Impact of interleukin‐1β single nucleotide polymorphisms and depressive symptoms in individuals with chronic viral hepatitis

Elevated levels of interleukin 1β (IL‐1β) have been identified in patients with chronic viral hepatitis and have been associated with depressive symptoms. Given the high prevalence of depression in this patient population, this study sought to explore the potential influence of IL‐1β genetic variations on the severity of depressive symptoms. In a cohort of 181 Taiwanese patients with chronic viral hepatitis, we investigated the impact of five common IL‐1β single nucleotide polymorphisms (SNPs), including rs16944, rs1143627, rs1143630, rs1143643, and rs3136558, on depressive symptoms using the Beck's Depression Inventory‐II. Additionally, we analyzed the primary domains of IL‐1β‐related depressive symptoms according to Beck's six symptom categories of depression. Our analysis revealed significant associations between depressive symptoms and three intronic IL‐1β SNPs. After controlling for age, sex, marital status, and education level, patients with the rs1143630 GG, rs1143643 CC, and rs3136558 AA genotypes demonstrated higher severity of depressive symptoms in the domains of indecision (p = 0.004), agitation (p = 0.001), and feelings of punishment (p = 0.005), respectively, compared to rs1143630 GA+AA, rs1143643 CT, and rs3136558 AG+GG genotypes. According to Beck's categorization, these symptoms can be classified into three dimensions: disturbances in emotion regulation, energy, and cognition. Our findings demonstrate the association between IL‐1β polymorphisms and depressive symptoms and suggest a potential underlying mechanism for specific depressive symptoms within the chronic viral hepatitis population. These insights could improve our understanding and treatment of depressive symptoms in individuals with viral hepatitis.

interleukin-6, 2 compared to their healthy counterparts. 3The association between the cytokine levels and the severity of depressive symptoms suggests elevated cytokines as unique pathogenic mechanisms underlying depression induced by viral hepatitis.Chronic viral infections result in increased pro-inflammatory cytokines capable of penetrating the blood-brain barrier and augmenting cytokine secretion by cerebral microglial cells. 4These signals modulate brain activity through various pathways linked to depression, including alterations in neurotransmitter metabolism, neuroendocrine function, and neural plasticity. 4,5ong the various pro-inflammatory cytokines released during chronic viral hepatitis infection, emerging evidence suggests that IL-1β may play a pivotal role in the pathogenesis of depression. 6creted by activated macrophages, IL-1β can activate, proliferate, and differentiate immune cells.The widespread expression of IL-1β receptor on numerous cell types, including immune cells, endothelial cells, and CNS cells, also suggests the profound impact of IL-1β. 7Previous research has suggested several single nucleotide polymorphisms (SNPs) of the IL-1β gene to be implicated in depression, such as rs16944, rs1143627, and rs1143643. 8[11][12][13] Therefore, this study aims to explore the potential influence of IL-1β on depressive symptoms in chronic viral hepatitis patients by examining whether IL-1β SNP genotypes affect depressive symptom phenotypes.

| Participants
A total of 181 participants with chronic hepatitis B virus (HBV), chronic hepatitis C virus (HCV), or both, were recruited from Kaohsiung Medical University Hospital between 2019 and 2021, and all participants were of Eastern Asian ethnicity.The inclusion criteria for participants with chronic HBV infection were a positive hepatitis B surface antigen test lasting over 6 months.For those with chronic HCV, criteria included a positive anti-HCV immunoglobulin test and a history of HCV presence, confirmed via polymerase chain reactions, for over 6 months.The patients' demographic information, including age, sex, and hepatitis status, was obtained from medical records.
Baseline characteristics, such as educational attainment, marital status, and past medical history (including malignancy, psychiatric disorders, and chronic kidney disease), were gathered through interviews.
The management of HBV and HCV in participants followed EASL 2017 18 and AASLD-IDSA guidelines, 19 respectively.Exclusion criteria included patients under 50 years of age, and those with a history of malignancy, severe psychiatric disorders, acute infection, or chronic kidney disease (stage IV or V).In accordance with the Helsinki Declaration's ethical principles, all participants provided written informed consent.The study procedures were approved by the ethical research committee of Kaohsiung Medical University Hospital.

| Assessment of liver fibrosis
The information on the Metavir score was available in 33 of the 181 participants (18.2%).Thus, we adopted the FIB-4 score to measure fibrosis staging.The FIB-4 score is a noninvasive scoring system based on the patient's age, platelet count, aspartate transaminase, and alanine transaminase.These were obtained within a 6-month period from the date of the assessment of depressive symptoms.

| Assessment of depressive symptoms
Depressive symptoms were evaluated using the Chinese BDI-II (C-BDI-II) questionnaire. 20The C-BDI-II is a self-report instrument consisting of 21 items that has been frequently employed as an indicator of depression severity with strong reliability and validity. 21The C-BDI-II employs a four-point (0-3) scale for its 21 items, with 0 indicating "no symptoms" and 3 indicating "severe symptoms."Overall scores range from 0 to 63 points, with higher scores reflecting more severe depressive symptoms.Participants responded to the C-BDI-II based on their daily situation within the 2 weeks preceding the assessment.Due to Chinese social customs, the data for the 21st item ("loss of interest in sex") was deemed potentially inaccurate and excluded from further statistical analysis to prevent bias, as almost a quarter of the participants did not provide an answer.For classification using BDI-II items, 22 Beck's categorization divided items into six main dimensions 23 : regulation of emotion (items 1, 4, 10, 13), motivation (items 2, 9), focused attention (items 12, 19), cognitive distortions (items 3, 5, 6, 7,8, 14), energy regulation (items 11, 15, 17, 20), and physiological/vegetative (items 16, 18, 21).

| Genotyping and linkage disequilibrium evaluation
Genomic DNA was extracted from participants' peripheral blood leukocytes using the Genomic DNA Extraction Kit (Geneaid, Taiwan).

| Genotype distributions
We conducted an investigation of 10 IL-1β SNPs, as shown in Table 1, and selected five SNPs with a minor allele frequency (MAF) exceeding 10% for further analysis for adequate sample size for each genotype.The genetic distribution of the selected SNPs was found to conform to the Hardy-Weinberg equilibrium (Table 1).Pairwise LD was computed among the seven IL-1β SNPs that have a non-zero MAF, and we observed the presence of one block (distance <1 kb) among them, as well as high LD between rs16944 and rs1143627 (D 0 = 1.0, r 2 = 0.957, p < 0.00001), as illustrated in Figure 1.The r 2 value of 0.957 between rs16944 and rs1143627 indicates strong LD between these SNPs.Therefore, we chose rs1143627, the SNP with a higher MAF, for association analysis.

| IL-1β SNP rs1143627 is not significantly associated with depressive symptoms
The frequency of the rs1143627 minor G alleles was 47.8% (Table 1).
Demographic characteristics were stratified based on the genotypes of the IL-1β SNPs rs1143627.Among the three groups, no significant differences were observed in age, sex, education, marital status, proportions of individuals achieving SVR, HBV/HCV infection rates, FIB-4 scores, or HCV genotypes (Table 2).We found no significant differences in any of the BDI score items (Table 2).Even after controlling for potential confounding factors such as age, sex, education, and marital status using Quade's test, the absence of significant differences persisted.These findings suggest that the rs1143627 SNP does not have a significant impact on the severity of depressive symptoms.

| IL-1β
SNPs rs1143630 GG, rs1143643 CC, and rs3136558 AA are associated with more severe depressive symptoms The frequency of the minor rs1143630 T allele was 18.5% (Table 1).
Since the number of individuals with the TT genotype of rs1143630 (n = 3) was relatively small, we combined individuals carrying the T allele rs1143630 (genotypes TG or TT) into one group for comparison with those possessing the GG genotype.We found no demographic characteristic differences between the two groups (Table 3).After controlling for confounding factors using Quade's test, individuals with the GG genotype of the rs1143630 SNP exhibited more severe symptoms for the "BDI-13: indecision" item compared to those carrying the T allele of rs1143630 ( p = 0.004; Table 3).Furthermore, based on Beck's classification of six depressive symptoms, the item can be categorized into dimension of emotion regulation.
The frequency of the minor rs1143643 C allele was 48.9% (Table 1).We observed no significant differences in demographic characteristics (Table 4).However, a statistically significant difference emerged in the "BDI-11: agitation" item among the different genotype groups ( p = 0.006; Table 4).To further investigate the specific genotype groups with significant differences in the "BDI-11: agitation" item, a Quade post-hoc analysis was conducted.This examination revealed that individuals with the CC genotype of rs1143643 exhibited more pronounced symptoms than those with the CT genotype (p = 0.001; Table 4).However, the post-hoc analysis indicated no significant differences between individuals with the CC and TT genotypes, or between those with the CT and TT genotypes ( p = 0.094 and p = 0.294, respectively).Employing the modified Beck's six-symptom categorization, based on the BDI-II, this item can be classified into a dimension reflecting high arousal type energy regulation.
The frequency of the minor rs3136558 G allele was 39.5% (Table 1).Since the number of individuals with the GG genotype of rs3136558 (n = 21) was <30, we combined those with the GG genotype with those with the AG genotype as one group, those carrying the G allele, to compare with those with the AA genotype rs3136558.
Under similar demographic characteristics between the two groups (Table 5), Quade's test demonstrated that those with the AA genotype of rs3136558 were associated with more severe symptoms compared to those carrying the G allele in the item "BDI-6: feeling of being punished" ( p = 0.005; Table 5).Furthermore, this item can be classified into cognitive distortions based on Beck's six depressive symptoms.
Lastly, we conducted a haplotype analysis to determine the combined effects of rs1143630 GG, rs1143643 CC, and rs3136558 on the depressive symptoms of the participants.However, due to a limited number of participants in some haplotype groups, our results did not find any significant differences in depressive symptoms across the different haplotype groups (Table 6).Although these variations do not modify proteins directly, numerous studies have shown that variations in non-coding regions can be phenotypically important by affecting the systems that regulate protein expression.These mechanisms include impacts on splicing, transcription, translation, RNA processing, and chromatin interactions. 24r findings revealed that the rs1143630 GG and rs3136558 AA genotypes were associated with heightened severity in emotion regulation and cognitive distortions, respectively.Although previous studies have suggested the functional significance of these two SNPs (rs1143630 and rs3136558) in several diseases, 25,26 their potential effects on modulating mood symptoms remain unexplored.Additionally, we found the C allele of another SNP, rs1143643, 16,17 to be associated with more severe symptoms of energy regulation.
However, we observed no significant associations between the SNPs located in the promoter region, both rs16944 and rs1143627, and depressive symptoms, which contradicts previous assumptions of their relation to IL-1β levels 27 and the severity of depressive symptoms in Caucasian 10,12,13 and Taiwanese 11,14 populations.The rs1143627 polymorphism, situated within the promoter sequence, has been postulated to enhance transcription factor binding and increase IL-1β production due to the presence of the major A allele. 27nversely, research conducted on the Finnish population identified an association between the minor G allele and elevated IL-1β expression. 9Phenotypically, one study reported a significant association between the co-occurrence of rs1143627 AA and rs16944 GG genotypes and the incidence of major recurrent depression. 13is lack of association in our study can be primarily attributed to The rs1143630 is an intronic SNP of IL-1β, situated near the 5 0 region of the IL1B gene. 25Although no published research has specifically investigated the relationship between rs1143630 and depressive disorders or provided definitive evidence concerning the IL-1β levels associated with SNP functionality, some studies have suggested that rs1143630 may cause susceptibility to certain diseases. 25,26Phenotypically, one study found that individuals carrying the rs1143630 G allele were predisposed to osteoporosis in the Chinese population, 26 potentially due to the more potent effect of IL-1β on osteoclast precursor differentiation into mature osteoclasts.Moreover, the rs1143630 T allele has been identified as exerting a protective effect against cervical cancer susceptibility, 25 possibly attributable to the reduced pro-inflammatory nature of IL-1β, which reduces tumor invasion.In alignment with previous studies in the Chinese population, 25,26 our results suggest that the GG genotype of rs1143630 is associated with more severe depressive symptoms and elevated IL-1β expression, which is compatible with one previous in silico study. 253136558 is an intronic SNP of IL-1β located in the 5 0 region of the IL1B gene.Several studies have demonstrated the correlation between the IL-1β SNP and susceptibility to certain diseases. 25,28For example, the Chinese study showing the protective effect of the rs1143630 T allele also found the rs3136558 G allele to exhibit a protective effect against cervical cancer susceptibility. 25However, there has been no reported association between rs3136558 and depressive disorder.Few studies have reported the differences in IL-1β levels between individuals with different rs3136558 alleles, with one study suggesting that this SNP may be involved in the regulation of transcription. 25In summary, our data corroborate the research finding conducted within the Chinese population, 25 suggesting that the rs3136558 AA genotype is associated with higher IL-1β levels and more severe depressive symptoms.
The rs1143643 variant, an intronic polymorphism situated near the 3 0 terminus of the IL-1β gene, has yet to have its functional implications definitively elucidated.A prior investigation employing in silico techniques proposed a potential role of rs1143643 in gene transcription. 25Our findings revealed a significant association between the CC genotype of rs1143643 and the increased severity of depressive symptoms.In line with our results, previous research has identified the CC genotype of rs1143643 as a predictor of suboptimal treatment outcomes in antidepressant therapies, alongside diminished responses within the amygdala and anterior cingulate cortex during emotional stimulation. 17There is substantial biological evidence supporting our findings regarding the adverse impact of elevated IL-1β levels on the development of depressive symptoms.IL-1β is expressed in glial cells within the cerebral cortex and hypothalamus, as well as neurons in the hippocampus, whereas its receptors are predominantly located in the hippocampus, hypothalamus, anterior cingulate cortex, and dentate gyrus. 29,30The unique distribution of cytokines and their receptors in these specific brain regions contributes to their distinct effects.For example, IL-1β influences tryptophan metabolism in human hippocampal progenitor cells by inducing the upregulation of indoleamine-2,-3-dioxygenase (IDO) enzyme, thereby reducing tryptophan availability for serotonin synthesis. 31In addition, IL-1β has been implicated in impairing striatal dopamine neurotransmission. 32These findings shed light on the mechanisms through which IL-1β may disrupt emotion regulation, as observed in our study.
There is also a compelling biological basis for the association between cognitive distortion and elevated IL-1β levels.For instance, when IL-1β levels produced by microglial cells exceed physiological thresholds, they may adversely impact neural synaptic plasticity and compromise hippocampal learning and memory processes. 29Furthermore, IL-1β interferes with brain-derived neurotrophic factor signaling pathways, 33 disrupts glutamatergic synaptic transmission in hippocampal pyramidal neurons, 34 and accelerates norepinephrine and dopamine turnover. 35 addition, elevated IL-1β levels have been implicated in dysregulated energy regulation.IL-1β has been demonstrated to trigger hyperactivation of the hypothalamic-pituitary-adrenal axis. 36,37pothalamic-pituitary-adrenal axis hyperactivation give rise to heightened energy regulation, such as agitation. 38Moreover, sustained elevations in downstream glucocorticoid levels can contribute to the atrophy of hippocampal pyramidal neurons and the medial prefrontal cortex, potentially resulting in depression with cognitive symptoms. 39,40spite its merits, the current study acknowledges several potential limitations.Firstly, the sample size restricted our ability to catego-

4 |
DISCUSSIONThis study identified three SNPs within the intronic region of the IL-1β gene that influence the severity of depressive symptoms in individuals with chronic viral hepatitis.Previous studies have rarely examined the influence of the IL-1β SNPs on depressive symptoms among individuals with chronic viral hepatitis, or the specific domain affected.

F I G U R E 1
Haploview analysis of linkage disequilibrium among IL-1β SNPs.The pairwise r 2 measures of linkage disequilibrium were calculated using Haploview software v4.2, based on unrelated Taiwanese individuals.R 2 values are displayed in white for r 2 < 15%, with shades of gray indicating intermediate values (15% < r 2 < 80%), and black representing high values (r 2 > 80%).The squares contain the respective r 2 scores for pairwise linkage disequilibrium.A single block (distance <1 kb) was identified among IL-1β SNPs, with a high level of LD observed between rs16944 and rs1143627 (D 0 = 1.0, r 2 = 0.957, p < 0.00001).The r 2 value of 0.957 between rs16944 and rs1143627 signifies complete linkage disequilibrium in a reverse pattern (rs16944 A/rs1143627 G or rs16944 G/rs1143627 A).T A B L E 2 Clinical characteristics and BDI-II scale results stratified by rs1143627 genotypes.
two factors.First, individuals from diverse racial backgrounds may have unique mechanisms for modulating cytokine production.As a result, the increased IL-1β expression associated with the rs1143627 A allele does not necessarily cause a corresponding elevation in IL-1β levels within our study population.Second, the potential modulatory impact of viral hepatitis on the influence of the rs1143627 SNP could play a role in the development of depressive symptoms.
rize participants based on their HBV or HCV status, thus hindering the investigation of IL-1β SNPs' distinct impacts on either HBV or HCV.Additionally, our study cohort included individuals with untreated chronic HCV as well as those who achieved SVR posttreatment.This heterogeneity in HCV infection status may have influenced participants' depressive symptoms.Nonetheless, χ 2 test results confirmed an equal distribution of patients achieving SVR across each analyzed IL-1β SNP group, suggesting that this heterogeneity does not compromise the study's validity.Lastly, in vivo IL-1β levels were not assessed in the current study.Future research incorporating laboratory data, such as IL-1β concentrations in blood or cerebrospinal fluid, would more effectively clarify the cytokine's role in developing depressive symptoms.While validating the results in an independent cohort with a larger number of sample size would strengthen our conclusions, it remains beyond the scope of this study.Subsequent research with a more extensive cohort can offer further validation.To conclude, we have comprehensively examined the relationship between depressive symptoms and IL-1β SNPs in individuals with chronic viral hepatitis.Our analysis revealed a significant association between three intronic IL-1β SNPs (rs1143630, rs1143643, and rs2853550) and the manifestation of depressive symptoms.Moreover, we have delineated three distinct dimensions of depressive symptoms influenced by these genetic variants, including disturbances in emotion regulation, cognitive distortions, and energy regulation.By establishing this correlation, we may optimize personalized medicine to prevent depression in chronic hepatitis patients proactively.
Clinical characteristics and BDI-II scale results stratified by rs1143630 genotypes.
T A B L E 5 Clinical characteristics and BDI-II scale results stratified by rs3136558 genotypes.