Am. J. Trop. Med. Hyg., 102(5), 2020, pp.

943–950
doi:10.4269/ajtmh.19-0487
Copyright © 2020 by The American Society of Tropical Medicine and Hygiene

Cytokine Expression in Dengue Fever and Dengue Hemorrhagic Fever Patients with Bleeding and
Severe Hepatitis
Hisham Ahmed Imad,1 Weerapong Phumratanaprapin,1* Benjaluck Phonrat,1 Kesinee Chotivanich,1
Prakaykaew Charunwatthana,1 Sant Muangnoicharoen,1 Srisin Khusmith,2 Terapong Tantawichien,3 Juthamas Phadungsombat,4
Emi Nakayama,4,5 Eiji Konishi,6 and Tatsuo Shioda4,5
1
Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 2Department of Microbiology and
Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 3Division of Infectious Diseases, Department of Medicine,
Chulalongkorn University, Bangkok, Thailand; 4Mahidol-Osaka Center for Infectious Diseases, Faculty of Tropical Medicine, Mahidol University,
Bangkok, Thailand; 5Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; 6BIKEN Endowed Department of Dengue Vaccine
Development, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Abstract. Dengue is the most common mosquito-borne flaviviral infection in the world today. Several factors con-
tribute and act synergistically to cause severe infection. One of these is dysregulated host immunological mediators that
cause transient pathophysiology during infection. These mediators act on the endothelium to increase vascular per-
meability, which leads to plasma leakage compromising hemodynamics and coagulopathy. We conducted a prospective
study to explore the expression of pro- and anti-inflammatory cytokines and how they relate to clinical dengue mani-
festations, by assessing their dynamics through acute dengue infection in adults admitted to the Hospital for Tropical
Diseases, Bangkok, Thailand. We performed cytokine analysis at three phases of infection for 96 hospitalized adults
together with serotyping of confirmed dengue infection during the outbreaks of 2015 and 2016. The serum concentrations
of seven cytokines (interleukin [IL]-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha, and interferon gamma) were
measured in duplicate using a commercial kit (Bio-Plex Human Cytokine Assay). In this study, the cytokine profile was
suggestive of a T-helper 2 response. Most patients had secondary infection, and the levels of viremia were higher in
patients with plasma leakage than those without plasma leakage. In addition, we observed that bleeding and hepatitis
were associated with significantly higher levels of IL-8 during the early phases of infection. Furthermore, IL-6 levels in the
early phase of infection were also elevated in bleeding patients with plasma leakage. These results suggest that IL-6 and
IL-8 may act in synergy to cause bleeding in patients with plasma leakage.

INTRODUCTION The endothelium plays a pivotal role in the pathophysiology

of dengue infection.6 Activation of the endothelium by the
The dengue virus is a flavivirus of the family Flaviviridae and expression of interleukin (IL)-6, IL-8, and tumor necrosis factor
has been characterized into four serotypes (Dengue viruses 1 alpha (TNFα) have been previously described.3,10 This acti-
to 4). Transmitted by Aedes species, dengue infection is the vation of the endothelial system not only contributes to the
most commonly occurring arthropod-borne viral infection bleeding, which is common in dengue fever (DF) and DHF,11
globally. Several factors such as globalization, urbanization, but also to liver damage, leading to hepatitis complicating
and lack of effective vector control have facilitated the spread dengue.12 Bleeding manifestations are seen in 20–60%13 of
of this disease beyond the subtropics.1 cases, and hepatitis with a 100-fold increase in transaminase
It is estimated that 50–400 million infections occur annually. was reported in 7% of cases.14 Thrombocytopenia with impair-
In Thailand, the first epidemic of dengue hemorrhagic fever ment of the coagulation system is associated with bleeding in
(DHF), a severe form of dengue virus infection, occurred in dengue,15 and multiple factors contribute to the development of
1958. Multiple outbreaks with greater epidemic potential have hepatitis. These factors include hypoxic injury caused by de-
occurred since then, with a range of 15,000–105,000 cases creased perfusion, direct damage by the virus, and dysregulated
occurring each year in Thailand.2 The infection causes a sys- immune-mediated injury in response to dengue virus. Interleukin-
temic viral infection, and complications are associated with the 8 is expressed by hepatocytes during dengue infection,16 with
pathogenic role of endothelial cells.3–5 Cytokines6,7 increase reports of cytokines correlating with elevated transaminase lev-
vascular permeability and hemorrhage during dengue infection. els17 and the expression of TNFα following the apoptosis of
These molecules are proteinaceous and are secreted during hepatocytes in dengue infection.18 Others reported a correlation
innate and adaptive immunological responses, acting as in- between severity and IL-6 and TNFα levels.19 However, only
flammatory mediators or modulatory molecules during dengue limited data are available for the relationship between the in-
infection. Viral infection activates the innate immune response creased expressions of cytokines with hepatitis and bleeding. In
to express pro-inflammatory cytokines that recruit and activate the present study, we investigated the expressions of cytokines
cells involved in inflammation and the induction of adaptive during dengue infection with bleeding and hepatitis and assessed
immunity. Cytokines also help distinguish between T-helper 1 the degree of viremia with plasma leakage.
and T-helper 2 (Th2) cells, and their expression in dengue has
been reviewed extensively.8 The dengue virus is tropic and
MATERIALS AND METHODS
replicates in human monocytes, macrophages, and hepato-
cytes, which are known to express cytokines.9 This was a prospective study of hospitalized adults with
symptomatic dengue infection at the Hospital for Tropical
* Address correspondence to Weerapong Phumratanaprapin, Faculty
Diseases, Bangkok, Thailand. Ethical approval to conduct this
of Tropical Medicine, 420/6 Rajchawithi Rd., Bangkok 10400, Thai- study was obtained from the Ethics Committee, Faculty of
land. E-mail: [email protected] Tropical Medicine, Mahidol University.

943
944 IMAD AND OTHERS

TABLE 1
Demographic data, clinical findings, and laboratory parameters in dengue infection
Parameters Dengue fever = 59, n (%) Dengue hemorrhagic fever = 37, n (%) P-value

Females 20 (33.89) 18 (48.64) 0.2

Males 39 (66.10) 19 (51.35)
Age (years), mean ± SD 31 ± 11 33 ± 11 0.44
Primary infection, age mean ± SD 25 ± 11 0 NA
Secondary infection, age mean ± SD 32 ± 11 33 ± 11 0.11
Duration of illness, mean ± SD 6.95 ± 0.9 7.8 ± 0.8 0.01
Fever 59 (100) 37 (100) NA
Headache 53 (89.83) 37 (100) 0.02
Myalgia/arthralgia 43 (72.88) 34 (91.89) 0.03
Nausea/vomiting 29 (49.15) 20 (54.05) 0.75
Lymphadenopathy 21 (35.59) 22 (59.45) 0.02
Hepatomegaly 18 (30.50) 15 (40.50) 0.31
Bleeding 20 (32.78) 20 (54.05) 0.05
Mean ± SD Mean ± SD P-value

Hemoglobin (g/dL) 14.3 ± 1.3 14.2 ± 1.9 0.53

Hematocrit (%) 42.4 ± 3.8 41.9 ± 5.3 0.54
Leukocytes (103/μL) 3.6 ± 1.7 3.4 ± 1.5 0.5
Neutrophils (%) 50.7 ± 16.5 51.3 ± 16.8 0.85
Lymphocytes (%) 26.7 ± 12.5 25.3 ± 11.6 0.58
Platelets (103/μL) 81 ± 46 63 ± 43 0.06
Atypical lymphocytes (%) 14 ± 10 13 ± 12 0.75
Sodium (mmol/L) 136 ± 3 134 ± 2 0.06
Potassium (mmol/L) 4.2 ± 3.9 3.7 ± 0.4 0.85
Bicarbonate (mmol/L) 23.7 ± 2.3 23.9 ± 2.9 0.71
Blood urea nitrogen (mg/dL) 10.1 ± 5.0 11.2 ± 4.6 0.23
Creatinine (mg/dL) 0.9 ± 0.3 0.9 ± 0.3 0.9
Total protein (g/dL) 6.9 ± 0.5 8.9 ± 12.7 0.25
Albumin (g/dL) 3.9 ± 0.3 3.9 ± 0.5 0.53
Total bilirubin (mg/dL) 0.5 ± 0.2 0.6 ± 0.5 0.14
Direct bilirubin (mg/dL) 0.19 ± 0.1 0.3 ± 0.3 0.16
Aspartate transaminase (U/L) 224 ± 399 308 ± 528 0.24
Alanine transaminase (U/L) 127 ± 192 122 ± 184 0.93
NA = not applicable. The data in the table represent the demographic data and clinical findings in DF and DHF. The clinical findings are represented as the actual number and percentage. The
laboratory parameters are represented as the median ± SD. The chi-square test was used to determine the differences in clinical findings in both groups. The Mann–Whitney U-test was used to
determine the P-value between two groups using the median value. Bold indicates statistically significant P-values.

During the dengue outbreak in 2015 and 2016, we recruited infection included bleeding and severe hepatitis (aspartate
adults who presented within 5 days after developing symp- aminotransferase/alanine aminotransferase > 400 U/L).
toms and with a positive non structural protein 1 antigen or a Cytokine measurement. Blood specimens were collected
positive anti-dengue IgM test into our study. We excluded any for cytokine analysis during the three phases of infection. A
vulnerable groups and participants who had received a commercial assay (Bio-Plex Human Cytokine Assay; Bio-Rad
transfusion of blood products during the study period. Be- Inc., Hercules CA) was performed to detect the levels of IL-2,
cause this was an observational study, all consenting partic- IL-4, IL-6, IL-8, IL-10, interferon gamma (IFNγ), and TNFα. In
ipants meeting the study criteria were recruited during the 2- brief, sera obtained from participants during the three phases
year study period. The participants were grouped based on were mixed with beads coated with antibodies to cytokines
the WHO 1997 classification, and a chest X-ray was per- and a unique fluorescent intensity. Subsequently, the mix-
formed to detect pleural effusion. Complications in dengue tures were incubated with biotinylated anti-cytokine anti-
bodies. Finally, phycoerythrin-conjugated streptavidin was
added, and the fluorescent signals were detected using a
multiplex array reader (Bio-Plex 200 System, Bio-Rad Inc.,
Hercules, CA). Raw data were initially measured as the rel-
ative fluorescence intensity and then converted to cytokine
concentration based on a standard curve generated from
the reference concentrations.
The acute phase specimen corresponded to blood col-
lected within 5 days following the development of symptoms
of dengue infection. The defervescence specimens were
obtained 8 hours after the participants had remained afebrile
with a recorded body temperature of 37.7°C or lower. The
convalescence specimens were obtained during the follow-up
within or later than 2 weeks after the onset of symptoms. The
standard provided by the manufacturer was used as a control,
FIGURE 1. Cytokine expression during the three phases of dengue and the observed readings were determined as the detection
infection. limits in pg/mL.
 CYTOKINE EXPRESSION IN PATIENTS WITH DENGUE COMPLICATIONS 945

FIGURE 2. Cytokine profile during the three phases of infection dengue fever vs. dengue hemorrhagic fever.

Quantification of RNA and sequence of infection. To when only anti-dengue IgM was positive together with the
determine the dengue serotype, a commercially available appearance of the control band. Secondary infections were
dengue subtyping multiplex kit by the Genesig company determined when both anti-dengue IgM and IgG were posi-
(Chandler’s Ford, United Kingdom) was used as per the tive, including the appearance of the control band.
manufacturer’s instruction.20 Then, RNA quantification was Statistical analysis. The Mann–Whitney U-test was used
performed using a One-Step SYBR® Prime Script RT-PCR Kit to assess differences in the cytokine levels between DF and
II, Takara Bio Inc., (Kusatsu, Japan); cDNA synthesis from RNA DHF. To establish the correlation between cytokine levels
was performed using reverse transcriptase Prime Script and clinical parameters/findings, a correlation matrix was
RTase; and polymerase chain reaction amplification was applied. Results are given as the correlation coefficient; r
performed by TaKaRa Ex Taq HS. This technique was pre- (range, from −1 to +1). A two-tailed P-value < 0.05 was
viously described.21 For dengue serology, commercially considered significant for all tests performed. One-way
available dengue IgM and IgG immunochromatography kits analysis of variance was used to evaluate differences
from Panbio Dengue Duo Cassette Abbott Inc., (Chicago, IL) among cytokine levels in the different groups. All statistical
were used in accordance with the manufacturer’s protocol. A analyses were performed using Microsoft Excel, SPSS
serum aliquot representing the day of defervescence was version 18, and GraphPad Prism 7 for Windows, version
used in these test kits. Primary infections were diagnosed 7.30 (San Diego, CA).

RESULTS
In this study, 96 hospitalized adult patients with a con-
firmed dengue infection were recruited during the outbreak
in 2015 and 2016. The demographic and clinical data as
well as laboratory parameters of the study population and
clinical details are shown in Table 1. Our results indicate that
symptoms of systemic illness including headache (P = 0.02),
myalgia (P = 0.03), lymphadenopathy (P = 0.02), and bleeding
(P = 0.05) were more common in DHF. A longer duration of
hospitalization was observed in DHF cases than DF cases
(P = 0.01). The hematological profile was typical for dengue
infection, with leukopenia and thrombocytopenia (platelet
count < 100 × 103/μL) being obvious findings in our subjects.
From the analysis of liver function tests, transaminitis
FIGURE 3. Viral load during the acute phase in dengue fever and was observed in 11% of subjects. From the analysis of
dengue hemorrhagic fever. liver function tests, transaminitis was observed in 11% of
946 IMAD AND OTHERS

FIGURE 4. Cytokine profile for bleeding during the three phases of infection.

subjects. The mean ± SD levels of aspartate aminotransfer- Cytokines profile with bleeding. Interleukin-8 expression
ase and alanine aminotransferase for DF were 224 ± 399 U/L was significantly elevated during the acute phase and at de-
and 127 ± 192 U/L, and for DHF were 308 ± 528 U/L and 122 ± fervescence. The IL-8 median value (interquartile range) was
184 U/L, respectively. 27.65 (range, 17.42–37.42) pg/mL for the group with bleeding
Cytokine expressions during dengue infection. Figure 1 during the acute phase and 17.98 (range, 13.06–25.95) pg/mL
shows the trend of the expressions of IL-4, IL-6, IL-8, IL-10, for the group without bleeding (P = 0.00, Figure 4). Similarly, at
TNFα, and IFNγ during the three phases of dengue infection. defervescence, the IL-8 median value (IQR) was 21.76 (range,
The expressed cytokines peaked during the acute phase. 16.86–30.14) pg/mL for the group with bleeding and 16.19
Interleukin-10 was most expressed during the acute phase (range, 11.18–25.30) pg/mL for the group without bleeding
followed by TNFα, IL-8, IL-6, IFNγ, and IL-4. Interleukin-2 was (P = 0.01, Figure 4). These data represent the detectable levels
not detectable. At defervescence, TNFα was the most of cytokines in 96 subjects during the three phases of dengue
expressed, and during convalescence, IL-4 had diminished to infection. The cytokine levels are shown as the median and IQR
low levels and was undetectable in most cases. We in- in pg/mL. The Mann–Whitney U-test was used to determine the
vestigated the cytokine profile by grouping our subjects P value between two groups using the median value.
based on their clinical classification. These included 59 Similarly, the levels of IL-8 were significantly higher in DHF
subjects with DF and 37 with DHF, of whom 17 were of DHF grade II–IV patients than DHF grade I patients (Figure 5). In
grade I, 19 were of DHF grade II, and a single participant was addition, IL-6 levels during the acute phase were significantly
of DHF grade IV as per the WHO 1997 classification.22 Most elevated in DHF grade II–IV compared with DHF grade I
patients had secondary dengue infection and had an un- (Figure 5). The levels of IL-8 were also significantly higher in DF
eventful milder clinical course during hospitalization. Com- with bleeding than DF without bleeding during the acute
plications identified in our study were shock in a single phase of infection (Figure 6).
subject, and bleeding in 42% and hepatitis in 11% of study Cytokine profile with hepatitis. In the cytokine profile
subjects. The bleeding manifestations were mucosal bleeding generated for severe hepatitis (AST/AST > 400 U/L), we ob-
in 31.6%, menorrhagia in 27.5%, epistaxis in 5.1%, melena in served that in addition to IL-8 being significantly elevated
two subjects, and hematemesis in a single subject. Our results during the acute phase (P = 0.00) and at defervescence (P =
revealed no significant differences in cytokine levels during the 0.01), TNFα was significantly elevated during the acute phase
three phases of infection between subjects with and without (P = 0.01, Figure 7).
plasma leakage (Figure 2). Nevertheless, we did observe higher The levels of IL-8 were significantly higher during the acute
viremia in those with plasma leakage (Figure 3). phase and at defervescence in patients with an increase in
 CYTOKINE EXPRESSION IN PATIENTS WITH DENGUE COMPLICATIONS 947

FIGURE 5. Cytokine profile for bleeding in dengue hemorrhagic fever during the three phases of infection.

liver enzymes (AST/ALT ³ 1,000 U/L). The median value of response was supported by our undetectable levels of se-
IL-8 was 30.28 pg/mL (IQR, 25.18–86.58) in subjects with creted IL-2, in addition to the very low levels of IFNγ, with an
AST/ALT ³ 1,000 U/L, which was significantly elevated increased expression of IL-10. This trend of cytokine ex-
during the acute phase (P = 0.03). At defervescence, in pression also suggests immunoregulatory effects consis-
subjects with AST/ALT ³ 1,000 U/L, the median IL-8 value tent with secondary infection.27 Memory B cells are primed to
was 32.29 pg/mL (IQR, 23.80–57.67), which was significantly produce antibodies by the expression of IL-10,28 and with
elevated compared with subjects with AST/ALT < 1,000 U/L its proteolytic properties, IL-10 inhibited IFNγ expression
(P = 0.01). and deregulated the expressions of IL-6, IL-8, and TNFα in
our study subjects and as previously described.29 Unlike
DISCUSSION previous reports that showed an association of IL-4 and
disease severity,30 our study failed to reveal such findings
In the present study, the levels of cytokines during probably because IL-4 was detected in only a few cases
dengue infection were measured, and we analyzed the partly because of the small number of study subjects.
profiled cytokine patterns to determine the immuno- We demonstrated endothelial activation by the detection
pathophysiological events that occur during dengue in- of IL-6, IL-8, and TNFα as previously described.3,10 This
fection leading to complications, such as bleeding and activation of the endothelial system not only contributes to
hepatitis. To the best of our knowledge, few studies have bleeding, which is common in DF and DHF,11 but also in-
performed cytokine analysis during the three phases of in- volved in liver damage, leading to hepatitis complicating
fection. In particular, we designed our study to measure the dengue.12 Our results were consistent with previous stud-
cytokine levels at defervescence and identified elevated ies in respect to higher viremia in DHF,31 and we observed
cytokine levels with complications. In viral infections, cyto- more bleeding and symptoms of systemic illness in DHF,
kines can interfere with the interferon signaling pathway,23 reflecting the degree of severity when compared with DF.
which is the host’s primary antiviral response to an acute However, we did not find any association between viremia
infection.24 Our study subjects had a diminished IFNγ re- and bleeding as previously reported.32 Interleukin 8 is
sponse, which might have resulted from a secondary dengue expressed by hepatocytes in dengue infection16 and was
infection, where the Th1 response is prominent in primary associated with disease severity. 17 Similar to a previous
infection.25 This phenomenon was previously demonstrated study,33 our results regarding IL-8 levels could not distin-
in vitro, where dengue virus–infected cells failed to express guish DF from DHF. However, we demonstrated signifi-
or demonstrated a reduced expression of IFNγ on secondary cantly elevated levels of IL-8 during the acute phase and at
infection.26 The cytokine response reflecting a T-helper 2 defervescence in patients with bleeding and in those with
948 IMAD AND OTHERS

FIGURE 6. Cytokine profile for bleeding in dengue fever during the three phases of infection.

hepatitis. We also showed an association of an increased neutrophils to the liver, causing liver injury and impaired
IL-8 level with bleeding during both phases. Significantly, coagulation. Our results show increased levels of neutro-
elevated levels of IL-6 were observed during the acute phils during the two phases of infection with hepatitis. This
phase in patients with bleeding, with a background of increase in neutrophils with hepatitis might be a result of
plasma leakage, whereas others reported elevated levels of the elevated levels of IL-8 with hepatitis. Although a positive
IL-6 in DHF34 and bleeding.32 Other studies reported TNFα correlation of IFNγ and transaminase was reported,34 we
was not detectable or only detected in 30% of cases of observed no similar pattern of correlations. Nevertheless,
dengue infection.35 In our study, we detected TNFα ex- there were positive correlations between IL-6 and IL-8 and
pression in all our subjects. We highlighted other factors between IL-6 and IFNγ during the acute phase, and at de-
that contribute to bleeding in dengue, such as thrombo- fervescence IL-8 and TNFα were positively correlated, in-
cytopenia, 36 which can worsen bleeding in dengue dicating these cytokines may act in synergy. In summary,
infection. the immunoregulatory responses observed may be impli-
The liver impairment, which occurs in dengue infection,14 cated in the bleeding and hepatitis observed in dengue
impacts bleeding with a worsening of coagulation. Our results patients.
demonstrated that IL-8 and TNFα expressions were signifi-
cantly higher in patients with transaminases elevated 10-fold. CONCLUSION
Furthermore, we observed that IL-8 levels were significantly
elevated with a 100-fold increase in transaminase levels dur- In this study, higher viremia in DHF with a T-helper 2 re-
ing both phases. sponse determined by profiling the cytokine expression was
The properties of IL-8 contribute to platelet activation and demonstrated. The expression of IL-8 was significantly ele-
endothelial permeability to cause thrombocytopenia,34 vated during the acute phase in patients with bleeding in both
which worsens bleeding in dengue infection. We also ob- DF and DHF, with a further increase in IL-8 levels in DHF at
served that the platelet counts during both phases were defervescence. IL-6 acts in synergy with IL-8 for bleeding with
significantly decreased in patients with bleeding. These plasma leakage during the acute phase. Significantly, higher
elevated levels of IL-8 observed in our study might facilitate levels of TNFα, IL-6, and IL-8 were observed in cases of severe
the events preceding bleeding during dengue infection. A hepatitis.
similar observation of elevated levels of IL-8 with throm- Limitations. There were some limitations in this study in-
bocytopenia in dengue was described.34 During dengue cluding the small number of total study subjects and those
infection, infected hepatocytes express IL-8, attracting with severe disease.
 CYTOKINE EXPRESSION IN PATIENTS WITH DENGUE COMPLICATIONS 949

FIGURE 7. Cytokine profile during the three phases of infection for severe hepatitis (AST/ALT ³ 400 U/L).

Received June 28, 2019. Accepted for publication January 19, 2020. osaka-u.ac.jp and [email protected]. Eiji Konishi, BIKEN
Endowed Department of Dengue Vaccine Development, Faculty of
Published online March 2, 2020. Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mail:
Acknowledgments: We thank the staff at Bangkok Hospital for Trop- [email protected].
ical Diseases for the support provided in this study and Jaranit
This is an open-access article distributed under the terms of the
Kaewkungwal and Pimphen Choroen for their assistance with the Creative Commons Attribution (CC-BY) License, which permits un-
statistical analysis. We also acknowledge Edanz Group (www. restricted use, distribution, and reproduction in any medium, provided
edanzediting.com/ac) for editing a draft of this manuscript. the original author and source are credited.
Financial support: This study was funded by 2015 Dean MOCID/
BIKEN scholarships awarded by Professor Yaowalak Sukthana for
PhD research. The BIKEN Endowed Department of Dengue Vaccine REFERENCES
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