Genetic polymorphisms of regulatory T cell-related genes modulate systemic inflammation induced by viral hepatitis.

Clinical and laboratory characteristics stratified by the genotypes of IL‐2 SNPs rs2069763 and rs2069772 are shown in Tables  1 and 2 , respectively. No significant differences in the level of ESR, CRP, ALT, or FIB‐4 index were noted between the different genotypes of the two SNPs. These results indicate that the two IL‐2 SNPs had no significant impact on the levels of systemic inflammatory markers, hepatic injury markers, or FIB‐4 index in the population of viral hepatitis. Clinical and laboratory characteristics stratified by rs2069763 genotypes H (2) = 0.167 p  = 0.920 F = 0.163 p  = 0.849 H (2) = 2.798 p  = 0.247 F = 0.043 p  = 0.958 H (2) = 0.230 p  = 0.891 F = 0.263 p  = 0.769 Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; DM, diabetes mellitus; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus; HLD, hyperlipidemia; HTN, hypertension. One missing value. Clinical and laboratory characteristics stratified by rs2069772 genotypes H (2) = 1.713 p  = 0.425 F = 0.640 p  = 0.529 H (2) = 0.909 p  = 0.635 F = 0.369 p  = 0.692 H (2) = 2.846 p  = 0.241 F = 1.504 p  = 0.226 Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; DM, diabetes mellitus; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus; HLD, hyperlipidemia; HTN, hypertension. One missing value. For the TGF‐β1 SNP, rs1800469, no significant difference in ESR, ALT, and FIB‐4 index levels was noted. On the other hand, we found a statistically significant difference in the level of CRP (H (2) = 6.592, p  = 0.037) between the different genotypes (GG, GA, and AA). The mean values of the CRP level of the genotype groups were 1.28 ± 0.996 mg/L for GG, 0.89 ± 0.963 mg/L for GA, and 1.00 ± 1.119 mg/L for AA, respectively (Table  3 ). As the rs1800469 (A) allele was shown to cause increased plasma concentration of TGF‐β1, we divided the participants into two groups based on the presence or absence of the A allele and compared the CRP levels of the two groups. 14 We found that participants carrying the rs1800469 (A) allele had significantly lower CRP levels than those without the allele (U = 948.500, p  = 0.010), with the mean CRP levels for the GG group and the GA + AA group at 1.28 ± 0.996 mg/L and 0.93 ± 1.023 mg/L, respectively. The statistically significant difference remained after controlling the confounding factors with Quade's test (F = 6.744, p  = 0.010) (Table  4 ). These results suggest that the participants carrying the rs1800469 (A) allele had a higher level of TGF‐β1, which causes an increased level of FOXP3 in Tregs to enhance the suppression of inflammatory responses, resulting in a lower level of CRP. However, no statistically significant difference was observed in the ALT or FIB‐4 index level between the different genotype groups. The lack of significant associations in either the ALT or FIB‐4 index with rs1800469 genotype groups suggests that the TGF‐β1 SNPs' effect on systemic inflammation is independent of the hepatic inflammation or fibrosis induced by viral hepatitis. Clinical and laboratory characteristics stratified by rs1800469 genotypes H (2) = 1.053 p  = 0.591 F = 0.621 p  = 0.539 H (2) = 1.603 p  = 0.449 F = 0.441 p  = 0.645 H (2) = 6.592 p  = 0.037* F = 3.349 p  = 0.038* Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; DM, diabetes mellitus; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus; HLD, hyperlipidemia; HTN, hypertension. One missing value. Clinical and laboratory characteristics stratified by GG and GA + AA of rs1800469 genotypes U = 1217.000 p  = 0.307 F = 1.103 p  = 0.295 F = 0.404 p  = 0.526 U = 948.500 p  = 0.010* F = 6.744 p  = 0.010* Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus. One missing value. For the FOXP3 SNP, rs3761547, a statistically significant difference in the ESR levels between the two rs3761547 genotypes (CT vs. TT) (U = 343.500, p  = 0.032) in females was observed, with the mean ESR levels for the CT group and the TT group at 21.04 ± 10.542 mm/h and 15.98 ± 9.146 mm/h, respectively. After controlling the confounding factors with Quade's test, a significant difference was still observed (F = 4.706, p  = 0.034). However, no differences in ESR level in males and CRP and ALT in both genders were noted (Tables  5 and 6 ). Clinical and laboratory characteristics of male participants stratified by rs3761547 genotypes U = 366.000 p  = 0.870 F = 0.041 p  = 0.840 U = 318.000 p  = 0.379 F = 1.177 p  = 0.282 U = 309.500 p  = 0.315 F = 1.881 p  = 0.175 Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; DM, diabetes mellitus; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus; HLD, hyperlipidemia; HTN, hypertension. Clinical and laboratory characteristics of female participants stratified by rs3761547 genotypes U = 404.000 p  = 0.276 F = 1.669 p = 0.201 U = 343.500 p  = 0.032* F = 4.706 p  = 0.034* U = 496.000 p  = 0.895 F = 0.123 p  = 0.727 Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; DM, diabetes mellitus; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus; HLD, hyperlipidemia; HTN, hypertension. One missing value. Furthermore, the FOXP3 SNP rs3761547 caused a significant difference in the FIB‐4 index within both the male and the female groups (U = 225.000, p  = 0.024 for males; U = 320.000, p  = 0.026 for females). Noteworthy is the opposite effect of the FOXP3 SNP observed within the male and the female groups. Among the male participants, the mean values of the FIB‐4 index of those carrying the C allele and the T allele were 1.43 ± 0.672 and 2.06 ± 1.111, respectively, while the mean values of the females carrying the CT genotype and TT genotype were 3.06 ± 2.005 and 2.17 ± 1.199, respectively (Tables  5 and 6 ). To examine whether the opposite effect observed between the male and female groups was due to the moderation by sex, we performed a linear regression‐based analysis. The participants were subgrouped into two groups, participants carrying the C allele were in the C group (C for males and CT for females), and participants not carrying the C allele were in the T group (T for males and TT for females). We found a significant interaction between sex and FOXP3 rs3761547 SNP on the FIB‐4 index (b = 1.5060, p  = 0.0045). These results indicate that female participants carrying the rs3761547 C allele had a significantly higher Fibrosis‐4 (FIB‐4) index than the females carrying the TT genotype, while the rs3761547 C allele had the opposite effect in males. The opposite effect on FIB‐4 observed in different sex was due to the moderation by sex on the FOXP3 SNP genotype (Figure  1 ). The effect of the FOXP3 SNP genotypes on the Fibrosis‐4 (FIB‐4) index is moderated by sex. Female participants carrying the rs3761547 C allele had a significantly higher Fibrosis‐4 index than the females carrying the TT genotype, while the rs3761547 C allele had the opposite effect in males. A significant interaction between sex and FOXP3 rs3761547 SNP on the FIB‐4 index was found. SNP, single nucleotide polymorphism For the ADCY9 SNP, rs1967309 were listed. No statistically significant differences in the level of ESR, CRP, ALT, and FIB‐4 index were noted (Table  7 ). These results indicate that the ADCY9 SNP examined in this study had no significant impacts on the levels of systemic inflammatory markers, ALT (hepatic injury marker), or FIB‐4 index (index for liver fibrosis) in the population of viral hepatitis. Clinical and laboratory characteristics stratified by rs1967309 genotypes H (2) = 0.820 p  = 0.664 F = 0.495 p  = 0.611 H (2) = 0.010 p  = 0.995 F = 0.287 p  = 0.751 H (2) = 0.884 p  = 0.643 F = 0.682 p  = 0.507 Abbreviations: ALT, alanine aminotransferase; CRP, C‐reactive protein; DM, diabetes mellitus; ESR, erythrocyte sedimentation rate; FIB‐4, Fibrosis‐4; HBV, hepatitis B virus; HCV, hepatitis C virus; HLD, hyperlipidemia; HTN, hypertension. One missing value.

We recruited a total of 138 participants diagnosed with HBV or HCV according to established clinical diagnostic criteria from Kaohsiung Medical University Hospital (KMUH). Patients with HBV were diagnosed with positive serum HBsAg tests. Patients with HCV were diagnosed with positive serum anti‐HCV IgG tests, and 54 of these anti‐HCV IgG positive participants have also obtained sustained virologic response (SVR). The patients' demographic characteristics were obtained during the interview with the participants, including age, sex, metabolic syndromes (hypertension, diabetes mellitus, and hyperlipidemia), and lifestyle behaviors (smoking). Age, sex, and the status of hepatitis virus infection were confirmed with medical records. Levels of ESR, CRP, thyroid‐stimulating hormone (TSH), creatinine, aspartate aminotransferase (AST), ALT, and platelet count (PLT) of the participants were checked at KMUH. Laboratory values exceeding the reportable limit established by the laboratory were calculated as either upper reportable limit value or lower reportable limit value. The FIB‐4 index was calculated with the formula: age (years) × AST [IU/L]/(PLT [10 9 /L] × (ALT [IU/L]) 1/2 ). Patients with the following features were excluded from our study: a history of malignancy, psychiatric disorders, acute infectious conditions, chronic kidney disease (stage IV, V), and HBV and HCV co‐infection. The participants had all completed the informed consent before enrollment to our study. Similar to our previous studies, 12 peripheral blood leukocytes from 138 patients were collected, and DNA was extracted with the Genomic DNA Extraction Kit of Geneaid (Geneaid, New Taipei City, Taiwan). The genotyping of the selected SNPs of all participants was performed with C2‐58 Axiom Genome‐Wide TWB 2.0 Array Plate based on the Affymetrix GeneChip platform. The laboratory technicians were blinded to the patient's demographic characteristics and read the results. As FOXP3 is located on the X chromosome, the genotypes of the FOXP3 SNP rs3761547 of male and female participants were analyzed separately. Since the rest of the selected SNPs, including rs2069763 and rs2069772 of IL‐2, rs1800469 of TGF‐β1, and rs1967309 of ADCY9, were not located on the sex chromosomes, the genotypes of both male and female participants were combined for analysis. All statistical analyses were performed on SPSS (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.). A two‐sided p of ≤0.05 was considered statistically significant. The distribution properties of quantitative variables were expressed using means ± SD and qualitative variables using frequencies or percentages. Normality was examined with the application of the Kolmogorov–Smirnov test or Shapiro–Wilk test. Quantitative parametric variables were tested with the Student t ‐test, and quantitative nonparametric statistics analyses were tested with the Mann–Whitney U test or Kruskal‐Wallis test. Qualitative variables were tested with a chi‐square test between the stratified groups. All the genotype frequencies of the selected SNPs in our study followed the Hardy–Weinberg equilibrium (Table  A1 ). Variables with the potential to interfere with the levels of inflammatory markers (ESR and CRP) and hepatic injury maker (ALT), including age, gender, smoking, thyroid disorder, renal function, hypertension, diabetes mellitus, and hyperlipidemia, were set as covariates in Quade's test to eliminate the effect of confounding factors. We also combined the genotypes with low frequency during statistical analysis to increase the power of the test. A regression‐based analysis was carried out with PROCESS macro on SPSS to calculate the interaction between sex (“female” was coded as 0 and “male” was coded as 1) and FOXP3 rs3761547 genotype (group carrying “C” was coded as 1 and group carrying “T” only was coded as 2) on the FIB‐4 index. 13

Tregs play a pivotal but complicated role in regulating the immune system of individuals with viral hepatitis. The differences in the Treg‐related genetic polymorphisms may result in different genetic product levels and, consequently, modify Tregs' activities and functions. In the current study, we used ESR and CRP as markers to evaluate the participants' systemic inflammatory conditions induced by viral hepatitis, while ALT and FIB‐4 index as indicators of hepatic injury and fibrosis. For evaluation of inflammatory status, one study had proposed the priority of CRP over ESR. 15 However, serial measurement of the two systemic inflammatory markers is more widely accepted for various conditions in clinical practices. As ESR is substantially influenced by the subjects' plasma fibrinogen and protein levels with relatively long half‐lives, the values often remain elevated for days to weeks after the underlying inflammatory conditions resolve. On the other hand, CRP is mainly secreted from the subject's liver upon stimulation by cytokines (e.g., interleukin‐1, interleukin‐6, and tumor necrosis factor‐alpha), and the level of CRP will decrease rapidly after the resolution of inflammation. 16 Under circumstances of chronic inflammation, the two markers' levels are determined by the hosts' inflammatory (anti‐viral) and anti‐inflammatory (viral‐persistence) responses in viral hepatitis. 17 However, few studies have investigated the impact of the genetic factors associated with Tregs, which is crucial in anti‐inflammation responses. In the current study, we explored the effect of the SNPs associated with Tregs on two inflammatory markers, hepatic injury marker (ALT), and liver fibrosis indicator (FIB‐4 index), in individuals with viral hepatitis. The IL‐2 gene polymorphism rs2069763C > A, located at position 114 from the first exon of the IL‐2 gene, does not cause any amino acid change. On the other hand, the genetic polymorphism was demonstrated to lose sensitivity to the restriction enzyme Mwol. 18 In one study, the “A” allele carrier was shown to be susceptible to the development of autoimmune hepatitis compared to those carrying the “C” allele. 19 We hypothesized that the rs2069763C > A polymorphism might contribute to the fine‐tuning of IL‐2 secretion and modify the downstream cascades affecting the immune‐modulatory functions of Tregs in response to viral hepatitis. However, our results do not support a significant immune‐modulatory effect conferred by rs2069763. The other polymorphism of IL‐2 examined in this study, rs2069772T > C, was shown to be associated with cow's milk allergy, a disorder with an increased propensity toward the development of other allergic diseases later in life. 20 Also, for individuals with the “TC” genotype, a significantly higher level of serum IL‐2 was found in endometriosis patients but not in the healthy control counterparts. 21 Based on these studies' results, we hypothesized that this polymorphism would cause differences in Tregs' functions and lead to the modulation of the inflammation induced by the hepatotropic viruses. However, no significant associations between this polymorphism and immune‐modulatory effect were found. TGF‐β1 is a pleiotropic cytokine involved in various immunological functions through complex interactions and modulates immune homeostasis. 22 Previous studies had shown the effect of TGF‐β1 in reducing the inflammatory responses in the respiratory mucosa. 23 The TGF‐β1 gene polymorphism rs1800469G > A is in the promoter region and does not cause changes in the consequent amino acid sequence. 14 However, the SNP was predicted to exert its effect by altering the expression of TGF‐β1. 14 Previous studies have demonstrated that the expression of TGF‐β1 upregulates the expression of FOXP3 and facilitates the conversion of naïve CD4 + T cells into Tregs, which are central in immunological tolerance. 24 , 25 In this study, we examined the SNP's impact on the virus‐stimulated inflammatory responses. We found individuals carrying the “A” allele to have significantly lower CRP levels than those carrying the homozygous “GG” genotypes. The results of several previous studies support the finding. In one study, the “A” allele was shown to increase TGF‐β1 production by selectively preventing the binding of the down‐regulatory transcription factor “activator protein 1 (AP1)”. 26 Another study found the “A” allele to be associated with higher levels of plasma TGF‐β1 concentration than those without the “A” allele within this locus. 14 Previous studies also showed TGF‐β to orchestrate CRP production in an inverse dose‐dependent manner: higher concentration of TGF‐β inhibits the CRP production while lower concentration promotes CRP production. 27 Later, in vivo studies also validated the inverse relationship between TGF‐β1 and CRP. 28 All these studies support our findings and hypothesis that the SNP causes an inhibited binding of down‐regulatory transcription factor AP1 and results in an enhanced level of TGF‐β1 to inhibit inflammatory responses and reduce CRP production. Nonetheless, such an effect of the genetic polymorphism was not observed in ESR levels. The differences may result from a more dominant impact of TGF‐β1 on CRP generation. Although the mean values of the CRP level in the groups carrying different rs1800469 genotypes were all within the widely accepted reference range (0 ~ 5 mg/L), several studies had demonstrated the higher CRP levels, within the reference range, to be associated with various pathological conditions, such as Alzheimer's diseases, type 2 diabetes mellitus, and cardiovascular diseases. 29 , 30 , 31 Meanwhile, the lack of a significant effect of the SNP rs1800469 on ALT levels or FIB‐4 index suggests that the SNP's impact on systemic inflammation is independent of the hepatic inflammation or fibrosis induced by viral hepatitis. The polymorphism rs3761547T > C is located in the promoter region of the FOXP3 gene on the X chromosome. In one study, generalized vitiligo patients carrying the “CC” genotype were found to have significantly decreased FOXP3 transcript and protein expression compared with controls. It was speculated that the “C” allele of rs3761547 might prevent the binding of the transcription factor and cause reduced expression of FOXP3. 32 In this study, we found that female participants carrying the “C” allele had significantly higher levels of ESR than the females carrying the “T” allele alone. Although no study had investigated the effect of the SNP on ESR levels, the result is supported by the studies showing that the “C” allele causes a decreased level of FOXP3, and secondarily results in the reduced immune suppression by the Tregs, leading to more severe inflammation and higher ESR level. 32 Meanwhile, the female participants carrying the “C” allele of rs3761547 were found to have a significantly higher ESR level than the females carrying the “T” allele and the reference range. Nonetheless, the CRP level in individuals carrying the “C” allele in the female group did not cause significantly higher serum CRP levels than those carrying the “T” allele only. Similar to our finding, previous studies showed the FOXP3 SNP rs3761548C > A in the promoter region causing altered transcription level to be associated with higher IL‐35 levels, 33 and the IL‐35 levels are inversely correlated with the ESR levels in rheumatoid arthritis patients, in the absence of a significant correlation with the CRP level. 34 These results also suggest that the IL‐35 may be the mediator for the effect of the SNP on ESR. The lack of significant differences in ALT levels between different rs3761547 genotype groups also suggests that the effect of the Treg‐related gene SNPs is independent of the hepatic inflammation. More importantly, in the current study, we found the significant effect of the FOXP3 SNP rs3761547T > C on hepatic fibrosis in the population with viral hepatitis. The finding indicates the phenotype difference conferred by the FOXP3 SNP and the involvement of FOXP3 in the course of hepatic fibrosis induced by viral hepatitis. Moreover, the opposite effect of rs3761547 on the FIB‐4 index between male and female participants may be secondary to the modulation of Treg functions by sex hormones that have been shown in several studies investigating the impact of sex on the effect of FOXP3 in diseases ranging from autoimmune to infectious diseases. 35 , 36 The mechanisms underlying the modulation of Treg functions by sex hormones need further investigation but are beyond the scope of the current study. In this study, we also examined the effect of SNP rs1967309A > G, which is an intron variant of the ADCY9 gene. Previous studies showed the AA genotype of the SNP to be associated with a reduced risk for cardiovascular events and regression of ultrasonography‐determined carotid intimal‐medial thickness when treated with dalcetrapib. 37 The phenotype difference conferred by the SNP and the in vitro results suggesting the potential involvement of Treg ADCY9 in the cAMP‐mediated modulation of effector T cell functions triggered our interest to examine the effect of the ADCY9 SNP. However, no significant associations between the different genotypes and the levels of ESR, CRP, ALT, or FIB‐4 index were found in this study. The present study has several limitations. First, we examined only five SNPs among the four genes associated with Treg functions, and the lack of associations in our observation does not exclude the possibility of a significant role of these genes, such as IL‐2 and ADCY9, in the inflammatory processes induced by viral hepatitis. Second, 54 of the participants obtained SVR after successful HCV treatment. Admittedly, the elimination of active HCV viruses in these participants may have changed the inflammatory process. However, as the ongoing liver inflammation in patients with chronic HCV and SVR has been reported, 38 these participants were not excluded. Third, the evaluation of the severity of hepatic fibrosis is based on the non‐invasive FIB‐4 index. Further validation with histopathological proof will provide a more definitive conclusion. Fourth, the application of strict inclusion criteria for recruiting participants to minimize confounding factors limited the number of participants in the current study. The relatively small sample size may limit the power of this study and causes difficulty in conducting a multivariable analysis to determine the relative contributions of various factors to the levels of inflammatory markers, hepatic injury indicator, and fibrosis index. 39 The participant number also caused difficulty in evaluating the SNPs' effect on HBV and HCV, specifically. However, the number of participants should be enough to make the findings representative of the population of chronic viral hepatitis patients at our hospital. Finally, since our current study is based on the hypothesis‐driven planned comparisons, we did not apply the conservative Bonferroni or other multiple testing corrections to avoid type II error. 40 Future studies with a larger sample size in an independent cohort investigating immune‐related parameters and long‐term clinical events will be needed to validate the results of this exploratory study. To conclude, we explored Tregs' role in the inflammation induced by viral hepatitis by examining the differences in ESR, CRP, ALT, and the FIB‐4 index between different genotypes of Treg‐related SNPs. We found the GG genotype of a TGF‐β1 SNP, rs1800469, to be associated with higher CRP levels. The C allele of a FOXP3 SNP, rs3761547, is associated with higher ESR levels and exerts different effects on hepatic fibrosis between males and females. As previous results have shown the changes of the target gene activity conferred by the SNPs, the temporal precedence of the Mendelian randomization of the participants carrying different SNPs at birth suggests the cause‐and‐effect relationship of the SNPs on the inflammation induced by viral hepatitis, indicating the phenotypic differences conferred by the SNPs rs1800469 and rs3761547 and the significant roles of TGF‐β1 and FOXP3 in modulating systemic inflammation and hepatic fibrosis. Meanwhile, our study also showed the impact of sex on moderating the function of FOXP3 in inflammatory responses.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are hepatotropic viruses, causing chronic hepatitis in 5%–10% and more than 70% of the infected individuals, respectively. 1 Uncontrolled HBV and HCV might lead to cirrhosis, a preface to the high probability of carcinogenicity. Meanwhile, the interactions between innate immunity, adaptive immunity, and various treatment modalities would be of significant importance in preventing severe complications. Previous studies have suggested the involvement of regulatory T cells (Tregs) in the disease course of viral hepatitis. 2 Tregs' nature has been recognized as subcategorization of CD4 + CD25 + FOXP3 + T cells, which serve as suppressors for the immune system in preventing over‐active autoimmunity disrupting the immune homeostasis. 3 Furthermore, Tregs have been shown to play essential roles in controlling overactive inflammation in some infectious conditions. 4 Nevertheless, the trajectory from the local inflammation toward hepatocellular carcinoma formation in chronic viral hepatitis is diverse, and Tregs' role remains to be explored. Emerging evidence has shown that the viral‐specific Tregs have a two‐sided effect on viral hepatitis: preventing overactive immune response leading to irreversible hepatic damage and permitting the virus's existence via down‐regulating the T cell‐mediated immune response. 2 Additionally, the immune response induced by the hepatotropic viruses has been shown to interact with both intra‐ or extrahepatic Tregs through various mechanisms. 2 , 5 Therefore, the Tregs' role might be crucial in immunomodulatory function. Interleukin 2 (IL‐2) and transforming growth factor‐β (TGF‐β) were shown to promote naive CD4+ T cells' conversion into Tregs through the upregulation of forkhead box P3 (FOXP3) 6 that was shown to regulate immune responses in various pathological conditions ranging from autoimmune disorders to infectious diseases. 7 Besides, FOXP3 was shown to suppress the expression of miR‐142‐3p, which is a potent inhibitor of adenylyl cyclase 9 (ADCY9), resulting in the generation of cyclic adenosine monophosphate (cAMP). 6 The cAMP generated would then lead to the Treg‐mediated suppression of the effector T cells and antigen‐presenting cells via gap junction intercellular communication. 6 Despite the various effects of IL‐2, TGF‐β, FOXP3, and ADCY9 in modulating the downstream effectors associated with immune‐regulation, the impact of these genes in immune responses has rarely been investigated. With a pivotal role in orchestrating the Tregs' function, IL‐2 was shown to augment the immunosuppressive effect via the downstream molecules innate to Tregs. 8 TGF‐β, as an inhibitory cytokine, was demonstrated to modulate the transcription of FOXP3 upon signaling on Tregs via a dose‐dependent manner. 9 As FOXP3 is crucial in the development, stabilization, and functioning of Treg lineage cells, the activity and level of FOXP3 are vital in regulating immune responses. 6 Being the downstream effector molecule of FOXP3, ADCY9 regulates the second messenger cAMP level and is crucial in regulating immune responses. Furthermore, one single nucleotide polymorphism (SNP) of ADCY9 was demonstrated to influence the level of C‐reactive protein (CRP) in participants with atherosclerotic diseases, where cholesterol‐lowering agents were administered. 10 Few studies have investigated Tregs' role in conditions other than autoimmune diseases, 2 and only one study suggested the immunomodulatory effect of the FOXP3 SNPs (rs2232365, rs3761549, and rs3761548) in the population carrying viral hepatitis. 11 In this study, we hypothesized that genetic polymorphisms of FOXP3 and its correlated molecules, including IL‐2, TGF‐β, and ADCY9, cause phenotype differences in Treg‐mediated immunomodulatory functions in the individuals with viral hepatitis, and hence leads to the differences in the level of systemic inflammation, hepatic inflammation, and fibrosis. Therefore, we examined the effect of selected SNPs, including rs2069763 and rs2069772 of IL‐2, rs1800469 of TGF‐β1, rs3761547 of FOXP3, and rs1967309 of ADCY9, on the serum level of erythrocyte sedimentation rate (ESR), CRP (systemic inflammatory markers), alanine aminotransferase (ALT) (hepatic injury indicator) and Fibrosis‐4 (FIB‐4) index (hepatic fibrosis indicator) in the population carrying viral hepatitis. We believe that our investigations into the selected SNPs would be of value in elucidating the role of the immune regulatory functions associated with Tregs in the chronic inflammation status induced by viral hepatitis.

The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.