AhR may be involved in Th17 cell differentiation in chronic hepatitis B

Th17 cells which are crucial for host immunity have been demonstrated to increase HBV infection. However, the mechanism of the Th17 cell increase is unknown. Hence, the mechanism of Th17 cell enhancement is important to provide a theoretical foundation for chronic hepatitis B immunotherapy. This study included 15 instances in the healthy control (HC) and 15 cohorts in the chronic hepatitis B (CHB). Their CD4+T cells were isolated from their peripheral blood and then subjected to RNA transcriptome sequencing. Then, to identify target genes linked to Th17‐cell differentiation, DEGs associated with CHB were convergent with the Th17‐cell‐associated genes from the KEGG database. Hub genes of DEG and target genes linked to Th17 cells were analysed for correlation. The AhR‐related genes were located using the GeneMANIA database. To analyse the function of the genes, GO and KEGG pathways were employed. Protein–protein interaction network analysis employed the Metascape, STRING and Cytoscape databases. Finally, Western blotting and RT‐qPCR were used to validate AhR. A total of 348 differential genes were identified in CHB patients. CytoHubba was used for screening five hub genes associated with CHB: CXCL10, RACGAP1, TPX2, FN1 and GZMA. This study aimed to determine the mechanism of elevated Th17 cells in CHB. As a result, further investigation using the convergence of DGEs and Th17 cell‐related genes identified three target genes: AhR, HLA‐DQA1 and HLA‐DQB1, all of which were elevated in CHB. The three genes were primarily involved in immune response‐related processes, according to the GO enrichment analysis. Correlation analysis of CXCL10, RACGAP1, TPX2, FN1 and GZMA genes with AhR, HLA‐DQA1 and HLA‐DQB1 revealed that AhR was positively associated with CXCL10 and GZMA genes, which best respond to the severity of CHB disease. Combined with the role of AhR in Th17 cell differentiation, the genes AhR was chosen for confirmation by RT‐qPCR and WB in this study. The results showed that the CHB group had higher expression levels of AhR at both RT‐qPCR and WB levels. Furthermore, this study's findings revealed that AhR may contribute to the development of CHB by affecting the differentiation of Th17 cells.


| INTRODUC TI ON
Chronic hepatitis B is a chronic infectious disease that is still not completely curable and can progress to liver failure, cirrhosis and even liver cancer, posing a serious threat to human health.
In chronic HBV infection, the interplay between HBV and host immunity controls the course and evolution of the disease. 1 Among them, HBV-specific T lymphocytes, including CD4 + T lymphocytes and CD8 + T lymphocytes, are essential in viral clearance and immunopathogenic mechanisms of hepatitis. 2,3Helper T cells-17 (Th17) are differentiated from CD4 + T lymphocytes.Th17 cells release Retinoic acid-related orphan receptor gamma t (RORγt) and IL-17, promoting inflammation and virus clearance. 4,5In hepatic immunological homeostasis, Th17 and Treg cells are dynamically balanced.
However, when liver diseases develop, the balance between Th17 and Treg breaks down and can lead to disease progression. 6][9][10][11] According to recent studies, variations in Th17 cell numbers may be related to antiviral effectiveness in CHB. 12,13However, very little indepth research has been done on its mechanisms.
Therefore, this research aims to explore the mechanism of Th17 cell irregularity in chronic hepatitis B by employing RNA transcriptome sequencing (RNA-seq) of CD4 + T lymphocytes in peripheral vein blood to identify genes associated with CHB, homeostasis and validating Th17 cell-related target genes by using quantitative reverse transcription-PCR (RT-qPCR) and Western blotting (WB).

| Clinical research subjects
The research related to human use has complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and the study protocol was obtained from the First Affiliated Hospital of Kunming Medical University's ethics committee.Informed consent has been obtained from all individuals included in this study.PASS 15 was used to estimate the sample size.This study included the HC group 15 instances and 15 patients of the CHB group between March 2020 and May 2020.Three cases were selected from the HC and CHB groups and randomly selected to undergo RNA transcriptome sequencing.The CHB group satisfied the criteria for the diagnosis of CHB in China's Chronic Hepatitis B Prevention and Control Guidelines (2019 edition). 14Diagnoses of fatty liver disease, autoimmune disorders, other viral hepatitis, immunodeficiency diseases, alcoholic liver disease and current immunosuppressive or immunomodulatory medications were used as exclusion criteria.Prior to receiving therapy with nucleotide analogues and/or interferon α-2b, peripheral vein blood samples from CHB patients and the HC group were taken for RNA sequencing, RT-qPCR and WB, respectively.

| RNA extraction library construction and sequencing
Ficoll® was used as a density gradient medium to separate the peripheral blood mononuclear cells (PBMCs) from fresh peripheral venous blood that contained anticoagulants from the CHB and HC groups (5 mL).In order to separate CD4 + T lymphocytes from PBMCs, a Human Magnetic Bead Sorting Kit (Negative Sorting) (Bioegend, 480,013) was used.Trizol lysate was then added, rapidly frozen in liquid nitrogen and stored at −80°C.Cells were sequenced by a sequencing company.Following the manufacturer's instructions, the Trizol reagent (thermofisher, 15596018) was used to extract the total RNA from CD4 + T cells.The next step is to begin the RNA transcriptome sequencing; Figure S1's flow chart of the experiments illustrates this.

| Bioinformatics analysis
Through base recognition analysis, the raw picture data from highthroughput sequencing performed on the Illumina Novaseq TM 6000 sequence platform was transformed into the original sequence.Subpar sequences were filtered using Cutadapt to get clean data for further analysis. 15The detailed analysis is identical to Li's article. 16w sequence data are being submitted to the NCBI Gene Expression Omnibus (GEO).The record number is GSE221213.

Quantification of gene abundance
StringTie 17,18 was utilized to compose the default parameter set for each sample mapping read.Utilizing the gffcompare software, the transcriptomes of all samples were combined to reconstruct an entire transcriptome.Following the generation of the final [21] Differentially expressed genes (DEGs) and Th17 cell-related target genes analysis Differential gene expression analysis was carried out using the DESeq2 software package for two distinct groups.Genes with false discovery rate (FDR) parameters <0.05 and ≧2-fold change were thought to be differentially expressed genes.
To find the target genes associated with Th17 cells in the chronic hepatitis B sequencing genes, the differential genes analysed above converged with the Th17 cell-associated genes from the KEGG database.

Genes functional analysis
We then enriched for differentially expressed genes with respect to GO (Gene Ontology) function and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway. 22,23GO analysis of functional enrichment was obtained, satisfying this requirement with p below .05were defined as GO terms significantly enriched in DEGs.Pathway enrichment analysis was performed by KEGG pathways satisfying this requirement with p below .05were defined as pathways significantly enriched in DEGs. 24,25

Protein-protein interaction (PPI) network construction
In order to identify differential gene interactions, DEGs and Th17 cell-related target genes were analysed using the STRING database.
The study also analysed the hub genes and mapped the molecular interaction networks of top 50 DEGs using Cytoscape.

| AhR mRNA detection using RT-qPCR
First, TRIzol®Reagent was added to lysis of PBMC and then left the supernatant after centrifuging at 4°C, 12,000 g for 10 min.
Next, glacial acetic acid and the phase-separating reagent were added to the supernatant liquid, at 12,000 g at 4°C centrifuged for 15 min after leaving for 3 min at room temperature.Isopropanol was added to the new supernatant and −20°C overnight.
After centrifugation the next day, the precipitate was combined with 80% ethanol and kept on ice for 5 min.The supernatant was discarded 10 min after 12,000 g centrifugation at 4 C. RNA-free water was added to the total RNA.Retro-transcription of RNA into cDNA was performed strictly in accordance with SureScript™ First-Strand cDNA Synthesis Kit (GeneCopoeia, QP056) instructions.qPCR reaction solution configuration strictly followed SYBR Green qPCR (GeneCopoeia, QP031) instructions.Sequences of the PCR primers were given in Table S1.Finally, combine the amplification curve, melting curve, calculated the CT value according to the standard curve analysis, and finally calculated the 2 −△△C T value.

| AhR protein expressions measurement by WB
PBMC was added with 300 μL of RIPA lysate (containing 30 μL of protease inhibitor), shaken, and lysed on ice for 15 min.The cells were repeatedly puffed with a pipette gun, centrifuged at 13,958 g, 4°C for 15 min and collected supernatant.In other words, the total protein solution and the protein concentration were calculated according to the BCA protein concentration measurement kit (beyotime, P0009-1).The next step is SDS-PAGE protein electrophoresis followed by membrane transfer.Following step, the PVDF membrane was placed in 5% bovine serum albumin (BSA) and closed at temperature (20°C-30°C) for 1 h; after TBST rinsing, added separately the anti-AhR (affinity, AF6278), and place it in the refrigerator at 4°C overnight.The next day, removed the hybridization bag, rewarmed it at room temperature for 10 min, removed the primary antibody liquid, added the enzyme secondary antibody after TBST washing and incubated for 2 h at room temperature.After washing using TBST, added ECL solutions A and B of the kit and clicked live acquire to start image acquisition.As a loading control, β-actin (Abcam, ab6276) was used.

| Statistical analyses
Data analysis of RNA transcriptome sequencing was described in Section 2.3.2.Graphpad prism 8.0 and SPSS 26.0 (IBM) were used to analyse RT-qPCR and WB validation data.The information is presented in the form of median (interquartile range, IQR) or means ± standard deviation (SD).The independent samples t-test and Mann-Whitney U tests were utilized to identify differences between groups depending on whether the data had a normal or non-normal distribution.At a p-value of .05 or below, all two-tailed tests were deemed statistically meaningful.To minimize selection bias, the study selected the HC group that met the diagnostic criteria from the physical examination department and the CHB group that met the diagnostic criteria from the infection and gastroenterology departments.

| Analysis of clinical data included in the study subjects
As shown Table 1, There was no statistically significant difference in sex and age between the HC and CHB groups (p > .05).The levels of alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TB) increased obviously in CHB group (p < .05).

| Analysis of CHB differential gene expression
Results of differential gene expression of CD4 + T lymphocytes by RNA transcriptome sequencing are shown in Figure 1A,B.The HC group was screened for 263749 genes and the CHB group was screened for 268512 genes.Then the absolute value of log 2 FC (difference multiplicity) was ≥1 and q < 0.05 (|log 2 FC| ≥ 1 and q < 0.05) as the criteria, The screening of 348 differentially expressed genes revealed 224 genes up-regulation and 124 genes down-regulation.
Cluster analysis was performed for all differentially expressed genes (Figure 1C).In the clustering plot, the colours from blue to red and to white showed varying degrees of gene expression, with red designating genes with high expression and blue, low expression.
Figure 1C shows that the differential genes in the CHB group are mostly highly expressed.

| Functional enrichment analysis of CHB differentially expressed genes
According to the GO functional enrichment analysis, the majority of the differential genes were focused on biological processes, including immunological response, negative regulation of the T cell receptor signalling pathway, and other activities.The gene expression was higher in cellular components and molecular functions.The highest gene expression was found in cellular components constituting the cell membrane, plasma membrane and the highest gene expression was found in molecular functions involved in protein binding.
Terms for cellular components, molecular functions and biological processes were listed in order of differential genes number (S-gene count) and illustrated from largest to smallest, and accordingly the Top 15, Top 10, Top 25 terms were selected to be mapped in the GO enrichment classification bar chart for display.GO enrichment scatter plot displayed the Top 20 terms with the smallest p-value (Figure 1D,E).
Using KEGG to enrich the pathways involved in differentially expressed genes, as shown in Figure 1F, most genes were enriched in diseases, such as herpesvirus infection, and influenza virus infection; followed by organ system processes, including Th17 cell differentiation, signalling pathways of chemokine, mTOR, IL-17 and JAK/ STAT.

| Construction of PPI networks
Figure 1G shows PPI networks of 348 differential genes associated with chronic hepatitis B, most up-and down-regulated genes had interactions.Finally, according to using CytoHubba, the study identified five hub genes associated with CHB: CXCL10, RACGAP1, TPX2, FN1, GZMA, which were elevated (Figure 1H).

| Screening for Th17 cell-associated target genes from differential genes in CHB
According to convergence investigation of 348 differential genesrelated CHB with 108 Th17 cell-associated genes from the KEGG database, the study revealed three Th17 cell-related target genes: AhR, HLA-DQA1 and HLA-DQB1, which were all elevated (Figure 2A,B).

| Function analysis of target genes related to Th17 cells
AhR, HLA-DQA1 and HLA-DQB1 were functionally analysed using GO and KEGG.
GO functional enrichment analysis showed 21 GO enrichment terms.In biological processes, the three genes are primarily involved in immune response-related processes; in cellular components, they are primarily involved in the composition of MHC class II protein complex and endoplasmic reticulum membranes; and in molecular functions, they are primarily involved in MHC class II receptor activity, MHC class II protein complex binding and so on (Figure 2C).
In Figure 2D, the GO functional analysis of AhR, HLA-DQA1 and HLA-DQB1 in chronic hepatitis B is displayed.AhR, HLA-DQA1 and HLA-DQB1 were enriched for 18 GO terms in chronic hepatitis B. The three genes were mainly involved in immune response and immune system function in biological processes; they were primarily involved in cellular components such as the transport vesicle membrane, a part of the plasma membrane and the MHC class II protein complex in biological processes; and in molecular functions, they were only involved in DNA-binding transcription factor activity.
These results suggested that the three genes were mostly involved in biological processes related to immune responses and cellular membrane components.

| Correlation investigation of Th17 cell-associated target genes with chronic hepatitis B-associated hub genes
The

| Function analysis and PPI networks construction of AhR
The study searched for 20 AhR-related genes through GeneMA-NIA database (http://genem ania.org/)(Figure 4B).The Metascape database was used for functional enrichment analysis of 21 genes, including AhR, and it was discovered that they were primarily enriched in the AhR pathway (Figure 4C).The creation of a PPI network revealed that AhR interacted with the AHRNT and AHRNT2 proteins (Figure 4D).

| Differential validation analysis of AhR genes (RT-qPCR)
mRNA expression levels of AhR upregulated statistically in the CHB group compared to the HC (p < .05)(Figure 5A) and were consistent with the RNA-seq results.

| Differential validation analysis of AhR protein expression levels (WB)
Western blotting results were presented (Figure 5B, C).According to β-actin expression levels, we detected the relative levels of AhR protein expression and observed that the CHB group was expressed at a higher level than the HC group (p < .05),which was in accordance with the RT-qPCR results.

| DISCUSS ION
Chronic hepatitis B has a very complicated pathophysiology, and curing it is still difficult everywhere in the globe.The immune system's response to HBV infection among individuals is crucial to the prognosis and course of hepatitis B. The chronic hepatitis B-associated hub genes evaluated in this study include CXCL10, RACGAP1, TPX2, FN1 and GZMA.CXCL10 and GZMA have been found to be involved in the immune response.The inflammatory chemokine CXCL10 belongs to the CXC family and is involved in cell mobility and the inflammatory reaction.8][29] GZMA promotes inflammatory responses in viral and bacterial infections 30,31 and may be involved in the development of HCC. 32Treg 6,10 imbalances, to be related to liver fibrosis and cirrhosis and to be able to predict the severity of illness. 39These findings imply that Th17 cells play a role in the pathogenesis of HBV infection.
However, there are no studies on the mechanism of Th17 cell dysregulation in HBV infection.
By convergence analysis of DEGs with genes associated with Th17 cells coming from the KEGG database, it was discovered in this study that AhR, HLA-DQA1 and HLA-DQB1 were involved in Th17 cell differentiation and had elevated expression in CHB.
MHC class II molecule HLA-DQA1 and DQB1 genes were engaged in the control of immune-specific responses to common allergens in asthma 40 and skin cutaneous melanoma. 41Due to their MHC class II molecule antigen-presenting capacity, HLA-DQA1 and DQB1 have been found to support immunological responses in many diseases.HLA-DQA1 and DQB1 have been researched in chronic hepatitis B, 42 despite the fact that the functional mechanism has not been examined.
Numerous studies have demonstrated that the AhR is essential for controlling innate and adaptive immunity, including controlling the development and operation of Treg and Th17 cells.The AhRreceptor-ARNT complex binds to a genomic region containing particular DNA recognition sites in the nucleus, resulting in downstream gene expression. 43The aforementioned findings support the roles played by AhR immune responses in biological processes, transcription factor complexes in cellular components and DNA-binding transcription factor activity in molecular functions in different disorders.
However, except for us, there are no other reports concerning the role of AhR in chronic hepatitis B.
So AhR has been identified to be important in Th17 cell development via unique signalling pathways in various illnesses, [44][45][46][47] but it has not been examined in CHB, which is also a current research focus.
Correlation analysis of CXCL10, RACGAP1, TPX2, FN1, GZMA with AhR, HLA-DQA1, and HLA-DQB1 revealed that AhR and HLA-DQB1 were positively connected with CXCL10, AhR was positively connected with GZMA, but HLA-DQA1 and HLA-DQB1 had no correlation with GZMA.It was discovered that CXCL10 and GZMA stimulated an inflammatory response following viral infection, 26,31 and it is thought that CXCL10 and GZMA play a significant role in HBV infection, suggesting that AhR may play a significant role in the progression of chronic hepatitis B illness.
Because AhR was positively associated with CXCL10, GZMA, it might indirectly respond to CXCL10, GZMA expression in CHB.
Combined with the role of AhR in Th17 cell differentiation, AhR was chosen as a validation indicator in CHB peripheral blood in the study to create the groundwork for the next cellular experiment to explore the mechanism of AhR involvement in Th17 cell differentiation.
The Aryl Hydrocarbon Receptor (AhR) is a transcription factor that is a fundamental member of the helix-turn-helix family.It is ligand-dependent, meaning that it requires the binding of a specific molecule in order to be activated and regulate gene expression.It is made up of the Per-Arnt-Sim structure, which associates with a variety of both exogenous and endogenous compounds as well as certain cofactors such as heat shock protein 90 (HSP90) and proteins associated with HBV-X. 43,48AhR is essential for mediating the biological response to a range of physiological and environmental chemicals. 43,49AhR is present in intrinsic and specific immune cells and regulates immune cell proliferation, differentiation and cytokine production, which are important for infection and inflammation control. 50,51cently, it has been discovered that AhR plays an important role in controlling Th17/Treg equilibrium.Sun and his team demonstrated that The AhR-hypoxia-inducible factor 1 and AhR-GOT1 molecular pathways influence lung responses to PM2.5 by disrupting the balance of Th17/Treg cells. 52Xie et al. 53 showed that in an AhR-dependent way, Sema4D caused an imbalance of Th17/Treg and contributed to the pathogenesis of ankylosing spondylitis.Takei et al. 54  The existence of Th17/Treg imbalance in chronic hepatitis B has been previously reported in the literature, 9,56 but the exact mechanism is unclear, and whether and how AhR is involved in regulating Th17/Treg balance in chronic hepatitis B needs further study.
Current research has found that gut microbial dysbiosis is associated with a variety of diseases.The role of AhR and gut microbes in disease is also receiving increasing attention.In Alzheimer's disease (AD), the dysregulated gut microbes produced large amounts of tryptophan metabolites that activate AhR signalling in the brain, disrupting the blood-brain barrier and promoting inflammation and AD pathology. 57Human umbilical mesenchymal stem cells (HUMSCs) cured collagen-induced arthritis (CIA) in mice by modulating the interplay between host immunity and gut microbiota via the AhR. 58It suggested that AhR is essential for the control of immune responses and gut microbiota.
The presence of the gut-liver axis leads to a close relationship between gut microbiota and the development of liver disease.An increasing amount of research reveals a link between gut microbial dysbiosis and illness progression in CHB patients. 59,60Some research on alcoholic Liver Disease, 61 Nonalcoholic Fatty Liver Disease (3) It has not been examined how AhR affects Th17 transcription.
In conclusion, Th17 cell dysregulation may also be one of the immune mechanisms in the pathogenesis of chronic hepatitis B. AhR, as one of the many genes regulating Th17 cell differentiation, could be used as a biomarker in response to Th17 cell differentiation in the future.

AUTH O R CO NTR I B UTI O N S
Ruyi Zhang designed the study, implemented the experiment and wrote the first draft of the article.Huaie Liu reviewed the experimental process and experimental data.Jie Ling helped with data evaluation.Jing You and Jiawei Geng summarized the data and revised the manuscript.

ACK N O WLE D G E M ENTS
The Yunnan Provincial Fund Project (YNWR-MY-2019-072) and the National Natural Science Foundation of China (81760617 and 81760111) provided funding for this study.We thank all healthy volunteers and patients with chronic hepatitis B who were recruited into the study.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The information and content of this manuscript are free of any conflicts of interest, according to the authors.

Figure 3
Figure 3 depicts the KEGG signalling pathways for AhR, HLA-DQA1 and HLA-DQB1 involved in Th17 cell differentiation.

F I G U R E 1
correlations between AhR-, HLA-DQA1-, HLA-DQB1-related Th17 cell differentiation and CXCL10-, RACGAP1-, TPX2-, FN1-, Results of differential gene expression analysis by transcriptome sequencing of CD4+ T lymphocytes.(A) Bar graph of differential genes expressed.(B) Volcano map of differential genes expressed.(C) Cluster analysis of differential.(D) GO enrichment bar graph.(E) GO enrichment scatter plot.(F) KEGG pathway classification chart of differential genes expressed.(G) PPI networks of 348 differential genes associated with chronic hepatitis B. (H) Interaction network of hub genes among top 50 DEGs associated with CHB.Control: HC group; patient: CHB group.GZMA-associated CHB were analysed (Figure 4A).According to the study, AhR had a positive strong link with CXCL10 and GZMA; HLA-DQA1 had a positive relationship with RACGAP1 and TPX2 and HLA-DQB1 had a positive strong association with CXCL10.

F I G U R E 2
Screening and analysing Th17 cell-associated target genes from DEGs in CHB.(A) Venn diagram of Th17 cell-associated target genetic screening.(B) Cluster analysis of Th17 cell-associated target genes.(C) GO enrichment scatter plot.(D) GO enrichment plot of AHR, HLA-DQA1 and HLA-DQB1 in CHB.BP, biological processes; CC, cellular components; MF, molecular functions.

F I G U R E 3
KEGG signalling pathways for AhR, HLA-DQA1 and HLA-DQB1 are involved in Th17 cell differentiation.TPX2 might have a role in the development and progression of HBV-related HCC.It has the potential to predict the prognosis of HBV-associated HCC. 33-35Through affecting the viral protein NS5B polymerase's activity, RACGAP1 is essential for HCV replication 36 but has not been studied in CHB.Th17 cells are crucial for host immunity.Th17 cells play a role in the body's inflammatory response, tissue damage and disease development by secreting cytokines including IL-17 and IL-22, encouraging the release of IL-6 and TNFα 1 .Th17 cell levels have been reported to be increased in CHB, to have Th17/Th1 37,38 and Th17/ F I G U R E 4 AhR-related analysis.(A) Correlation analysis of the hub genes linked with chronic hepatitis B and the target genes related to Th17 cells (*p < .05,**p ≦ .01).(B) AhR-associated genes.(C) GO pathway and process enrichment analysis heatmap of AhR and AhRassociated genes.(D) PPI network of AhR and AhR-associated genes.

F I G U R E 5
found that in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, stimulation of AhR signalling attenuated pulmonary fibrosis by increasing Tregs and suppressing inflammatory T cell subsets.According to Quintana et al., 55 various AhR agonists differently controlled the differentiation of Th17 and Treg cells in the autoimmune mic model.Liu et al. 45 found that STAT3 was involved in AhR regulation of Th17 and Treg cell differentiation.Validation of AhR gene expression in peripheral blood of CHB patients [median (interquartile range, IQR)].(A) mRNA levels of AhR in HC group and CHB groups.(B) AhR protein expression levels relative to β-actin in HC and CHB groups (compared to β-actin).(C) AhR protein expression bands in HC and CHB groups.CHB, Chronic hepatitis B; HC, Healthy control.*p < .05,**p ≦ .01,***p ≦ .001,****p ≦ .0001.The aforementioned data demonstrate that AhR is engaged in Th17/Treg homeostasis via various pathways in various illnesses, either as a pathogenic factor or as a protective factor, emphasizing that AhR has an essential immunomodulatory function in disease development and prognosis.
62 and acute liver failure62 has concentrated on microbial ecology treatments to control AhR signallings, such as antibiotics, prebiotics/probiotics and faecal microbiota transplantation (FMT).These results clarify the innovative therapeutic strategy for CHB.However, more verification is still required.By using RT-qPCR and Western blotting validation of peripheral blood, the study discovered that AhR expression was considerably higher in the chronic hepatitis B group, indicating that AhR may be implicated in HBV infection.We hypothesized that AhR may contribute to the emergence of chronic hepatitis B by influencing the differentiation of Th17 cells in combination with the findings of bioinformatics research.However, this study also has shortcomings: (1) Further study of the impact of AhR on Th17 cell development did not involve in vitro cellular experiments.(2)The signalling pathway analysis related to AhR regulation of Th17 cell differentiation was not performed in the study.
Analysis of clinical data included in the study subjects.