key: cord-0002922-t40nnh7h authors: Yu, Ying; Chen, Ying; Wang, Feng-Ling; Sun, Jing; Li, Hai-Jun; Liu, Jia-Ming title: Cytokines Interleukin 4 (IL-4) and Interleukin 10 (IL-10) Gene Polymorphisms as Potential Host Susceptibility Factors in Virus-Induced Encephalitis date: 2017-09-22 journal: Med Sci Monit DOI: 10.12659/msm.904364 sha: 5f071d0350cb9013deb98f9a52070bbf0f70bb84 doc_id: 2922 cord_uid: t40nnh7h BACKGROUND: This study aimed to analyze and explore the relationship between the cytokines IL-4 and IL-10 in relation to gene polymorphism and their respective effects on the susceptibility to virus-induced encephalitis. MATERIAL/METHODS: From January 2012 to June 2013, 112 patients with virus-induced encephalitis (the case group and 109 healthy individuals (the control group) were recruited for the purposes of this study. The functional variations that IL-4 and IL-10 genes exhibit were detected through the use of a function analysis and selection tool for single-nucleotide polymorphisms (FASTSNP). The genotypes of IL-4 were rs2227283 and IL-4 rs2227288, and the genotypes of IL-10 were rs1800871 and IL-10 rs1800872. These genotypes were respectively assessed using direct sequencing. RESULTS: IL-4 rs2227283 and IL-10 rs1800871 have no correlation in with risk of virus-induced encephalitis (both P>0.05) GA and AA genotypes were related to IL-4 rs2227288 and GT, while TT and GT + TT genotypes were related to IL-10 rs1800872. These were highlighted as being risk factors in virus-induced encephalitis (all P<0.05). However, the duration of fever, white blood cell (WBC) count, C-reactive protein (CRP), neutrophils, and lymphocytes and monocytes of virus-induced encephalitis patients with IL-4 rs2227288 and IL-10 rs1800872 all displayed significant differences (all P<0.05). Frequencies of GAGT and CAGT haplotypes were evaluated and deemed to be of statistical significance and subsequently were highlighted as being risk factors in virus-induced encephalitis (all P<0.05). CONCLUSIONS: IL-4 rs2227288 and IL-10 rs1800872 may contribute to an increased risk for virus-induced encephalitis. Through use of direct sequencing, we showed that genotypes of IL-4 rs2227288 and IL-10 rs1800872 may have particular host susceptibility to virus-induced encephalitis. Virus-induced encephalitis is a life-threatening disorder characterized by inflammation of brain tissue. The inflammation associated with brain parenchyma is linked to and a consequence of viral infections [1] [2] [3] . Virus-induced encephalitis is associated with misdiagnosis and delays in recognition, which may have devastating consequences for patients [4] . Additionally, the efficacy of therapies is highly time-dependent due to high morbidity and mortality rates [5, 6] . Enterovirus is the main etiological agents of virus-induced encephalitis, followed by mumps, rubella, and Japanese encephalitis virus. However, the lack of unified guidelines in the assessment and management of the illness is a serious limitation [2, 7] . The most common symptoms and neurological signs of virus-induced encephalitis are fever, reduced consciousness, seizures, and focal neurological deficits [8] [9] [10] . Patients with virus-induced encephalitis suffer from various degrees of renal damage [11] . Additionally, virus-induced encephalitis may cause serious brain damage and bleeding; therefore, prompt diagnosis and early treatment are crucial to prevent the disease from developing, and delayed treatment often results in poor prognosis [1] . In patients who are able to survive the virus-induced encephalitis, impairments of neurologic defect and cognitive, emotional, and behavioral function impairments are common [12] . However, chronic encephalitis has been reported as being uncommon, possibly owing to its viral origins, which may result in the disease being exhibited in patients that have compromised immunity, as well as in healthy individuals [13] . Thus, difficulties are faced in correctly diagnosing patients and subsequently providing prompt therapies for individuals with virus-induced encephalitis [14] . Consequently, it is important to further explore the details and mechanisms involved. At present, increasing numbers of scientific and clinical studies are being initiated to further understand virus-induced encephalitis. Interleukin 4 (IL-4) and interleukin 10 (IL-10), are 2 cytokines that have been found to have strong links to encephalitis [15] [16] [17] . It has been suggested that IL-4 is crucial to the induction of the naive helper T (Th0) cells differentiating to type 2 helper T (Th2) cells [18, 19] . IL-4 is known to have 2 different receptor complexes: the IL-4Ra chain and the gc chain [20] . IL-4 has a role in maintaining physiological balance and repairing tissues [21] . Moreover, IL-4 has exhibited in anti-inflammatory functions. IL-4 derived by activated CD4+ T cells can promote allergic responses [20, 22, 23] . Additionally, IL-10 is a cytokine produced by T cells, B cells, and macrophages, exhibiting a role in anti-inflammation and immunosuppression [24] [25] [26] . Virusinduced encephalitis is reported to respond to IL-10 [27] . IL-4 and IL-10 gene polymorphisms have been widely investigated in inflammatory diseases, such as asthma, chronic polyarthritis, rhinovirus bronchiolitis, and type 2 diabetes mellitus (T2DM) [28] [29] [30] [31] . Patients previously diagnosed with virusinduced encephalitis formed the basis of the experimental exploration of this study. The IL-4 and IL-10 gene polymorphisms and their correlation to virus-induced encephalitis susceptibility, through direct sequencing, were analyzed during the study. The Ethics Committee of Taizhou Municipal Hospital approved the study. All subjects were given official consent documents that were subsequently agreed upon and signed by all. Between January 2012 and June 2013, 112 patients (63 males and 49 females) with a mean age of 39.89±15.98 years ranging from 14 to 78 years, participated in the study. All subjects had been previously diagnosed with virus-induced encephalitis and were evaluated during the study as a case group. The control group consisted of 109 healthy individuals. The inclusion criteria were as follows: (1) Patients previously diagnosed with symptoms such as fever of varying degrees, disorders of consciousness, seizure, meningeal irritation sign, pyramidal sign, and intracranial hypertension detected by computed tomography (CT) head scan, electroencephalogram (EEG), and cerebrospinal fluid (CSF), which corresponded to the seventh edition of the diagnostic criteria regarding virus-induced encephalitis [32] ; (2) Positive results were detected using reverse transcription-polymerase chain reaction (RT-PCR) of encephalitis virus nucleic acid in the stool of subjects as well as cerebrospinal fluid (CSF) samples. The exclusion criteria were as follows: (1) patients who had experienced mumps, meningo-encephalitis, epidemic encephalitis B, or herpes simplex encephalitis; and (2) Patients that exhibited abnormalities detected by brain CT or magnetic resonance imaging (MRI) Peripheral venous blood samples (3 ml) were collected from healthy individuals as well as virus-induced encephalitis patients in the acute phase (5-day duration) with empty stomachs. The general conditions, signs, symptoms, and other accessory examinations of virus-induced encephalitis patients were recorded within 13 to 20 days from clinical observation to admission. The blood samples (3 ml) were placed in tubes with ethylenediamine tetraacetic acid (EDTA)-K2 and shaken. DNA was extracted using the improved potassium iodide method. Using HapMap database, the genomic data was downloaded. The following methods were used: (1) literature review; (2) Tag 4542 SNP selection; and (3) functional variations in IL-4 and IL-10 genes detected by function analysis and selection tool for single-nucleotide polymorphisms (FASTSNP). After selection, IL-4 rs2243283/rs2243288 and IL-10 rs1800871/rsl800872 were eligible for further study. The PCR primers were designed by Primer Premier 5.0 and synthesized by Shanghai Sangon BioTech Co., Ltd. (Shanghai, China). The forward and reverse primers of IL-4 and IL-10 gene polymorphisms are shown in Table 1 and IL-10 rs1800871/rsl800872 shared a pair of primers. The total volume of PCR reaction system was 50 μl, including 0.5 μl of DNA template, 5 μl of forward primer and reverse primer, respectively, 25 μl of PCR-pfu mix enzyme, and 14.5 μl of water. After DNA was dissolved, 5-μl samples (concentration >0.1 μg/μl) were assessed using an ultraviolet spectrophotometer on the basis of the ratio (260/280) of the range between 1.8 to 2.1. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 45 s, annealing at 60°C for 45 s, extension at 72°C for 45 s, 39 cycles, and extension at 72°C for 5 min by proper adjustment of annealing temperature according to primer synthesis report. A total of 1.5 μl of PCR product was detected with 1.0% agarose gel using electrophoresis. The gel was transferred and photographs were taken with an ultraviolet analyzer for observational purposes. Selected PCR product was sequenced in Beijing ZhongKe Xilin Biotechnology Co., Ltd. to determine the genotypes of IL-4 rs2243283, IL-4 rs2243288, IL-10 rs1800871, and IL-10 rsl800872. Statistical methods SPSS 19.0 software was used for data analysis. The Hardy-Weinberg equilibrium (HWE) detection was performed for genotype distribution analysis. Measurement data are shown as mean ± standard deviation and compared by t test and categorical data are presented as percentage and rate and were examined by the c 2 test or Fisher's exact test. The odds ratio and 95% confidence intervals were estimated using nonconditional logistic regression analysis. Haplotype analysis of IL-4 and IL-10 genes was performed using Shesis software. There was no significant difference in age and sex of subjects between the case and control groups (P>0.05). However, remarkable differences were found in the number of white blood cells (WBC), C-reactive protein (CRP), neutrophils, lymphocytes, and monocytes between the 2 groups (all P<0.05, Table 2 ). Genotype frequencies of IL-4 rs2243283/rs2243288 and IL-10 rs1800871/rs1800872 were tested by HWE method. The observed and expected values of each genotype in the case and control groups were compared and chi-square analysis demonstrated no statistically significant difference (P>0.05) in the distribution of observed number and the expected number of each genotype in the 2 groups. This allowed for the achievement of genetic balance with group representation (Table 3) . As shown in Table 4 , IL-4 rs2227283 and IL-10 rs1800871 gene polymorphisms were not associated with the risk of virus-induced encephalitis (both P>0.05); however, GA, AA, and GA + AA genotypes in IL-4 rs2227288, and GT, TT, and GT + TT genotypes in IL-10 rs1800872, A allele in IL-4 rs2227288, and T allele in IL-10 rs1800872 were risk factors for virus-induced encephalitis (all P<0.05). The sex and age of virus-induced encephalitis patients with different genotypes in IL-4 rs2227283/rs2227288 and IL-10 rs1800871/rs1800872 polymorphisms showed no statistically significant difference (all P>0.05). There were no clear differences in the duration of fever, CRP, WBCs, neutrophils, lymphocytes, and monocytes in virus-induced encephalitis patients with IL-4 rs2227283 genotype and IL-10 rs1800871 (all P>0.05). However, the duration of fever, CRP, WBCs, neutrophils, lymphocytes, and monocytes of virus-induced encephalitis patients with different genotypes in IL-4 rs2227288 and IL-10 rs1800872 showed significant differences (all P<0.05, Table 5 ). Haplotype analysis in the case and control groups Haplotypes of IL-4 rs2227283/rs2227288 and IL-10 rs1800871/rs1800872 are shown in Table 6 , which were analyzed by using the Shesis software (haplotypes with the frequency under 0.05 in these 2 groups were excluded). The results revealed that the frequencies of haplotypes (CAAT, GAGT, GAAT, CAGT, and CGGT) showed statistical significance (all P<0.05) as the risk factors for virus-induced encephalitis, while the frequencies of several other haplotypes (CGAG, CGGG, GGAG, GGGT, CGAT, and CGAG) showed no significant differences between the 2 groups (all P>0.05). During the evaluation of the correlation between encephalitis and other viral infections at similar time points, impairments of the central nervous system and associated inflammation were observed (CNS). When virus-induced encephalitis was correlated with viral infections, the central nervous system (CNS) also shows associated impairment and inflammation [3, 33, 34] . Virus-induced encephalitis affected approximately 7.5 people out of every 100 000, with considerable morbidity and mortality rates and displaying an increased risk for development of seizures in approximately 22% of patients [35] . This being said it was of considerable importance that the mechanisms in which Table 6 . Haplotype analysis of rs2243283, rs2243288, rs1800871 and rs1800872 in the case and control groups. OR -odd ratio; CI -confidence interval. virus-induced encephalitis work were further explored and understood. Moreover, the diagnosis of encephalopathy can be achieved on the basis of higher serum thyroperoxidase antibody titer (including antithyroglobulin antibodies and antithyroid peroxidase antibody) and clinical manifestations such as brain MRI abnormalities, fever, headache, and stroke. [36] . Anti-thyroid antibodies also play significant roles in the pathogenesis of encephalopathy [14] . Using direct sequencing, our study determined the genotypes of IL-4 rs2243283, IL-4 rs2243288, IL-10 rs1800871, and IL-10 rsl800872 and analyzed the correlation of IL-4 and IL-10 gene polymorphisms with the susceptibility to virus-induced encephalitis, suggesting that IL-4 rs2227288 and IL-10 rs1800872 might enhance the risk for virus-induced encephalitis. Previous studies have suggested that IL-4 and IL-10 were 2 cytokines that have a relationship to encephalitis [15] [16] [17] 37] . Initially, we found during this study that the A allele, and GA, AA, and GA + AA genotypes in IL-4 rs2227288 were risk factors for virus-induced encephalitis. According to Anovazzi et al., there were strong relationships between the alleles, genotypes, and haplotypes of IL-4 gene polymorphisms and chronic periodontitis [38] . It was suggested that the IL-4-590 T/T and IL-4-33 T/T genotypes were potentially correlated with resistance to therapy in hepatitis C virus patients, and the CT/TT genotypes of IL-4 C-589T was correlated with wheezing without actually having a cold in African Americans infants [39, 40] . The etiology of systemic lupus erythematosus (SLE) and elevated risk for SLE were revealed as having correlations with IL-4 gene in a previous study [41] , and another study indicated that the IL-4-590 C/T polymorphisms might participate in controlling parasitemia and the result could affect the severity of malaria [42] . Promoter polymorphisms of IL-4, an immune-regulatory Th2 cytokine, were proposed as being related to T2DM and might play a role in the susceptibility to T2DM [43] . Furthermore, IL-4 polymorphisms potentially contribute to evaluation of severity of rheumatoid arthritis (RA), and IL-4-590 promoter polymorphism might be correlated with elevated risk and increased activity of RA [44] . An SNP of IL-4 rs2243250 was demonstrated as being correlated with increased risk of development of Clostridium difficile infection in patients with inflammatory bowel disease [45] . We found that T allele, and GT, TT, and GT + TT genotypes in IL-10 rs1800872 were risk factors for virus-induced encephalitis. Attempts to control the subsequent inflammation and decreased tissue destruction were of particular importance to CNS infections, including virus-induced encephalitis, as well as the regulation IL-10 expressions, could possibly diminish the tissue damage in acute encephalitis [37] . IL-10 rs1800872 polymorphism might be a risk factor for colorectal cancer development in European populations and the A/C allele of IL-10 rs1800872 might raise the risk for RA [46, 47] . Previous studies have revealed that polymorphisms in IL-10-592C/A (rs1800872) were associated with risk of acute myeloid leukemia and enhanced the likelihood of early-onset preeclampsia [48, 49] . IL10-592C/A polymorphisms were correlated with risk factors of coronary heart disease (CHD). Additionally, the A allele may be a risk factor for CHD [50] . Polymorphisms of IL-10 rs1800872 were recorded in order to draw links and data-related correlations with the increased production of IL-10 protein in peritoneal fluid (PF) in endometriosis patients [51] . Furthermore, haplotypes of IL10-rs1800872 had significant correlation with the increase of risk for early pregnancy loss [52] . In conclusion, IL-4 rs2227288 and IL-10 rs1800872 may lead to increased risk in relation to virus-induced encephalitis. This being said, the sample size of this study was relatively small and the range of research should be extended for future research in this particular area. It remains unknown in our study whether IL-4 rs2227288 and IL-10 rs1800872 gene polymorphisms affect the levels of IL-4 and IL-10 and subsequently participate in the pathogenesis of encephalopathy. Further prospective studies should be performed to provide stronger evidence of the underlying mechanisms of IL-4 rs2227288 and IL-10 rs1800872 gene polymorphisms in virus-induced encephalitis. None. Etiology and clinico-epidemiological profile of acute viral encephalitis in children of western Uttar Pradesh, India Epidemiology of viral encephalitis in 2011 Early recognition of encephalitis in acute settings Treatment of viral encephalitis A brain slice culture model of viral encephalitis reveals an innate CNS cytokine response profile and the therapeutic potential of caspase inhibition Consensus guidelines on evaluation and management of suspected acute viral encephalitis in children in India Viral encephalitis Viral encephalitis in the ICU IL-4 and IL-10 polymorphisms and susceptibility to virus-induced encephalitis Clinical presentation of severe viral encephalitis with known causative agents in children: A retrospective study on 16 patients hospitalized in a pediatric intensive care unit Diagnostic value of serum Cystatin C in renal function impairments in children with viral encephalitis Viral encephalitis: Neuropsychiatric and neurobehavioral aspects Probable chronic viral encephalitis with microglial nodules in the entire brain: A case report with necropsy A case of hashimoto's encephalopathy misdiagnosed as viral encephalitis Role of IL-4 in an experimental model of encephalitis induced by intracranial inoculation of herpes simplex virus-1 (HSV-1) Tick-borne encephalitis is associated with low levels of interleukin-10 in cerebrospinal fluid Association of interleukin 10 and interferon gamma gene polymorphisms with enterovirus 71 encephalitis in patients with hand, foot and mouth disease Interleukin-4 is required for the induction of lung Th2 mucosal immunity Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells Untangling the complex web of IL-4-and IL-13-mediated signaling pathways Interleukin-4-and interleukin-13-mediated alternatively activated macrophages: roles in homeostasis and disease Inflammatory monocytes recruited to allergic skin acquire an anti-inflammatory M2 phenotype via basophilderived interleukin-4 Characterization of the acute inflammatory response after irradiation in mice and its regulation by interleukin 4 (Il4) Inflammatory bowel disease and mutations affecting the interleukin-10 receptor Differential expression in lupus-associated IL-10 promoter single-nucleotide polymorphisms is mediated by poly(ADPribose) polymerase-1 Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation Shifting hierarchies of interleukin-10-producing T cell populations in the central nervous system during acute and persistent viral encephalomyelitis Association of IL4R gene polymorphisms with asthma in Chinese populations IL-4 VNTR gene polymorphism in chronic polyarthritis. The rare allele is associated with protection against destruction IL-10 gene polymorphism at -1082 A/G is associated with severe rhinovirus bronchiolitis in infants Association of IL-6, TNF-alpha and IL-10 gene polymorphisms with type 2 diabetes mellitus Case definitions, diagnostic algorithms, and priorities in encephalitis: Consensus statement of the international encephalitis consortium Immune system's role in viral encephalitis CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis Viruses and the immune system: Their roles in seizure cascade development MR imaging of adult acute infectious encephalitis Highly activated cytotoxic CD8 T cells express protective IL-10 at the peak of coronavirus-induced encephalitis Polymorphisms and haplotypes in the interleukin-4 gene are associated with chronic periodontitis in a Brazilian population Interleukin-4 polymorphisms and response to combination therapy in Egyptian chronic hepatitis C patients Environmental tobacco smoke and interleukin 4 polymorphism (C-589T) gene: Environment interaction increases risk of wheezing in African-American infants Interleukin 4 and STAT6 gene polymorphisms are associated with systemic lupus erythematosus in Chinese patients Relative levels of IL4 and IFN-gamma in complicated malaria: Association with IL4 polymorphism and peripheral parasitemia Association of interleukin-4 promoter polymorphisms in Taiwanese patients with type 2 diabetes mellitus Influence of interleukin-4 gene polymorphisms and interleukin-4 serum level on susceptibility and severity of rheumatoid arthritis in Egyptian population An interleukin-4 polymorphism is associated with susceptibility to Clostridium difficile infection in patients with inflammatory bowel disease: Results of a retrospective cohort study Meta-analysis of epidemiological studies of association of two polymorphisms in the interleukin-10 gene promoter and colorectal cancer risk Association between polymorphisms of interleukin 10 with inflammatory biomarkers in East Chinese Han patients with rheumatoid arthritis Association between Interleukin-10 gene polymorphisms and risk of early-onset preeclampsia Interleukin-10 polymorphisms associated with susceptibility to acute myeloid leukemia Interleukin-10 gene promoter polymorphisms and their protein production in peritoneal fluid in patients with endometriosis Interleukin 10 gene promoter polymorphisms in women with pregnancy loss: Preferential association with embryonic wastage IL-4 and IL-10 polymorphisms and susceptibility to virus-induced encephalitis This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)