Dysregulation of T Cell Differentiation and the IL17A(+)Foxp3(+)Treg Subset in Chronic Hepatitis B Patients with Hepatitis Flare

Abstract

The regulatory T (Treg) and T helper 17 (Th17) cells modulate the immune response in chronic hepatitis B virus (HBV) infection by promoting immune tolerance and restricting liver damage or stimulating inflammatory response and rendering hepatocyte injury. These cells act through signaling transcription factors and secreting cytokines. We aimed to observe the percentages of Treg, Th17 cells, and their messenger RNA (mRNA) level of forkhead box protein 3 (Foxp3) and retinoid orphan receptor γt (RORγt) in the chronic hepatitis B (CHB)-infected group and CHB patients with hepatitis flare (HF). We recruited 103 participants, including 88 CHB-infected cases and 15 healthy controls (HCs) in Ho Chi Minh City. CHB cases were enrolled into two groups: HBeAg+ CHB infection (e+CHBI; n = 42) and HF (including 20 mild HF and 26 severe HF [sHF]). The Foxp3(+)Treg and Th17 cells were measured by flow cytometry. The mRNA levels of Foxp3 and RORγt were analyzed by real-time polymerase chain reaction. The percentages of Foxp3(+)Treg, of Th17, and the Foxp3(+)Treg/Th17 ratio were significantly higher in the sHF compared to the e+CHBI group. The sHF and e+CHBI groups had significantly higher mRNA levels of Foxp3 and RORγt compared to the HC group. Furthermore, a special subset, interleukin 17A(+)Foxp3(+)Treg cells, were observed with a significantly higher percentage in the sHF compared to the e+CHBI group. This finding revealed the contributions of this new subset on the severe flare cases. Our results explained the diversity of T cells and their subsets in the immune response in CHB. This subset should be further investigated as a specific tool in HBV immune response.

Introduction

Hepatitis B virus (HBV) is a noncytopathic virus (Tong and Revill, 2016). Liver injury in people with HBV infection is due to complex nonspecific and specific, innate, and adaptive immune responses. The response of the antigen-presenting cells that capture HBV antigen can activate innate immunity and early control of the viral replication (Maini and Gehring, 2016). Subsequently, adaptive immunity activates HBV-specific antibody-producing B cells, the effector T cells, and this determines the outcomes of HBV infection (Bertoletti and Ferrari, 2016). Many people can tolerate and silently sustain the virus without liver damage for up to several decades during this immune tolerant phase, while others progress into multiple crises of active hepatic inflammation that might subsequently fall into hepatitis flare (HF) or liver failure and death (Wan et al, 2020).

Nowadays, there are increasing considerations on T cell dysregulation in HBV infection, especially two subsets were differentiated from CD4⁺ T cells as regulatory T (Treg) and T helper 17 (Th17) cells. The percentages of Treg and Th17 cells in peripheral blood mononuclear cells (PBMCs) of chronic HBV patients were higher compared to that value in healthy individuals (Feng et al, 2015; Niu et al, 2013). Treg cells play crucial roles in preventing hepatocyte damage, either by delaying viral clearance or sustaining chronic infection (Wan et al, 2020).

Several studies described the predominate Treg cells in chronic HBV carriers with persistently normal alanine aminotransferase (ALT) compared to acute-on-chronic liver failure (ACLF) patients (Liu et al, 2017; Xue-Song et al, 2012). Meanwhile, Th17 cells have been considered for nonspecific inflammatory cell recruiting or interleukin (IL)17A and other cytokines secretion in liver damage (Wan et al, 2020). In ACLF, peripheral Th17 cells were predominately present compared to chronic HBV carriers (Liu et al, 2017; Niu et al, 2013; Xue-Song et al, 2012). Inversely, there were conflicting reports that related to a higher number of these cells in the ACLF compared to chronic hepatitis B (CHB) patients (Niu et al, 2013).

Transcription factors and cytokines of Treg and Th17 cells were recently investigated. The Forkhead box protein 3 (Foxp3) was uniquely expressed on Treg cells and the retinoid orphan receptor γt (RORγt) on Th17. Former studies have recorded different messenger RNA (mRNA) levels of Foxp3 expression among different groups of chronic HBV infection such as hepatitis B carriers, CHB, and ACLF. A higher mRNA level of Foxp3 had been reported in patients with ACLF in several studies (Tan et al, 2021; Xia et al, 2018) but it was not proven in another study (Liu et al, 2017). The RORγt mRNA levels among different groups of CHB were not well assessed (Tan et al, 2021; Xia et al, 2018).

Our study aims to describe Foxp3(+)Treg, Th17 populations, and their transcription factors among different Vietnamese CHB-infected groups: the high replicative, noninflammation (HBeAg+ chronic HBV infection)—e+CHBI group and HF—HF group. We hypothesized that the e+CHBI group had a higher percentage of Treg cells and mRNA level of Foxp3; inversely, the HF group had a higher percentage of Th17 cells and mRNA level of RORγt.

Materials and Methods

Patient recruitment

From December 2018 to December 2021, 103 participants (88 CHB cases and 15 healthy controls [HCs]) were recruited from the Liver Clinic of the University Medical Center and Hospital for Tropical Diseases in Ho Chi Minh City. These CHB cases were composed of three separated groups based on their levels of liver injury and liver failure: the HBeAg-positive chronic HBV infection (e+CHBI) group (42 cases), the mild hepatitis failure (mHF) group (20 cases), and severe hepatitis failure (sHF; 26 cases).

The inclusion criteria for each group were as follows: (1) HC group included HBsAg negative and normal ALT individuals. (2) e+CHBI group composed of HBeAg-positive cases with former persistently normal ALT (upper limit of normal [ULN] of ALT ≤40 U/L); HBV DNA >5 × 10⁶ copies/mL, normal liver in ultrasound, and aspartate aminotransferase to platelet ratio index score <0.5 (Lampertico, 2017; Sarin et al, 2016), (3) mHF group composed of patients who had a current HF with ALT >5 ULN (Chang and Liaw, 2014; Sarin et al, 2016); international normalized ratio (INR) <1.5 and bilirubin <85 mmol/L; and (4) sHF composed of patients with current HF (ALT >5 ULN) with INR ≥1.5 or bilirubin ≥85 mmol/L.

The expression of transcription factors Foxp3 and RORγt was analyzed on 15/15 HC, 17/42 e+CHBI, 8/20 mHF, and 7/26 sHF cases. We excluded participants who had either non-HBV liver diseases such as hepatitis C virus-induced hepatitis, alcohol-induced hepatitis, ongoing infections, or pregnancy.

Biochemical and virological assessments

ALT, bilirubin, and INR were measured by an automatic biochemistry analyzer. Serum HBV DNA load was quantified by a real-time polymerase chain reaction (PCR) machine, with AccuPid HBV Quantification Kit, PCR MX 3005P system. The threshold of the HBV DNA detection limit was 250 copies/mL. HBeAg was assessed by electrochemiluminescence immunoassay technology with Roche reagents in Cobas e immunoassay analyzer.

Analysis of T cell population in PBMC

The peripheral blood of all participants was collected in a tube containing 2 mL of ethylenediaminetetraacetic acid. PBMCs were isolated from fresh whole blood by density gradient centrifugation using Histopaque 1.077 mg/mL (Sigma-Aldrich, St. Louis, MO). The isolated PBMCs were incubated with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich) and ionomycin (1 μg/mL; Sigma-Aldrich) in RPMI-1640 medium supplemented with 10% fetal calf serum for 4 h. Next, 4 μL of BD GolgiSTOP containing Monensin (BD GolgiStop™, USA) was added for every 6 mL of cell culture and mix thoroughly during the incubation period.

Cells were harvested and examined with IL17A and Foxp3 marker by HumanTh17/Treg Phenotyping Kit (BD Pharmingen™, USA). This cocktail contains FITC-conjugated anti-human CD4 antibodies, phycoerythrin-conjugated anti-human IL17A, and Alexa Fluor^® 647 conjugated anti-human PoxP3. In brief, cells were fixed by Human Foxp3 Buffer A (10 × ) containing formaldehyde and methanol and permeabilized by Human FoxP3 Buffer B (50 × ) containing nitrogen oxides.

Next, the fixed/permeabilized cells were suspended in 50–150 μL Stain Buffer (fetal bovine serum stain buffer), and a 20 μL/test of the cocktail was added to each tube. Cells were incubated at room temperature for 40 min in the dark throughout staining and storage. Flow cytometry was performed using FACS Canto II Flow Cytometer (BD Biosciences, San Jose, CA) and using FACS Diva (BD Biosciences) for analyzing the results. The cells with Foxp3 expression were defined as Foxp3(+)Treg and the cells with IL17A expression were defined as Th17. The percentage of Foxp3(+)Treg and Th17 cells were counted in their relative numbers to CD4⁺T cells.

Measurement of mRNA levels of Foxp3 and RORγt

PBMCs were isolated and total RNA was prepared using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's protocol. RNA was quantified at 260 nm in a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The integrity of RNA was assessed using gel electrophoresis and measuring the ratio A260/A280 with appropriate purity values between 1.8 and 2.0. The reverse transcription was performed to obtain complementary DNA (cDNA) from 1 μg of purified RNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's protocol. PCR amplification was carried out on a 15 μL reaction volume containing 25 ng cDNA, 1 × TB Green Premix EX Taq II (Takara, Japan), and 250 nM of gene-specific forward and reverse primers.

The primer sequences are designed using CLC Main Workbench Software (QIAGEN, Germany) as follows: RORγt (5′-GCATGTCCCGAGATGCTGTC-3′ and 5′-GGGTCTTGACCACTGGTTCC-3′), Foxp3 (5′-TGAACCCAGGAAGGACAGCA-3′ and 5′-GGAAGTCCTCTGGCTCTTCG-3′) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-TCGTGGAAGGACTCATGACC-3′ and 5′-TGACCTTGCCCACAGCCTTG-3′). The reaction was run in the Mastercycler EP Realplex RealTime PCR system (Eppendorf, Germany). Calculation of gene expression was based on cycling determination and was analyzed using the modification of the ΔΔCt equation, taking into account the efficiency of the reaction. Melting curve analysis was carried out to assess template specificity. The mRNA level of Foxp3 and RORγt in PBMCs were normalized to GAPDH, which was used as an internal control gene.

Statistical method

Data were collected and analyzed by SPSS 20.0 software (Armonk, NY). Categorical variables were compared by Pearson's chi-square. Continuous variables with asymmetrical distribution were expressed as median and interquartile ranges and were compared by the Mann–Whitney U Rank Sum test (2 groups) or Kruskal–Wallis H test (>2 groups). The significant difference was calculated at a two-sided p-value <0.05.

Results

Patient's characteristics

Table 1 showed the age, gender, HBeAg marker, and clinical parameters of four study groups. Males were more prominent in two HF subgroups compared to the e+CHBI group. The sHF group had a higher median of age and had more HBeAg negative cases compared to the mHF group (p = 0.02 for both). The high values of HBV DNA were recorded in all CHB groups and were found not different between study groups.

Table 1.

Epidemiological and Clinical Characteristics of Participants

	Study group				p
	HC (n1 = 15)	e+CHBI (n2 = 42)	mHF (n3 = 20)	sHF (n4 = 26)	p
Age (years)	41 (32–50)	32 (19–43)	30 (27–43)	42 (34–51)	0.02^a
Gender (M/F)	10/5	16/26	18/2	22/4	<0.001
HBeAg (+/−)	−	42/0	15/5	12/14	0.02^a
HBV DNA (log₁₀ copies/mL)	−	8.74 (8.06–8.74)	8.65 (7.62–8.74)	8.32 (6.78–8.99)	NS
ALT (U/L)	25 (18–37)	27 (21–34)	631 (306–1,554)	1,279 (914–1,899)	NA
TBil (mmol/L)	−	−	25 (13–43)	194 (137–340)	NA
INR	−	−	1.09 (1.05–1.28)	1.27 (1.11–1.43)	NA

Continuous variables were expressed as median (interquartile range), compared by the Mann–Whitney U test. Binary variables were expressed as a ratio, compared by Pearson's chi-Square test.

Compared between the mHF and sHF group.

ALT, alanine aminotransferase; e+CHBI, HBeAg+ chronic HBV infection; HBV, hepatitis B virus; HC, healthy control; INR, international normalized ratio; M/F, male/female; mHF, mild hepatitis flare; NA, not applicable; NS, nonsignificance; sHF, severe hepatitis flare; TBil, total bilirubin.

Foxp3(+)Treg and Th17 cells distributions

Figure 1A–D showed the distribution of Foxp3 expression Treg cells [Foxp3(+)Treg] (blue dots) and Th17 cells (purple dots) in different groups. The red dots represented the cells that did not either produce IL17A or express Foxp3. The Foxp3(+)Treg cells appeared on the right lower and upper quarters. The IL17A expression cells were detected on the left upper quarter. The observation had recorded a predominant detection of Foxp3(+)Treg and Th17 cells in the e+CHBI (Fig. 1B), mHF (Fig. 1C), and sHF group (Fig. 1D), but had rarely recorded in the HC group (Fig. 1A). In addition, another special T cell subset was presented by blue dots on the right upper quarter. This subset represented the cells that expressed both Foxp3 and IL17A.

FIG. 1.

The percentages of Foxp3(+)Treg and Th17 cells and the Treg/Th17 ratio in the study groups. The flow cytometry displayed the representative Foxp3(+)Treg and Th17 cells among groups: HC (A), e+CHBI (B), mHF (C), and sHF (D). The percentage of subsets among groups were displayed in dot plots: Foxp3(+)Treg (E), Th17 cells (F), and Foxp3(+)Treg/Th17 ratio (G). Significant p-value was indicated (*p < 0.05). e+CHBI, HBeAg+ chronic HBV infection; Foxp3, forkhead box protein 3; HC, healthy control; IL17A, interleukin 17A; mHF, mild hepatitis flare; sHF, severe hepatitis flare; Th17, T helper 17; Treg, regulatory T.

The median of Foxp3(+)Treg percentage of the sHF was the highest and was significantly higher than that of the mHF (4.1 vs. 2.3; p = 0.02), the e+CHBI (4.1 vs. 1.3; p < 0.001), and the HC groups (4.1 vs. 1.1; p = 0.006) (Fig. 1E).

Differently, the median of Th17 percentage of the sHF was only significantly higher than that of the e+CHBI group (2.3 vs. 1.2; p = 0.01) (Fig. 1F).

The ratio of Foxp3(+)Treg/Th17 in the sHF group was recorded as the highest value and was significantly higher than the mHF (1.8 vs. 1.1; p = 0.03), the e+CHBI (1.8 vs. 1.0; p = 0.01), and the HC group (1.8 vs. 0.8; p = 0.003) in Figure 1G.

The correlation of the percentages of two cells with ALT and HBV DNA

Only one positive correlation had been found between the Foxp3(+)Treg percentage and ALT (p < 0.001) in this CHB population (Fig. 2A). The other correlations were not statistically significant [between the Foxp3(+)Treg and HBV DNA (Fig. 2B), Th17 and ALT (Fig. 2C), Th17 and HBV DNA (Fig. 2D)].

FIG. 2.

The correlation of the percentages of cells with ALT and HBV DNA. The correlation between Foxp3(+)Treg with ALT (A), HBV DNA (B), and between Th17 with ALT (C) and HBV DNA (D) in 88 CHB cases. ALT, alanine aminotransferase; CHB, chronic hepatitis B; HBV, hepatitis B virus.

The distribution of Foxp3(+)Treg and Th17 by HBeAg status

The percentages of Foxp3(+)Treg, of Th17, and the Foxp3(+)Treg/Th17 ratio in the HBeAg− and HBeAg+ subgroups of the HF (mHF and sHF) population were not statistically different (Fig. 3A–C).

FIG. 3.

The percentages of Foxp3(+)Treg and Th17 cells and the ratio of two HBeAg subgroups of the HF population. The percentages and ratio of Foxp3(+)Treg and Th17 in HBeAg− and HBeAg+ subgroups were displayed in dot plots: Foxp3(+)Treg (A), Th17 cells (B), and Foxp3(+)Treg/Th17 ratio (C); e−HF, HBeAg− hepatitis flare; e+HF, HBeAg+ hepatitis flare.

Foxp3 and RORγt mRNA: Transcription factors of Treg and Th17

The median levels of Foxp3 in the sHF [2.94 (1.87–4.75)], the mHF [2.85 (2.41–3.92)], and the e+CHBI [2.42 (1.53–3.05)] groups were significantly higher than that of the HC [1.22 (1.05–1.70)] group (p = 0.001, p = 0.001, and p = 0.004; respectively) (Fig. 4A).

FIG. 4.

Transcription factors of Treg and Th17 cells and the Foxp3/RORγt ratio in the study groups. The relative levels of Foxp3 (A), RORγt (B), in PBMCs, and the Foxp3/RORγt ratio (C) among study groups were shown. Significant p-value was indicated (*p < 0.05). PBMCs, peripheral blood mononuclear cells; RORγt, retinoid orphan receptor γt.

Meanwhile, the median of RORγt of the sHF [4.53 (3.49–5.03)] was the highest and was significantly higher than that of the mHF [2.43 (1.79–3.23)] (p = 0.04), the e+CHBI [1.88 (1.19–2.30)] (p = 0.006), and the HC groups [1.20 (0.59–1.49)] (p < 0.001). There were also higher levels of this transcription factor in comparison between the mHF versus the HC and between the e+CHBI groups versus the HC group (p = 0.002 and 0.007, respectively) (Fig. 4B).

The Foxp3/RORγt ratio of the sHF [0.82 (0.59–1.01)] was the lowest and was significantly lower than that of the mHF [1.20 (1.00–1.50); p = 0.02] and the HC group [1.16 (0.92–1.95); p = 0.03] and was marginally different to the e+CHBI group [1.20 (0.77–1.76); p = 0.05] (Fig. 4C).

IL17A(+)Foxp3(+)Treg subset

The distribution and value of IL17A(+)Foxp3(+)Treg among three CHB groups were presented in Figure 5A. This median of percentage value of this new detection subset on the sHF group [0.35 (0.10–0.60)] was statistically higher than that of the e+CHBI group [0.10 (0.00–0.30)] (p = 0.02), but was not enough statistically different compared with the mHF group [0.15 (0.10–0.45)].

FIG. 5.

The percentages of IL17A(+)Foxp3(+)Treg detection in three CHB groups and the percentages Foxp3(+)Treg separately presented among two IL17A detection subsets. The dot plot figures presented the percentage of IL17A(+)Foxp3(+)Treg (A) among the CHB subgroups, and percentages of Foxp3(+)Treg separately into two subgroups: absence of IL17A-producing (B, left ) and presence of IL17A-producing Foxp3(+)Treg (B, right). Significant p-value was indicated (*p < 0.05).

Figure 5B separately presents and compares the values of the Foxp3(+)Treg cell into two subgroups based on the detection of the IL17A(+)Foxp3(+)Treg subset of the Foxp3(+)Treg population. Most CHB cases were in groups with IL17A(+)Foxp3(+)Treg detection (Fig. 5B, right). There were minimal cases (11/42 e+CHBI; 4/20 mHF; 3/26 sHF cases) without IL17A(+)Foxp3(+)Treg detection, especially of the sHF group (Fig. 5B, left). Regarding, the subset with IL17A(+)Foxp3(+)Treg detection recorded higher percentage of values of Foxp3(+)Treg compared to those correspondent values on the IL17A(+)Foxp3(+)Treg nondetection group.

Discussion

In this study, we are the first group who detected and observed the Treg and Th17 cells and their mRNA expressions in the e+CHBI and HF groups. In contrast to a time-consuming, complex, and expensive flow cytometry, PCR is a time- and cost-saving and less technical skillful requirement.

Our study revealed that the sHF group had significantly higher percentages of both Foxp3(+)Treg and Th17 cells compared to the e+CHBI group. In addition, the sHF group showed a significantly higher value of Foxp3(+)Treg cell, but not Th17 value compared to the mHF group.

In addition to the increasing response of Treg and Th17 in the sHF group, the Treg/Th17 ratio was still found higher in the sHF compared to the e+CHBI group. This ratio among the sHF group exposed the dominant response of Treg compared to Th17 during severe hepatitis following previous findings about the vital necessities of immune suppression to reduce liver injuries, preserve liver cells, and progress to recovery in the survivor group (Niu et al, 2013).

In our study, the predominance of Th17 cells in the sHF compared to the e+CHBI and CHB group was in line with several previous studies (Liu et al, 2017; Niu et al, 2013; Tan et al, 2021; Wan et al, 2020; Xue-Song et al, 2012). Those observations supported the evidence on the modulating role of IL17A(+)-producing Th17 cells on the liver injury of HF individuals. However, the higher detection of Foxp3(+)Treg cells and Foxp3 expression simultaneously with the enhanced detection of Th17 and RORγt expression on the sHF group revealed that both anti-inflammatory protective response and inflammatory response to destroy infected cells and cure HBV infection had truly coexisted during severe liver injuries. Our findings were similar to the study conducted by Niu et al (2013).

A previous study recorded that Treg prevents hepatocyte damage but delays viral clearance (Wan et al, 2020). Therefore, the enhancing activities of the Treg subset that were found in our sHF group led to another hypothesis about whether Treg cells could play any other roles in acute inflammatory response besides their anti-inflammatory properties. Indeed, there was a subset of Treg that was described as IL17A(+)Foxp3(+)Treg cells that produce IL17A and play a vital role in liver damage need to state the research group (Jung et al, 2017). These specific Treg cells were found elevated in ACLF patients and correlated with the severity of hepatocyte injury, according to the research group of Zhang et al, 2012.

From our study, we found the significantly elevated Foxp3(+)Treg cells in the sHF group; we also detected the existence of the Foxp3(+)Treg population with IL17A production (presented by the blue dots on the upper right quarter). Furthermore, the higher detection value response of Foxp3(+)Treg cells in groups with the presence of IL17A(+)Foxp3(+)Treg subset compared to the groups without presenting IL17AFoxp3(+)Treg subset revealed the contributory role of this subset in the immune pathology of CHB and need to be further investigated. Further studies are mandatory to investigate and elucidate the role of IL17A(+)Foxp3(+)Treg cells in CHB patients.

Generally, our study group was the earliest team who demonstrated the dual anti- and proinflammatory function of Treg cells, in modulating the response to HFs in Vietnamese CHB patients. The number of our sHF cases was small and differed from the ACLF population. In previous studies, the difference in response of Treg and Th17 subsets between the ACLF and e+CHBI was obvious, especially since the ACLF group was represented by severe hepatitis (Liu et al, 2017; Xue-Song et al, 2012). In our study, by separating the case with ALT flare into the mHF and sHF groups, we also found that both Foxp3(+)Treg and Th17 subsets in the sHF group were significantly predominant compared to the e+CHBI group. This result revealed the stronger responses of both T cells in the severe group.

We found that the Treg/Th17 ratio was highest in the sHF group and lowest in the HC and e+CHBI groups. This highest ratio detected in the sHF group exposed the predominant and essential role of Treg in liver injury and protection. This result conflicted with previous studies (Liu et al, 2017; Xue-Song et al, 2012). The Th17 cells were the majority in the severe hepatic injury group in previous studies (Liu et al, 2017; Niu et al, 2013; Xue-Song et al, 2012) and also in our findings (Fig. 1F). However, the presence of IL17A(+)Foxp3(+)Treg in the sHF group could relate to the higher Treg/Th17 ratio. Our findings added the evidence and the complicated role of Treg/Th17 imbalance in the progression of CHB.

We had only found a positive correlation between Foxp3(+)Treg and ALT. That suggested either Foxp3 expressing T cells increased following or causing hepatocyte damage and need to be observed in cohort studies. In addition, our results showed that the percentage of Treg and Th17 in the HBeAg+ HF group was not different from that in the HBeAg− HF group. There was no difference in Treg percentage between the HBeAg+ and HBeAg− CHB groups in another study (Peng et al, 2008). Then, other studies are necessary to understand further immune responses in different HBeAg statuses.

We assessed Foxp3 and RORγt expression which was used as well as cytokines to measure the activities of Treg and Th17 cells. These transcription factors were investigated in different CHB groups in several studies (Liu et al, 2017; Tan et al, 2021; Xia et al, 2018). However, their findings conflicted with our results. Furthermore, our study showed that Foxp3 and RORγt levels in the sHF were highest and were higher compared to the mHF and e+CHBI groups. These trends of transcription factors were similar to that of their cells.

Our study is an observational study with a small sample size, and therefore, it may not be powered enough to conclude whether the mRNA T cell expression by PCR technique might get higher levels of confidentiality or could be used in interchanging or replacing the flow cytometry in study T cell response. Further studies are needed to investigate these markers for further understanding.

The limitation of this study was the small sample size, especially the small HC group, low number of cases that were analyzed for mRNA transcriptions. We also could not perform any cytokine measurement as well as cellular assays to determine the pathogenesis roles of these T cell subsets.

Conclusion

We reinforced the imbalance of Treg and Th17 cells in modulating the inflammation response and progression of CHB among Vietnamese patients. The IL17A(+)Foxp3(+)Treg subset was detected predominately in the sHF group, which suggested the dual and opposite functions of Treg cells on immune suppression as well as inflammatory enhancement in patients with HFs.

Footnotes

Acknowledgments

The authors acknowledge financial support from the research fund of the University of Medicine and Pharmacy at Ho Chi Minh City. The authors also thank the staff of the Center Molecular Biomedicine as well as patients in the University Medicine Center and Hospital for Tropical Diseases.

Authors' Contributions

H.L.T.T, A.L.B, H.K.T.T, T.N.T, T.P.V, and N.T.H contributed to conception and design. H.L.T.T and H.K.T.T analyzed and interpreted data. H.P.T.L contributed to reviewing and editing the original draft. All listed authors approved this article.

Ethics Statement

All participants gave informed consent. The protocol was approved by the Ethics Committees of the University of Medicine and Pharmacy at Ho Chi Minh City (No. 93/DHYD-HD).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This project was supported by the University of Medicine and Pharmacy at Ho Chi Minh City in Vietnam.

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