Differences with P < 0.05 were considered significant Fig RAP's antitumor activity is dependent on the immune system (A) Timeline of 4T1 tumor-bearing mouse model RAP was pre-treated for d before 4T1 cells were implanted into the mammary fat pads of BALB/c mice or nude mice (n = 8– 10 for each group) and then the treatment continued Mice were sacrificed after 21 d treatment with RAP (B and C) Tumor (B) and tumor weight (C) in BALB/c mice (D and E) Tumor (D) and tumor weight (E) in nude mice (F and G) Percentage of CD8+ T cells (CTL, F) and CD4+CD25+ T cells (Treg, G) in the tumor of BALB/c mice (H–K) Cytokine production in tumors isolated from BALB/c mice, including TGF-β (H), IL-10 (I), IFN-γ (J), and MCP-1 (K), Data are shown as mean ± SD Significant difference *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 breast tumor growth between BALB/c mice and the immune-deficient nude mice (Fig 1A) The results showed that 100 mg/kg RAP signifi cantly suppressed tumor growth (P < 0.001; Fig 1B and C) in BALB/c mice but became inactive in nude mice (Fig 1D and E) Body weight of mice did not show a big difference during the whole experiment (Sup plementary Fig 4) Thus, these findings suggest that RAP has a signifi cant antitumor effect, which is related to its immunoregulation functions We further screened immune cells and cytokines to reveal RAPinduced immune responses in tumor tissues by Flow cytometry and ELISA assay Our data indicated that RAP could induce antitumor im mune responses in tumor tissues, including the increase of CD3+CD8+ cytotoxic T lymphocyte (CTL) (P < 0.05; Fig 1F), the decrease of CD4+CD25+ regulatory T cells (Treg, P < 0.05; Fig 1G) and changes in antitumor-related cytokines TGF-β (P < 0.05; Fig 1H), IL-10 (P < 0.05; Fig 1I), IFN-γ (P < 0.05; Fig 1J), and MCP-1 (P < 0.05; Fig 1K) These findings suggest that the immune system plays a critical role in RAP's anti-tumor effects Furthermore, a big difference between the tumor tissues of BALB/c and nude mice was found in the population and differentiation of macrophages Flow cytometry results showed that total macrophages of tumor tissues were significantly decreased in RAP-treated BALB/c mice (P < 0.01, Fig 2B) but not in nude mice (Fig 2D) Further analysis of macrophage differentiation in BALB/C mice showed that RAP caused a noticeable increase of the F4/80+CD11bhigh M1-type macrophage (MTM, P < 0.001) and a significant decrease of F4/80+CD11blow tumorassociated macrophage (TAM, P < 0.01) in BALB/c mice (Fig 2A and B), both of which contributes to the inhibition of tumor growth (Franklin et al., 2014) While in the tumor tissue collected from nude mice, neither MTM nor TAM was affected by RAP treatment (Fig 2C and D) To confirm the effects of RAP on differentiation of macrophages, we also test the effects of RAP on RAW264.7 cells in vitro Interestingly, RAP promoted the differentiation of RAW264.7 cells into F4/ 80+CD80+CD86+ M1 type macrophages (Fig 2E-G) but had no effects on CD206 expression (Fig 2H) Besides, RAP also induced RAW264.7 cells to produce IL-6 and TNF-α production (Fig 2I and J) These in vitro findings suggest that RAP could induce macrophage differentiating into MTM Taken together, the above findings prove that RAP-induced antitumor effects are dependent on the immune system Fig Differentiation of macrophages induced by orally administrated RAP (A) Dot plots of flow cytometry showing F4/80+CD11b+ macrophages in 4T1 breast tumor-bearing BALB/C mice (B) Per centages of F4/80+ macrophages, M1 type macro phages (MTM, F4/80+CD11bhigh macrophages), and tumor-associated macrophages (TAM, F4/ 80+CD11blow macrophages) in the tumor of BALB/c mice (C) Dot plots of flow cytometry showing F4/ 80+CD11b+ macrophages in 4T1 breast tumorbearing nude mice (D) Percentage of F4/80+ mac rophages, MTM, and TAM in 4T1 breast tumorbearing nude mice (E-H) Flow cytometry histogram of F4/80 (E), CD80 (F), CD86 (G), and CD206 (H) expression on RAW264.7 cells RAW264.7 cells were treated with RAP (blue) or without treatment (con trol, red) (I and J) Cytokine IL-6 (I) and TNF-α (J) production from RAW264.7 cells with treatment of RAP at different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 100 μg/mL) Data are shown as mean ± SD Significant difference **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significance Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 3.2 Intact RAP is selectively transported into lymphatic system tracts after treating with the unlabeled RAP by oral administration The results showed that the carbohydrate quickly left the stomach in 1–2 h (Fig 3A), mainly stayed in the small intestine for 3–4 h (Fig 3B), but shortly occurred in the large intestine only at the third hour (Fig 3C) And the carbohydrate content detected in the large intestine was only around 1/6 of that in the small intestine These results are consistent with DOP's destiny where the carbohydrate polymers were quickly degraded to short-chain fatty acids in the large intestine (Li et al., 2019) Then we labeled RAP with FITC to improve the detection sensitivity for subsequent tracking its distribution in organs (Supplementary Fig 5) Analysis of fluorescence intensity demonstrated a similar We previously found the positive signal of FITC-RAP in the bone marrow (Bao et al., 2021) but did not find it in the serum So, we hy pothesized that there might be a blood-independent way for RAP to work in the body To test this possibility, we systematically tracked RAP in its oral route We already found that Dendrobium officinale poly saccharide (DOP), being indigestible and unabsorbed, ended in regu lating gut microbiota as a prebiotic (Li et al., 2019) So, DOP was used as a control polysaccharide We first monitored the carbohydrate contents in the gastrointestinal Fig Dynamic distribution of RAP in the gastrointestinal tract (A-C) Dynamic carbohydrate contents in the stomach (A), the small intestine (B), and the large intestine (C) collected from normal mice at 1– h after gavage with unlabeled RAP (10 mg/mouse); RAP was detected by the phenol‑sulfuric acid method Mice without RAP treatment is the control group (D) Fluorescence images of major organs, including stomach, small intestine, caecum, colon, liver, kidneys, and spleen collected from normal mice (n = 6) at to h after gavage with FITC-RAP (10 mg/mouse) Mice without RAP treatment is the control group (0 h) (E) HPGPC-FLD chromatograms of FITC-RAP in serum (F-K) Fluorescence intensity of small intestine (F), caecum (G), colon (H), liver (I), kidneys (J), and spleen (K) (L-N) HPGPCFLD chromatograms of FITC-RAP in the small intestine (L), caecum (M), and colon (N) Red dotted lines show the chromotographic retention time of original FITCRAP Significant difference *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 dynamic distribution of FITC-RAP signals in the gastrointestinal tract (Fig 3D and F-H), suggesting positive signals of FITC-RAP were detected in the small intestine (Fig 3F), caecum (Fig 3G), and colon (Fig 3H) And no positive signals were detected in the serum (Fig 3E), liver (Fig 3I), kidneys (Fig 3J), or spleen (Fig 3K), showing that FITC-RAP could not be absorbed into the blood system, liver, kidney, and spleen The HPGPC-FLD chromatograms further indicated that FITC-RAP remained undegraded in the small intestine (Fig 3L) but quickly degraded in the caecum (Fig 3M) and colon (Fig 3N) Therefore, the fluorescence signals in the caecum and colon were proved to be unre liable to detect carbohydrate polymers These data suggest that the small intestine is the main location where RAP remained an undegraded car bohydrate polymer for a long time Further examination of the gut-associated lymphoid tissues PPs, which are the immune sensors of the small intestine, offered interesting data The fluorescence images showed that FITC-RAP, but not FITCDOP, transiently accumulated in the PPs about 3–4 h (P < 0.001; Fig 4A and B) The procedure was highly consistent with RAP's stay in the small intestine As the fluorescence intensity reached maximum at h, HPGPC analysis was used to further confirm the integrity of FITCRAP The result indicated that FITC-RAP remained intact in PPs (Fig 4C), while FITC-DOP failed to enter PPs (Fig 4D) The confocal micrographs also clearly displayed the occurrence of FITC-RAP in PPs (Fig 4E) These results demonstrate that the PPs gateway for RAP is efficient and selective 3.3 RAP directly targets follicle dendritic cells and initiates immune responses in Peyer's patches After entering PPs, the cells directly targeted by RAP play a critical role in the beginning and initiation of its antitumor immune responses By screening immune cells in PPs using flow cytometry, we observed that clearly at h after FITC-RAP treatment, the positive signals of FITCRAP were only detected in the monocytes (Fig 5C) rather than CD8+ T cells (Fig 5A), CD4+ T cells (Fig 5B), or CD19+ B cells (Fig 5D) Furthermore, without a significant increase of CD11c+ DCs (Fig 5E and F), a noticeable portion (22.4%) of CD11c+ DCs among those monocytes could bind with FITC-RAP (Fig 5G and H), suggesting that CD11c+ DCs might be RAP's direct target cells Observations of frozen sections confirmed that FITC-RAP was directly captured by CD11c+ DCs in PPs (Fig 5I) These data suggest that RAP could directly target FDCs after being transported into PPs Further analysis of RAP-induced immune responses in PPs showed that the proportion of CD11c + DCs was significantly increased by RAP Fig Selective transportation and intact detection of RAP in PPs (A and B) Fluorescence images (A) and fluorescence intensity (B) of PPs separated from small intestines collected from normal mice at to h after gavage with FITC-RAP and FITC-DOP (10 mg/mouse) Mice without RAP treatment is the control group (0 h) (C and D) HPGPC-FLD chromatograms of PPs at h after gavage with FITC-RAP (C) and FITC-DOP (D) (E) Confocal microscopic images of PP sections isolated from mice treated with FITC-RAP (green) for h DAPI (blue) was used as a DNA-specific stain Scale bar, 40 μm Significant difference ****P < 0.0001, ns = no significance Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 Fig Direct contact of RAP with DCs in vivo (A-D) Flow cytometry histogram of RAP-bound immune cells, including CD8+ T cells (A), CD4+ T cells (B), monocytes (C), and CD19+ B cells (D) of PPs isolated from mice treated with FITC-RAP (10 mg/kg) for h (E and F) Flow cytometry analysis (E) and percentage (F) of CD11c+ DCs population in the monocytes of PPs isolated from mice of the control and FITC-RAPtreated groups (G and H) Flow cytometry histogram (G) and percentage (H) of RAP-binded CD11c+ DCs in the CD11c+ DCs shown in Fig 5E (I) Confocal images of PP frozen sections (dome zone) collected from mice treated with FITC-RAP for h FITC-RAP (green), CD11c (yellow), DAPI (blue), and scale bar, 40 μm Significant difference ****P < 0.0001, ns = no significance treatment at 24 h (Fig 6A), suggesting that RAP could increase the population of DCs in the PPs Flow cytometry analysis also indicated that CD80 and MHCII expressions of CD11c+ DCs were induced in 24 h after oral administration of RAP (Fig 6B), suggesting that RAP would quickly induce the maturation and differentiation of DCs in PPs As we previ ously found that RAP could quickly induce the phosphorylation of MAPKs and NF-κB signaling pathways (Wei et al., 2016), we next examined how orally administrated RAP affected these signaling path ways in PPs at different time points (1–4 h) Western blotting results showed that RAP could quickly induce the phosphorylation of ERK, p38, and p65, implying the activation of MAPKs and NF-κB signaling in the PPs in h (Fig 6C) In addition, cytokines production was also detected, and the results indicated that RAP was quickly transported into PPs to trigger immune responses The results of the ELISA assay indicated that 100 mg/kg RAP could up-regulate the production of IL-6 (P < 0.001; Fig 6D), TGF-β (P < 0.05; Fig 6F), and INF-γ (P < 0.05; Fig 6G), but down-regulate M-CSF (P < 0.01; Fig 6E) in PPs isolated from mice treated with RAP for 24 h Taken together, these results demonstrated that RAP quickly triggered immune responses in PPs by targeting and activating FDCs, which should be the initiation of RAP-induced immu noregulation in vivo 3.4 Follicle dendritic cells might further transport RAP in the lymphatic system To figure out the destiny of RAP after being transported into PPs, we determined the signal of FITC-RAP in the distant lymphatic organs using immunofluorescence staining assay Further tracking the binding of CD11c+ DCs and FITC-RAP using confocal microscopy revealed that, after the treatment of FITC-RAP for 24 h, positive signals of FITC-RAP were detected in mesenteric lymph node (MLN, Fig 7A), bone marrow (Fig 7B), and even tumor tissues (Fig 7C) Furthermore, the binding of CD11c+ DCs and FITC-RAP was demonstrated in the merged images of these organs, as found in PPs Thus, these observations collectively Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 Fig Immune responses induced by RAP in PPs (A) Flow cytometry histogram of CD11c+ DCs from PPs of mice treated with or without RAP for 24 h Red, control group; blue, RAP-treated group (B) Dot plots of cell surface expression of MHCII and CD80 in CD11c+ DCs shown in Fig 6A (C) Signaling path ways of NF-κB and MAPKs (p38 and ERK) of PPs at 1– h after oral administration with RAP (100 mg/ kg), as determined by Western blotting assay GAPDH was the control Gray value of protein was analyzed by ImageJ (D-G) IL-6 (D), M-CSF (E), TGF-β (F), and IFN-γ (G) production in PPs of the small intestine collected from mice 24 h after RAP treatment PP homogenate was collected and detected by ELISA kits Data are shown as mean ± SD Significant dif ference *P < 0.05, **P < 0.01, ***P < 0.001 suggest that RAP might be further transported to distant organs via the lymphatic system by FDCs, providing a chance for RAP to directly contact diverse cells and to exert beneficial effects In this regard, this lymphatic route successfully bridges the gap between the in vivo and in vitro investigations Discussion The current investigations of polysaccharide pharmacokinetics solely rely on interpreting fluorescence signals; however, this method needs to be validated before it can be considered reliable The fluores cence signal might arise from the fluorescence reagent itself if the sample is not pure enough or if the fluorescence flag is released some how So, the first step to ensuring valid fluorescent results is to confirm Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 Fig Distribution of FITC-RAP in other tissues (A–C), Confocal microscopic images of cell suspension isolated from mesenteric lymph nodes (MLN, A), bone marrow (BM, B), and tumor (C) The tumor-bearing mice were treated with FITC-RAP (green) for 24 h Cell suspensions were stained with CD11c-APC antibody (red) Scale bar, 20 μm the purity and stability of the labeled polymer In this study, we used HPGPC-FLD to check the purity (Supplementary Fig 5A and C) and HPGPC-CAD to determine whether the FITC-RAP polymer has been degraded after labeling The second concern is that the polymer might be degraded in the gut This concern is proved by the difference of the signals in the caecum and colon between Fig 3G/H and M/N It was clearly shown that the positive fluorescence signals observed in the caecum and colon came from the degraded chemicals instead of the intact polymer The fluorescence signal needs to be validated So, we not only tested the carbohydrate property using the phenol‑sulfuric acid method, but also checked the molecular size using HPGPC-FLD to see if RAP remains an intact carbohydrate polymer in the intestinal organs With these validations, the fluorescence signal observed in the confocal micrograph can be assigned to the polymer with confidence Here we present the first evidence to show that RAP remains intact in the lymphatic system after oral administration Most in vitro studies of polysaccharides were focused on macro phages (Guan et al., 2020; Kallon et al., 2013; L Zhou et al., 2017), while our findings highlight the importance of FDCs in the mechanism of polysaccharide's bioactivities The cells that polysaccharides directly target after passing through the gut cell wall have not been directly identified before (Wang et al., 2021; Yin et al., 2019; Yue Yu et al., 2018; Zhao et al., 2020) Our findings clearly reveal that FDCs are RAP's direct targets in PPs and further deliver RAP to other lymphatics and even tumor tissues These results may offer a further explanation about the initiation of polysaccharide-induced immune responses, which high lighted the important role of FDCs The multiple immunomodulatory effects of RAP in vivo might result from a combination of the lymphatic system-dependent effects in the small intestine and gut microbiota-related effects in the large intestine The interaction between prebiotics and gut microbiota needs time and cannot explain the fast immune responses in the small intestine induced by orally administrated polysaccharides (Kim et al., 2019; Sakai et al., 2019) This lymphatic route found in this study works h before RAP arrives the caecum where gut microbiota mainly works, therefore it is independent to gut microbiota But we cannot exclude the possibility of microbiota-related immune-regulation because a large amount of RAP entered the caecum and was quickly digested (Fig 3M and N) Consis tent with this finding, many studies have shown that gut microbiota could digest polysaccharides to produce short-chain fatty acids (SCFAs) which are the mediators of microbiota and the immune system (Hong et al., 2020; Li et al., 2019; Liu et al., 2019; Zhou et al., 2021) We speculate that polysaccharides' beneficial effects are a combination of activity in both the small and large intestines Although we here proved a blood/microbiota independent and se lective lymphatic route for RAP to work in vivo, one limitation of this study is that we are not sure how RAP passes through the gut cell wall As shown in the Fig 4A and B, the control polysaccharide DOP failed to enter PPs, suggesting that this lymphatic route is selective to poly saccharides There are several possible cell receptors known to poly saccharides, such as TLRs, which might also mediate this delivery route (Batbayar et al., 2012; Taylor et al., 2007; Wei et al., 2016) By screening surficial TLRs using flow cytometry (Supplementary Fig 6), we found TLR4 might be the targeting receptor of RAP on DCs, which is consistent 10 Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 with results shown in previous studies Interestingly, we also found TLR1 expression on DCs was induced by RAP, suggesting TLR1 is a possible related receptor of RAP as well We speculate the way of RAP passing through the gut cell wall might be receptor-dependent, which deserves further investigation Sciences Appendix A Supplementary data Supplementary data to this article can be found online at https://doi org/10.1016/j.carbpol.2022.119653 Conclusions References In summary, our results demonstrated that intact Radix astragali polysaccharide RAP could quickly enter the lymphatic system after oral administration and directly target FDCs to initiate immune responses, which plays a crucial role in the tumor suppression of RAP These findings indicate a blood/microbiota independent and selective lymphatic route for intact RAP to modulate the immune system This route helps bridge the gap between the in vitro and in vivo studies of natural polysaccharides and spires novel insights into how orally administered polysaccharides work in vivo Bao, W R., Li, Z P., Zhang, Q W., Li, L F., Liu, H B., Ma, D L., & Han, Q B (2019) Astragalus polysaccharide RAP selectively attenuates paclitaxel-induced 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hemopoietic system International Journal of Biological Macromolecules, 124, 505–514 CRediT authorship contribution statement Quanwei Zhang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft Lifeng Li: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft Shuang Hao: Methodology, Formal analysis, Visualization Man Liu: Methodology, Formal analysis, Visualization Chuying Huo: Methodology, Formal analysis, Visualization Jianjun Wu: Methodol ogy, Formal analysis, Visualization Hongbing Liu: Methodology, Formal analysis, Visualization Wanrong Bao: Methodology, Formal analysis, Visualization Hongming Zheng: Methodology, Formal anal ysis, Visualization Zhipeng Li: Methodology, Formal analysis, Visual ization Huiyuan Cheng: Methodology, Formal analysis, Visualization Hauyee Fung: Methodology, Formal analysis, Visualization Tinlong Wong: Methodology, Formal analysis, Visualization Pingchung Leung: Writing – review & editing Shunchun Wang: Writing – review & editing Ting Li: Writing – review & editing Ge Zhang: Writing – review & editing Min Li: Writing – review & editing Zhongzhen Zhao: Writing – review & editing Wei Jia: Writing – review & editing Zhaoxiang Bian: Writing – review & editing Timothy Mitchison: Writing – review & editing Jingchao Zhang: Writing – review & edit ing Aiping Lyu: Writing – review & editing Quanbin Han: Concep tualization, Funding acquisition, Project administration, Resources, Writing – original draft, Writing – review & editing Handong Sun: Writing – review & editing 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 Acknowledgments This work was funded and supported by HKSAR Innovation and Technology Fund (ITF), Tier 3, ITS/311/09, General Research Fund (12100615, 22100014, 12100818), UGC Research Matching Grant Scheme (2019-1-10, 2019-1-14, 2019-2-06), Health Medical Research Fund (11122531, 14150521, 17182681), National Natural Sciences Foundation in China (81473341, 82173948), the Science and Technol ogy Project of Shenzhen (JCYJ20160531193812867), the Key-Area Research and Development Program of Guangdong Province (2020B1111110007), Hong Kong Baptist University (RC-Start-up Grants, MPCF-001-2016/2017, MPCF-002-2021-22, RC-IRMS/14-15/ 06, IRMS-20-21-02, FRG2/17-18/060 and FRG2/16-17/002), and Vin cent & Lily Woo Foundation We appreciate the advice from Prof Ding Kan at Shanghai Institute of Materia Medica, Chinese Academy of 11 Q Zhang et al Carbohydrate Polymers 292 (2022) 119653 Yang, B., Xiao, B., & Sun, T (2013) Antitumor and immunomodulatory activity of Astragalus membranaceus polysaccharides in H22 tumor-bearing mice International Journal of Biological Macromolecules, 62, 287–290 Yin, J., Chan, B C., Yu, H., 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pathway Molecules, 24(10), 2016 Wei, W., Xiao, H T., Bao, W R., Ma, D L., Leung, C H., Han, X Q., & Han, Q B (2016) TLR-4 may mediate signaling pathways of astragalus polysaccharide RAP induced cytokine expression of RAW264.7 cells Journal of Ethnopharmacology, 179, 243–252 Xie, Y., Hu, X., He, H., Xia, F., Ma, Y., Qi, J., & Wu, W (2016) Tracking translocation of glucan microparticles targeting M cells: Implications for oral drug delivery Journal of Materials Chemistry B, 4(17), 2864–2873 12 ... bioavailable polysaccharide contact immune cells after oral administration (Barclay et al., 2019) Interestingly, we found that orally administrated RAP might be able to enter the bone marrow to. .. of an efficient lymphatic route for intact RAP to directly trigger immune responses after oral administration This interesting route might be also applicable to many other immunomodulatory carbohydrate... Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft Shuang Hao: Methodology, Formal analysis, Visualization Man Liu: