Dengue virus and Japanese encephalitis virus infection of the central nervous system share similar profiles of cytokine accumulation in cerebrospinal fluid

Introduction

Each year 50 million people worldwide are infected with dengue virus (DENV) [1], which consists of four antigenically distinct serotypes (DENV-1, 2, 3, and 4) [2]. DENV infection causes dengue fever, dengue haemorrhagic fever (DHF), and dengue encephalitis in both children and adults [3, 4]. Dengue fever has recently become one of the most challenging public health problems in China. In June 2014, the most severe outbreak of dengue fever was reported in Guangzhou [5].
The pathophysiology of DF/DHF in humans likely involves the interplay of host and viral factors that influence disease severity and clinical outcome [6]. Previous studies have suggested that cytokines secreted by dengue-infected monocytes/macrophages influence DHF physiopathology. Increased levels of interferon  (IFN-), tumor necrosis factor  (TNF-), interleukin 6 (IL-6), and IL-8 were detected in adult patients with dengue fever [7, 8], and elevated levels of IL-6, IL-10, IFN-, macrophage migration inhibitory factor (MIF), and chemokine C-C motif ligand 4 (CCL-4) were associated with severity of dengue infection [9-11].
Some animal experiments have implicated direct neurovirulence of DENV infection in dengue encephalitis. The blood-brain barrier can be damaged by cytokines such as TNF-y and IFN- [4, 12]. Other experiments indicated that elevated IFN-, TNF-, MIF, IL-8, IL-6, RANTES, cyclooxygenase 2 (Cox-2), IL-1, and MCP-1 levels were associated with poor outcomes in Japanese encephalitis (JE) patients [13-18].
However, the cytokine profiles of the cerebrospinal fluid (CSF) of patients with dengue encephalitis and JE remain to be established. This study aimed to investigate cytokine profiles in the cerebrospinal fluid (CSF) of children hospitalised with encephalitis-like syndrome and diagnosed with dengue infection of the central nervous system (CNS).

Material and methods

Patients

Between May 2014 and April 2015, 183 children (≤ 16 years of age) with encephalitis-like symptoms [19], admitted to The Children’s Hospital of Chenzhou, Hunan Province, China, were included into this study. Blood and paired CSF samples were collected upon admission. Control blood samples were collected from 26 age-matched children who underwent surgery without current or recurrent infectious diseases. The study protocol was approved by the Ethics Committee of the Hospital, and written consent was obtained from the parents of participants.

Virus detection

Dengue virus or Japanese encephalitis virus infection was confirmed by DENV IgM enzyme-linked immunosorbent assay (ELISA) (Virion/Serion, Germany) or JEV IgM ELISA assay (Boyan, Shanghai). The level of DENV RNA in each CSF sample from anti-DENV IgM-positive patients was tested by PCR, as described elsewhere [20]. Children with DENV and JEV co-infections were excluded from subsequent data analysis.

Cytokine measurement

We measured the level of IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN-, MCP-1, MIP-1, and TNF- in the sera of healthy controls, and in the serum and CSF of DENV and JEV infected patients by Bio-Plex assay (BIORAD, USA).

Statistical analyses

The significance of differences between continuous variables was evaluated by Student’s t test or Mann-Whitney U test, and the significance of differences between categorical variables was determined by 2 test. Statistical analyses were carried out with SPSS 18.0 software. The result was considered significant when two-sided p value was < 0.05.

Results and discussion

Clinical characteristics of children with DENV and JEV infection of CNS
Of 183 hospitalised children with acute viral encephalitis-like symptoms, 29 (15.85%) were diagnosed with DENV infection. The most common symptoms were vomiting (44.8%), headache (51.7%), and fever (72.4%), their median age was 60 months, and 16 (55.2%) were male (Table 1).
In addition, 26 (14.21%) were diagnosed with JEV infection of the CNS. The most common symptoms were vomiting (42.3%), headache (42.3%), and fever (76.9%), their median age was 24 months, and 18 (69.2%) were male (Table 1).
Of the children hospitalised with acute viral encephalitis-like symptoms in this cohort, 15.85% and 14.21% were diagnosed with DENV and JEV, respectively. The higher incidence of DENV infection may reflect the spread of DENV infection following the June 2014 dengue fever outbreak in Guangdong province, which is adjacent to Chenzhou. The incidence of JEV infection was similar to previously published studies [21].

Cytokine profiles in patients with dengue virus and Japanese encephalitis virus infection of CNS

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the serum of patients with dengue encephalitis was significantly lower than that in JE patients (Fig. 1A), and the levels of IL-6, IL-8, MCP-1, and other cytokines did not differ significantly between the two groups (Fig. 1B-D).
The levels of GM-CSF, IL-6, and MCP-1 in the CSF of patients with dengue encephalitis and JE were significantly higher than in matched serum samples (Fig. 1A-C), whereas the levels of IL-8 and other 13 cytokines (see in supplemental material 1 and 2) in the CSF did not differ significantly from matched serum samples from dengue encephalitis patients (Fig. 1D).
The CSF contained similar levels of cytokines in both dengue encephalitis and JE patients (Fig. 1A-D).
Interestingly, serum levels of GM-CSF were significantly lower in dengue encephalitis patients than in JE patients, but GM-CSF accumulated in CSF of patients with dengue encephalitis, exceeding levels in matched serum samples. GM-CSF has been reported to modulate immune responses in vivo [21], but the precise biological functions of elevated GM-CSF in the CSF warrants further investigation.
Interleukin 6 and MCP-1 accumulated in the CSF of both dengue encephalitis and JE patients, exceeding levels in matched serum samples, and IL-8 accumulated in the CSF of JE patients. Interleukin 6 is neurotrophic and neuroprotective and can increase blood-brain barrier permeability [17]. MCP-1 is the major monocyte chemoattractant produced during the inflammatory response in CNS infections. Interleukin 8 attracts and activates neutrophil leukocytes and is associated with blood-brain barrier breakdown [22]. Therefore, cytokine accumulation in the CSF may contribute to the immune response to dengue encephalitis and JE. The identification of similar CSF cytokine profiles in both dengue encephalitis and JE patients suggests that the cytokine response is non-specific and both dengue encephalitis and JE may share similar mechanisms of pathogenesis.

Concluding remarks

The results of the present study provide that DENV and JEV infection of the CNS was common among children, and induced expression of multiple cytokines. Several cytokines accumulated in the CSF, which may augment inflammation and other pathological changes in the infected brains. Effective treatment of dengue encephalitis and JE should take account of the high levels of inflammatory cytokines in affected brains, and aim to mitigate brain injury.

The authors thank all family members for enrolment in this study. The authors are appreciative of Department of Paediatrics of The First People’s Hospital of Chenzhou for collecting samples. This work was supported by The First People’s Hospital of Chenzhou, University of South China (Grant No. 2013–007, 2013–001, and 2014–003) The authors declare no conflict of interest.

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