In the present study, IL-17 levels in the cell culture supernatants of PBMCs from CAHB patients were significantly higher than those obtained from controls. Earlier studies showed that the level of IL-17 was elevated in CHB patients with high ALT level and suggested that IL-17 is associated with the active intrahepatic inflammation in CHB patients.
In recent studies of patients with autoimmune hepatitis
 and hepatitis B
, the degree of hepatic inflammation was directly correlated with Th17 cell infiltration. In our study, the frequency of peripheral Th17 cells in CAHB patients was found to be significantly higher than that of normal controls. The frequency of Th17 cells in the PBMC population was examined after stimulation with PMA and ionomycin, which are pharmacological T cell-activating agents that mimic signals generated by the T cell receptor (TCR) complex. However, this combinatory stimulation did not replicate the physiological conditions by which T cells are stimulated in vivo.
Our results are also consistent with those reported earlier in CAHB patients. Th17 cells were found to be highly enriched in the peripheral blood and liver, and exhibited a significant potential to exacerbate liver damage during HBV infection
. The frequency of peripheral Th17 cells also correlated with ALT, suggesting a potential role for Th17 cells in the immune activation of chronic HBV infection
. Recently, it was reported that the frequency of circulating Th17 cells in HBV-infected patients positively correlated with the severity of the disease and the extent of liver injury. Therefore, it has been hypothesized that Th17 cells might contribute to HBV disease progression and development of liver injury
IL-10 is an anti-inflammatory cytokine involved in the down-regulation of the immune response. Research has indicated that induction of IL-10 can limit proinflammatory Th17 responses in HBV infected patients
. The strong correlation between HBV load and the regulatory T cell (Treg)/Th17 ratio in CAHB indicates that the imbalance of these two cell types might contribute to HBV persistence. Th17-mediated inflammatory diseases
 strongly support the notion that Th17 cells play a proinflammatory role in the pathogenesis of hepatitis B and HBV infection-related injury.
The IFNs are a family of proteins with an important role in protection against viral infections, tumor growth, inflammation and angiogenesis
. The IFNs are divided into three classes whereby the first two are most important from an immunological point of view. In humans, the main Type I IFNs consist of IFN-α (with 13 subtypes) and IFN-β (1 subtype). IFN-α is produced in large amounts by plasmacytoid dendritic cells, whereas IFN-β is mainly produced by fibroblasts.
IFN-α has been successfully used for the treatment of hepatitis
 and we wondered if IFN-α has any effects on IL-17 and IL-10. In this study, we found that IFN-α 2a could inhibit IL-17 expression and increased IL-10 production by PBMCs from CAHB patients and controls. Further experiments using isolated CD4+ T cells from healthy controls showed similar results.
In our CAHB patients, we observed a stronger induction of IL-10 as compared with controls following incubation of PBMCs with IFN-α. This result seems to suggest that a latent negative regulatory mechanism already exists in these patients but that it is not triggered and is insufficient to suppress the overwhelming ongoing Th17 response. We also investigated whether IFN-α could exert its effects through up-regulation of IL-10. The results showed that IFN-α induced both PBMCs and CD4+ T cells to produce IL-10. We then questioned whether IFN-α could inhibit IL-17 expression through an up-regulation of IL-10. Therefore, an experiment using an anti-IL-10 antibody was performed to examine underlying mechanisms of action of IFN-α. Interestingly, we did find that the anti-IL-10 antibody blocked the inhibition of IL-17 production promoted by IFN-α. These results suggest that the function of IFN-α is at least partially mediated by IL-10.
Also our results concerning the mechanism of action of IFN-α are consistent with those reported earlier in patients with other diseases. Zhang et al.
 showed that IFN-α inhibited the differentiation of Th17 cells and production of IL-17 through up-regulating TLR7 expression in dendritic cells from patients with multiple sclerosis. It was reported that treatment with IFN-α could induce an increased frequency of CD4+CD25high T cells in PBMCs from MS patients
. However, Seya and coworkers
 found that IFN-α from polyI:C-stimulated dendritic cells (DCs) inhibited the expansion of CD4+CD25+FoxP3+ Tregs. Whether a down-regulatory effect of IFN-α on IL-17 production by T cells, as observed in CAHB, is associated with an increased CD4+CD25+FoxP3+ Tregs needs further studies.
We performed these experiments using PBMCs from healthy control patients and ideally the data should also be confirmed using cells obtained from CAHB patients. Other cytokines such as IL-27 that are known to be induced by IFN-α and that can block the differentiation of Th precursor cells into Th17 cells may also be involved but were not included in our study
[22, 23]. Our experiments using isolated CD4+ T cells from healthy controls showed levels of IL-17 and IL-10 in the culture supernatants much higher than in experiments with the same number of PBMCs. The absolute number of CD4+ T cells was much higher in such cultures and indicated that these cells are probably the main source of IL-17 and IL-10 in this in vitro model. On the other hand, δγ T cells have also been shown to produce IL-17 and may play an important role. Further studies are needed to establish which T-cell subpopulations are subject to IFN-α regulation.