Rheumatoid arthritis (RA) is a common, chronic autoimmune disease that affects approximately 1% of the general population (1). The main clinical features of RA patients are synovial inflammation, bone and cartilage erosion, and symmetric polyarthritis with joint swelling (2), however, the pathogenesis of this disease is not fully understood. In previous studies, the immunological dysfunctions of RA were identified in various immune cells, such as T cells, B cells, macrophages, and abnormal expression levels of inflammatory cytokines, including IL-1, IL-6, IL-10, TNF-α, were observed in RA patients (3). In addition to immunological findings, many studies have also found an influence of genomic and epigenomic features, and environmental factors on disease activity, prognosis, and therapy prediction of RA (4–6). Genome wide association study and candidate gene studies have identified a large number of RA related genes/loci in different ethnic groups, however, these genes/loci only account for a small part of RA phenotypic variation (7, 8). DNA methylation is a widely studied form of epigenetic modification, and some prior studies have suggested that an association between DNA methylation and inflammation-regulated immune pathways may play an important role in the pathogenesis of RA (9).

Previous studies confirmed that environmental pollutants from smoking and hydrocarbon burning were associated with RA disease risk, as both pollutants contained agonists or exogenous ligands for aryl hydrocarbon receptor (AHR) that may promote AHR activation (10). As a ligand-activated transcription factor, AHR is involved in regulating the gene activity in response to hydrophobic halogenated aromatic hydrocarbons in order to promote the metabolism, and elimination of xenobiotics. The role of AHR in cellular responses against a variety of endogenous and physiological ligands have been extensively studied, while increasing evidence indicates that AHR is involved in the regulation of innate and adaptive immune responses, and immunologic processes in autoimmune disease, including multiple sclerosis (MS) (11, 12). For example, AHR expression is abnormally elevated in synovial tissues of RA patients and could regulate the expression of cytokines such as growth factors (13). Over-activation of AHR signaling may promote inflammation and bone destruction in RA by activating macrophages, osteoclasts, dendritic cells, and inhibiting osteoblasts (10).

In the regulation of the AHR signaling pathway, ligand binding to AHR causes conformational changes that induce this receptor to migrate to the nucleus where it can generate a hetero dimer with AHR nuclear translocation protein (ARNT) to form aryl hydrocarbon receptor complex, inducing the transcription of relevant target genes (14). In addition, AHR repressor (AHRR) serves as a negative regulator within this pathway through inhibiting AHR-mediated signal transduction (15). Recent studies have investigated the association of common, potentially functional single nucleotide polymorphisms (SNPs) in AHR signaling pathway genes with MS, essential hypertension (14, 16). Given the importance of the AHR signaling pathway in the development of RA, this present study aimed to evaluate the association between SNPs in the AHR signaling pathway genes (AHR, ARNT, AHRR) and RA susceptibility. The methylation levels of these genes were also detected in these patients because DNA methylation is considered to be an important factor for RA.

In this study, the mean age of the 479 RA patients was 52.71 ± 12.42 years, including 391 females and 88 males, and the average age of the 496 healthy controls was 50.61 ± 14.76 years, including 384 females and 112 males. The genotype distributions of all SNPs in RA patients were in accordance with HWE (rs2066853, P = 0.997; rs2158041, P = 0.792; rs2282885, P = 0.555; rs10847, P = 0.747; rs1889740, P = 0.530; rs11204735, P = 0.914; rs2292596, P = 0.713; rs2672725, P = 0.346; rs349583, P = 0.913). In healthy controls, AHR rs2158041 did not conform to HWE (P = 0.023), while other SNPs conformed to HWE (rs2066853, P = 0.249; rs2282885, P = 0.401; rs10847, P = 0.871; rs1889740, P = 0.461; rs11204735, P = 0.603; rs2292596, P = 0.373; rs2672725, P = 0.918; rs349583, P = 0.855). Therefore, this study excluded rs2158041, and the allele and genotype frequency distributions of other SNPs in RA patients and healthy controls were shown in Tables 2 , 3 .

The impact of environmental factors on the pathogenesis of autoimmune diseases has attracted increasing attention (20). AHR is considered to be a crucial regulator of host-environment interactions, especially in immune and inflammatory responses (21, 22). Proper regulation of AHR-mediated signal transduction might be critical for maintaining immune cell function in autoimmunity, while abnormal AHR signaling has been closely related to the pathogenesis of autoimmune diseases (13). Clarifying the role of AHR in disease and health is crucial in understanding the disease occurrence process and individual differences in treatment response, both of which might contribute to the development of novel therapies (23). RA is a common autoimmune disease and AHR plays a role in regulating the response to the environment factors and may contribute to disease development. Clinical studies had demonstrated that AHR expression was approximately two-fold higher in RA patients than in controls (24). AHRR expression of the synovia was significantly increased from RA patients who smoke cigarettes, but not from the patients who do not smoke, which indicated that there was a potential interaction between AHR activation and cigarette smoking in RA patients (25). The murine models of RA suggested that AHR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could contribute to RA disease severity, disease progression, osteoclasts differentiation, and increase the numbers of IL17-expressing cells in the inflamed joints (10). In addition, previous studies had shown that several SNPs located near AHR-regulated genes might contribute to AHR-dependent disease mechanisms (26). Therefore, we investigated the association between multiple SNPs in AHR signaling pathway genes (AHR, ARNT, AHRR) and RA genetic risk, and detected the methylation levels of these genes in RA patients.

A large number of studies had discussed the association of the genetic polymorphism of AHR, ARNT, AHRR genes and human diseases such as Crohn’s disease (CD), lung cancer, male infertility, and autoimmune diseases (27–30). Zorlu et al. evaluated the relationship between multiple SNPs in the AHR signaling pathway genes, including AHR, ARNT, AHRR and MS susceptibility, and their results inferred that the AHR signaling pathway genetic variation might contribute to MS pathogenesis (14). However, our study did not indicate a significant association of AHR rs2066853, rs2282885 polymorphism with RA susceptibility, and rs2158041 was excluded as it did not satisfy HWE. Consistent with our findings, Cheng et al. demonstrated that rs2066853 polymorphism did not have any effect on RA susceptibility in Han Chinese populations (31). These results suggested that AHR gene variation might not be associated with RA, however, other functional SNPs required further investigation.

Our study also determined that ARNT rs11204735 polymorphism was significantly associated with an increased risk of RA, and that a decreased risk of rs11204735 polymorphism was observed under the dominant model. This result was similar to Schurman’s study (30), and further reflected the importance of rs11204735 polymorphism in the pathogenesis of autoimmune diseases. Haplotype analysis demonstrated that ARNT CCC haplotype frequency significantly increased in RA patients in comparison to controls. Therefore, this present study provided additional evidence that ARNT gene was an important susceptibility gene in RA, and reproducibility studies were needed to verify our results. A previous study demonstrated that rs2292596 genotype frequency was significant with genetic susceptibility to RA patients (31). Their study also demonstrated that the rs2292596 G allele might have a dangerous effect on RA (31), while we identified an increased frequency of rs2292596 G allele with no statistical significance. This difference might be due to sample size and experimental methods. Our result was also the first to show that AHRR rs2672725 GG genotype, G allele frequencies in RA patients were significantly increased, and that this SNP could be a novel susceptibility locus to RA.

In addition to DNA sequence, epigenetic variations also contained important genetic information, and the key role of epigenetic variation in the pathogenesis of human disease could not be ignored (32, 33). For example, DNA hypomethylation was associated with differentiation and proliferation of inflammatory processes, resulting in the increased transcription and secretion of inflammatory proteins (34). At present, studying the role of DNA methylation in autoimmune diseases offers an interesting perspective. One study detected the methylation status of lymphocytes in patients with systemic lupus erythematosus, RA, and found a significant hypomethylation in T cells (35). Due to the possible role of AHR, ARNT, AHRR genes in RA pathogenesis, we also examined the methylation status of these genes in RA patients. The results revealed that AHR methylation level was significantly increased, while AHRR methylation level was abnormally reduced in RA patients. These data confirmed that AHR, AHRR genes were involved in the development of RA, and provided valuable information for further revealing RA pathogenesis. RA patients could be divided into different subgroups according to the status of autoantibodies, including RF and anti-CCP, which was of great significance when selecting appropriate treatments. Our study also determined that ARNT methylation level was related to the anti-CCP status of RA patients. In addition, we also revealed that AHRR rs2672725 polymorphism was significantly associated with AHRR methylation level in RA patients. We assumed that rs2672725 might be involved in the pathogenesis of RA by affecting AHRR methylation level, however further verification was needed.

Our study had several limitations. First, the sample size was insufficient and might affect the power of this study. Second, this study failed to analyze the influence of the interaction between environmental factors and gene variation on RA occurrence due to a lack of information on environmental factors. Undoubtedly, a complete understanding of the precise role of AHR signaling pathway genes in RA pathogenesis will require additional experiments and clinical studies with larger sample sizes.

In conclusion, our results provided strong evidence that the ARNT gene rs11204735, AHRR gene rs2292596, rs2672725 polymorphisms were related to RA susceptibility in the Chinese population, while AHR genetic variation might not be associated with RA risk. These three SNPs might modify individual genetic susceptibility to RA, which could provide a basis for early detection of RA susceptible individuals. Furthermore, an increased methylation level of AHR, as well as a decreased methylation level of AHRR, was identified in RA patients, and ARNT methylation level was related to anti-CCP in RA patients. We can assume that AHR, AHRR methylation level might be used as novel auxiliary biomarkers for RA and ARNT methylation level could be used to distinguish the different serotypes of RA.

The data presented in the study are deposited in the dbSNP repository, accession number 1063327. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethical Committee of the First Affiliated Hospital of USTC (Hefei, Anhui, China). The patients/participants provided their written informed consent to participate in this study.

X-ML and G-SW designed the study. T-PZ and H-ML conducted the experiment. RL performed the statistical analyses. H-ML performed the statistical analyses. NX and ZT participated in the collection of samples. T-PZ drafted the manuscript. X-ML, G-SW and RL contributed to manuscript revision. All authors contributed to the article and approved the submitted version.

This work was supported by the Fundamental Research Funds for the Central Universities (WK9110000180, WK9110000148), Anhui Provincial Natural Science Foundation (2108085QH362), and National Natural Science Foundation of China (81871271).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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