ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 67
OBJECTIVE: In hypoxia, the most common mechanism
of injury is the increase in reactive oxygen species
and energy deficiency, which can cause mitochondrial
morphology and endogenous apoptosis. This study in-
vestigated whether autophagy was activated by a large
number of active regulatory processes to adapt cells to
hypoxia and help protect against hypoxia. Additionally,
the role of lipopolysaccharide (LPS) in this process was
assessed.
STUDY DESIGN: Mouse-derived dendritic cells were
cultured in a hypoxic environment. Cell viability and
expression of autophagy-related proteins LC3, Beclin1,
and hypoxia-inducible factor–1α (HIF-1α) were detect-
ed at different hypoxia time points. Formation of au-
tophagy bodies was observed at different hypoxia times
by transmission electron microscopy.
RESULTS: After 12 hours of hypoxia the effect of
autophagy was obvious, and the formation of auto­
phagosomes increased. The expression of HIF-1α, LC3-
II, and Beclin1 was significantly higher than that in
the control group, which reached the highest value at
24 hours, whereas the expression in hypoxic 48-hour
cells was lower than that in the 24-hour treated group
but was still higher than the control group. After LPS
was treated, autophagy flux in the hypoxia-LPS group
was enhanced as compared with the hypoxia group.
CONCLUSION: Hypoxia can induce autophagy in
mouse-derived dendritic cells, and simultaneous ad-
ministration of LPS under the condition of hypoxia
can enhance autophagy. The expression of HIF-1α is
consistent with the expression of autophagy-related
proteins LC3-II and Beclin1, which plays an impor-
tant role in hypoxia-induced activation of autophagy.
(Anal Quant Cytopathol Histpathol 2019;41:67–
73)
Keywords: autophagy; dendritic cells; HIF-1α; hy-
poxia; lipopolysaccharide.
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4102-0067/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Synergy Assessment of Hypoxia and
Lipopolysaccharide on Autophagy
in Dendritic Cells
Jie Zhang, M.M., Jingming Shi, Ph.D., Min Zhang, Ph.D.,
Hongyan Li, Ph.D., and Jinwang Ding, M.M.
From the Sections of Morphology and of Microbiology and Immunology, XiZang Minzu University Health Science Center, Xianyang;
and the Department of Oncological Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hang-
zhou, Zhejiang, China.
Drs. J. Zhang, Shi and Li are Instructors, Morphology Teaching and Research Section, XiZang Minzu University Health Science Center.
Dr. M. Zhang is Instructor, Microbiology and Immunology Teaching and Research Section, XiZang Minzu University Health Science
Center.
Dr. Ding is Clinician, Department of Oncological Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of
Medicine.
This work was supported by grants from the Natural Science Foundation of Tibet Autonomous Region (XZ2018ZR G-71), Project of
training young scholars in XiZang Minzu University (18MDX02), Applied Research Project of Commonweal Technology in Zhejiang
Province (2017C33180), Science Research Program of Hangzhou (20160533B05), Talents Scheme of “131” Program in Hangzhou, and
National Natural Science Foundation of China (81560668, 81860719).
Address correspondence to: Jinwang Ding, M.M., Department of Oncological Surgery, Affiliated Hangzhou First People’s Hospital,
Zhejiang University School of Medicine, Hangzhou 310006, Zhejiang, China (zjlsdjw@163.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Hypoxia exists in a variety of physiological and
pathological conditions, such as stroke and myo-
cardial infarction.1-3 In response to a hypoxic en-
vironment, cells demand an efficient system to
initiatively activate several signaling pathways
ranging from cytoprotective functions to elimi­
nating damaged substances. Misfolded proteins,
dysfunctional organelles, and invading pathogens
were cleared to provide new materials for cell
regeneration, reparation, and cell recycling. These
actions are carried out by macroautophagy. In
the autophagy pathway, double membrane vesi-
cles (autophagosomes) engulf regions of the cy-
toplasm and organelle. These vesicles then de-
liver cargo for lysosomal degradation, which is
a major component of the cellular stress re-
sponse.4-8 A number of studies in recent years
have demonstrated that autophagy is induced by
hypoxia to survive. Di Gui et al reported that
hypoxia induced pulmonary artery smooth mus-
cle cell (PASMC) proliferation and reverse apo­
ptosis resistance via the upregulation of au-
tophagy through both the AMPKα1-ULK1 and
AMPKα1-mTOR-ULK1 pathways.9 Bellot et al
demonstrated that hypoxia-induced autophagy
via BNIP3 and BNIP3L is clearly a survival mech­
anism that promotes tumor progression.10 Zhu
et al reported that the hypoxia-inducible factor–1
alpha (HIF-1α) induced autophagy in the conver­
sion of non-stem pancreatic cancer cells into can-
cer stem-like cells on the tumor.11 However, there
is little research focused on the the mechanisms
and principles of autophagy induced by hypoxia
in dendritic cells.
Dendritic cells are the highly specialized antigen-
presenting cells of the immune system that play
a key role in regulating immune responses. They
were first discovered by Steinman and his men­
tor Cohn in the spleen of mice in 1973.12,13 They
are the only professional antigen-presenting cells
that activate primary T cells and initiate adaptive
immune response. Dendritic cells are the hub for
innate and acquired immune responses in anti-
infective, antitumor, transplant rejection, and au-
toimmune, which play an important role in the
process of illness.14-17 In this study we first exam-
ined the effect of hypoxia and/or lipopolysac­
charide (LPS) on cell proliferation by MTT assay.
Then we detected the effectiveness of hypoxia
and/or LPS in changing expression levels of HIF-
1α, LC3-II, and Beclin1. Finally, we tested the re-
lationship between HIF-1α and autophagy and at-
tempted to clarify part of the mechanism. Our
data showed that hypoxia-induced autophagy re-
sulted from the elevated HIF-1α. However, the
exact mechanisms of how hypoxic niches induce
autophagy via HIF-1α remain largely unknown.
Materials and Methods
Reagents
The following materials and antibodies were
purchased: lipopolysaccharide (LPS) 1 mg/mL
(Sigma, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) (Sigma,
USA); anti-LC3 1:1000 (Santa Cruz Biotechnology,
USA); Beclin1 antibodies 1:500 (Santa Cruz Bio­
technology); HIF-1α antibody 1:1000 (Santa Cruz
Biotechnology); and b-actin antibody 1:2000 (Bio­
world Technology, USA).
Cell Culture
Mouse dendritic cells, logarithmic growth phase,
were inoculated in a 96-well plate at a density of
1×103–1×104 cells/well and maintained in me-
dium (DMEM; HyClone, USA) in an incubator.
The cells were divided into 4 groups as follows:
the control group: oxygen(+)/LPS(-); the LPS
group: oxygen(+)/LPS(+); the hypoxia group:
oxygen(−)/LPS(−); and the hypoxia-LPS group:
oxygen(−)/LPS(+). All hypoxia groups were
kept in the hypoxia incubator at 37°C with 1%
oxygen, 5% CO2, and 94% nitrogen for 6 hours,
12 hours, 24 hours, and 48 hours for the time-
dependent study, whereas the normal oxygen
incubator was 5% CO2 and 95% humidified air.
For LPS stimulation, the cells were cultured at a
concentration of 5 μg/mL of LPS medium.
MTT Assay
For MTT assay the cells were cultured with 20
μL of MTT solution at 5 mg/mL for each well for
3.5 hours. Then the medium was removed and
200 μL of DMSO was added. The plates were
shaken for 10 minutes to allow the crystals to
dissolve sufficiently. Absorbance (A) at 490 nm
was detected with enzyme-linked immunosor­
bent assay instrument in accordance with the
manufacturer’s instructions.
Western Blot Analysis
The cells (5× 106) were lysed with 200 μL of cell
lysis buffer (Beyotime Institute of Biotechnology,
China) containing a 1× protease inhibitor per 10
mL of protein extraction. The protein content
68 Analytical and Quantitative Cytopathology and Histopathology®
Zhang et al

3. was measured by Coomassie Brilliant Blue assay
(Beyotime). The total cell lysates were suspended
by 1/4 volume of loading buffer (313 mM Tris-
HCl, pH 6.8; 10% SDS; 2-ME; 50% glycerol; and
0.01% bromophenol blue) and boiled for 5 min-
utes, cooled, and stored at −20°C for detection.
Protein samples were separated on 6% and 12%
SDS-PAGE and transferred onto a polyvinyli­
dene fluoride membrane (Pall Laboratory, USA)
using the Trans-Blot Turbo Transfer system (Bio-
Rad) with Trans-Blot Transfer Packs. The second
antibodies (1:8000) were horseradish peroxidase-
conjugated secondary antibody: anti-rabbit (Ther­
mo Scientific). Western blots were processed on
PowerPac300 Western System (Bio-Rad). The incu-
bated membranes were developed and the band
density was quantified using an enhanced chemi­
luminescent autoradiography system (Clinx).
Transmission Electron Microscopic Examination
For transmission electron microscopy, cells were
fixed in fixation solution (Servicebio) for 2 hours
at 4°C and post-fixed with 1% citrate 0.1 M phos­
phate buffer (pH 7.4) for 2 hours at room temper­
ature and dehydrated stepwise with ethanol and
acetone. The dehydrated pellets were embedded
in Spurr resin for sectioning. Transmission elec­
tron microscopy images were captured using a
transmission electron microscope (Hitachi) on
sectioned cells (60–80 nm) stained with uranium
lead double staining.
Statistical Analysis
Statistical analysis was carried out by one-way
analysis of variance (ANOVA) followed by Stu­
dent’s t test (between 2 groups). Data are pre-
sented as means±standard error of the means.
P values ≤0.05 were considered statistically sig­
nificant.
Results
Autophagy Was Involved in Hypoxia-Induced Cell
Proliferation and Apoptosis Resistance
We first assessed the effects of hypoxia and/or
LPS on the proliferation of dendritic cells based
on the MTT test. There was no significant dif­
ference between the control group from 6 hours
to 48 hours. Compared with the control group,
higher proliferation rates were clearly observed
by LPS matured with time (**p<0.01). However,
the proliferation was obviously inhibited by hy-
poxia after 12 hours. An increased rate of pro­
liferation was observed at 48 hours as compared
to that at 24 hours. After LPS was given, the cells
inhibited by hypoxia were stimulated to maturity
(##p<0.01) (Figure 1).
Enhanced Autophagy Flux May Underlie the
Upregulation of HIF-1α
In order to evaluate if autophagic flux could be
induced upon hypoxia treatment, the autophagy
level after hypoxia from 6 hours to 48 hours was
determined. The results showed that there was
no significant difference between all groups at
6 hours. The expression of autophagy-related
protein LC3-II, Beclin1, and HIF-1α was signifi­
cantly higher than that in the control group after
12 hours of hypoxia, which reached the highest
value at 24 hours, whereas the expression in hy-
poxic 48-hour cells was lower than that in the
24-hour treated group but was still higher than
the control group (##p<0.01). After LPS was treat­
ed, autophagy flux in the hypoxia-LPS group was
enhanced as compared with that in the hypoxia
group (**p<0.01) (Figures 2 and 3). In addition,
to further measure autophagy flux, the formation
of autophagosomes was determined by electron
microscopy so that similar results were obtained.
Compared with the control group, whose autoph­
agic vacuoles were rarely seen, we noticed ac-
Volume 41, Number 2/April 2019 69
Hypoxia and LPS on Autophagy in Dendritic Cells
Figure 1 Effect of hypoxia and LPS on the proliferation of
dendritic cells. The MTT assay was employed to assess the cell
proliferation after treatment with or without hypoxia or LPS for
6, 12, 24, and 48 hours, respectively. For hypoxia administration,
hypoxia was given at a condition of 1% oxygen, 5% CO2,
and 94% nitrogen. For LPS administration, LPS was given at a
concentration of 5 μg/mL. Bar represents mean±SD in every
group. *p<0.05, **p<0.01 compared with the control group;
#p<0.05, ##p<0.01 compared with the hypoxia group.

4. crual of membrane vacuoles, and cytosolic com­
ponents or organelles were sequestered in some
of these vacuoles after 12 hours of hypoxia, which
reached the highest flux at 24 hours. With the
stimulation of LPS and hypoxia, the formation of
autophagosomes was also significantly more than
the hypoxia group (Figure 4).
Discussion
Autophagy is an important biological process in
70 Analytical and Quantitative Cytopathology and Histopathology®
Zhang et al
Figure 2 Hypoxia-induced autophagy activity via HIF-1α. Protein expression and quantitative analysis of HIF-1α, LC3II, and Beclin 1.
Following LPS stimulation (5 μg/mL) or hypoxia for 6, 12, 24, or 48 h, mouse dendritic cells were lysed and subjected to western blotting
with the antibodies indicated. Levels of b-actin protein were used as the loading control. The data were from the same polyvinylidene
difluoride membrane and were representative of at least 3 independent experiments. Bar represents mean±SD in every group. *p<0.05,
**p<0.01 compared with the hypoxia group; #p<0.05, ##p<0.01 compared with the control group.
Figure 3 Time-dependent effect of protein expression of HIF-1α, LC3II, and Beclin 1 induced by hypoxia and/or LPS. Quantitative
analysis of immunoblotting of HIF-1α, LC3II, and Beclin 1 controlled by β-actin at 6, 12, 24, and 48 h in dendritic cells. Bar represents
mean±SD in every group. **p<0.01 compared with the control group; ##p<0.01 compared with the hypoxia group.

5. that proper regulation of autophagic flux is es-
sential for the homeostasis of cells under physio­
logical conditions, particularly in response to
damage.18,19 In our study, dendritic cells were
used because several issues reported that den­
dritic cell–based therapies are widely being used
for the treatment of cancer and the prevention of
liver transplant rejection and autoimmune dis­
eases.20-24 We generated a proper hypoxia model
to imitate a hypoxia environment in vivo. The
activation of autophagy after hypoxia initiation
was confirmed by the induction of LC3-II and
Beclin 1. Additionally, autophagosomes, C-shaped
double-membrane structures, and engulfment of
cytoplasmic materials by autophagosomes were
also observed synchronously.
The inducer LPS was identified as a positive
control to check the role of autophagy in hypoxia
establishment.25-27 Dendritic cells were treated in
the hypoxia incubator with the stimulation or
nonstimulation of LPS for 48 hours. In the present
study, hypoxic injury was proved by the inhibi­
tion of cell proliferation, while LPS could increase
cellular activity partially blocked by hypoxia and
upregulate the autophagy flux induced by hypox­
ia in LPS-matured cells at 12 hours, which reached
Volume 41, Number 2/April 2019 71
Hypoxia and LPS on Autophagy in Dendritic Cells
Figure 4 Hypoxia results in autophagic vesicle formation. The cells were cultured as described in Materials and Methods for 6 h,12 h,
24 h, and 48 h. The formation of autophagosomes were examined with an electron microscope. Scale bar=1 μm. Black arrows indicate
autophagosomes. Autophagosomes in the images were enlarged in order to observe the morphology clearly.

6. the highest value at 24 hours. However, we found
that the proliferation and autophagy flux were
both downregulated at 48 hours, which did not
further enhance autophagy activation, suggesting
that a ceiling effect of autophagy activation to
avoid excessive induction resulting in cell apopto-
sis under physiological conditions may exist.
Numbers of studies have provided indirect or
circumstantial evidence for HIF-1α in the molec­
ular mechanism for the collateral induction of
autophagy in hypoxia. Wang and Li revealed that
in ischemic kidney injury, there is a signaling
axis of HIF-1α, miR-20a-5p, and ATG16L1 in the
autophagic process induced by hypoxia.28 Yue et
al reported that GRIM-19 inhibition inducted of
autophagy was activated through ERK and HIF-
1α, not STAT3 in Hela cells.29 Lőw et al also
showed that HIF-1α/Sima may contribute to up-
regulation of autophagy by impaired proteasomal
activity in Drosophila.30 In this study we con­
firmed that the activation of the expression of HIF-
1α, which is closely related with the autophagic
process, has an important effect on the cellular
activity of dendritic cells, which may contribute to
a theoretical basis for clinical treatment.
In conclusion, we clearly show that, to adapt
to hypoxia, autophagy is a biological process that
actively regulates cell homeostasis. Although fur­
ther detailed studies are necessary to resolve the
pathway by which this phenomenon occurs, our
results suggest that HIF-1α was involved in the
regulation of autophagy in dendritic cells.
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