ORIGINAL INVESTIGATION
The impact of KIR–HLA genotype on hepatitis B virus clearance in Iranian infected individuals
Alireza Shah‑Hosseini1 · Mohammad Jafari1,2 · Asadollah Mohammadi1,3 · Roozbeh Sanaei1 · Seyed Moayed Alavian4 · Amin Doosti‑Irani5 ·
Mehrnoush Nooradeh Keykavousi6 · Nader Tajik1
Received: 2 July 2017 / Accepted: 21 September 2017 / Published online: 14 October 2017
© Springer-Verlag GmbH Germany 2017
significantly elevated in recovered individuals when com- pared with both control and patient groups. Also, KIR2DL5, and KIR3DP1 full were escalated in recovered individuals in comparison with patient groups. In addition, HLA-Bw4 ligand and HLA-A Bw4 were highly frequent in recovered individuals compared with healthy controls. Furthermore, the KIR3DS1 + HLA-Bw4, KIR3DS1 + HLA-Bw4Iso80, and KIR3DS1 + HLA-A Bw4 genotypes were significantly more common in recovered individuals than both healthy control and patient groups. Interestingly, AA genotype had less frequency and Bx had higher frequency in recovered individuals compared with both healthy control and patient groups. Our findings suggest a potential impact of the NK cells’ activating phenotype that leads to the HBV clearance in infected individuals.
Keywords Genotyping · HBV infection · HLA ligand genes · KIR genes · NK cells
Introduction
Hepatitis B virus (HBV) infection is one of the serious issues in the world and almost 2 billion people around the world have encountered this virus. This virus infection is one of the top 10 causes of mortality throughout the world and infec- tions of 686 thousand people are dying from this infection, annually [WHO 2016, available at: http://www.who.int/
mediacentre/factsheets/fs204/en/ (Accessed 28 July 2016)].
HBV infection could be seen in different forms including acute and chronic infection, cirrhosis and finally cause Hepa- tocellular carcinoma. Because HBV is not cytopathogenic, the interactions between virus and immune system play an important role in pathogenesis, inflammation, necrosis and fibrosis of the liver tissue [1, 2]. It is commonly accepted Abstract Killer cell immunoglobulin like receptors (KIRs)
have a principal role in regulating the effector functions of NK cells, particularly in viral infections. The major ligands for KIRs are human leukocyte antigen (HLA) class I mol- ecules. The aim of this study is to investigate the possible association of KIR genes, their known HLA ligands and com- pound KIR–HLA genotypes with hepatitis B virus (HBV) infection. Our study group consisted of 202 Iranian HBV- infected patients (52 spontaneously recovered, 50 asympto- matic carriers, 50 chronic sufferers and 50with liver cirrho- sis) and 100 ethnic-matched healthy control subjects. KIR and HLA genotyping was performed by a polymerase chain reaction–sequence-specific primer (PCR–SSP). The frequen- cies of the KIR2DL5A, KIR2DS1, and KIR3DS1 genes were
Alireza Shah-Hosseini and Mohammad Jafari are contributed equally to this work.
* Nader Tajik [email protected]
1 Immunology Research Center (IRC), Institute of Immunology and Infectious Disease, Iran University of Medical Sciences, Tehran, Iran
2 Cellular and Molecular Research Center, Gerash University of Medical Science, Gerash, Iran
3 Inflammation and Inflammatory Disease Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
4 Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran, Iran
5 Department of Epidemiology, School of Public Health, Hamadan University of Medical Science, Hamadan, Iran
6 Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
that HBV-specific T cells play an important role in chronic HBV infection-induced hepatocellular damage; [3, 4] how- ever, recent studies suggest that other innate immune effector mechanisms could be responsible for HBV recognition, viral clearance and liver pathogenesis [5, 6]. As a crucial element of innate immunity, NK cells fight against viral infection.
Previous studies have indicated increased cytotoxicity of NK cells in acute phase of HBV infection, which in animal models could prevent HBV proliferation through TNF-α and IFNγ secretion [7–9]. The elevated level of IFNγ seems to be a definite factor of activating the adaptive immunity which could affect the result of HBV infection [10, 11].
Killer cell immunoglobulin-like receptors (KIRs) are a family of highly polymorphic activating and inhibitory receptors found on the cells surface of NK cells and a certain subsets of T cells which interact with HLA class I antigens [12]. The regulation of NK cells performance is partially done by inhibitory and activating KIRs. Thus, the outcome of these receptors’ signaling determines the appropriate type of performance in NK cells [8]. The KIR genes are located in the leukocyte receptor complex (LRC) that spans a region of about 150 kb on chromosome 19q13.4 [13–15]. There are seventeen different KIR genes recognized in human body including 9 inhibitory genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B, 3DL1, 3DL2, 3DL3), 6 activating genes (2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1) and 2 pseudo- genes (2DP1, 3DP1) [8, 9]. The KIR genes are arranged on the inherited haplotypes that differ in number and type, and considerably vary among different populations. The KIR haplotypes could be divided into two groups based on gene content which includes: group A and group B KIR haplo- types. The group A haplotypes are the most prevalent KIR haplotypes in human body that have a fixed content of seven KIR genes [six inhibitory KIR (iKIR) genes plus one activat- ing KIR (aKIR) gene] and two pseudogenes, which are diver- sified through allelic polymorphisms [16]. The group B hap- lotypes include more than one aKIR gene and are diversified by both gene content and allelic polymorphisms. NK cells of individuals with B haplotypes may express more activating receptors, therefore, could have stronger responses to patho- genic agents [9]. The KIR receptors interact with HLA class I molecules on the surface of target cells and provide activat- ing or suppressing signals to regulate the activation of NK cells, thereby playing an important role in antiviral immunity [17]. HLA-C antigens are the main ligands for the inhibitory KIRs. In terms of KIR recognition, all the allelic variants of HLA-C could be divided into two groups on the basis of alternative amino acids at position 80 of the extracellular domain; HLA-C group 1 alleles (HLA-C1) have an aspara- gine at this position, whereas HLA-C group 2 alleles (HLA- C2) have a lysine. KIR2DL1 interacts with HLA-C2 mol- ecules, whereas KIR2DL2 and KIR2DL3 prefer HLA-C1 [18]. Inhibitory signals launched by KIR2DL1 + HLA-C2
interaction are stronger than KIR2DL2/3 + HLA-C1 inter- action caused signals. KIR3DL1 recognizes the Bw4 motif, that is present on 40% of the HLA-B allotypes [19, 20], and certain HLA-A molecules [21, 22]. Existing dimorphism at position 80 among Bw4 molecules may influence the reac- tion with KIR3DL1; such that HLA-B allotypes with isoleu- cine at position 80 (Bw4Iso80) may apply stronger inhibitory action than Bw4 allotypes with threonine at this position (Bw4Thr80) [23]. Activating receptors of KIR2DSl, KIR2DS2 and KIR3DS1 share similar sequence in their extracellular domains with their inhibitory types (KIR2DL1, KIR2DL2/
KIR2DL3, and KIR3DL1, respectively). Therefore, they could also have properties of linking to the same HLA ligands as well [24]. KIR2DS1 exhibits weak recognition of HLA-C2 and a possibility of having functional impor- tance [25]. KIR2DS2 weakly links to HLA-C1 as well [26].
High polymorphisms in KIR and HLA genes beside the independent segregation of these genes due to their dis- similar position on the assigned chromosomes (19 and 6 chromosomes, respectively) could cause diversity in both quantity and types of KIR–HLA combinations among dif- ferent individuals. This diversity in KIR–HLA genes pairs can cause different activation points in NK cells and may affect the immune response against viral infections. Previ- ous studies on KIR–HLA genes performed on viral infected patients indicated that various NK cell responses caused by various KIR–HLA combinations could impact the outcome of disease as well [8]. In this study, we aimed to investigate whether the difference in the frequency of inhibiting/acti- vating KIR genes, KIR genotypes and haplotypes, and gene combinations of KIR–HLA could cause susceptibility or a protection against HBV infection in Iranian population.
Materials and methods Patients and controls
Two hundred and two unrelated Iranian people with HBV infection were enrolled from Baghiatallah Research Center of Liver and Gut Diseases. So we collected a group of patients of which, 52 were spontaneously recovered individ- uals, 50 were asymptomatic carriers, 50 patients had active chronic hepatitis B, and 50 had liver cirrhosis. Patients were categorized based on their medical files, conduction of sero- logic tests, determination of viral load, and by verification of clinical consultation. Only patients who had negative serologic test for HCV, HDV, and HIV infections and had no evidence for other disease such as diabetes and autoim- mune diseases were included in this study. The age range of patients was 20–60 years.
A control group of 100 healthy unrelated individuals, who were ethnically matched, were also included (between 20
and 60 years old). These individuals did not have any medi- cal history of hepatitis B, C or D viruses and HIV infection, autoimmunity or alcohol consumption. In addition to the participating agreement, informed consents were obtained from all subjects before blood sampling.
Approximately 5 ml of whole venous blood was collected in ethylene diamine tetra acetic acid (EDTA) tubes from each subject. Then DNA was extracted using salting out method [27] and their quality and quantity were determined by ultraviolet spectrophotometry.
Genotyping
KIR and HLA genotyping was performed by PCR–SSP in all the recruited subjects as previously described [28, 29].
In the present study, DNA samples were typed for the pres- ence or absence of 17 KIR genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B,2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1,2DP1 and 3DP1) and their three major HLA class I ligands (-C1, -C2 and -Bw4: -B Bw4Ile80, -B Bw4Thr80 and A Bw4), in a combined genotyping assay.
The KIR–HLA combinations would be classified into following groups based on the specification of the ligand–receptor interactions: (i) KIR2DL2/3 or KIR2DS2 with alleles of HLA-C1 group, (ii) KIR2DL1 or KIR2DS1 with alleles of HLA-C2 group, and (iii) KIR3DL1 or KIR3DS1 with HLA-B and HLA-A allotypes including Bw4 (Alleles HLA–Bw4) motif. In addition, we considered two sub-groups of Bw4Ile80 and Bw4Thr80 when analyzing the HLA-B Bw4. For each individual the KIR gene content was used to indicate group A and B KIR haplotypes and to assign each person to one of two genotypes. Individuals having only the activating gene KIR2DS4 were homozygote form of AA. All other individuals had one or more B haplotype specific genes (2DS1, 2DS2, 2DS3, 2DS5, 3DS1 and 2DL5) and, therefore, had either one (AB heterozygotes) or two (BB homozygotes) B haplotypes. Such individuals were assigned the KIR genotype Bx [30].
The frequency of KIR and HLA genes, activating and inhibitory KIR–HLA combinations, and KIR haplotypes were counted directly. In addition, the frequencies of KIR genotypes (including AA, AA-zero and Bx), HLA ligand genotypes, activatory and inhibitory KIR+HLA combina- tions, and genotype status of iKIR+HLA/aKIR+HLA and iKIR+HLA/aKIR (>, =, <) were defined.
Statistical analysis
All genes and genotypes among patients and control group were reported as frequency and percent. The Chi-squared test with Yate’s correction or Fisher’s exact test was used for comparison of genotypes between patients and control group. The Bonferroni correction was conducted, and only
comparisons with “p < 0.05/number of comparisons” were considered as significant. In addition, the univariate logistic regression model was used to determine the association of KIR genes, KIR genotype, HLA ligand genes, and compound KIR–HLA genotype with HBV infection. The Stata software version 11 (Stata Corp., College Station, TX, USA) was used for data analysis.
Results
We analyzed the frequencies of KIR genes, HLA genes, KIR genotype, and combination of KIR–HLA genotypes among three categories of patients (150), recovered patients (52), and healthy controls (100).
Analysis of KIR gene content
The frequency of various KIR genes in the control group and different sub-groups of patients are presented in Table 1.
Our study indicated that the frequency of KIR2DL5A (Pc:
0.0002 < 0.005, OR: 4.07, 95% CI: 1.95–8.46), KIR2DS1 (Pc: 0.0001 < 0.006, OR: 4.93, 95% CI: 2.22–10.90) and KIR3DS1 (Pc: 0.0001 < 0.006, OR: 7.3, 95% CI:
3.11–17.27) genes significantly increased in recovered indi- viduals compared with controls. In addition, KIR2DL5(Pc:
0.0033 < 0.005, OR: 3.21, 95% CI: 1.49–6.87), KIR2DL5A (Pc: 0.0023 < 0.005, OR: 3.02, 95% CI: 1.51–6.03), KIR 2DS1(Pc: 0.0011 < 0.006, OR: 3.57, 95% CI: 1.67–7.65), KIR3DS1 (Pc: 0.0001 < 0.006, OR: 5.64, 95% CI:
2.49–12.80) and KIR3DP1 full (Pc: 0.0079 < 0.01, OR:
2.6, 95% CI: 1.32–5.09) gene frequencies were significantly higher in recovered individual compared with patient groups (Table 1).
Analysis of HLA ligand
The frequencies of HLA ligand genes were not sig- nificantly different between patient groups and controls (Table 1). Comparison between recovered individuals and controls revealed that the frequencies of HLA-Bw4 (Pc:
0.0017 < 0.008, OR: 8.78, 95% CI: 1.99–38.67) and HLA- A Bw4 (Pc: 0.0007 < 0.008, OR: 3.59, 95% CI: 1.73–7.46) genes were significantly increased in recovered individuals.
The HLA-A Bw4 (Pc: 0.0001 < 0.008, OR: 4.41, 95% CI:
2.07–9.41) was significantly higher in asymptomatic carri- ers than healthy controls (Table 1). In addition HLA–Bw4 (Pc: 0.0044 < 0.008, OR: 7.32, 95% CI: 1.69–31.68) had considerably higher frequency in recovered individual than patients groups.
Analysis of KIR genotypes
The evaluation of KIR genotypes showed that in recovered individuals Bx (Pc: 0.0012 < 0.016, OR: 9.24, 95% CI:
2.10–40.69) genotypes were significantly increased; but, AA (Pc: 0.0012 < 0.016, OR: 0.10, 95% CI: 0.02–0.47)
and AA-Zero (Pc: 0.0019 < 0.016) genotypes were sig- nificantly decreased compared with controls. Similarly, Bx (Pc: 0.0002 < 0.016, OR: 10.71, 95% CI: 2.49–45.94) genotypes were significantly higher in recovered indi- viduals compared with patient groups; however, AA (Pc:
Table 1 Comparison of frequencies for KIR genes, their HLA ligands, and AA, AA-zero and Bx genotypes in controls and patients with HBV infection
aPc, p value for the comparison between each patient groups and healthy controls, Pc was significant when p value < 0.05/number of compari- sons
bPr, p value for the comparison between patient groups and recovered individuals, Pr was significant when p value < 0.05/number of compari- sons
KIR-Gene Controls
(n = 100) Asymptomatic
carriers (n = 50) Chronic hepatitis
(n = 50) Liver cirrhosis
(n = 50) Recovered indi-
viduals (n = 52) Patient groups (n = 150) No. (%) Pca No. (%) Pca No. (%) Pca No. (%) Pca No. (%) Pca Prb Inhibitory
KIR 2DL1 95 50 (100) 0.1077 49 (98) 0.3767 48 (96) 1 52 (100) 0.1658 147 (98) 0.2724 0.5706 KIR 2DL2 55 25 (50) 0.5656 29 (58) 0.729 20 (40) 0.119 30 (57.6) 0.8855 74 (49.3) 0.4542 0.3802 KIR 2DL3 87 46 (92) 0.3622 47 (94) 0.1909 48 (96) 0.1492 44 (84.6) 0.8875 141 (94) 0.0919 0.2120
KIR 2DL4 100 50 (100) 50 (100) 50 (100) 52 (100) 150 (100)
KIR 2DL5 63 28 (56) 0.4095 29 (58) 0.5541 28 (56) 0.5169 42 (80.7) 0.039 85 (56.7) 0.3864 0.0033 KIR 2DL5A 40 26 (52) 0.1625 23 (46) 0.4839 22 (44) 0.7641 38 (73) 0.0002 71 (47.3) 0.3101 0.0023 KIR 2DL5B 35 16 (33) 0.7184 22 (44) 0.2835 14 (28) 0.4976 26 (50) 0.1061 52 (34.7) 0.9203 0.0731 KIR 3DL1 91 49 (98) 0.1051 46 (92) 0.8374 50 (100) 0.0605 46 (88.4) 0.8414 145 (96.7) 0.1035 0.0350
KIR 3DL2 100 50 (100) 50 (100) 50 (100) 52 (100) 150 (100)
KIR 3DL3 100 50 (100) 50 (100) 50 (100) 52 (100) 150 (100)
Activating
KIR 2DS1 46 26 (52) 0.4884 29 (58) 0.1658 26 (52) 0.6033 42 (80.7) 0.0001 81 (54) 0.2674 0.0011 KIR 2DS2 56 26 (52) 0.6390 29 (58) 0.8230 24 (48) 0.4502 28 (53.8) 0.8640 79 (52.7) 0.6985 1.0 KIR 2DS3 37 17 (35) 0.7184 20 (40) 0.7184 14 (28) 0.3593 26 (50) 0.1703 51 (34) 0.7290 0.0599 KIR 2DS4 91 49 (98) 0.1051 46 (92) 0.8374 50 (100) 0.0605 46 (88.4) 0.8414 145 (96.7) 0.1035 0.035 KIR 2DS4 full 32 16 (33) 1 8 (16) 0.0584 26 (52) 0.0282 10 (19.2) 0.1389 50 (33.3) 0.9203 0.0817 KIR 2DS4 var 82 46 (93) 0.1029 41 (82) 1.0 36 (72) 0.2301 38 (73) 0.2835 123 (82) 0.8624 0.2384 KIR 2DS5 41 15 (30) 0.1896 19 (38) 0.7184 18 (36) 0.6801 22 (42.3) 1 52 (34.6) 0.3504 0.4130 KIR3DS1 43 23 (46) 0.729 23 (46) 0.7290 28 (56) 0.1833 44 (84.6) 0.0001 74 (49.3) 0.3928 0.0001 Pseudogenes
KIR 2DP1 95 50 (100) 0.1077 49 (98) 0.3767 48 (96) 1 52 (100) 0.1658 147 (98) 0.2724 0.5706
KIR 3DP1 100 50 (100) 50 (100) 50 (100) 52 (100) 150 (100)
KIR3DP1 full 30 14 (28) 0.8064 7 (14) 0.0521 12 (24) 0.5656 22 (42.3) 0.1809 33 (22) 0.2017 0.0079 KIR 3DP1 var 97 50 (100) 0.216 49 (98) 0.72 48 (96) 1 52 (100) 0.3203 147 (98) 0.6858 0.5706 HLA ligands
HLA-C1 75 35 (70) 0.5119 35 (70) 0.5119 38 (76) 1 44 (84.6) 0.2470 108 (72) 0.7082 0.1029 HLA-C2 72 36 (72) 1.0 44 (88) 0.0455 40 (80) 0.3896 40 (76.9) 0.6467 120 (80) 0.1884 0.7913 HLA Bw4 74 44 (88) 0.0782 31 (62) 0.1302 42 (84) 0.2418 50 (96) 0.0017 116 (77.3) 0.6467 0.0044 HLA Bw4i 57 26 (52) 0.5598 21 (42) 0.0832 30 (60) 0.8624 30 (57.6) 0.9203 77 (51.3) 0.4542 0.5270 HLA Bw4T 10 2 (4) 0.2016 0 (0) 0.0311 2 (4) 0.3385 4 (7.6) 0.7726 4 (2.6) 0.0284 0.2079 HLA A Bw4 43 40 (80) 0.0001 15 (30) 0.1228 28 (56) 0.1833 38 (73) 0.0007 83 (55.3) 0.0750 0.0370 KIR genotypes
AA 27 30 (15) 0.6985 16 (32) 0.5219 14 (28) 1 2 (3.8) 0.0012 45 (30) 0.7082 0.0002
AA-Zero 19 7 (14) 0.4463 6 (12) 0.2773 4 (8) 0.1512 0 (0) 0.0019 17 (11.3) 0.1319 0.0163 BX 73 35 (70) 0.6985 34 (68) 0.5219 36 (72) 1 50 (96.2) 0.0012 105 (70) 0.7082 0.0002
0.0002 < 0.016, OR: 0.0933, 95% CI: 0.02–0.40) genotype was significantly lower in this comparison (Table 1).
Combinatorial analysis of KIR–HLA pairs
The frequency of inhibitory (KIR2DL2/3 + HLA-C1, KIR2DL1 + HLA-C2 and KIR3DL1 + HLA-Bw4) and acti- vating (KIR2DS2 + HLA-C1, KIR2DS1 + HLA-C2 and KIR3DS1 + HLA-Bw4) combinations were analyzed in HBV-infected individuals and controls (Table 2). Compari- son between recovered individuals and controls revealed that the KIR3DL1 + HLA-A Bw4 (Pc: 0.0051 < 0.008, OR: 2.83, 95% CI: 1.41–5.69), KIR2DS1 + HLA- C2(Pc: 0.0058 < 0.008, OR: 2.76, 95% CI: 3.42–16.05), KIR3DS1 + HLA-Bw4 (Pc: 0.0001 < 0.008, OR: 7.41, 95% CI: 2.71–20.28), KIR3DS1 + HLA-Bw4Iso80(Pc: 0.013 < 0.008, OR: 3.34, 95% CI: 1.63–6.84), and KIR3DS1 + HLA-A Bw4(Pc: 0.0001 < 0.008, OR: 5.81, 95% CI: 2.76–12.22) were significantly elevated in recovered
individuals. Coinheritance of KIR3DL1 + HLA-A Bw4 (Pc:
0.0001 < 0.008, OR: 5.31, 95% CI: 2.43–11.59) in asymp- tomatic carriers were significantly increased compared with controls. In recovered individuals the activating combina- tion of the KIR3DS1 + HLA-Bw4(Pc: 0.0001 < 0.008, OR:
5.00, 95% CI: 2.42–10.30), KIR3DS1 + HLA-Bw4Iso80(Pc:
0.0034 < 0.008, OR: 2.75, 95% CI: 1.43–5.28), and KIR3DS1 + HLA-A Bw4 (Pc: 0.0003 < 0.008, OR: 3.39, 95% CI: 1.76–6.52) were significantly increased compared with patient groups (Table 2).
To determine the relationship between iKIR+HLA and aKIR+HLA pairs, we examined the combined frequen- cies of these genotypes in the study groups (Table 2).
The iKIR+HLA>aKIR+HLA genotype was signifi- cantly less common in recovered individuals than both healthy controls and patient groups (Pc: 0.0084 < 0.016, OR: 0.37, 95% CI: 0.18–0.75; Pc: 0.0033 < 0.016, OR:
0.37, 95% CI: 0.19–0.69, respectively). However, the iKIR + HLA < aKIR + HLA (Pc: 0.0099 < 0.016, OR: 6.39,
Table 2 Frequencies of coinheritance for inhibitory and activating KIR genes, their HLA ligands, and frequencies of different compound KIR–
HLA genotypes in controls and patients with HBV infection
aPc, p value for the comparison between each patient groups and healthy controls, Pc was significant when p value < 0.05/number of compari- sons
bPr, p value for the comparison between patient groups and recovered individuals, Pr was significant when p value < 0.05/number of compari- sons
Combination (KIR–HLA) Controls
(n = 100) Asymptomatic
carriers (n = 50) Chronic hepati-
tis (n = 50) Liver cirrhosis
(n = 50) Recovered indi-
viduals (n = 50) Patient groups (n = 150) No. (%) Pca No. (%) Pca No. (%) Pca No. (%) Pca No. (%) Pca Prb Inhibitory
2DL1 + C2 68 36 (72) 0.6170 43 (86) 0.0298 38 (76) 0.4095 40 (76.9) 0.3348 117 (78) 0.1055 1.0 2DL2/L3 + C1 77 35 (70) 0.3537 35 (70) 0.3537 38 (76) 1.0 44 (84.6) 0.3710 108 (72) 0.4624 0.1029 3DL1 + Bw4 70 42 (85) 0.0632 30 (60) 0.2206 0.2044 0.1669 44 (85) 0.0754 114 (76) 0.3651 0.2713 3DL1 + Bw4i 53 25 (50) 0.7290 21 (42) 0.2044 0.2044 0.5219 26 (50) 0.8624 76 (50.6) 0.8230 0.9203 3DL1 + Bw4T 8 1 (2) 0.1446 0 (0) 0.0528 2 (4) 0.4970 4 (7.6) 1.0 3 (2) 0.0296 0.074 3DL1 + A Bw4 40 38 (78) 0.0001 14 (28) 0.1492 28 (56) 0.0925 34 (65.3) 0.0051 81 (54) 0.0413 0.2059 Activating
2DS1 + C2 33 20 (41) 0.3994 23 (46) 0.1205 20(40) 0.5071 30 (57.6) 0.0058 63 (42) 0.1844 0.0727 2DS2 + C1 45 17 (34) 0.1976 23 (46) 0.9203 16 (32) 0.1761 28 (53.8) 0.3864 56 (37.3) 0.2814 0.0550 3DS1 + Bw4 31 21 (43) 0.1821 13 (26) 0.527 26 (52) 0.0203 40 (77) 0.0001 60 (40) 0.1884 0.0001 3DS1 + Bw4i 23 12 (24) 0.8875 8 (16) 0.3173 20 (40) 0.0360 26 (50) 0.0013 40 (26.7) 0.6101 0.0034 3DS1 + Bw4T 6 1 (2) 0.2735 0 (0) 0.0771 0 (0) 0.0993 4 (7.6) 0.7361 1 (0.67) 0.0175 0.0162 3DS1 + A Bw4 19 18 (36) 0.0378 9 (18) 0.8875 16 (32) 0.1167 30 (57.6) 0.0001 43 (28.7) 0.1131 0.0003 iKIR+HLA/aKIR+HLA
iKIR+HLA>aKIR+HLA 66 34 (68) 0.8064 32 (64) 0.8064 34 (68) 1.0 22 (42.3) 0.0084 100 (66.7) 1.0 0.0033 iKIR+HLA=aKIR + HLA 27 15 (30) 0.6985 16 (32) 0.5219 16 (32) 0.6547 24 (46.1 0.0284 47 (31.3) 0.5541 0.0782 iKIR+HLA<aKIR+HLA 7 1 (2) 0.1988 2 (4) 0.4658 0 (0) 0.0958 6 (11.5) 0.3693 3 (2) 0.0943 0.0099 iKIR+HLA/aKIR
iKIR+HLA>aKIR 37 16 (32) 0.5485 14 (28) 0.2733 12 (24) 0.1572 10 (19.2) 0.0390 42 (28) 0.1737 0.2877 iKIR+HLA=aKIR 18 8 (16) 0.7641 6 (12) 0.3454 4 (8) 0.1658 2 (3.8) 0.0281 18 (12) 0.2542 0.1532 iKIR+HLA<aKIR 45 26 (52) 0.4165 30 (60) 0.0832 34 (68) 0.0129 40 (76.9) 0.0003 90 (60) 0.0276 0.0426
95% CI: 1.53–26.57) genotype was significantly more com- mon in recovered individuals than patient groups.
Further, we investigated the coexistence of iKIR+HLA pairs and aKIR genes in the same individuals (Table 2). The iKIR+HLA<aKIR pair was significantly higher in recov- ered individuals (Pc: 0.0003 < 0.016, OR: 4.07, 95% CI:
1.91–8.67) than controls.
Discussion
The role of genetic factors on susceptibility or resistance to infectious diseases has long been established. The numer- ous genetic polymorphisms of immune response genes, par- ticularly the HLA genes, have been investigated in different populations. Extensive allele variety was examined in HLA associations with susceptibility or resistance to the HBV infections and disease progression in different ecumenical ethnic populations [31]. On the other hand specific motifs on HLA class I molecules interact with KIRs and modulate NK cell activity [32]. However, little is known about KIR–HLA combinations in HBV infection. In the present study fre- quency of KIR genes, genotypes of KIR, HLA ligand genes, and compound KIR–HLA genotypes have been surveyed in Iranian individuals infected with HBV. The HBV-infected individuals were categorized in spontaneously recovered, asymptomatic carriers of HBV, chronic hepatitis B, and cir- rhosis patient groups.
In this study, the frequencies of KIR genes, KIR geno- types, and gene combinations of KIR–HLA in liver cirrhosis, chronic hepatitis and total patient groups (including the sum of asymptomatic carriers, chronic hepatitis, and liver cir- rhosis patients infected with HBV) were not significantly different when compared with healthy controls.
Comparison of the asymptomatic carriers and healthy controls
Comparison between two groups indicates that the fre- quency of KIR genes and genotypes were not significantly different. However, regarding HLA-A Bw4 the difference between these groups was noteworthy. Asymptomatic car- riers had a significantly higher frequency of HLA-A Bw4 followed by higher occurrence of inhibitory combination of KIR3DL1 + HLA-A Bw4 genotype. A long period of normal liver function tests is a characteristic of the asymp- tomatic carriers, despite the presence of HBs antigen in the blood. These individuals are not able to clean their blood from HBV. Inhibitory combination of KIR3DL1 + HLA- A Bw4 may confer inhibitory signal to NK cell which leads to functional impairment of immune cells in HBV clearance. In contrast to our findings, Kalyanaraman et al.
reported that individuals with KIR3DS1 + HLA-BW4 are associated with HBV asymptomatism in south of India population [33].
Comparison of the recovered individuals and healthy controls
Our finding about recovered individuals was very strik- ing and interesting. We found that the inhibitory gene KIR2DL5A and the activatory genes KIR2DS1 and KIR3DS1 were considerably increased in recovered individuals com- pared with the controls. This report was similar to the pre- vious study in Chinese Han Population [34]. However, a study was recently performed by Di Bona et al. in Italian population indicate that the frequency of KIR2DL3 gene was significantly higher in recovered individuals than healthy controls [35]. Furthermore in the present study, HLA-Bw4 ligand, the presence of one or more -B Bw4Ile80, -B Bw4Thr80 and -A Bw4 specificities (especially -A Bw4), was signifi- cantly increased in recovered individuals compared with healthy controls. However, Di Bona et al. found no statisti- cally significant difference between recovered groups and healthy controls in the HLA-Bw4 allele frequency [35]. In our study, AA and AA-Zero genotypes were more frequent in controls than in recovered individuals. In contrast, Bx genotypes were more frequent in recovered individuals (Table 2). So far as we know, the influence of KIR geno- types in resolution of HBV infection has not been inves- tigated. Our data indicated that KIR3DL1+HLA-A Bw4, KIR2DS1+HLAC2, KIR3DS1+HLA-Bw4, KIR3DS1+HLA- Bw4Iso80, and KIR3DS1+HLA-A Bw4 combinations were significantly more frequent in recovered individuals than controls (Table 2). As you can see, activating combinations were more frequent in recovered individuals. Therefore, it seems that activating combination genotypes play a more prominent role in the increment of NK cell activity in these individuals which leads to HBV clearance. The present study shows that in recovered individuals iKIR+HLA>aKIR+HLA genotype is significantly decreased; but, iKIR+HLA<aKIR pair is significantly increased compared with controls (Table 2). These genotypes demonstrate a genetic tendency towards lower inhibition and higher activation of NK cell repertoire of recovered individuals that can contribute in elimination of the HBV infection. These findings, to our knowledge, are the first report highlighting the potential impact of the activating phenotype of NK cells lead to the HBV clearance of infected individuals. However, stud- ies conducted on HCV infected patients have shown that KIR2DL3, when present on a homozygous ligand back- ground (HLA-C1/C1), was associated with spontaneous resolution of HCV infection [36, 37].
Comparison of the recovered individuals and patients group
Our data showed that KIR2DL5, KIR2DL5A, KIR2DS1, KIR3DS1, and KIR3DP1 full genes were significantly more common in recovered individuals compared with patients group (Table 1). Also, the frequency of activatory combi- nation of KIR3DS1+HLA-Bw4, KIR3DS1+HLA-Bw4Iso80, and KIR3DS1+HLA-A Bw4 was significantly higher in recovered individuals. On the other hand, the frequency of iKIR+HLA>aKIR+HLA genotype was significantly lower in recovered individuals compared with patients group; how- ever, the frequency of iKIR+HLA<aKIR+HLA genotype was significantly higher in this comparison (Table 2). A previous study performed in Chinese Han population sup- ports our findings about the role of KIR2DL5, KIR2DS1, and KIR3DS1 genes in clearance of HBV [34]. However, findings of the current study performed on Turkish popula- tion by Kibar et al. did not support our results. They reported that KIR3DS1 could be the protector gene for HBV infec- tion which had lower frequency in patients with spontaneous remission [38].
The genotypes frequencies comparison between recov- ered individuals and patients group showed that AA geno- types, most frequent inhibitory genotypes, were extremely decreased, whereas Bx genotypes were greatly increased in recovered patients. Consequently, highly increased NK cells activity in these individuals leads to the resolution of HBV infection (Table 1). However, previous study on Turkish population did not show any statistically significant differ- ences between the frequency of AA and Bx genotypes of patients with spontaneous remission and control group [38].
As mentioned above, in the study performed on HCV- infected patients, the combination of KIR2DL3 + HLA-C1 was associated with spontaneous resolution of HCV infec- tion [37]. It was explained that inhibitory signals transmitted by KIR2DL3 and HLA-C1 interaction has weaker affinity (compared with KIR2DL1 and HLA-C2 interaction), and it would be easier to overcome this inhibitory interaction;
thus, NK cells in these individuals would be more easily activated during HCV infection resulting in a better out- come [8]. Several studies on HIV-infected patients showed the impact of KIR–HLA genes on HIV progression. Martin et al. reported that patients with activating KIR3DS1 + HLA- Bw4Iso80 combination will have slower progress toward AIDS during the disease [39]. Subsequent studies showed that activating combination is associated with a slightly lower viral load which could delay onset of opportunistic infections [8]. A recent study performed on respiratory pap- illomatosis patients demonstrated that the lack of KIR2DS1 and KIR3DS1 may promote the disease toward severe stage, and patients with these genes may remain in mild–moderate stage. Also, they suggested that the presence of KIR2DS1,
KIR2DS5, and KIR3DS1 correlate to the protection against HPV [40].
Finally, regarding our results we conclude that activating KIR genes, combinations, and haplotypes could cause highly activation of NK cells that result in protection against HBV infection and facilitate HBV clearance in Iranian patients.
Overall, our study has demonstrated the potential impact of the immunogenetic background to address resolving of the HBV and development of HBV-related liver disorders. It seems that the balance between the activating and inhibitory combination of KIR–HLA genotypes will determine the risk of progress from infection to liver diseases. Accordingly, functional studies are required for better understanding of the pathogenic processes through which these genes might affect disease progress.
Acknowledgements This work has been supported by a Grant from Iran University of Medical Sciences.
Compliance with ethical standards
Conflict of interest The authors have no conflicts of interest that are directly relevant to the content of this article.
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