A lymphatic route for a hyperbranched heteroglycan from Radix Astragali to trigger immune responses after oral dosing

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.

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/)

A lymphatic route for a hyperbranched heteroglycan from Radix Astragali

to trigger immune responses after oral dosing

aSchool of Chinese Medicine, Hong Kong Baptist University, Hong Kong 999077, China

bThe First Affiliated Hospital, Zhengzhou University, Zhengzhou 450000, China

cState Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Hong Kong 999077, China

dInstitute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

eState Key Laboratory for Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau 999078, China

fLaboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston 02115, United States

gState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

A R T I C L E I N F O

Keywords:

Radix Astragali

Polysaccharide

Intact

Targeting route

Antitumor immune responses

A B S T R A C T

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 1 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/microbiota-

independent 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

1 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 anahydro-lysis, GC-MS, NMR spectra, SEM and AFM

mi-croscopy The backbone of RAP consists of 1,2,4-linked Rhap, α-1,4-

linked 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)

1 These authors contributed equally

Contents lists available at ScienceDirect Carbohydrate Polymers

journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2022.119653

Received 24 January 2022; Received in revised form 5 May 2022; Accepted 22 May 2022

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 T-

linked 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 1 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

2 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

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), GM- CSF, 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 ((16- 65) 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 4 ◦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 2 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 cut- off) 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 4 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

2.5 Carbohydrate detection of intestinal contents by phenol‑sulfuric acid method

The intestinal contents (i.e., stomach, the small intestine, and the

large intestine) of BALB/C mice that had received oral doses of RAP (10

mg/mouse) were collected at 1, 2, 3, 4, 5 h after oral administration (n

=4 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 min 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 5 min 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

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 5 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 FITC-

RAP 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

2.7 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 min PP collected from the ligated loop assay model

was homogenized using 200 μL PBS and centrifuged at 15,000 rpm for

10 min 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 4 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 min 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 1 h

then incubated with primary antibodies at 4 ◦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 1 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

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,

then homogenized and centrifuged at 15,000 rpm for 10 min to obtain the supernatant for cytokine production assessment using ELISA kits According to the manufacturer's instructions, cytokines interleukin 6 (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 1 h Sections were incubated with anti-mouse CD11c antibody overnight at

4 ◦C PPs sections were washed three times with PBS and then treated with Alexa Fluor 568 secondary goat anti-rat antibody for 1 h at RT in dark, followed by three PBS washes The cell nuclei were stained with 1

μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 15 min 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

2.11 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 1

To figure out the potential receptors of RAP on DCs, bone marrow- derived 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 2 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 3 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 3 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 1

Table 1

Antibodies used for flow cytometry analysis

2.12 Flow cytometry analysis

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 5 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 min 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 1

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

3 Results

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

Fig 1 RAP's antitumor activity is dependent on the

immune system (A) Timeline of 4T1 tumor-bearing

mouse model RAP was pre-treated for 7 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

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 RAP-

induced 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 tumor-

associated 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 2 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 tumor-

bearing nude mice (D) Percentage of F4/80+

mac-rophages, MTM, and TAM in 4T1 breast tumor-

bearing 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

3.2 Intact RAP is selectively transported into lymphatic system

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

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

Fig 3 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– 5 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 1 to 5 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) HPGPC- FLD chromatograms of FITC-RAP in the small intestine (L), caecum (M), and colon (N) Red dotted lines show the chromotographic retention time of original FITC-

RAP Significant difference *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance

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 FITC-

DOP, 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 2

h, HPGPC analysis was used to further confirm the integrity of FITC-

RAP 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 1 h after FITC-RAP treatment, the positive signals of FITC- RAP 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 4 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 0 to 5 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 2 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 2 h DAPI (blue) was used as a DNA-specific stain 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 1 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

Fig 5 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 1 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-RAP-

treated 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 1 h FITC-RAP (green), CD11c (yellow), DAPI (blue), and scale bar,

40 μm Significant difference ****P < 0.0001, ns =

no significance

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

4 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 fluoresfluores-cence 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

Fig 6 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– 4 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

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 2 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 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

Fig 7 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