Microbiol Immunol 2015; 59: 152–159 doi: 10.1111/1348-0421.12227
ORIGINAL ARTICLE
Cerebrospinal fluid Th1/Th2 cytokine profiles in children with enterovirus 71-associated meningoencephalitis Huajun Li1,2, Shuxian Li1, Jianfeng Zheng3, Chunyan Cai1,4, Bin Ye5, Jun Yang6,7 and Zhimin Chen1 1
Department of Pulmonology, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003,, 2Department of Pediatrics, Shaoxing People's Hospital, Shaoxing, Zhejiang 312000, 3Department of Pediatrics, Maternal and Child Care Service Centre, Cixi, Ningbo, Zhejiang 315300, 4Department of Infectious Disease, Hangzhou Children's Hospital, Hangzhou, Zhejiang 310014, 5 Department of Pediatrics, Taizhou Central Hospital, Jiaojiang, Taizhou, Zhejiang 318000, 6Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang 310013 and 7Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310016, China
ABSTRACT Enterovirus 71 (EV71) infection can cause severe neurological complications including meningoencephalitis (ME) in some patients with hand, foot and mouth disease (HFMD). However, to date no studies have reported changes in cytokine concentrations and their correlations with clinical variables in patients with ME following EV71 infection. In this study, responses of Th1/Th2 cytokine, including IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g, in cerebrospinal fluid (CSF) from patients with EV71related HFMD with ME and patients with febrile convulsions (FC) were analyzed using cytometric bead array technology. It was found that CSF IL-6 and IFN-g concentrations were significantly higher in patients with EV71-related ME than in those with FC. Additionally, both CSF IL-6 and IFN-g concentrations were correlated with CSF cytology, fever duration and duration of hospital stay. More interestingly, a positive correlation between CSF IL-6 and IFN-g concentrations was observed. Finally, receiver operating characteristic analysis revealed that when a cutoff value of 9.40 pg/mL was set for IL-6, the sensitivity and specificity were 84.5% and 85.5%, respectively, for discriminating EV71-related ME from FC. In conclusion, IL-6 and IFN-g may be associated with EV71-induced neuropathology. Key words
cytokines, enterovirus 71, hand, foot and mouth disease, meningoencephalitis.
Enterovirus 71 is an important causative pathogen of HFMD in children worldwide (1). Although HFMD is usually a mild disease, some patients develop neurological complications, such as aseptic meningitis, meningoencephalitis (ME), brainstem encephalitis, encephalomyelitis, acute flaccid paralysis and PE, especially when they have EV71 infection (2). In addition, most deaths from
EV71-induced HFMD occur within 2–3 days of onset of this acute disease and are attributable to fulminant PE (3– 4). In China, sporadic epidemics or outbreaks of EV71 infection have occurred since 1998 (5). In particular, in 2008, a large, unprecedented HFMD outbreak caused by EV71 infection occurred in Fuyang city, Anhui province of China and resulted in a high mortality rate (1).
Huajun Li and Shuxian Li contributed equally to this work. Correspondence Zhimin Chen, Department of Pediatric Pulmonology, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China. Tel: þ86 571 8887 3741; fax: þ86 571 8703 3296; email: [email protected] Additional correspondence: Jun Yang, Department of Toxicology, Hangzhou Normal University, Hangzhou, Zhejiang 310016, China. Tel/fax: þ 86 571 8820 8141; email: [email protected] Received 5 July 2014; revised 2 January 2015; accepted 13 January 2015. List of Abbreviations: CBA, cytometric bead array; CNS, central nervous system; CSF, cerebrospinal fluid; EV71, enterovirus 71; FC, febrile convulsion; HFMD, hand, foot and mouth disease; ME, meningoencephalitis; miRNA, microRNA; PE, neurogenic pulmonary edema; ROC, receiver operating characteristic; WBC, white blood cell count.
152
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
CSF cytokines in EV71-associated ME
However, there is currently no approved antiviral drug or effective vaccination against EV71. Thus, in order to develop future therapeutic modalities, a better understanding of the pathogenesis of EV71 infection is of great importance. Cytokines are low molecular weight proteins that mediate inflammation, immunity, cell growth, differentiation, migration and repair. Traditionally, cytokines can be divided into at least two functional subgroups: Th1 cytokines, including IL-2, IFN-g and TNF-a, which are mainly secreted by Th1 cells and considered as proinflammatory cytokines; and Th2 cytokines, such as IL4, IL-5 and IL-13, which are mainly derived from Th2 cells and exert anti-inflammatory effects (6). However, there are exceptions to this simple classification. For example, IL-6 has both pro- and anti-inflammatory functions, whereas IL-10, which is classified as an antiinflammatory cytokine, can be produced by either Th1 or Th2 cells (7). Generally, cytokines are regarded as humoral factors that regulate inflammatory events. For instance, TNF-a, IFN-g and IL-4 increase endothelial cell adhesiveness for T lymphocytes and act in combination at sites of inflammation to promote lymphocyte extravasation (8). TNF-a and IFN-g also have the potential to synergize with HIV-1 to induce CXCL10 expression through the JAK/STAT and mitogen-activated protein kinase pathways in astrocytes, underlining the molecular mechanisms for their involvement in HIV-related encephalitis (9). Similarly, in the past few years, the importance of cytokines in the pathogenesis of EV71 infection, especially in EV71related PE, has been gradually recognized (10–11). For example, during systemic inflammatory responses, EV71 patients with PE show significantly increased blood concentrations of IFN-g, IL-10 and IL-13, but not of TNF-a, IL-2 and IL-4 (10). Additionally, in the central nervous system (CNS), cerebrospinal fluid (CSF) concentrations of IL-6 and IFN-g, which can increase pulmonary vascular permeability, are dramatically higher in children with PE than in those with uncomplicated brainstem encephalitis (11). Together, these data indicate that cytokines in the CNS or blood may be involved in the pathogenesis of EV71-related PE. On the other hand, there have been no reports of changes in cytokine patterns in the patients with less severe EV71-related neurological complications such as ME. Still, given that neurological complications usually have severe consequences (4), the early recognition of HFMD patients with neurological complications is important for selecting optimal intensive care that might help prevent worsening of the disease. Hence, to further elucidate the neuropathogenic mechanism of EV71 infection and to confirm the © 2015 The Societies and Wiley Publishing Asia Pty Ltd
involvement of the cytokines, in the present study, we examined the expression of CSF Th1/Th2 cytokines (IL2, IL-4, IL-6, IL-10, TNF-a and IFN-g) in patients with EV71-related ME by CBA technology. We found that both IL-6 and IFN-g were overexpressed in these patients, which may contribute to the pathogenesis of EV71 infection.
MATERIALS AND METHODS Case selection and clinical samples This study was performed at Children's Hospital, Zhejiang University School of Medicine and Hangzhou Children's Hospital during an outbreak of HFMD that occurred between May and August 2012. This study was approved by the Ethics Committees of both hospitals and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from each child's parent or guardian before they were included in this study. The criteria for diagnosing HFMD were vesicular lesions on the hands, feet, mouth, and frequently, buttocks, which were often accompanied by fever. ME was defined as CSF pleocytosis with CSF WBC >5/mL indicating infection within the sub-arachnoid space together with neurological symptoms or signs (i.e., signs of meningeal irritation, convulsions or change in mental status) indicating involvement of the brain parenchyma. The following criteria were used to diagnose simple FCs: (i) seizure(s) occurring in the first 48 hrs of a febrile illness; (ii) generalized tonic–clonic seizures without any focal signs or recurring within 24 hrs; (iii) seizures lasting less than 15 min; and (iv) no associated evidence of infection of the CNS or a metabolic disorder. Real-time PCR using TaqMan technology was performed to detect the EV71 genome in all throat swabs and stool specimens collected from HFMD patients. In short, viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Then the EV71 sequence was specifically analyzed using a one-step RT-PCR Detection Kit (Da An Gene, Guangzhou, China). RT-PCR amplification was carried out using a 7500 real-time RT-PCR system (Applied Biosystems, Foster, CA, USA). Eventually, 97 HFMD patients with ME were confirmed to have EV71 infection and included in this study, and 20 patients with FC were chosen as controls. Provided there were no contraindications, lumbar puncture was performed immediately after admission to obtain an acute phase CSF sample from each patient. Recovery phase CSF samples were obtained from only 13 153
H. Li et al.
HFMD patients with ME. The CSF samples were aliquoted and subjected to biochemical analyses (glucose, protein and chloride), cytological analyses (cell count and white cell distribution) and microbiology tests or frozen at 80°C for further cytokine determination. The samples were only thawed once. Measurement of CSF cytokines The concentrations of IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g in CSF were determined using a CBA Human Th1/Th2 Cytokine Kit II (BD Biosciences, San Diego, CA, USA) according to the manufacturer's specifications. Following the acquisition of sample data using a FACScalibur flow cytometer (BD Biosciences), results were generated using BD CBA Software. A standard calibration curve (ranging from 0 to 5000 pg/mL) was established for each cytokine and six results were obtained from each test sample. Statistical analysis All statistical analyses were performed using SPSS software, version 17.0 (SPSS, Chicago, IL, USA). The nonparametric Mann–Whitney U-test was used for data
that did not show a normal distribution, whereas data with a normal distribution were tested by Student's t-test. Differences between proportional data were analyzed using x2 or Fisher's exact test as appropriate. The Wilcoxon rank sum test (nonparametric distribution) or paired t-test (normal distribution) were used to compare the variables in the acute and recovery phases. Correlations between variables were calculated by Spearman's rank correlation test (nonparametric data) or Pearson's correlation test (normal data). Data are expressed as mean SD. A p value < 0.05 was considered statistically significant.
RESULTS Patients' clinical features and laboratory variables One hundred and seventeen children were recruited into this study, 97 of whom had EV71-related ME, the remaining 20 patients having FCs (controls). These patients' relevant clinical and laboratory data are presented in Table 1. In short, there were no significant differences between the two groups with respect to age, sex and routine blood tests. However, the durations of
Table 1. Comparison of clinical and laboratory variables between patients with EV71-related ME and those with simple FCs Characteristic Age (months) Sex (male/female) Hospital stays (days) Fever duration before admission (days) Fever duration during hospitalization (days) Total fever duration (days) Blood routine measures WBC (109/L) Neutrophil (%) Lymphocyte (%) Hemoglobin (g/L) Platelet count (109/L) Blood glucose (mmol/L) CSF variables WBC (106/L) Neutrophil (%) Lymphocyte (%) Total protein (g/L) Glucose (mmol/L) Chloride (mmol/L) Humoral immunity Serum IgG (g/L) Serum IgA (g/L) Serum IgM (g/L) Serum C3 (g/L) Serum C4 (g/L)
Febrile convulsion (n ¼ 20)
Meningoencephalitis (n ¼ 97)
P value
26.95 17.61 12/8 6.30 2.11 1.65 1.20 1.76 1.22 3.41 1.74
29.31 13.18 58/39 9.61 2.47 2.50 1.56 2.45 1.29 4.94 1.62
0.494 0.986
ORIGINAL ARTICLE
Cerebrospinal fluid Th1/Th2 cytokine profiles in children with enterovirus 71-associated meningoencephalitis Huajun Li1,2, Shuxian Li1, Jianfeng Zheng3, Chunyan Cai1,4, Bin Ye5, Jun Yang6,7 and Zhimin Chen1 1
Department of Pulmonology, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003,, 2Department of Pediatrics, Shaoxing People's Hospital, Shaoxing, Zhejiang 312000, 3Department of Pediatrics, Maternal and Child Care Service Centre, Cixi, Ningbo, Zhejiang 315300, 4Department of Infectious Disease, Hangzhou Children's Hospital, Hangzhou, Zhejiang 310014, 5 Department of Pediatrics, Taizhou Central Hospital, Jiaojiang, Taizhou, Zhejiang 318000, 6Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang 310013 and 7Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310016, China
ABSTRACT Enterovirus 71 (EV71) infection can cause severe neurological complications including meningoencephalitis (ME) in some patients with hand, foot and mouth disease (HFMD). However, to date no studies have reported changes in cytokine concentrations and their correlations with clinical variables in patients with ME following EV71 infection. In this study, responses of Th1/Th2 cytokine, including IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g, in cerebrospinal fluid (CSF) from patients with EV71related HFMD with ME and patients with febrile convulsions (FC) were analyzed using cytometric bead array technology. It was found that CSF IL-6 and IFN-g concentrations were significantly higher in patients with EV71-related ME than in those with FC. Additionally, both CSF IL-6 and IFN-g concentrations were correlated with CSF cytology, fever duration and duration of hospital stay. More interestingly, a positive correlation between CSF IL-6 and IFN-g concentrations was observed. Finally, receiver operating characteristic analysis revealed that when a cutoff value of 9.40 pg/mL was set for IL-6, the sensitivity and specificity were 84.5% and 85.5%, respectively, for discriminating EV71-related ME from FC. In conclusion, IL-6 and IFN-g may be associated with EV71-induced neuropathology. Key words
cytokines, enterovirus 71, hand, foot and mouth disease, meningoencephalitis.
Enterovirus 71 is an important causative pathogen of HFMD in children worldwide (1). Although HFMD is usually a mild disease, some patients develop neurological complications, such as aseptic meningitis, meningoencephalitis (ME), brainstem encephalitis, encephalomyelitis, acute flaccid paralysis and PE, especially when they have EV71 infection (2). In addition, most deaths from
EV71-induced HFMD occur within 2–3 days of onset of this acute disease and are attributable to fulminant PE (3– 4). In China, sporadic epidemics or outbreaks of EV71 infection have occurred since 1998 (5). In particular, in 2008, a large, unprecedented HFMD outbreak caused by EV71 infection occurred in Fuyang city, Anhui province of China and resulted in a high mortality rate (1).
Huajun Li and Shuxian Li contributed equally to this work. Correspondence Zhimin Chen, Department of Pediatric Pulmonology, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China. Tel: þ86 571 8887 3741; fax: þ86 571 8703 3296; email: [email protected] Additional correspondence: Jun Yang, Department of Toxicology, Hangzhou Normal University, Hangzhou, Zhejiang 310016, China. Tel/fax: þ 86 571 8820 8141; email: [email protected] Received 5 July 2014; revised 2 January 2015; accepted 13 January 2015. List of Abbreviations: CBA, cytometric bead array; CNS, central nervous system; CSF, cerebrospinal fluid; EV71, enterovirus 71; FC, febrile convulsion; HFMD, hand, foot and mouth disease; ME, meningoencephalitis; miRNA, microRNA; PE, neurogenic pulmonary edema; ROC, receiver operating characteristic; WBC, white blood cell count.
152
© 2015 The Societies and Wiley Publishing Asia Pty Ltd
CSF cytokines in EV71-associated ME
However, there is currently no approved antiviral drug or effective vaccination against EV71. Thus, in order to develop future therapeutic modalities, a better understanding of the pathogenesis of EV71 infection is of great importance. Cytokines are low molecular weight proteins that mediate inflammation, immunity, cell growth, differentiation, migration and repair. Traditionally, cytokines can be divided into at least two functional subgroups: Th1 cytokines, including IL-2, IFN-g and TNF-a, which are mainly secreted by Th1 cells and considered as proinflammatory cytokines; and Th2 cytokines, such as IL4, IL-5 and IL-13, which are mainly derived from Th2 cells and exert anti-inflammatory effects (6). However, there are exceptions to this simple classification. For example, IL-6 has both pro- and anti-inflammatory functions, whereas IL-10, which is classified as an antiinflammatory cytokine, can be produced by either Th1 or Th2 cells (7). Generally, cytokines are regarded as humoral factors that regulate inflammatory events. For instance, TNF-a, IFN-g and IL-4 increase endothelial cell adhesiveness for T lymphocytes and act in combination at sites of inflammation to promote lymphocyte extravasation (8). TNF-a and IFN-g also have the potential to synergize with HIV-1 to induce CXCL10 expression through the JAK/STAT and mitogen-activated protein kinase pathways in astrocytes, underlining the molecular mechanisms for their involvement in HIV-related encephalitis (9). Similarly, in the past few years, the importance of cytokines in the pathogenesis of EV71 infection, especially in EV71related PE, has been gradually recognized (10–11). For example, during systemic inflammatory responses, EV71 patients with PE show significantly increased blood concentrations of IFN-g, IL-10 and IL-13, but not of TNF-a, IL-2 and IL-4 (10). Additionally, in the central nervous system (CNS), cerebrospinal fluid (CSF) concentrations of IL-6 and IFN-g, which can increase pulmonary vascular permeability, are dramatically higher in children with PE than in those with uncomplicated brainstem encephalitis (11). Together, these data indicate that cytokines in the CNS or blood may be involved in the pathogenesis of EV71-related PE. On the other hand, there have been no reports of changes in cytokine patterns in the patients with less severe EV71-related neurological complications such as ME. Still, given that neurological complications usually have severe consequences (4), the early recognition of HFMD patients with neurological complications is important for selecting optimal intensive care that might help prevent worsening of the disease. Hence, to further elucidate the neuropathogenic mechanism of EV71 infection and to confirm the © 2015 The Societies and Wiley Publishing Asia Pty Ltd
involvement of the cytokines, in the present study, we examined the expression of CSF Th1/Th2 cytokines (IL2, IL-4, IL-6, IL-10, TNF-a and IFN-g) in patients with EV71-related ME by CBA technology. We found that both IL-6 and IFN-g were overexpressed in these patients, which may contribute to the pathogenesis of EV71 infection.
MATERIALS AND METHODS Case selection and clinical samples This study was performed at Children's Hospital, Zhejiang University School of Medicine and Hangzhou Children's Hospital during an outbreak of HFMD that occurred between May and August 2012. This study was approved by the Ethics Committees of both hospitals and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from each child's parent or guardian before they were included in this study. The criteria for diagnosing HFMD were vesicular lesions on the hands, feet, mouth, and frequently, buttocks, which were often accompanied by fever. ME was defined as CSF pleocytosis with CSF WBC >5/mL indicating infection within the sub-arachnoid space together with neurological symptoms or signs (i.e., signs of meningeal irritation, convulsions or change in mental status) indicating involvement of the brain parenchyma. The following criteria were used to diagnose simple FCs: (i) seizure(s) occurring in the first 48 hrs of a febrile illness; (ii) generalized tonic–clonic seizures without any focal signs or recurring within 24 hrs; (iii) seizures lasting less than 15 min; and (iv) no associated evidence of infection of the CNS or a metabolic disorder. Real-time PCR using TaqMan technology was performed to detect the EV71 genome in all throat swabs and stool specimens collected from HFMD patients. In short, viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Then the EV71 sequence was specifically analyzed using a one-step RT-PCR Detection Kit (Da An Gene, Guangzhou, China). RT-PCR amplification was carried out using a 7500 real-time RT-PCR system (Applied Biosystems, Foster, CA, USA). Eventually, 97 HFMD patients with ME were confirmed to have EV71 infection and included in this study, and 20 patients with FC were chosen as controls. Provided there were no contraindications, lumbar puncture was performed immediately after admission to obtain an acute phase CSF sample from each patient. Recovery phase CSF samples were obtained from only 13 153
H. Li et al.
HFMD patients with ME. The CSF samples were aliquoted and subjected to biochemical analyses (glucose, protein and chloride), cytological analyses (cell count and white cell distribution) and microbiology tests or frozen at 80°C for further cytokine determination. The samples were only thawed once. Measurement of CSF cytokines The concentrations of IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-g in CSF were determined using a CBA Human Th1/Th2 Cytokine Kit II (BD Biosciences, San Diego, CA, USA) according to the manufacturer's specifications. Following the acquisition of sample data using a FACScalibur flow cytometer (BD Biosciences), results were generated using BD CBA Software. A standard calibration curve (ranging from 0 to 5000 pg/mL) was established for each cytokine and six results were obtained from each test sample. Statistical analysis All statistical analyses were performed using SPSS software, version 17.0 (SPSS, Chicago, IL, USA). The nonparametric Mann–Whitney U-test was used for data
that did not show a normal distribution, whereas data with a normal distribution were tested by Student's t-test. Differences between proportional data were analyzed using x2 or Fisher's exact test as appropriate. The Wilcoxon rank sum test (nonparametric distribution) or paired t-test (normal distribution) were used to compare the variables in the acute and recovery phases. Correlations between variables were calculated by Spearman's rank correlation test (nonparametric data) or Pearson's correlation test (normal data). Data are expressed as mean SD. A p value < 0.05 was considered statistically significant.
RESULTS Patients' clinical features and laboratory variables One hundred and seventeen children were recruited into this study, 97 of whom had EV71-related ME, the remaining 20 patients having FCs (controls). These patients' relevant clinical and laboratory data are presented in Table 1. In short, there were no significant differences between the two groups with respect to age, sex and routine blood tests. However, the durations of
Table 1. Comparison of clinical and laboratory variables between patients with EV71-related ME and those with simple FCs Characteristic Age (months) Sex (male/female) Hospital stays (days) Fever duration before admission (days) Fever duration during hospitalization (days) Total fever duration (days) Blood routine measures WBC (109/L) Neutrophil (%) Lymphocyte (%) Hemoglobin (g/L) Platelet count (109/L) Blood glucose (mmol/L) CSF variables WBC (106/L) Neutrophil (%) Lymphocyte (%) Total protein (g/L) Glucose (mmol/L) Chloride (mmol/L) Humoral immunity Serum IgG (g/L) Serum IgA (g/L) Serum IgM (g/L) Serum C3 (g/L) Serum C4 (g/L)
Febrile convulsion (n ¼ 20)
Meningoencephalitis (n ¼ 97)
P value
26.95 17.61 12/8 6.30 2.11 1.65 1.20 1.76 1.22 3.41 1.74
29.31 13.18 58/39 9.61 2.47 2.50 1.56 2.45 1.29 4.94 1.62
0.494 0.986
