Association between IL28B rs12979860 single nucleotide polymorphism and the frequency of colonic T_reg in chronically HCV-infected patients

Abstract

The IL28B gene is associated with spontaneous or treatment-induced HCV viral clearance. However, the mechanism by which the IL28B single nucleotide polymorphism (SNP) affects the extra-hepatic HCV immune responses and its relationship to HCV pathogenesis have not been thoroughly investigated. To examine the mechanism by which IL28B affects HCV clearance. Forty Egyptian patients with chronic HCV infection receiving an Interferon/ribavirin treatment regimen were enrolled into this study. There were two groups: non-responders (NR; n = 20) and sustained virologic responders (SVR; n = 20). The initial plasma HCV viral loads prior to treatment and IL28B genotypes were determined by quantitative RT-PCR and sequencing, respectively. Liver biopsies were examined to determine the inflammatory score and the stage of fibrosis. Colonic regulatory T cell (T_reg) frequency was estimated by immunohistochemistry. No significant association between IL28B genotypes and response to therapy was identified, despite an odds ratio of 3.4 to have the TT genotype in NR compared to SVR (95 % confidence interval 0.3–35.3, p = 0.3). Patients with the TT-IL28Brs12979860 genotype (unfavorable genotype) have significantly higher frequencies of colonic T_reg compared to the CT (p = 0.04) and CC (p = 0.03) genotypes. The frequency of colonic T_reg cells in HCV-infected patients had a strong association with the IL-28B genotype and may have a significant impact on HCV clearance.

Introduction

Chronic hepatitis C infection (CHC) is a global worldwide health problem, causing an increasing burden year by year, particularly in areas with high endemicity, such as Egypt [1–3]. The ultimate outcome of HCV infection is determined by the complex interaction of the host immune response, genetic predisposition and extrinsic modulators of clearance and progression.

Independent genome-wide association studies (GWAS) consistently identify variants within the IL28B gene region that are strongly associated with spontaneous and treatment-induced HCV clearance [4–9]. Of these, the rs12979860 single nucleotide polymorphism (SNP) is one of the strongest predictors [4]. However, the exact mechanism by which its protective effect are largely unknown.

The IL28B rs12979860 CC favorable genotype has a marked differential distribution between racial groups, being least frequent in African Americans, most frequent in Asians, and intermediately frequent in Hispanics and Caucasians [4, 9]. In HCV genotype-4-infected Egyptian patients, there appear to be conflicting reports on the association between IL28Brs12979860 polymorphism and responses to therapy or immune response. Some reports have supported the association with response to therapy [10–12]. Others have failed to identify an association with HCV-specific cell-mediated immune responses [13]. The reasons for these differences are not clear, but the number and the HLA types of the enrolled patients as well as the geographical site of the study with differences in genetic backgrounds may explain some of the controversy [14, 15].

The rs12979860 SNP is located in the promoter sequence, upstream of the IL-28B gene, which encodes a member of the IFN-λ family of cytokines [4], which includes IL-29 (IFN-λ 1), IL-28A (IFN-λ 2), and IL-28B (IFN-λ 3), all of which are encoded by a gene cluster on a chromosomal region mapped to 19q13 [16, 17]. More recently, a transiently induced gene near the IFN-λ3 gene has been described to contain a dinucleotide variant, ss469415590 (TT or ΔG), that is in high linkage disequilibrium with rs12979860. The ss469415590 [ΔG] allele is a frameshift variant that creates a novel gene, designated as IFN-λ4, which encodes the interferon-lambda 4 (IFN-λ4) protein [18, 19].

The ss469415590 [TT] allele, which abrogates the production of the IFN-λ4 protein, is considered a favorable haplotype for HCV clearance, while the ss469415590 [ΔG] allele is considered unfavorable [20].

Recent in vitro studies in mice [21] and humans [22], have suggested that IFN-λ promotes the generation of immature dendritic cells (DCs) with tolerogenic activity. IFN-λ-treated DCs specifically induced proliferation and expansion of regulatory T (T_reg) cells which play, a fundamental role in maintaining immune homeostasis. It has been reported that one of the potential mechanisms that might modulate HCV-specific immune responses is T_reg cells [23–25]. However, the relationship between the protective effect of IFN-λ during HCV infection and T_reg cells has not been explored. T_reg cells are characterized by the expression of a unique transcription factor called forkhead box protein 3 (FoxP3), which is highly expressed in the nucleus of T_reg cells and is generally accepted to be the single best marker for quantifying T_reg cells [25–28]. In cases of chronic hepatitis C (CHC), there is evidence that the frequency of T_reg cells is higher than in controls and is negatively correlated with the necro-inflammatory score [29–31]. The elevated frequency of T_reg cells may explain the weak HCV-specific T-cell responses in CHC [32]. Moreover, there is some evidence that individuals with CHC may harbour more T_reg cells in their peripheral circulation [33] and in the liver than those uninfected [34]. Recent data from our laboratory have suggested that colonic T_reg cells are negatively correlated with liver inflammation and HCV viral load, which supports a strong linkage between gut-derived T_reg and HCV infection [35, 36]. T_reg cells appear to assist in the maintenance of chronicity by inhibition of anti-HCV immune responses and consequently attenuate the intrahepatic tissue-damaging response to infection [26, 32].

The immunologic link between the gut and the liver through recirculation of a population of shared lymphocytes that are capable of homing to both the liver and gut via portal circulation has been reported [37].

The aim of our work was to determine the protective mechanism of the rs12979860 SNP in patients infected with HCV genotype 4 and its relationship to colonic T_reg cell frequency. In addition, the relationships between IL-28B polymorphism and viral persistence, degree of liver inflammation, and fibrosis were investigated.

Patients and methods

Patient recruitment

A cross-sectional study was conducted to evaluate the role of colonic T_reg cells and the IL28B rs12979860 SNP in CHC patients. All participants were recruited between January 2012 and September 2013 from Assiut Liver Institute outpatient clinics, Assiut, Egypt. Consent was obtained from all participants according to an IRB protocol approved by the Assiut University College of Medicine Institutional Review Board.

Forty patients with CHC who received interferon/ribavirin therapy were enrolled and divided into two groups according to the outcome of therapy. The first group included patients who were non-responders (NR group; n = 20), and the second group included patients with sustained virologic response (SVR group; n = 20) to the standard of care in Egypt during the time of enrollment. Inclusion criteria for the patients with CHC were positivity for HCV antibodies by ELISA and HCV RNA by real-time PCR. Exclusion criteria were pregnancy, history of Schistosoma infection, inflammatory bowel disease (IBD), and autoimmune disease. There was no exclusion was due to race, age, or gender. Twenty milliliters (ml) of blood was drawn from each subject. Figure 1 shows a flow chart describing the study design.

Fig. 1.

Flow chart for study of the role of IL28B and T_reg in HCV infection. A schematic presentation of the study design and the number of subjects enrolled in each group are shown

Colonic tissue biopsies

After proper preparation, three random colonic biopsies (from the descending colon, 1–3 mm in size, using biopsy forceps) were obtained from each patient via flexible sigmoidoscopy. The colonic biopsies were taken within an average of 6 months after completion of the anti-HCV therapy.

Serological and liver function tests

Liver function tests were performed using a Hitachi 911 chemical analyzer (Boehringer Mannheim, Germany). HBsAg, anti-HCV and anti-HIV were tested using commercially available microparticle enzyme immunoassay kits (AXSYM, Abbott Laboratories, Germany) as specified by the manufacturer.

Determination of HCV viral load by real-time PCR

The HCV viral load in patients’ plasma was determined using real-time PCR as described [38]. Quantitative measurement of HCV RNA by real-time PCR was performed using a PCR kit (Artus® HCV RG RT-PCR supplied by QIAGEN GmbH, Germany, Cat. No. 4518263) as specified by the manufacturer.

Assessment of liver inflammation and fibrosis

Liver biopsy specimens were obtained from all patients with CHC prior to treatment and used for assessment of liver inflammation and fibrosis. Hepatic histopathological findings were interpreted independently of clinical and biochemical data by a pathologist, according to the criteria described for the METAVIR scoring system [39].

Assessment of IL28B genotypes

Peripheral blood mononuclear cells (PBMCs) were used for DNA extraction and subsequent sequencing. A DNA Mini Kit (QIAGEN, GmbH, Germany) was used for DNA extraction from the PBMCs. A Taq PCR Master Mix Kit (QIAGEN) and IL28B forward and reverse primers were added to the extracted DNA to amplify the rs12979860 SNP, and the amplified DNA was then subjected to gel electrophoresis. The samples with visible bands were purified using a QIAquick PCR Purification Kit (QIAGEN) and sent to the DNA Core Facility (Massachusetts General Hospital, Cambridge, MA) for rs12979860 SNP sequence analysis.

Examination of the frequency of T_reg cells

The frequencies of T_reg and CD3⁺ T cells in colonic biopsies were determined using fluorescent immunohistochemistry as described previously [35, 36]. The number of colonic T_reg and CD3⁺ T cells was determined by using a Zeiss LASER scanning confocal microscope (LSM710) with high-power field (1HPF = 0.4 mm²). Ten HPFs were examined for each slide and averaged. Data were calculated as the percent frequency of T_reg as follows:

$$\%\text{ Frequency of }{\mathrm{T}}_{\text{reg}}\text{ cells}=\frac{\text{Avrerage number of }{\mathrm{T}}_{\text{reg}}\text{ cells}/\mathrm{H}\mathrm{P}\mathrm{F}}{\text{Avrerage number of }\mathrm{C}\mathrm{D}{3}^{+}\mathrm{T}\text{ cells}/\mathrm{H}\mathrm{P}\mathrm{F}}\times 100$$

Statistical analysis

Data for clinical and demographic characteristics are presented as range (minimum, maximum) or mean ± SD. Continuous variables (such as T_reg frequencies, liver fibrosis and inflammation, ALT, AST, platelet counts and viral load) were compared among the enrolled patients using Student’s t-test, or the Wilcoxon rank sum test, as appropriate, with significance set at p ≤ 0.05. The ANOVA test was used to examine differences between groups of enrolled subjects. Correction for multiple testing was done using the Tukey-Kramer method. Correlations between the parameters measured were calculated using Spearman’s correlation coefficient. All statistical analyses were done using Graph Pad Prism 6 Software (San Diego, California, USA).

Results

Characteristics of the study subjects

Demographic and clinical characteristics of the enrolled subjects are summarized in Table 1. Both the SVR and NR groups had a predominantly male patient population at 85 % each. There was no significant difference in platelet count (p = 0.4), ALT (p = 0.6), or AST (p = 0.4) levels between the two groups. Regarding the histopathologic grading, there was no significant difference in their METAVIR score prior to treatment.

Table 1.

Demographic and clinical data for the enrolled subjects

Demographic characteristics	NR group (N = 20)	SVR group (N = 20)
Sex
Male:female	17:3	17:3
Age
Range	21–54	20–51
Mean ± SD	43.5 ± 8.6	40.3 ± 7.8
HCV viral load [106 IU/ml, (Mean ± SD)]	0.9 ± 0.8	NA
Distribution of liver inflammatory grade
Grade 1–2	11	14
Grade 3–4	9	6
Distribution of liver fibrosis stage
Stage 1–2	13	18
Stage 3–4	7	2
ALT [IU/ml (Mean ± SD)]	37 ± 22.5	32.5 ± 26.7
AST [IU/ml (Mean ± SD)]	32.2 ± 23	25.9 ± 18.9
Platelet count [×103/µl (Mean ± SD)]	202.1 ± 64	217.7 ± 47

Relationship between the IL28Brs12979860 genotype and response to therapy

All 40 samples examined were sequenced for IL28B. Overall, 67.5 % of the patients were genotype CT, 22.5 % were genotype CC, and 10 % were genotype TT. A previous study suggested that the frequencies of these IL28B genotypes in healthy controls in Egypt are CT (52.1 %), followed by CC (29.2 %) and TT (18.8 %), which are comparable to those observed in our study [40]. The NR genotyped group was distributed as 4 (20 %) CC, 13 (65 %) CT, and 3 (15 %) TT. Likewise, the SVR group included 5 (25 %) CC, 14 (70 %) CT, and 1 (5 %) TT. The T allele was less frequent (75 %) in the SVR group, while the favorable C allele was more frequent (95 %) in the same group when compared to the NR group. There was no significant association between the IL28B genotype and response to therapy, despite an odds ratio of 3.4 to have the TT genotype in NR compared to SVR (95 % confidence interval 0.3–35.3, p = 0.3) (Table 2).

Table 2.

Correlation between IL28B genotypes and clinical parameters of HCV4-infected patients

Clinical parameter	IL28B genotype			P-value
	TT	CT	CC	TT vs CT	TT vs CC	CT vs CC
CD3+ T cells (Avg ± SD)	14 ± 9.2	12.37 ± 6.58	13.11 ± 7.94	0.66	0.86	0.78
Total Treg (Avg ± SD)	4 ± 4.08	1.81 ± 2.29	1.33 ± 2.24	0.12	0.15	0.59
Treg frequency (Avg ± SD)	0.58 ± 0.11	0.15 ± 0.16	0.17 ± 0.15	0.04	0.03	0.76
Viral load (Copies/ml) (Avg ± SD)	958000 ± 1354816	687434 ± 984094	97350 ± 90110	0.72	0.80	0.26
Biopsy-inflammation grade (Avg ± SD)	2.0 ± 1.15	2.1 ± 0.83	1.9 ± 0.93	0.83	0.87	0.55
Biopsy-fibrosis stage (Avg ± SD)	2.0 ± 1.15	1.8 ± 0.79	1.6 ± 0.73	0.66	0.46	0.51
ALT (SGPT) (Avg ± SD)	36.8 ± 21.36	36.2 ± 25.97	29.4 ± 22.53	0.97	0.59	0.49
AST (SGOT) (Avg ± SD)	19.0 ± 11.34	30.5 ± 20.61	29.0 ± 25.74	0.29	0.48	0.86
Platelet count (Avg ± SD)	219.3 ± 52.94	209.4 ± 61.58	207.4 ± 46.25	0.76	0.69	0.93
No response to therapy (NR)	31	13	4
Response to therapy (SVR)	1	14	5

Bold means significant values (P < 0.05)

¹Odds ratio = 3.4, p = 0.3, 95 % confidence interval 0.3–35.3

Correlation between the IL28B genotype and HCV pathogenesis

No significant differences between the CD3⁺ T cell count, METAVIR inflammation and fibrosis score, ALT, AST, and platelet counts were observed when comparing TT vs. CT, TT vs. CC, and CT vs. CC, as shown in Table 2.

Correlation between the IL28B genotype and colonic T_reg cell frequency

Forty patients were examined for colonic T_reg cell frequency, 20 from the NR group and 20 from the SVR group. Colonic T_reg cells were detected by indirect fluorescent immunohistochemical staining of CD3⁺ and FoxP3. Bivariate analysis showed significant differences in the frequency of colonic T_reg in patients with TT vs. CT (p = 0.04) and TT vs. CC genotypes (p = 0.03). The average T_reg cell frequency was almost threefold higher in the TT genotype group (mean ± SD = 0.58 ± 0.11 %) compared to the CT genotype (0.15 ± 0.16 %) and CC genotype groups (0.17 ± 0.15 %; Fig. 2C). From a univariate regression model, we confirmed a significant effect of IL28B genotype on the frequency of colonic T_reg cells (Table 2). On the other hand, there was no significant difference in the number of CD3⁺ T cells or the total number of T_reg cells (Table 2).

Fig. 2.

Characterization of colonic T_reg cells by double IF staining with anti-CD3 and anti-FoxP3 antibody. Colonic T_reg cells were detected by indirect fluorescent immunohistochemical staining of CD3 and FoxP3 using mouse monoclonal anti-human CD3 antibody [PS1] and rabbit monoclonal anti-FoxP3 antibody [SP97]. A. T_reg cells identified by green surface CD3 (by Alexa Fluor® 488-donkey anti-mouse secondary antibody) or red nuclear FoxP3 (by Alexa Fluor® 594 donkey anti-rabbit secondary antibody). (B.). Examples of colonic T_reg in TT, CT and CC genotype patients. C. The frequency of colonic T_reg cells in the study groups in relationship to IL28B genotype

Discussion

In this study, we specifically examined the gut immune response and its relationship to HCV infection. The IL28B rs12979860 SNP is known to have a substantial effect on HCV pathogenesis. There is strong evidence from our laboratory that colonic T_reg cells are negatively correlated with liver inflammation and HCV viral load, which suggests a strong linkage between gut-derived T_reg cell populations and HCV infection [35, 36]. To investigate the mechanism by which the IL28B genotype affects the HCV gut immune response, we examined both the association between HCV clearance and IL28B rs12979860 genotype and the frequency of colonic T_reg cells in treated HCV-4-infected patients.

The rs12979860 SNP has been identified as a significant predictor of SVR in chronic HCV patients that have undergone standard IFN-α therapy [4]. The rs12979860 SNP can also predict natural clearance of HCV [9]. Moreover, it has been shown that IL28B induction upon IFN stimulation is significantly lower in patients with IL28B-unfavorable genotypes (rs12979860 CT/TT) than those with an IL28B-favorable genotype (CC). IL28B induction was also lower in non-responders than in relapsers, and lower in non-sustained virologic responders to triple therapy including NS3 protease inhibitors [41]. This suggests that specific genetic differences in humans play a significant role in modulating the HCV-specific immune response. Our data showed a trend towards the TT genotype in NR subjects (OR = 3.3), but it was not statistically significant (Table 2), probably due to the small number of subjects examined. Prior research on the relationship of IL28B genotypes to treatment responses in HCV-4-infected patients has shown that patients with the TT genotype had a lower cure rate, similar to that observed in our study [42].

The exact mechanism by which IL28Brs12979860 SNP affects the immune system or facilitates specific HCV antiviral responses is unclear. To identify the potential role of IL28Brs12979860 and its relationship to colonic T_reg cells in patients infected with HCV, we investigated the complex interplay between anti-HCV immune responses and genetic polymorphisms. We found a statistically significant association between colonic T_reg frequencies and rs12979860 genotype TT compared with both CC and CT. The T_reg cell frequencies are higher in TT compared to the CT and CC genotypes. This significant relationship was verified using a univariate regression model for IL28B genotypes on colonic T_reg cells. A recent report suggested that the TT genotype is associated with an increase in IL28B induction upon IFN stimulation and lower levels of several mRNAs of genes [43]. Some of those genes, such as SOCS1, SOCS3, and IRF1, are associated with T_reg induction and maintenance [44–46]. Therefore, suppression of those genes leads to an increase in T_reg frequency, with subsequent suppression of the HCV-specific immune response and failure of anti-HCV therapy in patients with the IL28B TT genotype.

Additionally, the mechanism responsible for elevated colonic T_reg in patients with the TT genotype may be related to the precise location of rs12979860 in the promoter region of the IL28B gene. The TT genotype might favor increased IL28B gene expression and secretion of interferon λ, resulting in stimulation of immature DCs and a consequent increase in T_reg frequencies. Mennechet and Uze reported that interferon λ specifically stimulates DCs differentiation and migration to lymphoid organs and induces the proliferation of IL-2-dependent T_reg cells [21]. This relationship between IL28B polymorphisms and T_reg cell frequency may provide insight into the immunologic differences engendered by the genetic makeup of each patient.

T-cells also play a pivotal role in hepatic necro-inflammation and subsequent fibrosis in an effort to limit viral replication [47–50] by suppressing HCV-specific immune responses [51]. Since IL-28B rs12979860 SNP interacts intimately with the immune system, we investigated the correlations between liver histopathology and IL28B genotypes. No statistically significant relationship between IL28B and liver inflammation could be identified. Prior reports examining IL28B rs12979860 SNP association with liver inflammation and fibrosis in HCV-4 patients are controversial. Some researchers have found no substantial correlation between the rs12979860 polymorphism and necroinflammatory scores [10, 11, 42, 52]. However, other researchers found that the T allele is associated with liver fibrosis [53]. In contrast, D’Ambrosio et al. found the CC genotype to be independently associated with more severe portal inflammation [52], albeit in HCV-1 patients. The reasons for those differences are not clear, but the genetic backgrounds of the studied patients as well as the genotype of the HCV may explain some of the controversy [14, 15]. Larger studies are needed to carefully dissect the relationship between liver inflammation and IL28B genotypes in HCV-4-infected patients.

To the best of our knowledge, our study is the first to link IL28B genotypes with the adaptive arm of the gut immune response in HCV patients. The importance of this study lies within the discovery of complex interactions between the underlying human genomics, the inherent immune response, and its defense against viral pathogens. In this study, we establish a strong relationship between high colonic T_reg cell frequencies and IL-28B rs12979860 TT.

Limitations of this study include the relatively small sample size and the cross-sectional nature of our study cohort. Our results may serve as a benchmark for future investigations that are longitudinal in nature and explore the natural history of HCV infection and clearance. Based upon our findings, we recommend a more in-depth exploration of the effect of IL28B genotypes on the liver-gut immune axis. In conclusion, the frequency of colonic T_reg cells in HCV-4-infected patients had a strong association with the IL-28B genotype.

Abbreviations

GWAS

Genome-wide association studies

SNP

Single-nucleotide polymorphism

PBMCs

perIpheral blood mononuclear cells

T_reg

Regulatory T cells

CHC

Chronic hepatitis C

HCV

Hepatitis C virus

HCV-4

Hepatitis C virus genotype 4

SVR

Sustained virologic response

DCs

Dendritic cells

T_reg

T regulatory

FoxP3

Forkhead box protein P3

IBD

Inflammatory bowel diseases

NR

Non-responder

Peg-IFN + Rib

Pegylated interferon-α and ribavirin

HPF

High-power field, 1 HPF=0.4 mm²

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

IF

Immunofluorescence