Dengue Virus (DENV)-1 Induces High Expression of Anti- and

Pro-inflammatory Cytokines in Human Macrophages

Winda Yulia

, Benediktus Yohan

, Febrina Meutiawati

, Alida R. Harahap

2,3

R. Tedjo Sasmono

Department of Microbiology, Faculty of Medicine, Universitas Syiah Kuala, Aceh, Indonesia

Eijkman Institute for Molecular Biology, Jakarta, Indonesia

Department of Clinical Pathology, Faculty of Medicine, Universitas Indonesia, Jakarta

Keywords: Dengue Viruses, Cytokines, Monocyte-Derived Macrophages (MDMs).

Abstract: Dengue is caused by dengue virus (DENV) infection. The pathological mechanism of dengue infection is

still poorly understood. Serotype variation and host’s immune response are thought to have roles in disease

severity. This study compared the ability of four DENV serotypes (DENV-1, -2,-3,-4) isolates from

Indonesia to induce the expression of anti- and pro-inflammatory cytokines in Monocyte-Derived

Macrophages (MDMs) differentiated from a healthy human. Monocytes were isolated from healthy human

donors and differentiated into macrophages under the stimulation of Macrophage Colony Stimulating Factor

(M-CSF). Mature MDMs were infected with four different serotypes of DENV isolates from Indonesia. The

resulting expression kinetics of Interferon alpha (IFN-2) and Interleukin-10 (IL-10) as anti-inflammatory

cytokines and IL-1 as pro-inflammatory cytokine was measured at six different time points using Luminex

immunoassay. The presence of DENV Non-Structural Protein 1 (NS1) as a marker of virus replication was

also measured using ELISA. DENV-1 showed a tendency to induce the expression of IFN2 and IL-10

more rapidly and at a higher level than other serotypes albeit of lower NS1 expression. The expression of

IL-1 was down-regulated in response to all DENV serotypes infection. This study demonstrates the

differences in the ability of four DENV serotypes in regulating the expression of cytokines in MDMs with

DENV-1 showed a superior inducing capability compared to other serotypes. This data provides

information of possible serotype-specific role on disease severity.

1 INTRODUCTION

Dengue is a febrile illness caused by dengue virus

(DENV) infection.(Gubler, 1998)

DENV is a

member of the Flaviviridae family, which includes

four serotypes identified as DENV-1, DENV-2,

DENV-3, and DENV-4.(Lindenbach & Rice,

2003)(Kuno, Chang, Tsuchiya, & Karabatsos, 2014)

The mechanisms that lead to clinical manifestations

of dengue are believed to be multifactorial. There

are two main factors which have been shown to

contribute to disease severity, i.e., viral and host

factors.(Halstead, 2008)

Viral factors (viral load and variations of viral

serotypes) have been shown to play critical roles in

the emergence of symptoms.(Halstead, 2008)(Clyde,

Kyle, & Harris, 2006) Previous research has argued

that the role of immune mediators such as anti- and

pro-inflammatory cytokines may be as important as

host immune factors.(Green & Rothman, 2006) In

humans, macrophages have been determined as

DENV targets.(Chen & Wang, 2002) The targeting

of these cells by DENV may then lead to the

immunological modulation,(Sun & Kochel, 2013) as

evidenced by the expression of various cytokines.

This study aimed to compare the ability of the

four serotypes of DENV in inducing the expression

of anti- and pro-inflammatory cytokines in MDMs

by looking at the kinetic patterns of cytokine

expression in vitro.

Previously, we have described the

cytokine/chemokine expression of four DENV

serotypes in the human A549 cell line.(Yohan,

Kendarsari, Mutia, Bowolaksono, & Harahap, 2014)

Here, we study the expression profile of MDM cells,

which more reflecting the natural pathogenesis

mechanism of DENV infection in human.

Yulia, W., Yohan, B., Meutiawati, F., Harahap, A. and Sasmono, R.

Dengue Virus (DENV)-1 Induces High Expression of Anti- and Pro-inﬂammatory Cytokines in Human Macrophages.

DOI: 10.5220/0008790500440048

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 44-48

ISBN: 978-989-758-438-1

2 METHODS

2.1 Monocyte Isolation and MDM

Differentiation.

Monocytes were isolated from venous blood of

healthy human volunteers. Ethical clearance was

obtained from the Eijkman Institute Research Ethics

committee.

Peripheral blood mononuclear cell (PBMCs)

isolation was performed using Ficoll gradient

centrifugation (Ficoll-Paque PLUS, GE Healthcare).

Monocytes fraction from PBMC were enriched by

overnight cell adherence into the surface of cell

culture flasks in RPMI-1640 medium (Gibco-

Thermo Scientific) supplemented with 10% FBS

(Gibco), 2 mM L-glutamine (Gibco), 100 U/ml

Penicillin and 100 μg/ml streptomycin (Gibco).

The enriched monocytes were further

differentiated into macrophages by stimulation with

10 ng/ml Macrophage Colony Stimulating Factor

(M-CSF) (Sigma) for eight days. Differentiation of

monocytes into macrophages was monitored based

on morphological observation and the expression of

c-fms gene as macrophage marker by RT-PCR. β-

actin gene was also amplified as RNA loading

control. The primer pairs used were as follows:

c-fms forward, 5-ACACTAAGCTCGCAATCCC-3,

and

revese 5’-GTATCGAAGGGTGAGCTCAAA-3’; β-

actin forward,

5’-CATCTCTTGCTCGAAGTCCA-3’, and

reverse,5’-ATCATGTTTGAGACCTTCAACA-

3’.(Jia et al., 2010)

2.2 DENV Infections

Four DENV clinical strains representing all four

serotypes were isolated from Indonesian dengue

patients’ sera. Viruses were cultured in Vero cells.

Viral titers were measured in plaque forming

units/ml (PFU/ml), using a modified plaque assay

method.(Lambeth, White, Johnston, & Silva, 2005)

MDMs were infected by four serotypes of DENV

using 0.1 multiplicity of infection (moi). Controls

included non-infected and lipopolysaccharide (LPS)-

stimulated cells. For cytokines and NS1 antigen

measurement, the cell culture supernatant was

collected in 12-hour intervals for a total of 72 hours

and immediately stored at -80°C. DENV NS1

antigen expression was measured in cell culture

supernatants by using Panbio Dengue Early NS1

ELISA kit (Alere, USA).

2.3 Cytokines Assay

The level of IFN-2, IL-10 and IL-1β were

measured using Milliplex MAP Human Cytokine kit

(Merck Millipore, MPXHCYTO-60K, Germany).

Multiplex fluorescent microbead immunoassay

containing fluorescent microspheres, conjugated

with specific monoclonal antibodies for the target

protein were used to detect and quantify the

cytokines from 25 µl of culture supernatant,

simultaneously, as described elsewhere.(Yohan et

al., 2014) Results were obtained in Median

Fluorescent Intensity (MFI) and further analyzed

using MasterPlex QT software to measure the

cytokines concentration in

pg/ml.(www.ReaderFit.com)

3 RESULTS

3.1 Isolation and Differentiation of

MDMs

In the early isolation of PBMC, the monocytes were

round upon microscopic inspection. Monocytes were

observed as small and uniformly distributed on the

tissue culture flask. Morphological changes were

observed during the eight days of differentiation and

can be seen in FIGURE 1. Cell development was

observed to have accelerated rapidly from day five

onward. Morphological observation of MDMs on

day eight indicated that the cells were fully

differentiated into macrophages, demonstrating cell

adherent, fibroblast-like morphology, and the

appearance of pseudopodia.(Abbas, Lichtman, &

Pillai, 2012)(Sasmono & Hume, 2004) Detection of

the c-fms gene, a marker of macrophage

differentiation(Sasmono & Hume, 2004), was

prominent at day 8 using RT-PCR. This gene was

not detected in fibroblasts control cells.

Dengue Virus (DENV)-1 Induces High Expression of Anti- and Pro-inﬂammatory Cytokines in Human Macrophages

Figure

1. Morphology MDMs isolation and differentiation on day 0 to day 8. Cell growth appears slow

from day 0 to 4 of isolation, and rapid growth seen up to day 8. 20 x magnification.

Figure 2. Expression of c-fms gene in the

MDMs. Comparison of the macrophage colony-

stimulating factor receptor (c-fms gene) and human

actin (β-actin gene) as an RNA loading control

using RT-PCR from isolation day one to day eight.

M, DNA Marker (100 bp) (Invitrogen); N,

negative control; F, Fibroblast.

Figure 3. Detection of DENV 1-4 NS1 antigen in

the MDMs. DENV NS1 antigen was measured

using commercial NS1 ELISA tested to culture

supernatant collected 72 hours post-infections.

3.2 DENV NS1 Antigen Detection

NS1 protein secretion is one marker of dengue virus

replication in the host cell. The ability of dengue

virus to replicate in infected MDMs was evidenced

by the detection of DENV NS1 antigen in all four

serotypes, observed at 72 hours post-infection. NS1

antigen of DENV-4 appeared to predominate,

whereas DENV-1 NS1 expression was the lowest

among all serotypes.

The absorbance levels were considered as

Positive using the Panbio Unit calculation. Non-

infected cell control showed no detection of NS1

(FIGURE 3).

3.3 Kinetics Profile of Cytokines

Expression in the MDMs

Our study demonstrated that IFN-2, IL-10 and IL-

1 were expressed as a result of the exposure of

MDM to the four serotypes of DENV. The kinetics

of the expression patterns of cytokines is shown in

FIGURE 4. The intensity of IFN-α2 expression on

MDMs exposed by DENV-1 was significantly

different in comparison with the other serotypes.

IFN-α2 expression is seen to rise sharply after

24 hours, then decreased sharply after 60 hours. The

pattern of cytokine expression seen in DENV-4

appears to increase at a slower rate, which then starts

to increase sharply at 60 and 72 hours post-infection.

In DENV serotype -2 and -3, the increase in IFN-α2

was overall not as high as in the two other serotypes.

The induction of IL-10 expression against

DENV-1 infection tended to be higher than the other

serotypes, despite lower levels of DENV NS1

antigen being detected. The IL-10 expression

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

patterns showed a sharp increase at about 36

hours post-infection in all serotypes, and then

plateaued or declined.

Expression of IL-1β slightly increased and then

gradually decreased after 36 hours. All serotypes

showed decreasing patterns until 60 hours and then

continue to slightly increase after 72 hours, except

for DENV-1. In those two cytokines, there was a

tendency that DENV-1 induces relatively highest

expression of cytokines among the DENV serotypes.

Figure 4. Cytokines expression kinetics in MDMs

in response to infection of four DENVserotypes.

4 DISCUSSION

In this study, differences in the pattern of expression

of cytokines in MDMs (FIGURE 4) was

demonstrated, with DENV-1 exposed MDMs

expressing the highest levels of cytokines. The

increase in cytokine expression is inversely related

to the detection of DENV NS1 antigen (FIGURE 3).

The response to DENV-4 was observed to be

lower than most other serotypes and virus NS1

antigen was detected at highest levels than with

exposure to DENV-1 and -3. This raises the question

of whether there are intrinsic factors in DENV-1

virus titers which, although less prominent than

other serotypes, triggers an increase in cytokine

expression compared to other serotypes. Further

study is needed to determine the possible existence

of DENV-1 intrinsic factors, especially when

associated with viral genetic traits that may be

observed with the complete genome sequence of the

virus.

Interferon (IFN) is a host defense system that is

activated during the initial stages of infection.

Previous in vitro studies showed that DENV

infection in human cells could be inhibited by initial

therapy using IFN-α which inhibits translation of

viral RNA kinetics.(Diamond & Harris, 2001) The

results of this study demonstrate that the expression

pattern of IFN-α2 in MDMs infected by DENV-1

and DENV-4 may demonstrate a correlation

between the concentrations of IFN-α2 and

expression of DENV NS1 antigen ELISA (FIGURE

3 and 4). The high-level of IFN-α2 expression

during the early phase of DENV-1-infected MDM

(up to 60 hrs post-infection) resulted in the low level

of virus titer, measured as NS1 level at 72 hrs.

Inversely, the relatively low level of IFN-α2

expression in the initial stages of DENV-4 infection

yielded increased level of NS1. However, the

patterns were not clearly observed in DENV-2 and

DENV-3-infected MDM. These figures indicate a

possible role of IFN-α2 in inhibiting DENV

replication in the host and the serotype-specific

induction mechanisms.

In DENV pathogenesis, resistance against IFN

can be caused by IL-10 led immunosuppression,

followed by failure in achieving viral clearance by

the immune system and persistent infection in acute

viral infections.(Diamond & Harris, 2001) The

results of this study demonstrate that the expression

pattern of IL-10 was increasing during the

progression of DENV infection. This may be due to

the immunosuppression mechanisms intrinsic to IL-

10 as the anti-inflammatory cytokine. The increase

in IL-10 expression may be correlated to the role of

the DENV in regulating the host’s immune system.

IL-1β is a pro-inflammatory cytokine produced

by macrophages. This cytokine plays a role in the

cellular activity, including proliferation and

differentiation kinetics of cytokine expression, and

Dengue Virus (DENV)-1 Induces High Expression of Anti- and Pro-inﬂammatory Cytokines in Human Macrophages

apoptosis.(Abbas et al., 2012) In this study, most

serotypes showed a gradually decreased expression

at later stages, post-infection. The role of this

cytokine in DENV infection may need to be

explored more.

5 CONCLUSION

In conclusion, cytokines expression in MDM

infected by various DENV serotypes showed a

marked difference in expression. These findings are

useful to assess the ability of serotypes in inducing

the host immune response by demonstrating the

variations in patterns of cytokines expression. In line

with previous research showing that cytokine IFN-

α2 has viral inhibition characteristic, our findings

suggest that IFN-α2 may contribute to DENV

inhibition. Further studies are needed to assess the

roles of infecting DENV serotype in disease

severity.

ACKNOWLEDGMENT

We thank the member of Dengue Laboratory

Eijkman Institute for support and suggestion.

REFERENCES

Abbas, A. K., Lichtman, A., & Pillai, S. (2012).

Cellular and Molecular Immunology. 7th ed.

United States of America: Elsevier.

Chen, Y., & Wang, S. (2002). Activation of

Terminally Differentiated Human Monocytes /

Macrophages by Dengue Virus : Productive

Infection , Hierarchical Production of Innate

Cytokines and Chemokines , and the Synergistic

Effect of Lipopolysaccharide †, 76(19), 9877–

9887. https://doi.org/10.1128/JVI.76.19.9877

Clyde, K., Kyle, J. L., & Harris, E. (2006). Recent

Advances in Deciphering Viral and Host

Determinants of Dengue Virus Replication and

Pathogenesis , 80(23), 11418–11431.

https://doi.org/10.1128/JVI.01257-06

Diamond, M. S., & Harris, E. (2001). Interferon

Inhibits Dengue Virus Infection by Preventing

Translation of Viral RNA through a PKR-

Independent Mechanism, 311, 297–311.

https://doi.org/10.1006/viro.2001.1114

Green, S., & Rothman, A. (2006).

Immunopathological mechanisms in dengue and

dengue hemorrhagic fever, 19, 429–436.

Gubler, D. J. (1998). Dengue and Dengue

Hemorrhagic Fever, 11(3), 480–496.

Halstead, S. B. (2008). Dengue. Imperial College

Press.

Jia, J.-B., Wang, W.-Q., Sun, H.-C., Zhu, X.-D., Liu,

L., Zhuang, P.-Y., Tang, Z.-Y. (2010). High

Expression of Macrophage Colony-Stimulating

Factor-1 Receptor in Peritumoral Liver Tissue

Is Associated with Poor Outcome in

Hepatocellular Carcinoma After Curative

Resection. The Oncologist, 15(7), 732–743.

https://doi.org/10.1634/theoncologist.2009-0170

Kuno, G., Chang, G. J., Tsuchiya, K. R., &

Karabatsos, N. (2014). Phylogeny of the Genus

Flavivirus Phylogeny of the Genus Flavivirus,

(February 1998).

Lambeth, C. R., White, L. J., Johnston, R. E., &

Silva, A. M. De. (2005). Flow Cytometry-Based

Assay for Titrating Dengue Virus, 43(7), 3267–

3272. https://doi.org/10.1128/JCM.43.7.3267

Lindenbach, B., & Rice, C. (2003). Molecular

biology of flaviviruses. Adv. Virus Res, 59, 23–

61.

Sasmono, R. T., & Hume, D. A. (2004). The Biology

of Macrophages in the Innate Immune Response

to Infection. Washington DC: ASM press.

Sun, P., & Kochel, T. J. (2013). The Battle between

Infection and Host Immune Responses of

Dengue Virus and Its Implication in Dengue

Disease Pathogenesis, 2013.

Www.ReaderFit.com. (n.d.). MasterPlex QT

readerfit for four parameters logistic (4PL) and

five parameters logistic (5PL) nonlinear

regression models with many options.

Yohan, B., Kendarsari, R. I., Mutia, K.,

Bowolaksono, A., & Harahap, A. R. (2014).

Growth characteristics and cytokine /

chemokine induction profiles of dengue viruses

in various cell lines, 36, 20–27.

https://doi.org/10.4149/av

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SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences