Royal jelly fermented by honeybee queen (Apis mellifera)-derived Lactobacillus panisapium M1 enhances macrophage phagocytosis through the TLR2/MAPK/SR signaling pathway

Abstract

Background

Royal jelly (RJ) has traditionally been used to maintain health, and its fatty acids (10-hydroxy decanoic acid and 10-hydroxy-2-decenoic acid) are reported to have immunomodulatory effects. We recently developed fermented RJ (fRJ) by fermenting RJ with honeybee queen (Apis mellifera)-derived lactic acid bacterium, Lactobacillus panisapium M1. fRJ increased the 10-hydroxy decanoic acid content five-fold and facilitated macrophage phagocytosis. In this study, we explored the components involved in the fRJ-induced enhancement of macrophage phagocytosis and assessed the biological mechanisms of these components.

Methods

The effects of fRJ on the phagocytosis of negatively charged carboxylate-modified latex beads, zymosan, IgG-coated beads, and apoptotic cells, Toll-like receptor 2 (TLR2) and phagocytic receptor expression, and p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) phosphorylation were assessed in the murine macrophage cell line J774.1. To explore the mechanisms of fRJ-induced macrophage phagocytosis, we used TLR2, mitogen-activated protein kinases (MAPKs), cluster of differentiation 36 (CD36), scavenger receptor inhibitors, and small interfering RNA. Phagocytosis was assessed using fluorescence microscopy or flow cytometry, whereas mRNA, protein, and cell surface antigen expression was assessed using quantitative polymerase chain reaction, western blotting, and flow cytometry, respectively.

Results

fRJ enhanced macrophage phagocytosis of carboxylate-modified latex beads and apoptotic cells. Additionally, L. panisapium M1 enhanced macrophage phagocytosis. Moreover, the assessment of lactic acid bacteria-mediated signaling pathways revealed that fRJ enhanced TLR2 expression, promoted p38 and JNK phosphorylation, and facilitated activator protein-1 nuclear translocation. However, fRJ-induced macrophage phagocytosis was reduced by the presence of inhibitors targeting TLR2, p38, and JNK. fRJ facilitated the gene expression of scavenger receptors, including Msr1 and Cd36, and this upregulation was suppressed by TLR2 inhibition. Surprisingly, fRJ-induced augmentation of macrophage phagocytosis was not suppressed by CD36 inhibition. However, it was significantly attenuated by the broad-spectrum scavenger receptor inhibitor polyinosinic acid.

Conclusions

The results indicate that fRJ enhances macrophage phagocytosis through TLR2/MAPK/SR, and L. panisapium M1 primarily contributes to immune function.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-04976-x.

Introduction

Royal Jelly (RJ)—a creamy substance secreted by honeybees (Apis mellifera)—is widely used to maintain health since ancient times. Its chemical composition includes water (60–70%), proteins (9–18%), sugars (7.5–23%), lipids (3–8%), and other minor components [1]. RJ contains two characteristic medium-chain fatty acids (10-hydroxy-2-decenoic acid [10H2DA] and 10-hydroxy decanoic acid [10HDAA]) that account for 60–80% of RJ lipids [2].

Previous studies have demonstrated that RJ and these fatty acids regulate immune functions. RJ administration in rodents regulates innate immunity in the small intestine [3], restores neutrophil phagocytosis [4], and suppresses allergic reactions [5]. Additionally, RJ supplementation extends the survival period during bacterial infections in aged Caenorhabditis elegans models [6]. Recently, we reported that RJ administration increases the number of hematopoietic stem cells that are crucial for maintaining immune function in middle-aged and elderly individuals [7]. Additionally, we demonstrated in vitro that RJ facilitates the differentiation of M cells [8] involved in antigen uptake and enhances the phagocytosis of latex beads by murine macrophages, J774.1 cells [9].

10H2DA and 10HDAA exhibit anti-inflammatory effects on lipopolysaccharide (LPS)-induced dendritic cells and macrophages in vitro [2, 10]. Additionally, 10H2DA facilitates macrophage phagocytosis [11]. However, there is limited data on the effect of 10HDAA on macrophage phagocytosis. 10HDAA facilitates the differentiation of M cells in vitro, but not 10H2DA [12]. Moreover, 10HDAA administration increased the number of M cells in the Peyer’s patches of the small intestine and enhanced antigen-specific IgA production in a primate model [12]. These results indicate that RJ and 10HDAA enhance the innate immune system.

Recently, we discovered that Lactobacillus panisapium M1—lactic acid bacteria (LAB) derived from honeybee queens (A. mellifera)—completely converts 10H2DA to 10HDAA [9]. We developed fermented RJ (fRJ) containing five-fold more 10HDAA than that of RJ by fermenting it with L. panisapium M1. fRJ significantly enhanced the M cell differentiation and macrophage phagocytosis of latex beads than that of RJ [9]. However, the components involved in the fRJ-induced enhancement of macrophage phagocytosis and the underlying mechanisms remain unclear.

Certain LAB have been reported to facilitate macrophage phagocytosis. The administration of Lactobacillus fermentum, Weissella kimchii, and Lactobacillus plantarum strains facilitates the phagocytosis of peritoneal macrophages in rodent models [13]. Heat-killed Latilactobacillus sakei, Latilactobacillus curvatus, and Lactobacillus brevis enhance the phagocytosis of RAW264.7 cells [14, 15]. L. panisapium, a novel and Gram-positive bacterium of the genus Lactobacillus, was recently discovered in the bee bread and gastrointestinal tracts of Apis cerana and A. mellifera, respectively [9, 16], and the functional studies of L. panisapium in mammals are limited.

These findings resulted in a hypothesis that both 10HDAA and honeybee queen-derived L. panisapium M1 may synergistically contribute to the fRJ-induced enhancement of macrophage phagocytosis. This study aimed to assess the effects of 10HDAA and L. panisapium M1 on phagocytosis and elucidate the mechanism of fRJ-induced enhancement of phagocytosis using murine macrophage cell line J774.1 and carboxylate-modified latex beads (CB).

Methods

Materials

L. panisapium M1 and fRJ were prepared as previously described [9]. Briefly, L. panisapium M1 strain was cultured in de Man, Rogosa, and Sharpe broth (Oxoid, Hampshire, UK) at 35 °C under anaerobic conditions for 48 h. For the preparation of fRJ, the raw royal jelly was digested with protease YBFII (Yamada Bee Company Inc., Okayama, Japan) at 50 °C for 2 h, and then anaerobically incubated with L. panisapium M1 at 35 °C for 120 h. Furthermore, the solution was heat-sterilized at 80 °C for 20 min. The fRJ was standardized to contain > 5.2% of 10HDAA. Cultured L. panisapium M1 strain was heat-killed at over 80 °C for 20 min, washed thrice with sterile distilled water, lyophilized, and suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Kyoto, Japan) for in vitro assay. The suspension was filtered through a 0.2 µm filter (TOYO ROSHI KAISHA, Tokyo, Japan). 10H2DA was purchased from Hangzhou Eastbiopharm, Co., Ltd. (Hangzhou, China), whereas 10HDAA was purchased from Combi-Blocks, Inc. (San Diego, CA, USA). Lipopolysaccharide from Escherichia coli O55:B5 (LPS) and scavenger receptor inhibitor polyinosinic acid (Poly-I) were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). Toll-like receptor 2 (TLR2) inhibitor TLR2-IN-C29, p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, and ERK inhibitor U0126 were purchased from Selleck Chemicals (Houston, TX, USA). The antibodies used in Western blotting are listed in Supplementary Table 1.

Cell culture

Murine J774.1 macrophages (RCB0434) were obtained from the RIKEN Bioresource Center (Ibaraki, Japan) and human Jurkat T cells (TIB-152) were obtained from the American Type Culture Collection (Bethesda, MD, USA). J774.1 and Jurkat cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 100 μg/mL of streptomycin, and 100 U/mL of penicillin at 37 ℃ and 5% CO₂.

Immunofluorescence staining

J774.1 cells (2.0 × 10⁴ cells/well) were seeded in a cover glass chamber, incubated overnight, and subsequently treated with 1 mg/mL of fRJ for 15 min. The cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 15 min. The cells were washed with phosphate-buffered saline (PBS) and incubated with a blocking buffer (5% normal goat serum and 0.3% Triton X-100 in PBS) for 1 h. Subsequently, the cells were incubated overnight at 4 °C with an anti-phospho-c-Jun (Ser73) primary antibody (Cell Signaling Technology, Danvers, MA, USA). The cells were washed with PBS and incubated with Alexa Fluor® 488 Conjugated anti-rabbit IgG antibody (Cell Signaling Technology) for 1 h at room temperature. After washing with PBS, the cells were stained with 4', 6-diamidino-2 phenylindole-Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were captured and analyzed using a BZ-X800 microscope (Keyence, Osaka, Japan).

Phagocytosis assay

J774.1 cells were seeded in a 12-well plate (5.0 × 10⁴ cells/well) and incubated overnight. Subsequently, the cells were treated with 1 mg/mL of fRJ, 10–200 µg/mL of L. panisapium M1, 250 µM of 10H2DA, 10HDAA, or 100 ng/mL of LPS (Sigma-Aldrich) as a positive control for 24 h. CB (2.0 µm) (Sigma-Aldrich), pHrodo Red Zymosan (Invitrogen, Carlsbad, CA, USA), and fluorescein isothiocyanate (FITC)-labeled IgG-opsonized latex beads (Cayman Chemical, Ann Arbor, MI, USA) were incubated with macrophages for 1 h. The cells were washed thrice with PBS and dissociated from the plate using an ice-cold fluorescence-activated cell sorting (FACS) buffer (2% FBS and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in Hank's balanced salt solution). For flow cytometry analysis, the cells were stained with Hoechst 33,242 (Invitrogen) for 5 min and washed with PBS. The percentage of Hoechst 33,242-positive phycoerythrin- or FITC-positive cells and mean fluorescence intensity (MFI) were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) as indicators of the phagocytosis rate and target uptake, respectively. For fluorescence microscopy, the cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 15 min. After washing with PBS, the cells were permeabilized with PBS containing 0.1% Triton X-100 and stained with Hoechst 33,242 and phalloidin-iFluor 488 conjugate (Cayman Chemical) for 30 min. Images were obtained using a BZ-X800 microscope (Keyence).

Apoptosis induction

Jurkat cells were resuspended in RPMI1640 and treated with ultraviolet (UV) irradiation at 254 nm (Analytik Jena AG, Jena, Germany) for 1 min and incubated for 4 h at 37 °C, 5% CO₂. Apoptotic cells were detected using the MEBCYTO Apoptosis Kit (MBL Life Science, Nagoya, Japan), following the manufacturer’s protocol. Briefly, the cells were stained with propidium iodide (PI) and annexin V-FITC for 15 min at room temperature in a binding buffer before efferocytosis assay. Apoptotic cells were defined as annexin V⁺ PI⁻ cells based on flow cytometry.

Efferocytosis assay

J774.1 cells were seeded in a 24-well plate (1.0 × 10⁴ cells/well) and treated with 1 mg/mL of fRJ for 24 h. Apoptotic cells were labeled with pHrodo-succinimidyl ester (Invitrogen) for 30 min at room temperature and washed thrice with PBS. J774.1 cells were incubated with pHrodo-labeled apoptotic cells at a 1:10 (macrophage to apoptotic cells) ratio for 1 h. Subsequently, the cells were washed thrice with PBS to remove non-engulfed apoptotic cells and dissociated from the plate using the FACS buffer. J774.1 cells were stained with Hoechst 33,242 for 5 min and washed with PBS. The cells were assessed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec) and the percentage of fluorescence-positive events and MFI were analyzed.

Quantitative real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted from the J774.1 cells using the NucleoSpin RNA Plus kit (Takara Bio, Shiga, Japan), and cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). qPCR was performed using the TB Green Premix Ex Taq™ (Takara Bio) or SsoAdvanced Universal Probes Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with CFX Opus real-time PCR system (Bio-Rad). Transcript levels were normalized to those of murine ribosomal protein S18. Primer sequences were described in Supplementary Table 2.

Flow cytometric analysis of cell surface

To quantify the cell surface expression of TLR2, CD36, and macrophage scavenger receptor 1 (MSR1), J774.1 cells were stained with anti-human/mouse TLR2-allophycocyanin (APC) (Miltenyi Biotec), anti-mouse CD36-APC (Miltenyi Biotec) or anti-mouse MSR1-FITC antibody (Miltenyi Biotec) for 15 min at 4 ℃. The cells were washed with FACS buffer and assessed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec).

Western blot analysis

Total cellular protein was extracted from J774.1 cells using radioimmunoprecipitation assay buffer (Nacalai Tesque) containing protease Nacalai Tesque) and phosphatase (Nacalai Tesque) inhibitor cocktails. The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For electrophoresis, 20 μg of protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes using a Trans-Blot Turbo transfer system (Bio-Rad). The membranes were washed with Tris Buffered Saline (Takara Bio) including 0.1% Tween 20 (Nacalai tesque) (TBST) and incubated with a blocking buffer (5% skim milk in TBST) for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with the primary antibodies listed in Supplementary Table 1. The membranes were washed with TBST and incubated with Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology) for 1 h at room temperature. Following immunoreaction, protein bands were visualized using a Clarity Western ECL Substrate (Bio-Rad). Band intensity was quantified using an image analyzer LAS-2000 (Fujifilm, Tokyo, Japan). The full-length membranes are illustrated in Supplementary Fig. 1.

Small interfering RNA (siRNA) transfection

J774.1 cells were transfected with 20 nM of negative control siRNA or siCd36 (Invitrogen, siRNA ID: s63620) for 24 h using Lipofectamine RNAiMAX (Thermo Fisher Scientific), following manufacturer’s protocols.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, Inc., La Jolla, CA, USA). The experimental replicate numbers are demonstrated in the figure legends. The data are presented as the mean ± standard deviation. Statistical differences among groups were analyzed using a one-way analysis of variance with Dunnett's or Tukey’s multiple comparison test. For comparisons between two groups, significance was assessed using the Student’s t-test. Statistical significance was set at P < 0.05.

Results

fRJ enhances macrophage phagocytosis

Phagocytosis is crucial for maintaining tissue homeostasis and innate immune balance, in which phagocytes eliminate various particles, including foreign pathogens, cancer cells, and negatively charged anionic ligands, such as pathogen components and self-apoptotic and necrotic cells [17]. fRJ enhances macrophage phagocytosis of latex beads [9]. However, the fRJ-facilitated specificity of phagocytic targets has not been assessed. In this study, CB was primarily used as a phagocytosis model for the anionic particles. To confirm the specificity of the targets engulfed by phagocytes, we used zymosan, IgG-modified beads, and UV-induced apoptotic Jurkat cells as models for microbial phagocytosis, opsonization, and apoptotic cell clearance (Fig. 1a). Initially, we assessed whether murine macrophage J774.1 treated with fRJ facilitated CB internalization under a microscope. LPS-treated macrophages engulfed more CB than that of the control [18]. Additionally, we observed that fRJ facilitated CB uptake by macrophages (Fig. 1b). To determine whether fRJ enhances macrophage phagocytosis of CB, we quantified the percentage of macrophages that engulfed CB (phagocytosis rate) and the MFI of CB uptake using flow cytometry. Consistent with microscopic observations, fRJ significantly enhanced the phagocytic rate and CB uptake (Fig. 1c). Subsequently, we assessed whether fRJ enhanced the phagocytosis of zymosan, IgG beads, and apoptotic cells using flow cytometry. We observed that fRJ did not affect the phagocytosis of zymosan and IgG beads (Fig. 1d, e). However, it significantly enhanced phagocytic rate and apoptotic cell uptake (Fig. 1f). These results indicate that fRJ specifically enhanced macrophage phagocytosis of CB and apoptotic cells.

Fig. 1.

Effect of fermented royal jelly (fRJ) on macrophage phagocytosis of various particles. a Schematic representation of the phagocytosis assay. Murine macrophage J774.1 cells are treated with 1 mg/mL of fRJ for 24 h and subsequently incubated with various types of particles for 1 h. b Phagocytosis of carboxylate-modified latex beads (CB) is observed using fluorescent microscopy. The cells are immunostained with fluorescein isothiocyanate (FITC) for phalloidin and Hoechst 33,342 to label the nuclei. Scale bar: 50 μm. c-f Phagocytosis of CB, zymosan, IgG-coated beads, or UVC-induced apoptotic Jurkat cells is detected using flow cytometry. Phagocytosis rate and uptake (MFI) are expressed as the percentage of phycoerythrin-positive or FITC-positive cells in Hoechst-positive cells and uptake (mean fluorescence intensity), respectively. Results are presented as the mean ± standard deviation (*P < 0.05 and **P < 0.01; Dunnett’s test, n = 3–5)

L. panisapium M1 is involved in fRJ-induced enhancement of macrophage phagocytosis

Because L. panisapium M1 completely converts 10H2DA to 10-HDAA in RJ (Fig. 2a) and L. panisapium M1 was heat-killed post-RJ fermentation [9], we hypothesized that 10HDAA and heat-killed L. panisapium M1 were associated with fRJ-induced enhancement of macrophage phagocytosis. To test this hypothesis, we assessed the effect of these components on CB phagocytosis by J774.1 cells using flow cytometry. The concentration of 10HDAA in 1 mg/mL of fRJ was approximately 250 μM. The effects of 250 μM of 10HDAA and 10H2DA on macrophage phagocytosis of CB were not observed (Fig. 2b). The concentration of L. panisapium M1 in 1 mg/mL of fRJ was approximately 100 μg. Heat-killed L. panisapium M1 facilitated the phagocytic rate and uptake in a concentration-dependent manner, which was significant at 200 µg/mL (Fig. 2c). These results indicate that L. panisapium M1 is the primary component of fRJ-induced macrophage phagocytosis.

Fig. 2.

Effect of royal jelly (RJ)-derived fatty acids and Lactobacillus panisapium M1 on macrophage phagocytosis. a Schematic representation of RJ fermentation by L. panisapium M1. L. panisapium M1 completely converts 10-hydroxy-2-decenoic acid (10H2DA) to 10-hydroxydecanoic acid (10HDAA) in RJ. J774.1 cells are treated with (b) 250 µM of 10H2DA, 10HDAA or (c) 0–200 µg/mL of heat-killed L. panisapium M1 for 24 h and subsequently incubated with carboxylate-modified latex beads for 1 h. Phagocytosis rate and uptake (mean fluorescence intensity: MFI) are measured using flow cytometry. Results are presented as the mean ± standard deviation (**P < 0.01; Dunnett's test, n = 3)

fRJ enhances macrophage phagocytosis through the TLR2/MAPKs signaling pathway

Because L. panisapium M1 facilitated macrophage phagocytosis, we focused on the signaling pathways through which LABs act. TLRs recognize pathogen-associated molecular patterns (PAMPs) and activate intracellular signaling pathways, including MAPKs (Fig. 3a). TLR2 is known to recognize PAMPs derived from gram-positive bacteria, such as LABs, including lipopeptides, peptidoglycans, and lipoteichoic acids [19, 20]. These findings led us to hypothesize that fRJ may enhance macrophage phagocytosis through the TLR2 signaling pathway. To test this hypothesis, we initially assessed whether fRJ affected TLR2 in J774.1 cells. We observed that fRJ significantly upregulated the mRNA levels of Tlr2 and its expression on the cell surface (Fig. 3b). The TLR2 inhibitor (C29) completely blocked the effect of fRJ on macrophage phagocytosis of CB (Fig. 3c). Additionally, we assessed the effect of fRJ on MAPKs, including p38, JNK, and ERK 1/2, which are downstream signals of TLR2 and involved in the regulation of macrophage phagocytosis [19, 21–26]. Western blot analysis demonstrated that fRJ significantly increased the protein levels of phosphorylated p38 and JNK compared to those of the control, but did not affect ERK (Fig. 3d, Supplementary Fig. 1). Moreover, we observed that fRJ facilitated the activation of activator protein-1 (AP-1) (nuclear translocation of phosphorylated c-Jun), which is downstream of MAPK (Fig. 3e). Consistently, p38 (SB203580) and JNK (SP600125) inhibitors significantly suppressed the effect of fRJ on macrophage phagocytosis of CB, but the ERK inhibitor (U0126) did not have this effect (Fig. 3f-h). These results indicate that fRJ enhances macrophage phagocytosis through the TLR2/p38 and JNK signaling pathways.

Fig. 3.

Effect of fermented royal jelly (fRJ) on the Toll-like receptor 2/mitogen-activated protein kinase (TLR2/MAPK) signaling pathway. a Schematic representation of the TLR2/MAPK signaling pathway. b TLR2 mRNA and cell surface expression (mean fluorescence intensity: MFI) in J774.1 cells treated with 1 mg/mL of fRJ for 24 h. c J774.1 cells are incubated with 1 mg/mL of fRJ and 50 µM of TLR2 inhibitor C29 for 24 h. Phagocytosis rate and uptake (MFI) are measured using flow cytometry. d Immunoblots for extracellular signal-regulated kinase (ERK)1/2 and p-ERK1/2, p38, p-p38, c-Jun N-terminal kinase (JNK) and p-JNK in whole cell lysates from J774.1 cells treated with 1 mg/mL of fRJ for 15 min. β-actin is used as a reference control. e J774.1 cells are stimulated with 1 mg/mL of fRJ for 15 min, and subsequently, activator protein-1 (AP-1) translocation to the nucleus is analyzed using fluorescence microscopy. The cells are immunostained with fluorescein isothiocyanate for AP-1 and Hoechst 33,342 to label the nuclei. Scale bar: 20 μm. J774.1 cells are pre-incubated with 10 µM of (f) ERK inhibitor U0126, (g) p38 MAPK inhibitor SB203580, or (h) JNK inhibitor SP600125 for 30 min and subsequently treated with 1 mg/mL of fRJ for 24 h. Phagocytosis rate and uptake are determined using flow cytometry. Results are presented as the mean ± standard deviation (*P < 0.05 and **P < 0.01; Tukey's multiple comparisons test, Dunnett's test, or Student’s t-test, n = 3–5)

fRJ-induced macrophage phagocytosis is mediated by scavenger receptors

Individual phagocytic receptors on the surface of phagocytes recognize particles as targets and facilitate phagocytosis through signaling pathways [27]. These receptors include Fcγ receptors that recognize IgG and complement receptor CR3 [17], lectin-like recognition receptors, mannose receptors, and scavenger receptors (SRs) that recognize polyanionic ligands, such as microbial ligands, apoptotic cells, modified lipoproteins and latex beads, and receptors for apoptotic cells [27–29]. Because fRJ enhanced macrophage phagocytosis of anionic particles, including CB and apoptotic cells, we hypothesized that SRs are involved in the fRJ-induced enhancement of phagocytosis. To test this hypothesis, we assessed whether fRJ affected SRs in J774.1 cells. Initially, we confirmed the expression levels of representative SRs (Msr1, Marco, and Cd36) in J774.1 cells. Owing to the extremely low expression of Marco in J774.1 cells, we focused on Msr1 and Cd36 for subsequent experiments (Supplementary Fig. 2). The mRNA levels of Msr1 and Cd36 in the J774.1 cells were significantly upregulated by fRJ (Fig. 4a). Because fRJ facilitates the phagocytosis of CB through the TLR2 signaling pathway, and TLR stimulation regulates the gene expression of phagocytic receptors [30], we assessed whether the fRJ-induced upregulation of phagocytic receptor expression was mediated by TLR2. The TLR2 inhibitor (C29) completely suppressed the effect of fRJ on the gene expression of Msr1 and Cd36 (Fig. 4b). Additionally, fRJ significantly increased the CD36 levels on the surface of J774.1 cells (Fig. 4c). Subsequently, we assessed whether CD36 contributed to the fRJ-induced macrophage phagocytosis of CB. CD36 was inhibited using siRNA and CD36 antagonist sulfosuccinimidyl oleate (SSO) in J774.1 cells (Fig. 4d). Compared to the control siRNA, the knockdown of Cd36 (siCd36) significantly suppressed CD36 expression and the percentage of CD36-positive cells (Fig. 4e). However, fRJ-induced facilitation of macrophage phagocytosis of CB was not suppressed by siCd36 transfection (Fig. 4f). SSO did not suppress the effect of fRJ on macrophage phagocytosis (Fig. 4g). Multiple phagocytic receptors interact with other receptors during phagocytosis [31–34]. These findings led us to hypothesize that multiple SRs contribute to the fRJ-induced macrophage phagocytosis of CB. To test this hypothesis, we used Poly-I—the broad-spectrum SR inhibitor [35]. Poly-I significantly suppressed the effect of fRJ on macrophage phagocytosis (Fig. 4h). These results indicate that fRJ upregulates the expression of multiple SRs through TLR2 and that these receptors enhance macrophage phagocytosis.

Fig. 4.

Effect of fermented royal jelly (fRJ) on scavenger receptors (SRs). a Relative mRNA levels of SR genes in J774.1 cells treated with 1 mg/mL of fRJ for 24 h. b J774.1 cells are incubated with 1 mg/ml of fRJ and 50 µM of Toll-like receptor 2 inhibitor C29 for 24 h. The relative mRNA expression levels of SRs is measured using quantitative polymerase chain reaction. c The cell surface expression (mean fluorescence intensity: MFI) of macrophage scavenger receptor 1 (Msr1) and cluster of differentiation 36 (Cd36) in J774.1 cells treated with 1 mg/mL of fRJ for 24 h is quantified using flow cytometry. d Schematic representation of phagocytosis assay. e, f For Cd36 knockdown, J774.1 cells are transfected with siCd36 for 24 h before 1 mg/mL of fRJ treatment, and subsequently CD36 expression on the cell surface and percent of CD36-positive cells to Hoechst-positive cells are assessed using flow cytometry. g For CD36 inhibition, J774.1 cells are pre-treated with 50 µM of sulfosuccinimidyl oleate (SSO) for 1 h before carboxylate-modified latex bead treatment. h For the inhibition of SRs, J774.1 cells are pre-incubated with 5 µM of polyinosinic acid (Poly-I) for 15 min before 1 mg/mL of fRJ treatment. Phagocytosis rate and uptake (MFI) are determined using flow cytometry. Results are presented as the mean ± standard deviation (*P < 0.05 and **P < 0.01; Tukey's multiple comparisons test, Dunnett's test, or Student’s t-test, n = 3)

Discussion

This study aimed to assess the effects of 10HDAA, L. panisapium M1, and fRJ components on macrophage phagocytosis and the mechanisms by which fRJ enhances phagocytosis. We demonstrated that fRJ enhances macrophage phagocytosis through the TLR2/MAPK/SR signaling pathway and indicated that L. panisapium M1, rather than 10HDAA, may be involved in the enhancement of fRJ-induced macrophage phagocytosis.

In this study, we demonstrated that 10HDAA and 10H2DA did not affect macrophage phagocytosis. At a concentration similar to that used in our study, 10H2DA facilitates neutral red phagocytosis by murine peritoneal macrophages [11]. These results indicate that 10H2DA may facilitate macrophage phagocytosis in a target-specific manner.

L. panisapium M1 was observed to stimulate macrophage phagocytosis. LABs facilitate macrophage phagocytosis [13–15]. PAMPs, such as peptidoglycan (PGN), lipoteichoic acid (LTA), cell wall teichoic acid, and exopolysaccharides (EPS) that constitute the cell surface of lactic acid bacteria are recognized by TLR2 [19, 20, 36, 37], with PGN and EPS specifically reported to facilitate macrophage phagocytosis [30, 38–40]. Western blot analysis and phagocytosis assays using TLR2 and MAPK inhibitors revealed that fRJ facilitates macrophage phagocytosis through the TLR2, p38, and JNK signaling pathways. The stimulation of TLR2 by PGN enhances macrophage phagocytosis through the MAPK signaling pathway [30], and activation of p38 and JNK is crucial for enhancing phagocytosis [21–24]. Specifically, the activation of p38 MAPK induces remodeling of the actin cytoskeleton involved in phagocytosis [41]. Although LAB-derived LTA activates MAPKs, the intensity of p38, JNK, and ERK activation varies based on LAB strains—indicating that the strain-specific glycolipid structure of LTA may be associated with MAPK activation [42]. These findings indicate that the complex and diverse structures of PAMPs derived from L. panisapium M1 induce the specific activation of p38 and JNK. However, further research is required to understand the characteristics of the PAMPs of L. panisapium M1 and their involvement in the activation of the TLR2/p38 and JNK signaling pathways.

fRJ treatment increased the mRNA expression levels of SRs, including Cd36 and Msr1 in J774.1, which was suppressed by the TLR2 inhibitor C29. The expression of phagocytic receptors is regulated by TLR stimulation [30]. Although fRJ increased the expression of CD36 on the cell surface, it did not increase MSR1 expression. CD36 is crucial for the uptake of various targets, such as latex beads, apoptotic cells, and oxidized low-density lipoproteins, which are polyanionic ligands [43–46]. A phagocytosis assay with CD36 inhibition disproved the hypothesis that fRJ facilitates macrophage phagocytosis of CB through CD36. Multiple phagocytic receptors or other receptors interact during target phagocytosis [31–34]. In this study, the broad-spectrum SR inhibitor Poly-I inhibited the fRJ-induced enhancement of macrophage phagocytosis of CB [35]. These results indicate that multiple SRs upregulated by fRJ may cooperatively facilitate macrophage phagocytosis. Further studies using pharmacological inhibitors and siRNA are required to identify the factors crucial in fRJ-induced macrophage phagocytosis.

In this study, we used negatively charged CB as a model to mimic anionic ligands, including exogenous pathogen components and apoptotic cells. We confirmed that fRJ enhanced the macrophage phagocytosis of CB. Additionally, we observed that fRJ treatment facilitated the phagocytosis of apoptotic cells by macrophages, but not by IgG-coated beads and zymosan. Phagocytes recognize IgG at the Fcγ receptor [17]; however, the effects of fRJ on these receptors are limited. Zymosan, a yeast-derived β-glucan, is a TLR2 agonist [47]. β-glucan is recognized by TLRs through interactions with Dectin-1 and TLR2 in macrophages [48]. Laminarin, a type of soluble β-glucan, inhibits zymosan phagocytosis by macrophages in a concentration-dependent manner [49]. These findings indicated that ligands derived from L. panisapium M1 competitively bind to TLR2, thereby inhibiting the recognition and internalization of zymosan by macrophages.

Impaired phagocytosis is associated with various diseases. Defects in the clearance of apoptotic cells are associated with atherosclerosis, age-related inflammation, cancer, and infections [50, 51]. The significance of macrophage phagocytosis of apoptotic cells during influenza virus infection has been demonstrated. Depletion of alveolar macrophages and reduced phagocytosis because of Axl deficiency, a phagocytic receptor for apoptotic cells, exacerbated the severity of influenza infection and significantly affected mortality [52, 53]. Influenza viruses induce apoptosis in infected cells [54]. Co-culturing influenza virus-induced apoptotic cells with macrophages completely suppresses the amount of virus released into the culture medium [55]. In this study, we confirmed that fRJ-treated macrophages exhibit enhanced phagocytosis of apoptotic cells. The effectiveness of LABs in suppressing influenza virus infection has been reported in mouse models [56–58]. These findings indicate that fRJ and L. panisapium M1 may inhibit viral infection by efficiently eliminating infection-induced apoptotic cells. However, due to the limited data on the viral infection-suppressing effects of fRJ, it is essential to further investigate its resistance to infection in vivo and confirm its efficacy through clinical trials using supplements.

Conclusions

In this study, we demonstrated that fRJ enhances macrophage phagocytosis through the TLR2/MAPK/SR signaling pathway. These findings indicate that fRJ may be crucial for maintaining and enhancing the immune function.

Abbreviations

RJ

Royal jelly

fRJ

Fermented royal jelly

10H2DA

10-Hydroxy-2-decenoic acid

10HDAA

10-Hydroxy decanoic acid

LAB

Lactic acid bacteria

PAMP

Pathogen-associated molecular pattern

TLR

Toll-like receptor

SR

Scavenger receptor

TLR2

Toll-like receptor 2

JNK

C-Jun N-terminal kinase

ERK

Extracellular signal-regulated kinase

MAPKs

Mitogen-activated protein kinases

CD36

Cluster of differentiation 36

CB

Carboxylate-modified latex beads

LPS

Lipopolysaccharide

RPMI

Roswell Park Memorial Institute

Poly-I

Polyinosinic acid

FBS

Fetal bovine serum

PBS

Phosphate-buffered saline

FITC

Fluorescein isothiocyanate

FACS

Fluorescence-activated cell sorting

MFI

Mean fluorescence intensity

UV

Ultraviolet

PI

Propidium iodide

PCR

Polymerase chain reaction

MSR1

Macrophage scavenger receptor 1

APC

Allophycocyanin

siRNA

Small interfering RNA

PAMPs

Pathogen-associated molecular patterns

AP-1

Activator protein-1

SSO

Sulfosuccinimidyl oleate

PGN

Peptidoglycan

LTA

Lipoteichoic acid

EPS

Exopolysaccharides

Authors’ contributions

Conceptualization and design of the work, S.N. and H.O.; the acquisition and analysis of data, S.N.; writing—original draft preparation, S.N.; writing—review and editing, H.I. and T.I.; and supervision, A.Y. and N.O. All authors have read and approved the final manuscript.

Funding

No funding was received.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.