Gene Expression and Promoter Region Polymorphisms of Interleukin-10 in Meningitis Patients

Abstract

Meningitis is an inflammatory disease caused by bacteria, fungi, and viruses with various clinical symptoms. Interleukin-10 (IL-10) levels have been shown to be increased in blood or cerebrospinal fluid of patients with meningitis, but the association of IL-10 gene promoter polymorphisms or gene expression with meningitis has not been evaluated. IL-10 gene promoter polymorphisms A-592C, T-819C, and A-1082G in 61 patients with meningitis and 64 healthy controls were determined by real-time polymerase chain reaction analysis. mRNA from blood and cerebrospinal fluid samples was extracted, and real-time polymerase chain reaction was performed for IL-10 gene expression. No statistically significant differences were found in the allele and genotypic frequencies between patients and control subjects. Expression of IL-10 in meningitis at mRNA levels was detected in the infiltrating leukocytes. IL-10 gene expression in blood from patients was significantly higher than the control group. Our results suggest that there was no association between promoter polymorphisms of IL-10 and meningitis, but a significant increase of IL-10 gene expression was present in patients with meningitis.

Introduction

Central nervous system (CNS) infections continue to be an important cause of mortality and morbidity. Bacterial infections of the CNS, such as meningitis, induce an extensive inflammatory response, which in turn may damage neurons. Gram-positive bacterial cell wall induces glial inflammatory activation and leads to the production of proinflammatory cytokines (Kim, 2009).

Interleukin-10 (IL-10) is an 18-kDa, homodimeric cytokine, which is produced by both T/B cells and monocytes/macrophages, microglia, myeloid dendritic cells, and mast cells (Mosser and Zhang, 2008). IL-10 possesses both anti-inflammatory and immunosuppressive properties. So, IL-10 plays a central role in vivo in restricting inflammatory responses. It antagonizes the production of proinflammatory cytokines, including tumor necrosis factor (TNF), IL-1, and IL-6, inactivating antigen presentation and cell-mediated immune response. Conversely, IL-10 has some proinflammatory activity or immunostimulatory effects because it promotes activation, proliferation, and differentiation of B-lymphocytes and induces immunoglobulin synthesis and autoantibody production (Llorente et al., 1994). The B-cell-stimulating property of IL-10 is thought to be the basis of several antibody-mediated autoimmune disorders (Groux and Cottrez, 2003). The critical role of IL-10 as a negative regulator of inflammation is illustrated by studies that revealed that IL-10^−/− mice spontaneously develop inflammatory bowel disease in response to bacteria (Khanna et al., 1991).

The human IL-10 gene is located on chromosome 1 (1q31-q32) and consists of five exons and four introns encoding for a 160-amino acid protein (Kim et al., 1992). The 5′ flanking region of the IL-10 gene, which regulates transcription, is polymorphic. The proximal promoter contains three common single-nucleotide polymorphisms (SNPs) at positions −1082 (A/G) (rs1800896), −819 (T/C) (rs1800871), and −592 (A/C) (rs1800872) from the transcription start site (Moore et al., 2001). The polymorphisms in the promoter region of the IL-10 gene on chromosome 1 (1q31) determine the amount of cytokine production (Eskdale et al., 1998). Many disease association studies using markers at the IL-10 promoter have shown IL-10 to be important in the susceptibility to infections or inflammatory diseases (Haukim et al., 2002).

IL-10 is synthesized in the CNS and acts to limit clinical symptoms of meningitis. IL-10 also limits inflammation in the brain by reducing synthesis of proinflammatory cytokines, suppressing cytokine receptor expression, and inhibiting receptor activation (Strle et al., 2001). IL-10 has been detected in sera of patients with sepsis and in most cerebrospinal fluid (CSF) samples from patients with bacterial meningitis, but found in only 10% of CSF samples from patients with viral meningitis (Attallah and Ibrahim, 2004), and may play an immunoregulatory role in childhood meningitis (Kornelisse et al., 1996). A recent study showed that the IL-10 levels in the CSF were markedly higher in patients with eosinophilic meningitis associated with angiostrongyliasis when compared with the controls (Intapan et al., 2008). Although these studies showed increased IL-10 blood or CSF levels in patients with meningitis, the association of IL-10 gene promoter polymorphisms or gene expression with meningitis has not been evaluated.

It was reported that the polymorphism of IL-10 gene may modify the inflammatory response in the host and influence the development of meningococcal infection (Brandtzaeg and van Deuren, 2002). The A allele in the IL-10 promoter region at position −1082 has been reported to be associated with decreased IL-10 production in patients with Crohn's disease (Koss et al., 2000). Although we have shown recently an increase in endothelial NOS expression in meningitis patients (Oztuzcu et al., 2010), to our knowledge, there is no study to show the association of IL-10 polymorphisms with meningitis susceptibility.

IL-10 production is under strong genetic influence and controlled at the transcriptional level. It was reported that between 50% and 75% of the observed interindividual variability in IL-10 production can be explained by genetic factors (Eskdale et al., 1998). Because cytokine production is regulated at the genetic level, it has been hypothesized that SNPs in the IL-10 gene may be relevant to the development of meningitis. Moreover, there were no data on the frequency of IL-10 promoter polymorphisms in Turkish meningitis patients. The aim of this study was to determine the gene expression and promoter region polymorphisms of IL-10 in Turkish meningitis patients.

Materials and Methods

Patients

Sixty-one patients with meningitis (38 bacterial, 17 tuberculous, and 6 viral) admitted to the Departments of Pediatrics and Infectious Diseases, Gaziantep University Hospital, and Gaziantep Child Disease Hospital (age: 0.25-73 years, mean: 23.86 ± 24.04 years) were included in the study. Sixty-four patients admitted to these clinics but not diagnosed with meningitis were also included and constituted the control group, with age ranging from 0.75 to 73 years (mean: 27.06 ± 24.23 years). Patients and controls were of the same ethnic origin and from the same geographical area (southeastern Turkey). Informed written consent was obtained from all patients or close relatives. The study protocol was approved by the local ethics committee at the University of Gaziantep.

The diagnosis of meningitis was based on a typical clinical presentation and a marked neutrophilic pleocytosis in the CSF. Blood samples and the CSFs taken for the diagnosis of meningitis were used in this study. Patients with meningitis showed leukocytosis in the CSF (>1000 cells/μL). All the patients with acute pyogenic meningitis met the established diagnostic criteria (Pfister et al., 1992): detection of bacteria in the CSF by microscopic examination of Gram-stained smear and/or CSF culture; or CSF pleocytosis of >1000 white blood cells/μL with >60% polymorphonuclear leukocytes and symptoms and signs of acute bacterial meningitis, including headache, photophobia, meningismus, fever, and systemic signs of infections; or both. The diagnosis of tuberculous meningitis was established on the basis of the presence of symptoms and/or signs suggestive of meningitis plus one of the following criteria: (1) positive culture of Mycobacterium tuberculosis, positive smear for acid-fast bacilli, or positive polymerase chain reaction (PCR) from CSF; (2) positive culture of M. tuberculosis, positive smear for acid-fast bacilli, or positive PCR from other body fluids or organs; and (3) negative CSF culture for virus, bacteria, and fungi, plus clinical response to antituberculosis therapy. Diagnosis by the first criterion was considered definite, whereas diagnosis by the second and third criteria was presumptive. Acute aseptic (viral) meningitis was diagnosed with the presence of pleocytosis with leukocytes >5 × 10⁶ per liter, negative bacterial culture from the CSF, no acute signs of parenchymatous brain dysfunction plus two of the following findings: headache, nausea, and vomiting, light sensitivity, neck stiffness, fever >38°C. CSF and brain imaging studies were performed at admission in all patients. Patients with meningitis due to brain abscess, intracranial empyema, or spinal epidural abscess were excluded from the study.

Blood and CSF samples and DNA isolation

From all, 5 mL of peripheral blood samples were collected by venipuncture into sterile siliconized Vacutainer tubes with 2 mg/mL disodium ethylenediaminetetraacetic acid. Immediately after collection, whole blood was stored at 4°C until use. CSF samples were taken in syringes and transferred to tubes. Genomic DNA was extracted from whole blood or CSF samples by using a standard proteinase K digestion and the salt-chloroform method (Müllenbach et al., 1989) and stored at −20°C.

Genotyping

The detection of IL-10 gene polymorphisms was achieved with real-time PCR by using a Light-Cycler Instrument (Roche Diagnostics). The Light-Cycler instrument measures the emitted fluorescence of the light cycler-red 640. The hybridization probes in combination with the Light-Cycler DNA Master Hybridization Probes Kit (Roche Diagnostics) were used to determine the genotype by using a melting curve analysis after the amplification cycles were completed.

All related gene regions were amplified in 20 μL PCR capillary tubes. After preparation of the master mixture, 19 μL of the reaction mixture and 1 μL of genomic DNA or control template were added to each Light-Cycler capillary tube. For negative control, PCR-grade water was added instead of template. The cycling program was carried out after a denaturation step at 95°C for 10 min through 50 cycles (denaturation at 95°C for 10 s, annealing at 50°C for 10 s, extension at 72°C for 15 s), with a maximum ramp rate of 20°C/s. Fluorescence was measured at the end of the annealing period of each cycle to monitor amplification. After amplification was complete, a final melting curve was recorded by 90 s denaturation at 95°C followed by a continuous temperature increase from 40°C to 85°C in increments of 0.2°C/s. The fluorescence signal was converted to a melting peak by plotting the negative derivative of the fluorescence with respect to temperature versus temperature (−dF/dT vs. T). The resulting melting peak allowed discrimination among the homozygous as well as heterozygous genotypes for each exon. Genotyping was conducted in a blinded fashion.

RNA isolation and gene expression

To confirm the presence and expression of IL-10 in brain and blood, we extracted mRNA from blood and CSF samples. RNA was extracted from leukocytes using the High Pure RNA Isolation Kit (Roche Diagnostics) as described by the manufacturer (www.roche-applied-science.com/pack-insert/1828665a.pdf). cDNA was produced with the First Strand cDNA Synthesis Kit (Roche Diagnostics) according to manufacturer's protocol. Real-time PCR was performed using the Light-Cycler Instrument (Roche Diagnostics) with IL-10 primers (Integrated DNA Technologies) (Table 1), HPRT1 (housekeeping gene), TaqMan MGB probes (FAM dye-labeled), and TaqMan Universal PCR Master Mix. All samples were prepared twice and each preparation was set up in triplicate. Data were analyzed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001), according to the following formula: −ΔΔC_T =−(ΔC_T,IL-10 ΔΔC_T,HPRT1), where C_T = threshold cycle. We calculated the content of IL-10 mRNA using the 2^−ΔΔCt formula.

Table 1.

Primer Sequences for IL-10 and HPRT1 (Housekeeping) Gene for Gene Expression Study

Gene	Primer sequence	UPL probe	NCBI nucleotide no.
IL-10	5′-tgggggagaacctgaagac-3′	Probe 30	NM_000572.2
	5′-ccttgctcttgttttcacagg-3′
HPRT1	5′-tgaccttgatttattttgcatacc-3′	Probe 23	NM_000194.1
	5′-cgagcaagacgttcagtcct-3′

Statistical analysis

Results are expressed as median (min-max) or percentage. Statistical analysis was performed using GraphPad Instat (version 3.05). Polymorphisms were tested for deviation from Hardy-Weinberg equilibrium by comparing the observed and expected genotype frequencies using the χ² test. For between-group analyses, the analysis of variance test was used; for calculation of the significance of differences in genotype and allele frequencies, the χ² test or Fisher's exact test were used. For comparisons of the differences between mean values of two groups, the unpaired Student's t-test was used. The Mann-Whitney U test was performed to compare gene expression data. The effects of genetic polymorphisms on the risk of meningitis were estimated with odds ratio and its 95% confidence interval. The haplotype analysis was performed using an online software, SHEsis (http://analysis.bio-x.cn/myAnalysis.php). For all statistical tests, p-values were two-sided, and p < 0.05 was considered statistically significant.

Results

No difference in the distributions of IL-10 promoter region genotype and allele frequencies was seen between patients and control subjects (Tables 2 -5). Further, the expected genotype frequencies under the assumption of the Hardy-Weinberg equilibrium were similar with the observed ones in our patient and control groups (p > 0.05).

Table 2.

Genotype and Allele Frequencies of IL-10 Promoter Region A-1082G Polymorphism Among Cases and Controls

Genotype/allele	Control (n = 64) n (%)	Cases (meningitis) (n = 61) n (%)	p	OR (95% CI)
A/A	34 (53.1)	40 (65.5)		1.00
A/G	27 (42.2)	17 (27.9)	0.105	0.535 (0.250-1.144)
G/G	3 (4.7)	4 (6.6)	1.000	1.133 (0.237-5.424)
A	95 (74.2)	97 (79.5)		1.00
G	33 (25.8)	25 (20.5)	0.322	0.742 (0.411-1.341)

CI, confidence interval; OR, odds ratio.

Table 3.

Genotype and Allele Frequencies of IL-10 Promoter Region T-819C Polymorphism Among Cases and Controls

Genotype/allele	Control (n = 64) n (%)	Cases (meningitis) (n = 61) n (%)	p	OR (95% CI)
C/C	26 (40.6)	31 (50.8)		1.00
C/T	26 (40.6)	22 (36.1)	0.383	0.710 (0.328-1.534)
T/T	7 (10.9)	8 (13.1)	1.000	0.959 (0.306-2.999)
C	83 (64.8)	84 (68.9)		1.00
T	45 (35.2)	38 (31.1)	0.501	0.834 (0.492-1.415)

Table 4.

Genotype and Allele Frequencies of IL-10 Promoter Region A-592C Polymorphism Among Cases and Controls

Genotype/allele	Control (n = 64) n (%)	Cases (meningitis) (n = 61) n (%)	p	OR (95% CI)
C/C	26 (40.6)	31 (50.8)		1.00
C/A	31 (48.5)	20 (32.8)	0.115	0.541 (0.251-1.165)
A/A	7 (10.9)	10 (16.4)	0.788	1.198 (0.400-3.592)
C	83 (64.8)	82 (67.2)		1.00
A	45 (35.2)	40 (32.8)	0.693	0.900 (0.533-1.520)

Table 5.

Haplotype Frequencies of IL-10 Promoter Region Among Cases and Controls

Haplotype	Control (n = 64) n (%)	Cases (meningitis) (n = 61) n (%)	p	OR (95% CI)
A C C	50.0 (39.1)	57.0 (46.7)	0.217	1.375 (0.829-2.280)
A T A	44.0 (34.4)	38.0 (31.1)	0.589	0.864 (0.508-1.469)
G C C	32.0 (25.0)	25.0 (20.5)	0.397	0.773 (0.426-1.403)

We performed the haplotype analysis for evaluating the haplotype frequencies of polymorphisms in the IL-10 promoter, trying to derive haplotypes specifically correlated with meningitis. Haplotypes based on the A-1082G, T-819C, and A-592C SNPs were constructed. Haplotype analysis detected only three variants: ACC, ATA, and GCC. However, no significant differences were observed in haplotype distributions of the IL-10 promoter region between patients with meningitis and control subjects (Table 5).

There was evidence that IL-10 gene expression was present in the blood and CSF samples from patients with meningitis. Our results showed that mRNA content in blood was markedly higher in the patients with meningitis when compared with the control group (Table 6). Additionally, IL-10 mRNA content in the CSF samples was significantly increased when compared with the blood samples in meningitis patients (Table 7).

Table 6.

Comparison of mRNA Content in Blood for the Controls and Patients with Meningitis

IL-10	Control (n = 64) median (min-max)	Cases (n = 61) median (min-max)	Fold ΔΔC_T	p
ΔC_T = C_T (target) - C_T (housekeeping)	6.51 (3.09-12.62)	5.47 (−0.16 to 7.52)	1.04	<0.0001
Content = 2^−ΔCT	1.10 (0.02-11.74)	2.26 (0.55-111.73)	0.49	<0.0001

Table 7.

Comparison of Interleukin-10 mRNA Content in Blood and Cerebrospinal Fluid Samples from the Patients with Meningitis

IL-10	Blood (n = 61) median (min-max)	Cerebrospinal fluid (n = 61) median (min-max)	p
ΔC_T = C_T (target) - C_T (housekeeping)	5.47 (−0.16 to 7.52)	1.98 (−7.31 to 5.30)	<0.0001
Content = 2^−ΔCT	2.26 (0.55-111.73)	25.35 (2.54-15,868.00)	<0.0001

Discussion

In view of the pivotal role that cytokines play in the immune response, we evaluated the influence of genetic variants of the IL-10 gene promoter region on meningitis. We studied polymorphic allele variants of the IL-10 gene in patients with meningitis, but no association was noted between A-592C, T-819C, and A-1082G polymorphisms and meningitis. However, we have found that IL-10 gene expression was markedly elevated in meningitis.

Twin and family studies have suggested that about 50% or 75% of the variation in IL-10 production is genetically determined, and IL-10 production appears to be controlled at the transcriptional level (Eskdale et al., 1998). IL-10 5′ flanking region contains numerous polymorphisms that directly influence the expression of the protein. The IL-10 gene has three, well-characterized SNPs in the promoter region (-1082A/G, −819T/C, −592A/C) that have been shown to affect transcription and cytokine production (Hoffmann et al., 2002). IL-10-1082GG genotype has been found to be associated with higher IL-10 levels and transcriptional activity (Westendorp et al., 1997; Rad et al., 2004). High IL-10 levels were also associated with poor prognosis in acute infectious diseases, particularly meningococcal meningitis (Westendorp et al., 1997).

Our population consisted of Turkish Caucasians. The genotype and allele frequencies found in this study were similar to the previously reported frequencies in a Turkish population (Ates et al., 2008). However, the frequency of the IL-10-1082 GG genotype (4.7%) in our population was lower than the reported range for other Caucasian populations (20%-28%) (Michaud et al., 2006). The prevalence of the IL-10-592A allele varies from population to population, with the greatest rate found in Japanese (67.2%), southern Chinese from Hong Kong (67%), Chinese healthy subjects from Taiwan (61.0%), and British Caucasians (21%). In our study of Turkish population, IL-10-592A allele frequency was 35%, which is close to the frequency in Caucasians, but markedly different from Korean and Chinese.

Our findings support the results of previous studies (Haukim et al., 2002) showing that three polymorphisms in the promoter of the IL-10 gene at positions −1082A/G, −819T/C, and −592A/C are in strong linkage disequilibrium, forming three haplotypes common in Caucasian populations: GCC, ACC, and ATA. These haplotypes have been shown to be associated with IL-10 production (Haukim et al., 2002).

Although three SNPs (A-592C, T-819C, and A-1082G) are defined relative to their positions in the IL-10 gene sequence upstream of the transcription initiation site, the molecular effects of these SNPs in the IL-10 gene promoter region are not precisely understood (Mosser and Zhang, 2008). The SNP at position −1082 is within a putative e-twenty-six-like transcription factor-binding site (Lazarus et al., 1997). The SNP at −592 is located in a region that mediates negative regulatory function and lies within a possible STAT3-binding site, whereas the SNP at −819 may affect an estrogen receptor element (Lazarus et al., 1997).

IL-10 influences the immune response via downregulation of proinflammatory cytokine release. The substantial IL-10 production during meningitis seems like a reasonable response for suppression of the inflammatory process maintained by ongoing production of proinflammatory cytokines. Endogenously formed IL-10 is important for limiting the production of TNF-α by leukocytes. It has been proposed that IL-10 in CSF will decrease the inflammatory reaction associated with meningitis and will result in the development of fewer sequelae because of its inhibitory effect on the production of TNF-α (van Furth et al., 1995). IL-10 is necessary to counterbalance proinflammatory reactions, but IL-10 overexpression might increase mortality because of unresolved infection (Schaaf et al., 2003). Schaaf et al. (2003) showed that the IL-10-1082 G genotype associated with increased IL-10 release seems to be a risk factor for septic shock in pneumococcal infection. They suggest that a strong anti-inflammatory response during severe bacterial infection could be harmful by causing immunosuppression. Therefore, manipulating host IL-10 responses may represent a two-edged sword that is not without considerable risk. Blocking IL-10 introduces the risk of autoimmunity, whereas inducing IL-10 overexpression can lead to immunosuppression (Mosser and Zhang, 2008).

Expression of IL-10 is elevated during the course of most major diseases in the CNS and promotes survival of neurons and all glial cells in the brain by blocking the effects of proapoptotic cytokines and by promoting expression of cell survival signals. Stimulation of IL-10 receptors regulates numerous life- or death-signaling pathways, ultimately promoting cell survival by inhibiting both ligand- and mitochondrial-induced apoptotic pathways (Strle et al., 2001).

In conclusion, the results of this study suggest that there is a marked increase in IL-10 gene expression in meningitis. However, we found no genetic association of the IL-10 A-1082G, T-819C, and A-592C polymorphisms with meningitis. Nevertheless, additional studies with larger sample sizes are necessary to confirm our findings. As genetic polymorphisms often vary among ethnic groups, further studies are needed to clarify the association of the IL-10 polymorphisms with the risk of meningitis in diverse ethnic populations.

Footnotes

Disclosure Statement

No competing financial interests exist.

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