American Journal of Infectious Diseases 4 (1): 22-31, 2008
© 2008 Science Publications
Dengue Virus Infection Induced NF-κB-dependent Macrophage
Migration Inhibitory Factor Production
1Lien-Cheng Chen, 2Huey-Wen Shyu, 3Huan-Yao Lei, 3Shun-Hua Chen,
3Hsiao-Sheng Liu, 3Yee-Shin Lin, and 4Trai-Ming Yeh
1Institute of Basic Medical Sciences, and 2Department of Medical Technology,
Fooyin University, Kaohsiung, Taiwan 3Department of Microbiology and Immunology,
and 4Department of Medical Laboratory Science and Biotechnology,
National Cheng Kung University, Tainan, Taiwan,
Corresponding Author: Dr. Trai-Ming Yeh, Department of Medical Laboratory Science and Biotechnology,
College of Medicine National Cheng Kung University, Tainan 701, Taiwan (ROC)
Tel. +886 6 2353535 ext. 5778 Fax +886 6 236 3956
Abstract: Dengue virus (DV) infection can cause mild dengue fever or severe dengue hemorrhage
fever and dengue shock syndrome. Macrophage migration inhibitory factor (MIF) is a cytokine that
plays an important role in the modulation of inflammatory and immune responses and serum levels of
MIF are correlated with disease severity in dengue patients. However, the mechanism that induces MIF
production during DV infection is unclear. In this study, we showed that DV infection, but not UV-
inactivated DV stimulation, dose-and time-dependently induced MIF secretion in human A649
epithelial cells. MIF promoter assays and RT-PCR demonstrated that MIF gene transcription was
activated during DV infection. Furthermore, DV infection induced NF-κB activation, and the NF-κB
inhibitors dexamethasone and curcumin inhibited DV-induced MIF production. Finally, we found that
different cells have different abilities to release MIF after DV infection. Interestingly, DV infection and
MIF production in the human monocytic cell line THP-1 and peripheral blood mononuclear cells
increased in the presence of antibodies against DV. Taken together, these results suggest that DV
infection of human cells induces NF-κB activation and MIF production, which can be increased in the
presence of pre-existing antibodies.
Key words: Cytokine; inflammation; infection
flaviviruses subgrouped into four antigenically related
serotypes: DV types 1, 2, 3, and 4. It is estimated that
over 50 million DV infections occur globally each
year. DV infection generally causes mild symptoms
such as fever, headache, and muscle and joint pain,
which is called dengue fever (DF). In some cases,
especially during secondary infection with a different
serotype of DV, the infection may progress to dengue
hemorrhagic fever (DHF) or dengue shock syndrome
(DSS). DHF is a severe febrile disease characterized
by abnormalities in homeostasis and increased capillary
leakage that can progress to hypovolemic shock
(DSS). Even though the process leading to DHF/DSS
is not fully understood,
enhancement (ADE) has been proposed to explain the
mechanisms by which heterogeneous serotype DV
infection can induce DHF/DSS in secondary
Dengue are mosquito-borne
infection[5, 6]. According to the ADE hypothesis, the
antibody generated during primary dengue infection can
neutralize only DV with the same serotype but not
those with different serotypes during secondary
infection. In addition, the non-neutralizing antibodies
may increase the virus uptake by macrophages through
Fc receptor and even worsen the disease. However,
antibody against DV pre M protein (anti-pre M Ab) can
also augment dengue virus infection in a concentration-
dependent but serotype and FcR-independent manner.
Macrophage migration inhibitory factor (MIF) is a
cytokine important in the modulation of inflammatory
and immune responses. MIF is released by different
cells in many tissues in response to a variety of
stimuli. Once released, MIF augments the secretion of
TNF-α and counteracts the anti-inflammatory action of
glucocorticoids. Interestingly, low concentrations of
glucocorticoids induce rather than inhibit MIF
production in macrophages. In our previous study,
Am. J. Infect. Dis., 4 (1): 22-31, 2008
we found that serum levels of MIF were correlated with
disease severity in dengue patients. The mechanism that
induces MIF production during DV infection is unclear,
however. In this study, we show that DV infection of
the human epithelial cell line A548 directly induced
MIF production via NF-κB activation. We also
explored the effects of DV infection on the production
of MIF in other human cell lines, as well as in primary
cells such as human umbilical vein endothelial cells
(HUVEC) and peripheral blood mononuclear cells
(PBMC), with or without pre-existing antibodies
MATERIAL AND METHODS
Preparing virus stock and virus titration: Dengue
type 2 (DV2) strain PL046 and JEV (strain RP-9)
viruses were propagated in C6/36 cells. Briefly,
monolayers of C6/36 were inoculated with the virus at a
multiplicity of infection (MOI) of 0.1 and incubated at
26°C and 5% CO2 for 5 days. The culture medium was
harvested, and cell debris was removed using
centrifugation at 900 × g for 10 min. After further
centrifugation at 16,000 × g for 10 min, the virus
supernatant was collected and stored at -80°C until use.
Virus titer was determined using a plaque assay with
the BHK-21 cell line. Briefly, a 10-fold serial dilution
of virus was added to BHK-21 monolayer and then
incubated at 37°C at 5% CO2 for 5 days. Plaque
numbers were counted after crystal violet staining. UV
inactivation of DV (UV-DV) was done in a UV
crosslinker (Stratalinker; Stratagen, La Jolla, CA) using
short wavelength ultraviolet radiation (UVC, 254 nm)
at a distance of 5 cm for 30 min on ice, as previously
Cell culture: A human lung cancer epithelial cell line
(A549) and a human hepatoma cell line (HepG2) were
cultured in DMEM (Invitrogen, Carlsbad, CA)
supplemented with 10% fetal bovine serum (FBS)
(Invitrogen), and 2 mM L-glutamine. A human T cell
line (Jurkat), a myelomonocytic cell line (K562), and a
monocytic cell line (THP-1) were grown at 37°C in 5%
CO2 in RPMI 1640 medium with 10% FBS. Human
peripheral blood mononuclear cells (PBMC) from
healthy blood donors with no antibodies against DV2
were isolated using lymphocyte separation medium
(Histopaque-1.077; Sigma-Aldrich Co., St Louis, MO).
Human umbilical vascular endothelial cells (HUVEC)
were isolated from human umbilical cord veins and
cultured as previously described.
Viral infection and ADE: Cells were pretreated with
or without dexamethasone
(Sigma-Aldrich) or curcumin
(Sigma-Aldrich) for 1 h before incubation with DV2 at
an MOI of 10 or as indicated. Viruses were allowed to
be absorbed by the cells for 2 h at 37°C. After that,
unbound viruses were removed by washing with
medium. Infected cells and culture supernatants were
collected at different time intervals. Human PBMC or
THP-1 cells were infected with DV2 at the MOI of 10
in the presence of the diluted dengue type 3 patient
serum (1:6000) or anti-pre M monoclonal antibody 70-
21 (1 µg).
Immunofluorescent staining and flow cytometric
assay: Cells (1 × 106) were infected with DV2 at an
MOI of 10 for 24 h. After they had been incubated, the
cells were fixed with 4% paraformaldehyde for 30 min
and then permeabilized with 0.5% Triton X-100 for 10
min. The cells were then washed with PBS and blocked
with 0.05% BSA in PBS. Fixed cells were stained with
primary antibody at 4°C for 1 h. After being washed,
the cells were incubated with secondary antibody and
observed under a fluorescent microscope (Olympus,
Tokyo, Japan) or for FACSCalibur analysis (Becton
Dickinson Immunocytometry Systems, San Jose, CA)
and analyzed using WinMDI 2.8 software. Anti-dengue
mAb (anti-E, anti-pre M, and anti-NS1, a gift from Dr.
Huan-Yao Lei), MIF rabbit antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), and NF-κB p65 rabbit
antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
were used as primary antibodies. FITC-conjugated
goat-polyclonal anti-mouse IgG antibody (1:200
dilution; Jackson ImmunoResearch, West Grove, PA),
Alexa Fluor 594 conjugate goat-polyclonal anti-Rabbit
IgG antibody (1:200 dilution; Molecular Probes, Inc.,
Eugene, OR) or Alexa Fluor 488 conjugate goat-
polyclonal anti-Rabbit IgG antibody (1:200 dilution;
Molecular Probes) were used as secondary antibodies.
Reverse-transcription polymerase chain reaction
(RT-PCR): RNA was extracted using an isolation
reagent (TRIzol; Invitrogen) and quantified at 260 nm.
Reverse-transcription (RT) was done using a kit
(Invitrogen) according to
dehydrogenase (GADPH) was a control. The primer
MIF (sense): 5’-TCCTTCTGCCATGCCGA-3’
MIF (antisense): 5’-TGCGGCTCTTAGGCGAAGGT-3’
GADPH (sense): 5’-CACGGCAAGTTCAATGGCACA-3’;
GADPH (antisense): 5’-GAATTGTGAGGGAGAGTGCTC-
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