Correlation analysis of IL‐37 gene polymorphisms and susceptibility to chronic HBV infection among Han people in Central China

Hepatitis B virus (HBV) is responsible for various liver diseases, such as chronic hepatitis B (CHB), liver fibrosis, liver cirrhosis (LC) and hepatocellular carcinoma (HCC), which pose a significant threat to human health. An ineffective immune response to HBV can result in viral chronicity. Interleukin‐37 (IL‐37), an immunomodulator, is capable of inhibiting both innate and adaptive immune responses. It is believed that single nucleotide polymorphisms (SNPs) within the IL‐37 gene could contribute to the regulation of HBV clearance. Our aim to conduct this study was to investigate whether SNPs in the IL‐37 gene were associated with the risk of chronic HBV infection in adults. A total of 342 participants, consisting of 171 cases and 171 controls, were recruited for this study. Sanger sequencing was employed for genotyping six SNPs (rs3811042 G/A, rs3811043 G/C, rs2466449 A/G, rs3811045 C/T, rs3811046 T/G and rs3811047G/A). There was no significant difference in allele and genotype distribution between the two groups, and the constructed haplotypes were not found to be associated with the risk of chronic HBV infection. Our results revealed that there was no relationship between these six SNPs (rs3811042G/A, rs3811043G/C, rs2466449A/G, rs3811045C/T, rs3811046T/G and rs3811047G/A) in the IL‐37 gene and susceptibility to chronic HBV infection among Han people in Central China.

HBV infection (Ben Selma et al., 2021;Zeng, 2014).In terms of host immunity, chronic HBV infection can lead to the suppression of innate immune cells (such as monocytes/macrophages, dendritic cells, and natural killer (NK) cells) as well as adaptive immune cells (such as CD4+ and CD8+ T cells) (T.Y. Li et al., 2019).HBV can induce the formation of an immunosuppressive cascade by promoting the expression of inhibitory molecules (such as PD-L1, PD-1, and IL-10) on immunosuppressive cells such as, NK-reg cells and T-reg cells, which contribute to the persistence of HBV infection (T.Y. Li et al., 2019).HBV can induce exhaustion of both HBV-specific T and B cells through peripheral tolerance mechanisms, particularly with regard to virus-specific T cells (Kuipery et al., 2020).These can ultimately result in the virus evading surveillance by the immune system, allowing it to persist and continue to cause damage to the liver.The liver is regarded as an indispensable immune organ due to its exceptional anatomy and immunological structure.The liver synthesizes a variety of cytokines that bind to particular receptors on target cells, initiating downstream signalling pathways and playing a pivotal part in the host's defence response to HBV (Shoraka et al., 2019;Zhong et al., 2021).Currently, tremendous efforts have been made to look for host factors that influence the susceptibility to chronic HBV infection, clinical outcomes, treatment response and responses to hepatitis B vaccination (Zhang et al., 2019).The single nucleotide polymorphisms (SNPs) located in the coding sequence of cytokine genes, which are associated with their structure, could exert an influence on their function, and those located in regulatory sequence, such as 3′ untranslated region and promoter region, could impact the gene transcription rate and subsequently modify cytokine production (Ben Selma et al., 2021).Earlier studies have established an association between SNPs in cytokine genes and susceptibility to chronic HBV infection (Ben Selma et al., 2021;El-Maadawy et al., 2019;M. Li et al., 2021).
Interleukin-37 (IL-37) is an anti-inflammatory cytokine that inhibits innate and adaptive immune responses by constraining the production of pro-inflammatory cytokines activated by Toll-like receptor (TLR) agonists and increasing the production of tolerant dendritic cells (DCs) (Gamal, 2020;Zhu et al., 2021).IL-37 limits the capacity of DCs to activate antigen-specific T cells while boosting the development of T-reg cells by inhibiting the production of MHC-II and CD40 (Davarpanah et al., 2020).IL-37 is expressed at low levels in leukocytes, epithelial cells, lymph nodes, thymus, lung and intestine but is upregulated in response to inflammation, serving as a self-protective mechanism against excessive inflammation (Su et al., 2021).Abnormal expression of IL-37 is observed in some inflammatory or autoimmune disorders (Y.Li et al., 2014;Ye et al., 2015), and IL-37 may be involved in the pathogenesis of these diseases.In infectious diseases, IL-37 plays a significant role in immunity against viral, bacterial and fungal infections by preventing inappropriate immune activation and suppressing inflammation caused by these infectious agents (Allam et al., 2020).
For instance, elevated IL-37 levels in Helicobacter pylori (HP)-infected patients can serve as a significant indicator of peptic ulcer (PU) development (Davarpanah et al., 2020).Similarly, CHB patients have been observed to have significantly higher IL-37 abundance in their serum and increased expression of IL-37 in their peripheral blood mononuclear cells (PBMCs) compared to healthy controls (Huang et al., 2017;Meng et al., 2020).After antiviral therapy, the serum level of IL-37 is demonstrably lower (Meng et al., 2020), suggesting that IL-37 may be associated with the pathogenesis of HBV.
Previous studies have demonstrated a robust correlation between SNPs within the IL-37 gene and susceptibility to inflammatory and autoimmune diseases, such as systemic lupus erythematosus, coronary artery disease and autoimmune thyroid diseases (Wu et al., 2021;Yan et al., 2015;Yin et al., 2017).Nevertheless, limited studies investigate the potential association between IL-37 gene polymorphisms and chronic HBV infection.Hence, we conducted a preliminary investigation to explore the possible link between SNPs within the IL-37 gene and chronic HBV infection among the Han population in Central China.

Populations studied
The study was performed in accordance with the Declaration of

DNA extraction
Each participant provided 2 ml of whole blood, which was collected using a tube containing ethylenediaminetetraacetic acid anticoagulant (Genenode).Genomic DNA was extracted from peripheral venous

SNP genotyping
The full sequence and SNP data for the IL-37 gene were obtained Sanger sequencing was performed to determine the genotype of the SNPs (Sangon Biotech).

Statistical analysis
SPSS version 25 (IBM Corporation) was used for statistical analysis.

Participant's characteristics
This study enrolled a total of 342 participants, consisting of 171 patients with chronic HBV infection and 171 healthy controls.The analysis showed no significant differences in sex (p = .187)or age (p = .913)between the two groups (Table 1).However, the two groups differed significantly in AFP, DBIL, IBIL, TBIL, ALT and AST (Table 1).

Association between IL-37 polymorphisms and chronic HBV infection
We successfully genotyped the tagged SNPs but only showed sequencing peak figures for rs3811042 and rs3811047 (Figure 2).The genotype distribution of rs3811042, rs3811043, rs2466449, rs3811045, rs3811046 and rs3811047 was consistent with HWE in both groups (Table 2), suggesting that the subjects were representative of the population.The six SNPs did not differ significantly in genotype and allelic frequencies between the groups (Table 2).Moreover, dominant and recessive models showed no significant associations of IL-37 gene polymorphisms with chronic HBV infection (all p > .05)(Table 2).

Haplotype analysis
Four haplotypes with frequencies > 0.03 identified by haplotype analysis.However, these haplotypes showed no association with the risk of chronic HBV infection (Table 3).

DISCUSSION
Cytokines are molecular messengers of the innate and adaptive immune systems, facilitating communication between immune cells over short distances in both paracrine and autocrine ways (Conlon et al., 2019).Polymorphisms in cytokine genes can cause changes in the expression levels and molecular structures of corresponding  proteins, which may affect the immune response to HBV and disease phenotypes (Ben Selma et al., 2021;Dondeti et al., 2022).The IL-37 gene, located in human chromosome 2 (2q14.1),harbours many SNPs.
Among them, rs3811042, rs3811043, rs2466449 and rs3811045 are located in the intronic region.So far, no studies have found any disease associations with these variants.This study also confirms that they are not linked to the risk of chronic HBV infection.Rs3811046 and rs3811047, two missense mutations in exon 2, lead to different amino acid codons (Gly/Val at rs3811046 and Thr/Ala at rs3811047) (Mookherjee et al., 2016).Although it has not yet been confirmed, this change could potentially alter the function of the protein (Mookherjee et al., 2016), resulting in a negative modulation of the immune response to HBV, which could increase the risk for chronic HBV infection.This study shows that SNP rs3811046 is not associated with susceptibility to chronic HBV infection, which implies that this presumption could not be true in the case of HBV infection.Earlier investigations suggest that SNP rs3811046 is unrelated to open-angle glaucoma, tuberculosis, RA and Hashimoto's thyroiditis (Ali et al., 2022;El-Sayed et al., 2018;Mookherjee et al., 2016).However, SNP rs3811046 is associated with severe chronic periodontitis, 10-year incidence tooth loss and aggressive periodontitis (Offenbacher et al., 2018).Similarly, another study in a Brazilian population demonstrates that the rs3811046 GG genotype is associated with an increased risk for moderate periodontitis (Cirelli et al., 2021).The rs3811046 G allele is significantly associated with a reduced risk for Graves' disease in female patients (Yan et al., 2015).The aforementioned data show that SNP rs3811046 seems to play a role in susceptibility to inflammation-related and/or immunerelated diseases.However, this conclusion is not applicable to chronic HBV infection, which could be a result of the diversity of the pathologic mechanisms involved in the various diseases.
Regarding rs3811047, researchers initially investigated the association between SNP rs3811047 and susceptibility to chronic HBV infection in a Saudi Arabian population, and no significant association was found (Al-Anazi et al., 2019).This study is the first to discuss the relationship between this locus and susceptibility to chronic HBV infection in the Chinese Han population, and we also did not obtain statistically significant results.However, evidence suggests that rs3811047 is associated with susceptibility to PU, ankylosing spondylitis, RA, autoimmune thyroid diseases and Behcet's disease (Davarpanah et al., 2020;Lin et al., 2018;Özgüçlü et al., 2019;Tan et al., 2016).Interestingly, a similar study found a significant association between the two SNPs and susceptibility to COVID-19 among Iraqi patients (Ahmed & Ad'hiah, 2022).In addition, we do not find the haplotypes associated with an increased risk for chronic HBV infection in this study, Several limitations should be considered in this case-control study.
First, the sample size is small, and all samples are obtained from a single hospital, which may limit generalizability and raise concerns about potential internal variation.Second, we did not collect comprehensive clinical data from participants, such as information on smoking and alcohol use, medication history and other factors that could interact with genetic factors to influence susceptibility to chronic HBV infection.As a result, caution should be exercised when interpreting the findings of this study.
To summarize, no significant correlation was found between IL-37 gene polymorphisms and the risk of chronic HBV infection among the Han Chinese population in Central China.However, due to the limited sample size and potential genetic variations among different populations, further studies with larger sample sizes are necessary to confirm whether IL-37 gene polymorphism is associated with the susceptibility to chronic HBV infection in other regions or ethnic groups.
Helsinki and was approved by the Ethics Committee of Jingzhou Hospital Affiliated to Yangtze University (ethical approval number: 2022-090-01).The cases and controls were recruited from Jingzhou Hospital affiliated to Yangtze University between May 2021 and August 2022.Patients with chronic HBV infection were those who had HBsAg positivity for over 6 months.Exclusion criteria consisted of (1) cirrhosis, liver failure, alcoholic hepatitis or autoimmune hepatitis; (2) liver cancer or other primary malignant tumours and (3) impaired kidney, lung or heart function.The healthy controls were randomly selected from healthy individuals without HBsAg, HBsAb, HBeAg, HbeAb and HBcAb in their serum.The study samples consisted entirely of Han Chinese participants.The following data were collected from all participants: age, sex, alpha-fetoprotein (AFP), direct bilirubin (DBIL), indirect bilirubin (IBIL), total bilirubin (TBIL), alanine transaminase (ALT) and aspartate transaminase (AST).
blood using the blood genomic DNA extraction kit (TIANGEN) following the manufacturer's instructions.The purity and concentration of DNA were assessed by a spectrophotometry apparatus (BioDrop).For DNA extraction, a concentration of at least 20 ng/μL is required, and an A260/A280 absorbance ratio ranging from 1.7 to 1.9 indicates highquality DNA with minimal protein contamination.The extracted DNA was stored at −80 • C.F I G U R E 1 Diagram of amplified fragment (chr2:112913079∼112914042) (GRCh38.p14).The blue portion represents introns, and the green portion represents exon.
from the Ensembl database (http://asia.ensembl.org/index.html).Six SNPs (rs3811042 G/A, rs3811043 G/C, rs2466449 A/G, rs3811045 C/T, rs3811046 T/G and rs3811047G/A) with a minor allele frequency > 15% in the East Asian population were selected as the tagged SNPs (https://www.ncbi.nlm.nih.gov/snp/).Since the SNPs were situated in close proximity, a pair of primers was designed to amplify them together.The forward primer sequence was 5′-CAGTGCTGCTTAGAAGGTAA-3′, and the reverse primer sequence was 5′-GCTCATCTTTCCCAGAGTTAT-3′, resulting in an amplified fragment of 964 bp in length (Figure1).The PCR system (25 μL) consisted of 2 μL of DNA extraction, 12.5 μL of 2×Taq PCR MasterMix (Takara), 1 μL of each primer (Sangon Biotech, China) and 8.5 μL of nuclease-free water.The PCR program involved an initial denaturation at 95 • C for 3 min, followed by 35 cycles of denaturation at 98 • C for 10 s, annealing at 62.8 • C for 30 s and extension at 72 • C for 60 s.The specificity of the PCR products was verified by 2% agarose electrophoresis.
Continuous variables were expressed as the means ± standard deviations or median (25th-75th percentile) based on their distribution, while categorical variables were expressed as frequencies and percentages.Demographic characteristics and clinical laboratory parameters were compared using t-test or Kruskal-Wallis test for continuous variables and Pearson chi-squared test for categorical variables.The Hardy-Weinberg equilibrium (HWE) was analysed using the Pearson chi-squared goodness of fit test.Logistic regression analysis was used to examine the difference in genotype and allele distribution between cases and controls by computing the adjusted odds ratios and 95% confidence intervals.Haplotype analysis was performed using the SNPstats program (http://bioinfo.iconcologia.net/SNPstats),with a minimum frequency threshold of 0.03.All p values were two-tailed, and statistical significance was set at p < .05.

F
I G U R E 2 DNA sequence peak figures of IL-37 gene single nucleotide polymorphisms (SNPs) (rs3811042 and rs3811047).The arrows in (A), (B) and (C) indicate rs3811042 GG genotype, GA genotype and AA genotype, respectively.The arrows in (D), (E) and (F) indicate rs3811047 GG genotype, GA genotype and AA genotype, respectively.
but the haplotype G-C-A-T-T-A (in the order: rs3811042, rs3811043, rs2466449, rs3811045, rs3811046 and rs3811047) is associated with a 10.23-fold increased risk of COVID-19.Due to the involvement of different populations and diseases in the aforementioned studies, the differences in the results may be attributed to genetic background variations or diverse pathological mechanisms of the diseases.
The characteristics of the included subjects.
Allele and genotype distribution between cases and controls.Haplotype frequencies of interleukin-37 (IL-37) between the cases and controls.
TA B L E 2Abbreviations: CI, confidence interval; HWE-P, Hardy-Weinberg probability; OR, odds ratio; SNP, single nucleotide polymorphism.aAdjusted for age and sex.TA B L E 3 a Adjusted for age and sex.