Chronic hepatitis from HCV infection is characterized by ongoing immune activation and a low-grade inflammation in the liver and in the periphery that accompanies the liver injury [13
]. While numerous studies reported the presence of inflammatory cascade activation, the mechanisms that trigger and sustain the increased TNFα and inflammatory cell activation remain elusive [2
]. Micro-RNAs have emerged as key regulators of inflammation of which miR-155 has a unique positive regulatory effect on TNFα production [19
]. In this report, we tested the hypothesis that micro-RNAs are dysregulated in chronic HCV infection. We found that treatment-naïve patients with chronic HCV infection have increased expression of miR-155 in their circulating monocytes that are the major sources of TNFα production. Importantly, miR-155 levels were not increased in monocytes from patients who have successfully cleared HCV infection after therapy, suggesting a possible correlation between increased miR-155 and HCV viral presence and/or replication. We also found that cHCV monocytes have increased mRNA expression of TLR8, a receptor for single-stranded RNA and showed that in normal monocytes not only TLR4 and TLR8 ligands but HCV core, NS3 and NS5 proteins induced miR-155 upregulation. MiR-155 was elevated both in the monocytes and in the serum compartment in chronic HCV infection, and this occurred in the presence of increased serum miR-122. Together, our results suggest that miR-155 upregulation in HCV infection in monocytes may contribute to the increased pro-inflammatory state and raises the possibility that circulating miRNAs could serve as disease markers.
Increased miR-155 levels in circulating monocytes of HCV patients may have several implications for disease development and progression. First, miR-155 over-expression in monocytes and macrophages has been shown to sensitize to stimulation with LPS [17
]. Indeed, we not only found increased baseline miRNA-155 expression but also a significant upregulation of miR-155 after in vitro LPS challenge in cHCV infection. Previous studies from our and other laboratories demonstrated that monocytes from chronic HCV infected patients have increased baseline TNFα production as well as hyper-responsiveness to ex vivo stimulation with LPS, suggesting that increased miR-155 levels may contribute to the over-activation of the pro-inflammatory phenotype of monocytes. This notion is supported by reports where knock-down of miR-155 or miR-155-deficient mice showed attenuated TNFα production to LPS stimulation [17
]. Intriguingly, various studies have demonstrated TNFα as a positive regulator of miR-155, indicating a positive feed back regulation [32
]. Based on our results, it is tempting to speculate that increased miR-155 sensitizes monocytes to a TLR-induced trigger for TNFα production in active HCV infection. In parallel to our work, a recent study showed that expression of BIC gene, a precursor of mature miR-155, was increased in PBMCs of HCV-infected patients [33
]. They further demonstrated that the highest expression of BIC was found in patients harboring HCV RNA in serum and PBMCs whereas lowest BIC expression was observed in patients that eliminated HCV RNA from both serum and PBMCs after anti-viral treatment, suggesting a correlation between HCV RNA and precursor levels of miR-155 [33
]. Moreover, a correlation between miR-155 expression and HCV replication has been demonstrated in PBMCs of chronic hepatitis C patients [34
]. Our findings further indicate an increase of mature miR-155 in monocytes and serum of cHCV patients. Although we found no significant changes in miR-146a levels in monocytes of cHCV patients, some of patients who had higher miR-146a levels also showed higher levels of miR-155 and miR-125b. A large-scale clinical study might answer if the variability between miRNAs correlates with HCV viral load or progression of HCV infection.
Our observations also suggest that HCV-related TLR ligands provide the possible mechanisms for in vivo induction of miR-155 and monocyte activation in cHCV patients [13
]. For example, high levels of HCV core protein as well as LPS are present in patients with cHCV [6
]. Our data show that both HCV core protein and LPS induce miR-155 in normal monocytes. It has been shown that HCV core protein can activate monocytes and DCs via TLR2 and the complement C1q receptor [9
]. In addition, HCV NS3 protein that activates TLR2, also increased monocyte miR-155 in our hands. A recent study reported the induction of TNFα in response to NS5 (non-structural viral protein) treatment in human Kupffer cells [11
]. In agreement with this study, we found induction of miR-155 and TNFα in monocytes treated with NS5. NS5 has been shown to activate the TLR4 pathway [36
]. Previously, it has been shown that viral E2 protein interacts with CD81 and increases the expression of activation-induced cytidine deaminase and TNFα production in B cells [12
]. We also found a moderate increase of TNFα in monocytes treated with E2 protein without any change in miR-155 levels. Our data suggest that viral E2 protein can induce TNFα independent of miR-155, however, further studies are needed to confirm these findings. Finally, we found that TLR8 ligand activation can increase miR-155 and TNFα production and it is feasible that HCV, as a single-stranded RNA, induces miR-155 in monocytes via TLR8. Together our data demonstrate that multiple, HCV-associated TLR ligands are likely to be involved in miR-155 induction in cHCV infection.
We not only found increased miR-155 and miR-122 levels in the serum of cHCV patients but levels of miR-125b and miR-146a were also increased. Increased circulating miR-125b levels have been found in various cancers and recently, it has been reported that increased circulating miR-125b levels were associated with chemotherapeutic resistance in breast cancer [37
]. The significance of increased miR-125b in HCV patients awaits further investigation. Circulating miR-146a levels were shown to be increased in patients with type 2 diabetes [38
]. Interestingly, HCV is known to induce type 2 diabetes [39
], and it is tempting to speculate that increased miR-146a in the circulation might predispose the individuals to HCV infection or vice versa.
Although, there are many studies depicting the increased circulating miRNAs in various etiologies, the source of circulating miRNAs is not as clear. The liver is a complex organ where various cell types reside and interact in close vicinity. In our study, there could be multiple factors that contribute to induction of miRNAs (miR-122, miR-155, miR-146a and miR-125b) in the circulation during HCV infection. The possibility that these miRNAs are either released from immune cells in a non-specific manner or are released from different cell types in a cell-specific manner in response to HCV infection cannot be ruled out. It is most likely that miR-122 is released from damaged hepatocytes. In agreement with this, we found increased levels of miR-122 in the supernatant of JFH-1 or J6/JFH-1 infected HuH7.5 cells (data not shown). Recent studies suggest that miR-155 is not only limited to immune cells (dendritic cells, Kuffper cells, monocytes, NK cells, T cells), but also prevalent in non-immune cells (hepatocytes, endothelial cells). We have recently reported that chronic alcohol feeding increases miR-155 not only in Kuffper cells but also in hepatocytes [40
], and it has been shown that alcoholic hepatitis patients have a higher prevalence of HCV infection [41
]. Our preliminary results suggest a moderate increase of miR-155 in the supernatant of JFH-1 or J6/JFH-1 infected cells compared to uninfected HuH7.5 cells (data not shown). On the basis of these findings, it is most likely that both immune cells and hepatocytes contribute to increase of miR-155 in the circulation in HCV infection. Further studies are warranted to investigate the cellular source of circulating miRNAs.
Growing evidences suggest that circulating miRNAs may be affected by age and body weight and it was reported that levels of circulating miRNA-155 inversely correlated with age in patients with coronary artery disease [42
]. Whether these factors affect the outcome of circulating miRNAs in HCV patients needs to be investigated. Finally, we found increased miR-122 levels in the serum of chronic HCV infected patients, which strongly correlated with liver damage markers such as ALT and AST. The correlation of serum miR-122 was more robust with ALT than AST. Our results support the previous findings where a strong correlation between serum miR-122 and ALT/AST increases has been reported [43
]. Interestingly, our data indicates a positive correlation between serum miR-155, an inflammation-related miRNA, and miR-122, a hepatocyte-specific micro-RNA. We speculate that this could be related to inflammation-induced hepatocyte damage that occurs in chronic HCV infection. In our patient cohort, there was no significant correlation between ALT values and serum miR-155 expression. Further investigation of serum miRNAs and their role as diagnostic biomarkers or biological mediators deserves attention.