Abstract
Background/Aim: Recent studies reported that lipopolysaccharide (LPS) exhibits beneficial effects on prevention of immune-related diseases by activating macrophages. We previously demonstrated that pre-treatment with LPS derived from Pantoea agglomerans (LPSp) activated amyloid β (Aβ) phagocytosis in mouse primary microglia. In the present study, we further examined the promotory effect on phagocytosis of phagocytic particles in the C8-B4 microglia cell line. Materials and Methods: Phagocytic analysis of C8-B4 cells was evaluated using phagocytic particles (latex beads or HiLyte™ Fluor 488-conjugated Aβ1-42). Results: The phagocytic activity of latex beads was dependent on the concentration of beads and incubation time. LPSp, at as low as 100 pg/ml, significantly increased phagocytosis against the beads. In the experiment of Aβ1-42 phagocytosis, LPSp significantly increased Aβ phagocytic activity. Conclusion: LPSp treatment was confirmed to enhance Aβ1-42 phagocytosis by mouse microglia. It is suggested that the use of LPSp may be a potential promising candidate for the prevention of Alzheimer's disease.
Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacterium. LPS has generally only been thought of as an endotoxin because it is a strong inducer of immune response in humans and animals. However, recent studies have demonstrated some beneficial effects on health. Braun-Fahrländer et al. reported that a higher exposure to environmental LPS in childhood was correlated with a lower incidence of asthma (1). Yoshida et al. demonstrated that pre-treatment with low-dose LPS via intravenous injection enhanced resistance to Staphylococcus aureus in a mouse model of infection with S. aureus and its possible mechanisms appear to be an activation (priming) of macrophages via epigenetic changes induced by LPS-mediated toll-like receptor (TLR) 4 signaling pathways (2). In our previous study, oral administration of LPS derived from Pantoea agglomerans (LPSp), enhanced phagocytic activity by priming of peritoneal macrophages via TLR4 signaling pathways in mice (3). Our clinical studies demonstrated that oral treatment with LPSp would be effective for therapy of malignant tumors (4) and prevention of osteoporosis (5).
Alzheimer's disease is an age-dependent neurodegenerative pathology characterized by a progressive and irreversible deterioration of cognitive functions. Although the etiology of Alzheimer's disease remains unknown, a consensus has emerged regarding the amyloid hypothesis. This widely accepted hypothesis posits that an imbalance between production and clearance of amyloid β (Aβ), a main component peptide of the amyloid plaques found in brain of patients with Alzheimer's disease, is involved in neuronal network dysfunctions (6). Microglia, the brain-resident type of macrophage, plays a crucial role in the immune defense of the neural parenchyma (7). Microglial cells lacking TLR2, TLR4, or the co-receptor cluster of differentiation (CD) do not exhibit an enhanced phagocytic response to Aβ (8). Furthermore, treatment with LPS (via intracranial route) (9) or monophosphoryl lipid A derived from Salmonella minnesota R595 LPS (MPL; via intraperitoneal route) (10) was found to reduce the cerebral Aβ accumulation in a mouse model of Alzheimer's disease. Our previous study demonstrated that LPSp pre-treatment enhanced Aβ1-42 phagocytotic activity by primary mouse microglia (11). The aim of this study was to further examine the promotory effect on phagocytosis of phagocytic particles in microglia cell line C8-B4.
Analysis of C8-B4 cell phagocytosis of fluorescent latex beads. C8-B4 cells (2×105 cells/well) were incubated with different ratios of cells to fluorescent latex beads for different times at 37°C. Before adding the beads, cells were pre-treated for 30 min with 50 μM cytochalasin D (CyD), an inhibitor of phagocytosis. The geometric mean fluorescence intensity (MFI) of beads was measured and the fluorescence of internalized beads was evaluated as: (MFI of sample without CyD treatment) – (MFI of the sample with CyD treatment). Data are presented as the mean±SEM of four samples (one-way ANOVA with Tukey-Kramer's test). Values without a common letter are significantly different at p<0.05.
Materials and Methods
Cell culture and treatment. Mouse microglial cell line C8-B4 (CRL2540, American Type Culture Collection, Manassas, VA, USA) was grown in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 units/ml penicillin–streptomycin and 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). The C8-B4 cells (2.0×105 cells/well) were seeded in a 48-well tissue culture plate then incubated with LPSp (Macrophi, Kagawa, Japan) for 18 h at 37°C. Negative control cells were treated with medium only. After the incubation, the cells were washed twice with Dulbecco's modified Eagle's medium and incubated with phagocytic particles for 3 h at 37°C. In the present study, we used fluorescent latex beads (Fluoresbrite® YG Microspheres 2.0 μm; Polysciences, Warrington, PA, USA) at cell/bead ratios between 1:1 and 1:10 or HiLyte™ Fluor- 488 labeled Aβ1-42 peptide (HF488-Aβ1-42; Anaspec, Fremont, CA, USA) at 10 ng/well. The latex beads, formed from polystyrene microspheres, were used to evaluate a non-selective phagocytosis. Cytochalasin D (CyD; Wako, Osaka, Japan), an inhibitor of phagocytosis, was used at 50 μM and cells were pre-treated or not for 30 min before adding the phagocytic particles. To remove non-internalized particles, cells were washed twice with ice-cold Phosphate-buffered saline. Cells were detached by 0.25% trypsin treatment and collected by centrifugation for 5 min at 300 × g. Cell pellets were re-suspended in hosphate-buffered saline containing 0.5% bovine serum albumin and 2 mM EDTA. The geometric mean fluorescence intensity (MFI) of phagocytosed beads in the cells was measured using a Beckman Coulter Gallios flow cytometer using Kaluza software (Beckman Coulter, Indianapolis, IN, USA). The internalized fluorescence of phagocytosed beads was evaluated as follows: (MFI of sample without CyD treatment) – (MFI of sample with CyD treatment), and the relative phagocytic activity was calculated by dividing the internalized fluorescence of phagocytosed beads of samples by that of the sample without LPSp treatment, as described previously (11).
Statistical analysis. Data are expressed as the mean±SEM. Statistical analyses were performed using StatMate V (ATMS, Tokyo, Japan). Statistical differences between multiple groups were determined using one-way ANOVA followed by Tukey-Kramer's test. A p-value of less than 0.05 was considered statistically significant.
Results
Phagocytic activity of C8-B4 cells toward fluorescent latex beads. The C8-B4 cells were incubated at cell/bead ratios of 1:1, 1:3, 1:5 or 1:10. As shown in Figure 1A, the phagocytic activity towards fluorescent latex beads was increased in a cell/bead ratio-dependent manner. In addition, the phagocytic activity increased in an incubation time-dependent manner at a cell/bead ratio of 1:5 (Figure 1B). The cell/bead ratio of 1:5 and 3 h incubation for the phagocytosis was selected for subsequent experiments.
Analysis of phagocysis of fluorescent latex beads by C8-B4 cells treated with lipopolysaccharides derived from Pantoea agglomerans (LPSp). C8-B4 cells (2×105 cells/well) were pretreated with LPSp for 18 h and then incubated with fluorescent latex beads at cell/bead ratio of 1:5 at 37°C for 3 h. Before adding the beads, cells were pretreated for 30 min with 50 μM cytochalasin D, an inhibitor of phagocytosis. The geometric mean fluorescence intensity of beads was measured and the relative phagocytic activity was calculated by dividing the fluorescence of internalized beads of the sample by that of the negative control sample. Data are presented as the mean±SEM of four samples (one-way ANOVA with Tukey-Kramer's test). Values without a common letter are significantly different at p<0.05.
Effect of LPSp on the phagocytic activity against fluorescent latex beads. We next examined whether LPSp affected phagocytic activity against fluorescent latex beads. The phagocytic assay was performed using C8-B4 cells pre-incubated with LPSp for 18 h. As shown in Figure 2, treatment with LPSp, at as low as 100 pg/ml, significantly enhanced the relative phagocytosis of the fluorescent latex beads.
Effect of LPSp on the phagocytic activity against Aβ. We further examined whether LPSp promoted HF488-Aβ1-42 phagocytic activity in the C8-B4 microglial cells. The cells were incubated with HF488-Aβ1-42 at 10 ng/well for 3 h. As shown in Figure 3, LPSp treatment (1 ng/ml) significantly enhanced the relative phagocytosis of HF488-Aβ1-42.
Discussion
In the present study, we demonstrated that pre-treatment with LPSp enhanced the phagocytic activity of fluorescent latex beads or HF488-Aβ1-42 by the mouse microglial cell line C8-B4 cells. Our previous study also indicated that the HF488-Aβ1-42 phagocytosis by the mouse primary microglia was enhanced by LPSp treatment (11). These results are consistent with recent studies reporting that Juzen-taiho-to (JTT), a Chinese herbal medicine, and LPS, a major macrophage-activating substances found in JTT, increased the microglial phagocytosis of Aβ1-42 in primary microglia (12), and monophosphoryl lipid A, and LPS, increased Aβ1-42 phagocytosis by mouse microglia cell line BV-2 (10). However, further studies were required for elucidating the molecular mechanisms underlying the promotory effect of Aβ phagocytosis by LPSp. We previously demonstrated that the sublingual administration of LPSp increased antibody responses to influenza vaccine-specific IgG (in serum) and IgA (in nasal mucosa) (13). Oral treatment with LPSp inhibited the development of atopic dermatitis in NC/Nga mice (14). In addition, oral administration of LPS derived from the subaleurone layer of rice (15) or acetic acid bacteria (16) prevented pollen allergy via activating macrophage activity in a mouse model of cedar pollen allergy. These findings suggest that the effects of oral or sublingual administration of LPS involved regulation of the systemic immune system. Some clinical trials indicated that enhancement of Aβ clearance by medical treatment led to a therapeutic effect on the brain level of Aβ (17, 18), implying that homeostatic regulation of immune responses in microglia by LPSp administration might be a potential preventive strategy for Alzheimer's disease.
Analysis of phagocytosis of Fluor 488-conjugated amyloid β (HF488-Aβ1-42) particles by C8-B4 cells treated with lipopolysaccharides derived from Pantoea agglomerans (LPSp). C8-B4 cells (2×105 cells/well) were pretreated with LPSp for 18 h and incubated with HF488-Aβ1-42 (10 ng/well) at 37°C for 3 h. Before adding the beads, cells were pre-treated for 30 min with 50 μM cytochalasin D, an inhibitor of phagocytosis, before adding HF488-Aβ1-42. The geometric mean fluorescence intensity of HF488-Aβ1-42 was measured and the relative phagocytic activity was calculated by dividing the fluorescence of internalized HF488-Aβ1-42 of the sample by that of the negative control sample. Data are presented as the mean±SEM of four samples (one-way ANOVA with Tukey-Kramer's test). Values without a common letter are significantly different at p<0.05.
Acknowledgements
This work was supported by the Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio-oriented Technology Research Advancement Institution, NARO).
Footnotes
Conflicts of Interest
The Authors have no financial conflicts of interest to declare interest in regard to this study.
- Received May 2, 2017.
- Revision received May 22, 2017.
- Accepted May 23, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
