1 INTRODUCTION

Hepatitis B virus (HBV), which is in the Hepadnaviridae family, still continues to be an important health problem due to a number of liver diseases such as acute and chronic hepatitis, cirrhosis, hepatocellular cancer (HCC).^1-3 With the integration of HBV into the host genome, nonfunctional T cell responses and deleterious immunopathology are triggered.⁴ Although the main mechanisms responsible for the natural course of HBV infection are not clear, the genetic factors of infected individuals play a crucial role in the immunopathogenesis of HBV infection.^{5, 6} Studies show that especially single nucleotide polymorphisms (SNP; substitution of one nucleotide with another nucleotide) in various immune cytokines and receptor genes play an important role in immunity, clinical course, and recovery from HBV infection.^{7, 8}

CC chemokine receptor 5 (CCR5) is expressed on the surface of natural killer (NK) cells, CD8+ T lymphocytes, and macrophages, and plays an important role in the migration of interferon-gamma (IFN-ɣ)-producing CD4+, CD8+, and NK cells to the liver after inflammation in chemotaxis and naturally throughout the virus clearance process.^{9, 10} In addition, CCR5 can contribute to the immunopathology of inflammation by taking part in the regulatory and memory T cell responses.¹¹ CCR5 function is altered by various polymorphisms, one of which is a 32-base pair (bp) deletion (Δ32) that causes a frameshift in the coding sequence of the receptor's second extracellular loop, resulting in loss of function of the prematurely truncated protein.¹² CCR5Δ32 polymorphism is found to be related for resistance or susceptibility to various viral diseases, such as hepatitis C, human immunodeficiency virus, influenza viruses.^13-15 Studies indicated that CCR5Δ32 polymorphism also can be involved in clinical outcomes, severity of hepatitis, and resistance/protective effect to HBV infection in different populations in many countries.^16-18 However, there is no scientific data about the relation of CCR5Δ32 polymorphism and clinical outcome of HBV infection in Turkish population.

Toll-like receptors (TLRs), one of the pathogen recognition receptors, recognize pathogen-associated molecular structures and activate innate immune mechanisms against pathogens.^{19, 20} TLR3, a member of the TLR family, has been shown to have a role in the recognition of HBV.²¹ A defective or decreased TLR3 response can lead to a weakening of the immune response against HBV.²² It is reported that a number of TLR3 polymorphisms including rs5743313 are associated with severity of some viral diseases, that is, enteroviruses, influenza viruses, and HBV by reducing the efficiency of the virus recognition mechanisms.^23-25 TLR3-rs5743313 is an intronic polymorphism, but is located near exon 4 which encodes transmembrane signal induction region of the receptor. It is thought to play an essential role in receptor function by reducing the efficiency of the virus recognition mechanisms.²³ A study showed that TLR polymorphisms including rs5743313 is significantly associated with Saudi Arabian chronic hepatitis B (CHB) patients.²⁵ However, relation between TLR3 rs5743313 polymorphism and clinical outcome of hepatitis B has not been investigated in Turkish population.

Non-cytopathic HBV-induced liver damage and viral control are regulated by the immune system.^{26, 27} The host coordinates the response against inflammatory diseases by regulating the proliferation, differentiation, and function of immune cells with the antiviral cytokines it produces to limit HBV infection.^26-28 It is well known that cytokines such as IFN-ɣ and TNF-alpha play a central role in the clearance of acute infection or the persistence of HBV.²⁹ Serum IFN-ɣ levels are found to be higher in CHB patients in comparison to healthy controls and increased serum levels of IFN-γ are correlated with HBV-related active hepatitis.^{30, 31} To date, there is no study for investigation for relation of serum IFN-ɣ levels of hepatitis B patients and CCR5Δ32 and TLR3 (rs5743313) polymorphisms by comparison the immune and CHB groups.

The aim of our study was to detect the association of CCR5 (CCR5Δ32) and TLR3 (rs5743313) functional gene polymorphisms and serum IFN-ɣ level in patients infected with HBV who are subsequently seroconverted (immune patients) and in patients receiving treatment for CHB disease (CHB patients). Due to lack of knowledge of the role of CCR5 (CCR5Δ32) and TLR3 (rs5743313) polymorphisms in HBV infected Turkish population, this study aimed to investigate the association of these polymorphisms with clinical course of HBV infection in Turkey.

2 MATERIALS AND METHODS

3 RESULTS

3.2 CCR5Δ32 polymorphism and TLR3 gene rs5743313 polymorphism results

For the CCR5Δ32 deletion Wt/Wt genotype was defined as homozygosity for the CCR5 wild-type allele; the Wt/Δ32 genotype was defined as heterozygosity for the CCR5 wild-type allele and the CCR5Δ32 deletion; the Δ32/Δ32 genotype was defined as homozygosity for CCR5Δ32 deletion (Figure 1). The genotype results of conventional PCR were confirmed by Sanger sequencing (data not shown). Wt/Wt genotype was detected in 95 out of 100 patients and 87 out of 100 patients in immune group and CHB group, respectively. Homozygous Δ32 genotype was detected only in 1 patient in CHB group (Table 3). The genotype distribution was in HWE (Wt/Wt 182 observed [O], 181.5 expected [E]; Wt/Δ32 17 O, 18.1 E; Δ32/Δ32 1 O, 0.5 E).

Table 3. Statistical evaluation of the distribution of CCR5Δ32 and TLR3_rs5743313 polymorphism genotypes in patients according to groups

CCR5 Δ32	Immune group	Chronic hepatitis B group	p Value	TLR3 rs5743313	Immune group	Chronic hepatitis B group	p Value
Wt/Wt	95	87	0.048a	CC	62	56	0.388a
Wt/Δ32	5	12	0.076a	CT	34	39	0.463a
Δ32/Δ32	0	1	-	TT	4	5	0.733a
Wt/Δ32 + Δ32/Δ32 versus Wt/Wt	5−95	13−87	0.048a	CT + TT versus CC	38−62	44−56	0.388a
Δ32/Δ32 versus Wt/Δ32 + Wt/Wt	0−100	1−99	-	TT versus CT + CC	4−96	5−95	0.733a
Total
Wt allele frequency	0.975	0.93	0.088a	C allele frequency	0.79	0.755	0.611a
Δ32 allele frequency	0.025	0.07	0.194a	T allele frequency	0.21	0.245	0.502a
OR; (95% CI)
Wt/Δ32 + Δ32/Δ32 versus Wt/Wt	2.839; (0.972−8.290)		0.056	CT + TT versus CC	1.282; (0.728−2.255)		0.388
Δ32/Δ32 versus Wt/Wt + Wt/Δ32	-		-	TT versus CT + CC	1.263; (0.329−4.848)		0.733
Δ32 versus Wt allele frequency	2.936; (1.037−8.311)		0.043	T versus C allele frequency	1.221; (0.764−1.951)		0.4

• Note: p value < 0.05 was considered statistically significant as bold.
• Abbreviations: CCR5, CC chemokine receptor 5; CI, confidence intervals; OR, odd ratio; TLR, toll-like receptors.
• ^a χ² test.

Wt/Wt genotype was statistically higher in immune group compared to CHB group (p = 0.048, χ² test) for CCR5Δ32 polymorhism. There was no statistically significant difference between the groups in Wt/Δ32 genotype (p = 0.076, χ² test). The total incidence of Wt/Δ32 and Δ32/Δ32 genotypes was 5% in immune group and 13% in CHB group, and this difference was statistically significant (p = 0.048, χ² test). The OR with 95% confidence intervals (OR, 95% CI) were calculated for each polymorphism using three genetic association models, including dominant, recessive, and allele models. There was no statistically significant difference between two groups for both polymorphisms, except; Δ32 allele frequency was statistically higher in immune group compared to CHB group (OR: 2.963, 95% CI: 1.037−8.311; p = 0.043) (Table 3).

For TLR3 gene rs5743313 polymorphism, CC, CT, and TT genotypes, including C wild type allele and T variant allele, were determined (Figure 2). Genotype and allele frequencies of TLR3 rs5743313 in immune and chronic hepatitis groups are shown in Table 3. For TLR3 gene rs5743313 polymorphism no statistically significant difference was found when CT, TT, and CC genotypes were compared for both groups. The genotype distribution was in HWE (p = 0.587).

There was a higher rate of the CCR5Δ32 Wt/Wt genotype in the HBeAg negative patients compared to the HBeAg positive patients (p = 0.007). There was no statistically significant difference between the viral load, ALT and AST levels, histological activity index (HAI), and fibrosis score with CCR5Δ32 and TLR3 rs5743313 genotypic polymorphisms distribution in patients of CHB group (Table 4).

Table 4. Statistical evaluation of CCR5Δ32 and TLR3 rs5743313 polymorphism genotype distributions according to HBeAg, viral load, HAI, fibrosis score, ALT, AST levels in patients of chronic hepatitis B group

	Genotypes of polymorphism	HBeAg positive	HBeAg negative	p Value	Viral load ≤105 copies/mla	Viral load >105 copies/mla	p Value
CCR5	Wt/Wt	8	79	0.007*	30	57	0.362*
	Wt/Δ32	2	10		5	7
	Δ32/Δ32	1	-		1	-
TLR3	CC	8	48	0.541*	22	34	0.684*
	CT	3	36		12	27
	TT	-	5		2	3

		HAI ≤ 5	HAI > 5		Fibrosis ≤ 2	Fibrosis > 2
CCR5	Wt/Wt	24	63	0.819*	63	24	0.244*
	Wt/Δ32	2	10		9	3
	Δ32/Δ32	1	-		1	-
TLR3	CC	11	45	0.244*	37	19	0.393*
	CT	12	27		31	8
	TT	2	3		3	2

		ALT ≤ 40 IU/Lb	ALT > 40 IU/Lb		AST ≤ 40 IU/Lb	AST > 40 IU/Lb
CCR5	Wt/Wt	52	35	0.067*	58	29	0.402*
	Wt/Δ32	8	4		10	2
	Δ32/Δ32	1	-		1	-
TLR3	CC	38	18	0.149*	37	19	0.723*
	CT	19	20		28	11
	TT	4	1		4	1

• Abbreviations: ALT, alanine transferase; AST, aspartate transferase; CCR5, CC chemokine receptor 5; HAI, histological activity index; HBV, hepatitis B virus; TLR, toll-like receptors.
• ^a Pretreatment HBV DNA.
• ^b Pretreatment ALT-AST.
• * χ² test.

4 DISCUSSION

In the clinical course of HBV infection, spontaneous clearance of HBV occurs in most of the HBV-infected patients, while the disease becomes chronic in some patients and causes severe liver damage.^{2, 3} This variable pattern of infection is thought to be due to environmental, virological, immunological, and host genetic factors. Although the effects of virological and immunological factors are well known, the effects of host genetic factors on HBV infection are not clearly understood.⁷ In this present study, CCR5Δ32 and TLR3 rs5743313 polymorphisms are investigated throughout the two different hepatitis B patient groups: one is spontaneously healed and became immune against HBV infection, and the other is CHB patients who are receiving antiviral therapy.

CCR5 plays a critical role in regulating T cell functions by mediating the migration and activation of antiviral type 1 cytokine-secreting T helper and cytotoxic T cells. It is thought that the inability of IFN-ɣ secreting T cells to function fully as a result of the CCR5Δ32 polymorphism, which leads to a decrease in the functional CCR5 receptor, increases the risk of CHB and C diseases.^{27, 34} In our study, CCR5Δ32 homozygosity was found only in 1 patient in CHB group, while CCR5Δ32 homozygosity and heterozygosity were found to be 5% in immune group and 13% in CHB group, and this difference was statistically significant (p = 0.048) and Δ32 allele frequency was higher than Wt allele frequency in chronic hepatitis group (OR: 2.936; 95% CI: 1.037−8.311) (Table 3). We can speculate that in our cohort, patients who had CCR5Δ32 allele has approximately three times the odds of having chronic HBV infection.

The significant elevation of the CCR5Δ32 polymorphism in CHB group supports the hypothesis that the CCR5 receptor cannot fully function and may increase the risk of CHB disease. Suneetha et al.¹⁶ reported that patients with CHB disease and healthy individuals were compared and CCR5Δ32 heterozygosity was found to be higher in patients with CHB compared to healthy controls (4.2% vs. 0.73%, p = 0.05), and CCR5Δ32 heterozygosity was associated with susceptibility to HBV infection. In some studies, however, such a relationship could not be demonstrated, possibly due to the very low frequency of CCR5Δ32 polymorphism in the ethnic populations studied.^{18, 35, 36} On the contrary to these findings, Thio et al.³⁷ reported that the CCR5Δ32 allele was detected more frequently in those who recovered from the infection compared to those with CHB infection (13% vs. 7.4%), and it was found that the CCR5Δ32 polymorphism reduced the risk of developing CHB infection by half. Abdolmohammadi et al.¹⁷ showed that CCR5Δ32 polymorphism was higher in healthy controls than in HBV-infected patients, and it was stated that CCR5Δ32 polymorphism may have a protective effect against HBV infection, at least in the Iranian population.

Because the frequency of CCR5Δ32 polymorphism is highly variable among different populations, the effect of this polymorphism in one population may not be valid for other populations.¹¹ In 1.3 million stem cell donors from 87 countries where the CCR5Δ32 polymorphism was investigated, the CCR5-Δ32 allele was found to be more common in European countries (e.g., 16% in Norway, 11% in Germany), and lower in Asian and African countries. In the same study, the frequency of CCR5-Δ32 allele was found to be 3.4% in 36 036 individuals in Türkiye.³⁸

Various factors such as the inhibitory effects of HBV, genetic variations, and epigenetic factors have been reported to affect TLR3 expression.²² SNPs in the TLR3 gene are thought to be among the factors affecting susceptibility to viral pathogens, including HBV.³⁹ In our study, assuming that TLR3 gene rs5743313 SNP may be associated with recovery from HBV infection or becoming chronic, we determined three genotypes for TLR3 gene rs5743313 polymorphism, namely CC, CT, and TT. However, we did not detect any significant difference between the groups (Table 4). We thought that TLR3 gene rs5743313 polymorphism had no significant effect on hepatitis B infection. Contrary to our study, some SNPs in the TLR3 gene have been associated with susceptibility to various diseases, including CHB and HCC. For example, in the study of Al-Qahtani et al.,²⁵ the relationship of 9 SNPs in the TLR3 gene with HBV infection was examined, and when haplotype analysis was performed, it was stated that the GCGA haplotype in 4 SNPs (rs5743313, rs5743314, rs5743315, rs1879026) had a significant effect on susceptibility to HBV infection. It was thought that the rs1879026 polymorphism may also have a protective role against the development of CHB infection. In the study of Ezzel-Din et al.,⁴⁰ the distribution of 6 SNPs, including rs5743313, in the TLR3 gene was examined, and GCTCCA and CCA haplotype frequencies were found to be significantly higher in the group with HBV clearance compared to CHB patients, and it was stated that these polymorphisms may have a protective role against HBV infection. In the study of Huang et al.,⁴¹ it was shown that TLR3 gene rs3775290 polymorphism may be a protective factor for CHB disease, HBV-associated liver cirrhosis and HCC. In the study of Fischer et al.,⁴² it was determined that the risk of developing CHB disease increased in the presence of TLR3 gene rs3775291 and rs5743305 polymorphisms. In the study of Chen et al.,⁴³ TLR3 gene rs377529 polymorphism was shown to be associated with a significant decrease in the risk of HBV-related HCC. In the study of Rong et al.,¹⁹ it was stated that the TLR3 gene C234T polymorphism is associated with a predisposition to CHB.

One can speculate that larger samples should be studied to fully understand the effect of TLR3 gene rs5743313 polymorphism and other TLR3 gene SNPs on HBV infection. Studies in different stages of HBV infection and in different ethnic populations are important in understanding this.

IFN-ɣ is produced from innate (e.g., NK cells, NKT cells, macrophages, and myelomonocytic cells) and adaptive (e.g., TH1 cells, cytotoxic T lymphocytes, and B cells) immune system cells.⁴⁴ Resolution of HBV infections is dependent on the killing of infected cells by viral antigen-specific cytotoxic T lymphocytes, and most likely interferons, including IFN-ɣ, and proinflammatory cytokines such as TNF-α, inhibiting viral replication by non-cytolytic pathways.⁴⁵ In our study, serum IFN-ɣ level was significantly higher in immune group patients (mean 75 ± 89 ng/ml) compared to CHB group patients (mean 4.35 ± 17.27 ng/ml) (p < 0.001) (Table 5). In two separate studies, IFN-ɣ levels were found to be higher in patients with HBsAg spontaneous seroconversion than in patients with CHB.^{46, 47} In the study of Chu et al.,³¹ IFN-ɣ level was found to be significantly higher in acute hepatitis B patients compared to CHB patients, and it was thought that this may be related to the sensitization of cytotoxic T lymphocytes by HBV antigens and then induction of IFN-ɣ production. Our results, which are consistent with these studies in which the IFN-ɣ level was found to be higher in patients with HBsAg seroconversion compared to the patients in the CHB group, suggests that IFN-ɣ is secreted from CD8+ T cells and helps to ensure clearance of the virus in acute hepatitis B recovery.

In conclusion, the higher CCR5Δ32 allele frequency in patients with CHB was considered as a sign of progression to chronic hepatitis. However, it is clear that disease stage, ethnicity, and many other determinants are also important in disease progression. Studies on larger populations with different polymorphisms in different regions are needed to clearly determine the effects of CCR5 and TLR3 genes and IFN-ɣ level on the severity of hepatitis B infection.

AUTHOR CONTRIBUTIONS

Burçin Tuncel, Emel Aksoy, and Ahmet Kürşat Azkur performed the experiments. Burçin Tuncel, Sedat Kaygusuz, Derya Beyza Sayın, Emel Aksoy, and Ahmet Kürşat Azkur analyzed the data, interpreted the results, and wrote the manuscript. Derya Beyza Sayın, Ahmet Kürşat Azkur, Burçin Tuncel, and Sedat Kaygusuz designed this study.

CONFLICT OF INTEREST

The authors declare no conflict of interest.