Association of Interleukin 18,Interleukin 2,and Tumor Necrosis Factor Polymorphisms with Subacute Sclerosing Panencephalitis

Introduction

S ubacute sclerosing panencephalitis (SSPE) is a progressive inflammatory and degenerative disorder of the central nervous system (CNS). It is caused by a mutant measles virus (MV) with an altered M protein, probably leading to a persistent viral infection (Gark, 2008). Several factors influence the risk of chronic brain infection with the mutant MV. Epidemiologic studies have shown that contracting the MV before 2 years of age increases the risk of SSPE. The early infection of an immature immune system and altered cellular and humoral factors reported in some patients suggest an abnormal immunologic host response with incomplete clearance of the first viral infection (Gascon, 1996; Tuxhom, 2004).

Most patients with SSPE exhibit a decreased MV-specific T helper (Th) 1 cytokine and preserved Th2 cytokine synthesis (Hara et al., 2000; Aydin et al., 2010). Additionally, previous immunohistochemical studies revealed that the cytokines mediating inflammation are expressed in SSPE brain lesions. The presence of interleukin (IL)-1β, IL-2, IL-6, tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) was demonstrated in the lesions (Anlar et al., 2001; Nagano et al., 2004; Aydin et al., 2010). These data indicated that these cytokines were produced in SSPE lesions and played an important role in the immunopathogenesis of SSPE.

IL-18 is a proinflammatory cytokine produced mainly by macrophages and monocytes, and it plays a crucial role in regulating both innate and acquired immunity and an important role in inflammatory and infectious diseases (Biet et al., 2002). There are two polymorphisms (–137 G/C and –607 C/A) in the IL-18 gene promoter region that seem to affect the expression of IL-18 at the transcription level (Giedraitis et al., 2001). IL-18, which is an important IFN-γ inducer, can activate immune cells, promoting the activation and proliferation of T and natural killer (NK) cells (Giedraitis et al., 2001; Biet et al., 2002). Stubblefield Park et al. (2011) suggested that IL-12 and IL-18 in the brain stimulate secretion of IFN-γ and reduce the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear the MV.

IL-2 is a pleiotropic cytokine that acts as a growth and differentiation factor for NK cells, some B lymphocytes, and lymphokine-activated killer cells. In addition, IL-2 activates monocytes to produce cytokines, particularly TNF-α (Pawlik et al., 2005; Elgert, 2009). IL-2 also increases the synthesis of other cytokines, including IL-4 and IFN-γ (Elgert, 2009). TNF-α is a powerful inducer of the inflammatory response, and it is a central regulator of innate immunity. Inflammatory responses to TNF-α are mediated both directly and through stimulation of other proinflammatory cytokines (Pawlik et al., 2005; Elgert, 2009; Pujhari et al., 2012).

The contributions of polymorphisms of many immunologically important genes encoding molecules such as the Toll-like receptor-3, IL-2, IL-4, IL-12, and MxA to susceptibility to the establishment of SSPE are controversial (Inoue et al., 2002; Torisu et al., 2004; Yilmaz et al., 2007). Evidence suggests that, as a result of genetic polymorphisms, individuals with SSPE exhibit an altered cellular response to common antigens, producing low levels of interferon, IL-2, IL-10, and IL-12, and higher levels of IL-4 and IL-1beta (Nagano et al., 1994; Yentur et al., 2005; Hara et al., 2000). The purpose of our study was to ascertain whether the IL-2, IL-18, and TNF-α gene polymorphisms contribute to the impaired immune response leading to the development of SSPE.

Materials and Methods

Results

The distribution of each genotype for IL-18, IL-2, and TNFα in the SSPE cases and controls is shown in Table 2. The distribution of genotypes in the controls was in the Hardy–Weinberg equilibrium. The frequencies of GG, GC, and CC genotypes for the IL-18 −137 G/C polymorphism were 46.3%, 46.3%, and 7.4%, respectively, in the cases and 75%, 22.2%, and 2.8%, respectively, in the controls. The distributions of the IL-18 −137 genotypes were significantly different between the cases and the controls (p=0.004). In the patients' group, the frequency of the IL-18 −607 AC genotype (50%) and AA genotype (18.5%) was higher than in the control group AC (25%) and AA (6.9%) genotype, and the increase was significant (p<0.001). However, there were no differences in the frequencies of the genotypes of IL-2 −330 and TNFα −308 between the two groups (p=0.406, p=0.950, respectively).

After grouping according to allele frequencies (Table 3), in the patients' group, the IL-18 −137 C allele and the IL-18 −607 A allele frequencies were found to be significantly different than in the control group (p=0.002, p<0.001, respectively). However, the frequencies of the IL-2 T and G alleles and the TNF-α G and A alleles were not significantly different between the two groups (p=0.79, p=1.0, respectively). When the haplotypes for the −137 and −607 polymorphisms were determined, the CA haplotype frequency was higher in the cases (26.9%) than in the controls (9.7%), and the increase was significant (p<0.001) (Table 4).

Discussion

Children with an inadequate primary immune response to the MV are at increased risk of developing SSPE (Schaulies and Reuter, 2010). Age is the strongest host factor associated with developing SSPE after exposure to aberrant measles infection (Gascon, 1996). In addition, people belonging to same age, sex, and ethnic group were exposed to the same MV strain, which could cause a broad spectrum of responses ranging from mild infection to severe or chronic SSPE infection. These data indicate that host genetic factors are responsible for the clinical outcomes of measles infection. Clearance of the MV requires a coordinated innate and adaptive humoral and cell-mediated immune response. A strong Th1 response, characterized by the production of IL-2, TNF-α, and IFN-γ, seems to be associated with the MV clearance (Hara et al., 2000; Schaulies and Reuter, 2010). In experimental MV-induced encephalitis, IFN-γ has been suggested to be a major antiviral cytokine (Patterson et al., 2002). IL-18 was first described as an IFN-γ-inducing factor, and it has multiple functions, including induction of synthesis of IFN-γ by T cells and NK cells, promotion of Th1-type immune responses, augmentation of proliferative response, and cytokine production of activated T cells (Strengell et al., 2003; Okamura et al., 1995).

In the present study, the people with the CC genotype at position −607 in the promoter of the IL-18 gene may be protected against persistent MV infection; moreover, the GC genotype at position −137 may be closely linked to SSPE. The A allele at position −607 and the C allele at position −137 of the IL-18 gene in the SSPE group were more frequent in cases than in controls and were associated with an increased SSPE risk. The risk for SSPE was more than 3.4 times higher in individuals with the GC genotype compared to the GG genotype for the IL-18 −137 polymorphism, and the risk of SSPE was more than 5.8 times higher in individuals with the AA genotype compared to the CC genotype for the IL-18 −607 polymorphism. In an attempt to confirm these findings, the haplotype frequencies were used to determine the genetic correlation between these SNPs. This analysis showed that the −137C/−607A haplotype frequency is higher in cases than in controls, and these results indicated that the −137C/−607A haplotype is a risk factor for developing the SSPE disease. To our knowledge, there is no published study of the role of the promoter of the IL-18 gene in SSPE, but some associations between the IL-18 gene polymorphisms and chronic infections such as hepatitis B and C have been described (Hirankarn et al., 2007; Bouzgarrou et al., 2008). Two SNPs in the promoter of the IL-18 gene, −607C/A and −137G/C, were predicted to be nuclear factor-binding sites for the cAMP-responsive element-binding protein and the H4TF-1 nuclear factor, respectively; moreover, mutation of the two sites can influence the expression of IL-18 and potentially of IFN-γ (Giedraitis et al., 2001). The IFN-γ signal is transduced within brain explants by the Jak/STAT signaling pathway. T cells directly clear the MV infection of the brain using a noncytolytic IL-12, IL-18, and IFN-γ-dependent mechanism in the CNS, and this mechanism relies upon Jak/STAT signaling (Stubblefield Park et al., 2011). Giedraitis et al. (2001) showed that the G allele of the −137 and the homozygous C at position −607 of the promoter of IL-18 were associated with elevated IL-18 expression and increased levels of IFN-γ mRNA in experiments in vitro. Considering our findings and those of Giedraitis et al., the −607C and −137G alleles of IL-18 gene appear to be related to high levels of IL-18, which may augment the production of IFN-γ, modulate the activity of NK and cytotoxic lymphocyte cells, and trigger complex immunological processes to eliminate the MV. We hypothesize that the −137C/−607A haplotype likely results in a lower transcription activity and a lower production of IL-18. A low production of IL-18 in the CNS leads to decreased elimination of the MV.

The results showed no significant differences between patients with SSPE and control subjects in the distributions of the genotypes or allelic frequencies for polymorphisms of the IL-2 and TNF-α gene promoters at positions −330 and −308. Carriers of the TNF-α −308A allele were known to produce a high level of TNF-α when compared with those of the G/G genotype (Abraham and Kroeger, 1999). In our study, the GG genotype at position −308 in the TNF-α promoter was very frequent in individuals with SSPE and the controls, but there was no significant association of SSPE with the TNF-α polymorphism. Some previous studies showed that the serum and cerebrospinal fluid levels of IL-2 and TNF-α were not different in SSPE patients and controls (Nagano et al., 1994; Anlar et al., 2001; Aydin et al., 2010). These findings, in conjunction with our polymorphism analysis, suggest that IL-2 and TNF-α are not associated with SSPE pathogenesis. However, Yilmaz et al. (2007) demonstrated that among the IL-2 −330 genotypes, the frequencies of the GG genotype and the G allele were low and suggested that a low frequency of IL-2 −330 G alleles may result in lower levels of IL-2 in SSPE patients.

In summary, to the best of our knowledge, this is the first analysis of polymorphisms of the IL-18 and TNF-α genes and risk for the SSPE disease. Our results revealed a significant association between the SSPE disease and the polymorphisms of the promoter region of the IL-18 gene at position −607 and −137 and the IL-18 CA haplotype. This finding may have implications for the theories of the pathogenesis of the disease as well as for therapeutic aspects. More studies are needed to confirm these findings in larger sample sizes and in SSPE patients from other ethnic groups.