Material & Methods
Monocyte-differentiated DC culture
Human monocyte cell line THP-1 were differentiated into DC using a
well-established protocol (25). THP-1 cells were cultured in complete
cell culture medium (RPMI-1640, Gibco, with 10% FBS, Sigma-Aldrich,
antibiotic-antimycotic solution, Gibco and 2-Mercaptoethanol, Gibco)
with 5.5 mM glucose, 11 mM glucose, and 25 mM glucose and supplements of
IL-4 (20 ng/mL) and GM-CSF (20 ng/mL) for 5 days to differentiate into
immature DC. Fresh media were replenished every 2 days. On Day 5,
suspended cells were collected and cultured in RPMI with 5.5 mM glucose,
11 mM glucose, and 25 mM glucose and supplements of IL-4 (20 ng/mL,
LifeSciences), GM-CSF (20 ng/mL, LifeSciences), TNF-\(\alpha\) (20
ng/mL, LifeSciences), and ionomycin calcium (20 ng/mL, Sigma-Aldrich) to
differentiated into mature DC for 24 hours. Dendrites formation and cell
adherences confirms the differentiation. On Day 6, differentiated DC
were seeded in a 6 well plate with a density of
1\(\times\)106 cells/2 mL cell culture medium per well
with supplements of GM-CSF (20 ng/mL), ionomycin calcium (20 ng/mL),P. gingivalis lipopolysaccharides (LPS, 1 \(\mu\)g/mL,
Invitrogen) and advanced glycation endproduct (AGE, 2 \(\mu\)g/mL,
Sigma-Aldrich) for 24 hours. Cells were cultured at 37 and
5% CO2.
BMDC culture
Bone marrow cells were extracted from femur and tibia bone of 6 – 12
weeks old C57BL/6 mice and plated on a 100 mm Petri culture dish at a
density of 4\(\times\)106 cell/10 mL with GM-CSF (20
ng/mL) in complete cell culture medium RPMI-1640 (Gibco) with glucose
concentration of 5.5 mM, 11 mM, and 25 mM. BM cultures were fed on Day 3
and 6 with 10 mL cell culture medium containing fresh GM-CSF (20 ng/mL,
Biolegend). On Day 9, BMDC were seeded in a 6 well plate with a density
of 1\(\times\)106 cells/2 mL cell culture medium per
well. P.gingivalis lipopolysaccharides (LPS, 1 \(\mu\)g/mL,
Invitrogen) and advanced glycation endproduct (AGE, 2 \(\mu\)g/mL,
Sigma-Aldrich) was added for 24 hours. Cells were cultured at 37
and 5% CO2.
Splenic T cell extraction
Spleens from C57BL/6 mice were minced and passed through a 40 \(\mu\)m
strainer prior to T lymphocyte isolation. Mice T cells were purified
from splenic cell suspension using EasySepTM Mouse T
Cell Isolation Kit (Stemcell Technologies) following the manufacturer’s
protocol.
DC-T cell coculture
0.5\(\times\)106 splenic T cells were cultured in a 12
well plate with inactivated BMDC cultures at 1:1 ratio supplemented with
GM-CSG (20 ng/mL, Biolegend) for three days with or without AGE (2\(\mu\)g/mL, Sigma-Aldrich) and LPS (1 \(\mu\)g/mL, Invitrogen). On Day
3, cell supernatant were collect for ELISA analysis. All cells were
cultured at 37 and 5% CO2.
Cell Morphology Assay
The differentiated mDC were grown in different glucose concentrations
with or without activation. The cells were fixed on glass coverslips at
a density of 0.8\(\times\)106 and stained with Hoechst
dye (Sigma), TRITC-conjugated phalloidin (Sigma), CD83 primary antibody
(Invitrogen), and FITC-conjugated secondary antibody (Invitrogen). Cells
were imaged using a fluorescent microscope system (Zeiss LSM 800,
Germany). Nine random areas were imaged at 40\(\times\) magnification
and the images were processed with Fiji ImageJ software.
Effect on DC Phenotype and Metabolism- Gene Expression Assays
Total RNA of inactivated or activated DC was extracted using RNeasy mini
kit (Qiagen) and the cDNA was synthesis using SensiFAST cDNA Synthesis
kit (Bioline) with random primers. Quantitative real time RT-PCR was
performed using SYBR Green Master Mix Reagent (Applied Biosystems) by
CFX Real Time System (Bio-Rad). Each sample was analyzed in triplicates
and normalized to control gene \(\beta\)-actin. Relative expression was
reported using the \(2^{\hat{}(\Delta Ct)}\) method. Table 1 and Table 2
presents the primer sequences used in the experiments.
Metabolic assay
The glycolysis activity of monocyte-differentiated DC was measured using
Agilent Seahorse XFe24 metabolic flux analyzer with glycolytic stress
test kit (Agilent Technologies). DC were seeded at a density of
1.75\(\times\)105 in the 24-well XF Cell Culture
Microplate. The extracellular acidification rate (ECAR) of the cells
were measured in triplicate following each injection of glucose,
oligomycin, and 2-deoxy-glycose. L-lactate from cell supernatant was
quantified following the manufacturers protocol (Cayman Chemical).
OXPHOS activity was measured using a commercial kit (Thermofisher).
Cytokine profile
The supernatants from monocyte-differentiated DC culture and BMDC
culture were collected on Day 7 and stored at -20 until further
use. Cytokine production by the DC were assessed using ELISA
(IFN-\(\gamma\), IL-6, IL-17, and IL-10) (Biolegend, California, United
States) following manufacturers protocol.
Phagocytosis assay
Inactivated BMDC were used in phagocytosis assay. Approximately
1\(\times\)106 cells were seeded in a 6 well plate
(Corning) with 2 mL complete cell culture medium RPMI-1640 (Gibco)
supplemented with GM-CSF (20 ng/mL, Biolegend). Cells were cultured with
5.68\(\times\)107 Fluoresbrite carboxylate
microspheres for 3 hours to assess cell phagocytosis. Cells were washed
and stained with Hoechst dye (Sigma) and imaged using a fluorescent
microscope system (Zeiss LSM 800, Germany). Six random areas were imaged
at 10\(\times\) magnification and the images were processed with Fiji
ImageJ software.
Statistical Analysis
The experiments were done in triplicates and repeated minimum of three
times. All data were analyzed with GraphPad Prism with unpaired t test
and ANOVA test. The difference between experimental groups was set to be
statistically significant at P \(<\) 0.05.