Effect of polyvinyl butyrate nanoparticles incorporated with immunesuppressing vitamins on alteration of population of intestinal immune cells

Yunmei Mu ,Yusuke Kinashi ,Akihiro Kishimura ,Takeshi Mori ,Koji Hase ,Yoshiki Katayama,g,＊＊＊

a Graduate School of Systems Life Sciences,Kyushu University,744 Motooka,Nishi-ku,Fukuoka,819-0395,Japan

b Division of Biochemistry,Faculty of Pharmacy,Keio University,Tokyo,105-8512,Japan

c Department of Applied Chemistry,Faculty of Engineering,Kyushu University,744 Motooka,Nishi-ku,Fukuoka,819-0395,Japan

d Center for Future Chemistry,Kyushu University,744 Motooka,Nishi-ku,Fukuoka,819-0395,Japan

e International Research Center for Molecular System,Kyushu University,744 Motooka,Nishi-ku,Fukuoka,819-0395,Japan

f Division of Mucosal Barrierology,International Research and Development Center for Mucosal Vaccines,The Institute of Medical Science the University of Tokyo,Tokyo,108-8639,Japan

g Department of Biomedical Engineering,Chung Yuan Christian University,200 Chung Pei Rd,Chung Li,32023,Taiwan

Keywords:Butyrate Polyvinyl butyrate Retinoic acid Vitamin D3 Tolerogenic dendritic cells Regulatory T cells

ABSTRACT The nanoparticles (NPs) from polyvinyl butyrate (PVBu) which can work as an orally applicable donor of butyrate for intestine were prepared.Immunotolerance inducing molecules such as vitamin D3 and all-trans retinoic acid (ATRA) were incorporated into PVBu NPs.The alteration in populations and numbers of DC103+dendritic cells (DC) and regulatory T (Treg) cells in intestinal immune tissues were examined after oral administration of NPs.It was found that NPs reduced the population of CD103+ DC and Treg cells in Peyer’s patched in lower part of the intestine and inversely increased the population of CD103+DC in mesenteric lymph node(MLN),while the population of Treg cells in MLN was unchanged.These observations indicate that NPs may enhance the immunotolerance at MLN and lamina propria toward luminal antigens,indicating the promising features of PVBu NPs as therapeutics of allergy and autoimmune diseases.

1.Introduction

Intestinal immune system is maintained to be tolerant against exogenous antigens such as food.To maintain the immunotolerance,food originated compounds such as butyrate [1],all-trans retinoic acid(ATRA) [2-4] and vitamin D3[5] affect gene expression in intestinal epithelial cells and immune cells such as dendritic cells (DC) and regulatory T cells(Treg)via working as an inhibitor of histone deacetylates or a ligand of transcription factors [6,7].The alteration of gene expression by these compounds contribute to tolerance induction toward exogenous antigens as well as maintenance of intestinal health.

Previously,we proposed polyvinyl butyrate (PVBu) as a donor of butyrate [8].PVBu is hydrophobic polymer and nanoparticles (NPs)prepared from PVBu could be degraded by enzymes such as lipases and esterases to release butyrate.After hydrolysis by enzymes,the main chain of PVBu is converted to polyvinyl alcohol which is a biocompatible polymer.We found that PVBu NP suppressed the activation of macrophagesin vitroalthough butyrate in free form inversely activated macrophages.Oral administration of PVBu NP ameliorated intestinal inflammation in mice model probably via suppressing immune activation.We found that incorporation of vitamin D3into PVBu NP showed synergetic effect on the amelioration of the intestinal inflammation.Thus,PVBu NP is a promising therapeutic of the intestinal inflammation not only as a donor of butyrate but also as a carrier of hydrophobic molecules to intestinal cells.

In the present study,we focused on the effect of PVBu NP on intestinal immune cells related to acquired immunity,which govern the induction of immunotolerance toward exogenous antigens (Fig.1).If PVBu NP has characteristics to enhance the intestinal immunotolerance,it may be useful as a therapeutic of allergy and autoimmune diseases,which are caused by antigen specific immune activation.We prepared PVBu NP containing vitamin D3or ATRA which enhance the immunotolerance [9-11],and examined the alteration of populations and numbers of DC and Treg cells in Peyer’s patches (PP) and mesenteric lymph nodes(MLN)in the intestine.PP are the lymphoid follicles in the intestine in which immune response to luminal antigens originated from food and intestinal bacteria is executed.

2.Experimental

2.1.Materials

Vinyl butyrate and all trans retinoic acid were purchased from Tokyo Chemical Industry Co.,Ltd.(Tokyo,Japan).Azobisisobutyronitrile,acetone,methanol and toluene were purchased from Wako Pure Chemical Industries,Ltd.(Tokyo,Japan).Pluronic F-127 was purchased from Sigma-Aldrich (St.Louis,USA).Calcitriol (1,25(OH)2D3;vitamin D3)was obtained from Cayman chemical(≥97%,Michigan,USA).

2.2.Preparation of PVBu NPs

PVBu was synthesized by radical polymerization according to our previous report [8].PVBu nanoparticles (NPs) were prepared by oil-in-water emulsion method following our previous report [8] with some modifications.2 ml of toluene solution containing PVBu(0.4 g/ml)[in case of incorporation,vitamin D3(1.25 μg/ml)or ATRA(31 μg/ml)was mixed into the toluene solution] was prepared.This solution was added to 20 ml aqueous solution containing Pluronic F-127 (5%) and 100 mg ascorbic acid.The two phases solution were mixed by homogenizer(T25 digital Ultra turax,IKA,Germany)at 12,000 rpm for 10 min and sonicated with a probe sonicator(UD-211(TOMY)equipped with a TP-040 tip) for 5 min (20% power,20 kHz,20 W).Then toluene was evaporated from the solution by stirring (300 rpm) overnight.NP dispersions were stored as prepared at 4°C.The size,polydispersity index(PDI) and ζ-potential of the obtained NPs were measured by Zeta Sizer Nano Series(Malvern Instrument,UK)at 25°C.To measure the size and PDI,the PVBu NP dispersions were diluted with water to a concentration of 0.27 mg/ml PVBu.For measurement of the ζ-potential,the PVBu NP dispersions were diluted with 10 mM HEPES buffer (pH 7.4) to a concentration of 0.27 mg/ml PVBu.

2.3.The entrapment efficiency of ATRA and vitamin D3in NPs determined by HPLC

PVBu NPs dispersion were centrifuged at 220,000 g for 30 min.After removing the supernatant,NPs were dissolved in methanol.The samples were analyzed by HPLC (HITACHI,Japan) equipped with a SunFire™Pre C18 column(10 mm×150 mm×5 μm).Analysis conditions are as follows:20 μl samples injection,mobile phase consisting of 90% methanol and 10%acetonitrile at a flow rate of 1.0 ml/min,the UV detector was set for ATRA and vitamin D3at 325 nm and 260 nm,respectively.The concentration of ATRA and vitamin D3were quantified from calibration curves.

2.4.Animal experiment

The C57BL/6 male mice were purchased from CLEA Japan.Mice were housed in a facility maintained in 25°C and 12 h light/dark cycle.All animal experiments were performed using protocols approved by Animal care of Keio University.The 4-week-old C57BL/6 mice were fed AIN93G-fomula diet(Oriental Yeast,Japan)for 21 days.From day 7,the mice received NP,NP-A or NP-D daily[10 ml/kg body weight,0.27 g/ml PVBu (2.4 M in butyrate) containing 8.8 μg/ml ATRA or 0.56 μg/ml vitamin D3]by oral administration for 14 days.At day 21,the mice were scarified,then the MLN and PP were collected from the intestine for the further experiment.

2.5.Preparation of MLN and PP cells

Mesenteric lymph nodes (MLN) were treated with RPMI 1640 medium (NacalaiTesque,Inc.,Kyoto,Japan) containing 10 mM HEPES(NacalaiTesque),100 U/mL penicillin,100 μg/mL streptomycin(NacalaiTesque),2% fetal bovine serum (MP Biomedicals),0.2 U/mL Liberase TM (Roche Diagnostics,Mannheim,Germany) and 0.125 mg/mL DNase I (Merck,Darmstadt,Germany) at 37°C for 20 min.The tissues then were disrupted through 100-μm nylon mesh cell strainers(Greiner Bio-One,Kremsmünster,Austria).After filtering,the single-cell suspensions were washed with HBSS and subjected to flow cytometry analysis.Peyer’s patches(PP)were minced and treated with RPMI 1640 medium (NacalaiTesque,Inc.,Kyoto,Japan) containing 10 mM HEPES(NacalaiTesque),100 U/mL penicillin,100 μg/mL streptomycin(NacalaiTesque),2% fetal bovine serum (MP Biomedicals),0.2 U/mL Liberase TM (Roche Diagnostics,Mannheim,Germany) and 0.125 mg/mL DNase I (Merck,Darmstadt,Germany) at 37°C for 30 min.After filtering,the single-cell suspensions were washed with HBSS and subjected to flow cytometry analysis.

2.6.Flow cytometry of MLN and PP cells

Cell staining was performed as previous study[12].MLN and PP cells were incubated with anti-mouse CD16/CD32 antibody(93;BioLegend,Inc.,San Diego,CA,USA) to block Fc receptors and then stained using antibodies conjugated with phycoerythrin(PE),allophycocyanin(APC),APC-eFlour 780,eFluor 450,Alexa Flour 700,PerCP-Cy5.5,Brilliant Ultraviolet (BUV)395,BUV737.Anti-CD45 (30-F11),anti-CD3(145-2C11),and anti-CD19 (1D3) antibodies were obtained from BD Biosciences (San Jose,CA,USA);anti-CD103 (2E7) antibody was obtained from Biolegend (San Diego,CA,USA);anti-CD45 R/B220(RA3-6B2),anti-CD11c (N418),anti-MHC class II (I-A/I-E)(M5/114.15.2),anti-CD4(RM4-5),anti-CD25(PC61.5),andanti-FOXP3(FJK-16s) antibodies were obtained from Thermo Fisher Scientific(Waltham,MA,USA).7-Aminoactinomycin D(BioLegend)was added to the cell suspension to label dead cells.To visualize intracellular Foxp3,the cells were stained for surface antigens,fixed,and permeabilized using Transcription Factor Buffer Set (BD Bioscience).Permeabilized cells were then stained withanti-Foxp3 antibodies.Dead cells were detected using the Fixable Viability Stain 780 (BD Biosciences) for intracellular staining.Stained cells were analyzed using an LSR II Flow Cytometer (BD Biosciences) and FlowJo software ver.10.4.2 (BD Biosciences).

2.7.Statistical data analyses

Statistical data analyses were performed using an analysis of variance ANOVA followed by Dunnett’s test.A p value of＜0.05 is minimal level of statistically significant.

3.Results and discussion

3.1.Preparation of PVBu NPs

PVBu NPs were prepared by oil-in-water emulsion method and characteristics of NPs are summarized in Table 1.Diameter of NPs was adjusted to be～200 nm by tuning the mixing ratio of PVBu and Pluronic F-127 and sonication condition.This size of the NPs has been reported to be taken up via Peyer’s patches [13].As shown in Fig.2,the size distribution of NP was almost constant during the storage as aqueous dispersion at least for 56 days.The surface charges of NPs were almost neutral due to Pluronic F-127 coating on their surface.To incorporate ATRA or vitamin D3into NP,each molecule was dissolved in organic phase and NP was prepared similarly(NP-A and NP-D).Although ATRA and vitamin D3were chemically unstable molecules,the relatively high entrapment efficacy of intact ATRA and vitamin D3in NPs were achieved by minimizing the sonication time and adding ascorbic acid as a radical scavenger(entrapment efficacy:42%of ATRA in NP-A and 66%of vitamin D3in NP-D,respectively).It was observed that NP released butyrate in the presence of pancreatic lipase [8].The releasing speed was much slower than monomeric vinyl butyrate,indicating that the limited access of the lipase to the hydrophobic PVBu chains in aqueous media.

Table 1 Characteristics of PVBu NPs.Data are given as mean ± SD(n=3).

3.2.Alteration of populations and numbers of intestinal immune cells by PVBu NPs

PVBu NPs were orally applied to mice daily for 14 days.After the treatment,the number of immune cells in intestinal MLN and Peyer’s patches in lower(LPP)and upper parts(UPP)of intestine were counted by flow cytometry using specific markers and gating procedure for each cell (Fig.S1).CD103+DC with tolerogenic phenotype (tDC) plays an important role in maintaining intestinal immune homeostasis and induce the T cell differentiation into Treg cells by secreting ATRA and TGF-β.These cells are required for the induction of tolerogenic immune response.As shown in Fig.3,the populations and numbers of tDC and Treg cells were not affected by the treatment of NPs in UPP,whereas marked changes were observed in LPP and MLN.tDC decreased in LPP and inversely tDC in MLN increased,indicating the migration of tDC from LPP to MLN.In the steady state,lamina propria-derived tDC migrate into MLN via C-C chemokine receptor type 7(CCR7)signaling,which is required for the induction of oral tolerance[14].The increment of tDC in MLN was enhanced in NPs containing vitamin D3or ATRA(Fig.3).Similar migration of tDC to MLN has been observed when tDC engulfed intestinal bacteria[15].Because migration of tDC in PP to MLNwould be accelerated by TLR ligands included in bacteria [16],the reason why NPs which do not contain TLR ligands also enhanced the migration of tDC is not clear.The authors previously found that PVBu NPs suppressed activation of macrophages by releasing butyrate inside of the cellsin vitro[8].Similar intracellular release of butyrate from PVBu NPs would occur in the intestinal tDC,which may be related to the activation of the migration of tDC.tDC migrated to MLN has been reported to induce Treg cells depending on ATRA which is secreted from tDC or supplied from food[17].Thus,the migration of tDC induced by NPs implies their promising feature as therapeutics to accelerate the tolerance induction toward luminal antigens.The ATRA-containing NP may be more suitable for this purpose because of the supplementation of ATRA to enhance the Treg induction.Another marked change observed by the NP treatment was reduction of Treg cells in LPP.This change was also enhanced by NPs containing vitamin D3or ATRA.Treg cells in LPP would not migrate to MLN because the population and number of Treg cells in MLN remain almost constant after the treatment of NPs.Thus,Treg cells in LPP were expected to migrate to lamina propria [18] to execute the tolerance toward luminal antigens.

Fig.2.Stability of NP during the storage as an aqueous dispersion at 4 °C(from day 1 to 56).0.27 mg/mL NP in 10 mM HEPES buffer (pH 7.4).

4.Conclusion

NPs containing immunotolerance inducing molecules (ATRA,vitamin D3) were prepared from PVBu,which can work as a donor of butyrate to intestine.Oral administration of PVBu NPs to mice for 14 days markedly alter the populations and numbers of tDC and Treg cells in LPP and MLN in the intestine.tDC is indicated to migrate from LPP to MLN,which contributes to the induction of Treg cells for antigenspecific immunotolerance toward luminal antigens.Because Treg cells in LPP did not seem to migrate to MLN,these Treg cells would migrate to lamina propria to exert tolerance induction.The migrations of tDC and Treg cells in LPP are suitable for tolerance induction,PVBu NPs have potential to be therapeutics of allergy and autoimmune diseases to accelerate the induction of the antigen-specific immunotolerance.

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.

Fig.3.PVBu NPs on the alteration of population (A) and number (B) of immune cells in UPP,LPP,and MLN.Mice were orally applied with PVBu nanoparticles (2.7 g/kg body weight) daily for 14 days (n=5 each group).Then the MLN and PP were collected for FACS analysis.Each immune cell subset was characterized based on the surface expression of specific markers.＊p ＜0.05,＊＊p ＜0.01,＊＊＊p ＜0.001.

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pnsc.2020.10.003.