Capable of recognition of pathogenic DNA, the innate immune defense system composed of intracellular DNA sensors plays an important role in preventing intracellular infection of pathogens such as DNA viruses. However, their role in HBV infection is still largely unclear. Published studies have shown that early in acute infection, HBV is poorly sensed by the innate immunity and does not or weakly induce innate immune responses [3][26][33–34]. It incubates stealthily in the liver for several weeks until elicitation of adaptive immune responses. Little is known about the involvement of DNA sensors in the process of acute and chronic HBV infection in humans. Collectively, our results reveal that (i) HBV can be detected by DNA sensors in acute or chronic HBV-infected patients; (ii) IFI16 expression is enhanced in PBMCs from both AHB and CHB patients and it is mainly expressed in monocytes; (iii) the IFI16-STING pathway in monocytes is attenuated by HBV infection.
The intracellular recognition of HBV infection by DNA sensors is still less understood. Moreover, it remains controversial in whether HBV can be sensed by intracellular DNA sensors or actively evades from the sensing pathways. To address this issue, we first analyzed the expression of the STING-dependent DNA sensors in PBMCs from chronic HBV-infected patients. The results showed the expression of IFI16, DDX41, and MRE11 was enhanced in CHB, IT, and IA patients. Published data showed that IFI16 was closely related to the degree of liver inflammation in CHB patients [35]. Upregulated expression of IFI16 only in chronic hepatitis patients suggests it maybe associate with inflammation within the liver. In fact, IFI16, predominantly nuclear as a transcription activator, is now recognized as a DNA sensor that dually functions as inducer of IL-1β and IFN-β through IFI16-inflammasome activation and STING, respectively [36][31]. IFI16 was found to be critical for inhibition of many viruses [37]. It was recently demonstrated that IFI16 could recognize and directly bind to HBV covalently closed circular DNA (cccDNA) via an interferon-stimulated response element (ISRE) in cccDNA in hepatocytic nuclei [38]. If this is the case, the enhanced expression of IFI16 in PBMCs occurs only with the blood exposure of cccDNA via inflammatory necrosis of hepatocytes, as HBV cccDNA only originates from hepatocytes, not from PBMCs [39]. This may explain why IFI16 expression in PBMCs is upregulated only in hepatitis patients. DDX41 and MRE11 were reported to serve as cytosolic dsDNA sensors by Liu’s and Kawai’s group, respectively [40–41]. DDX41, a member of the DEXDc helicase family, directly binds dsDNA and STING via its DEADc domain in dendritic cells and monocytes to trigger type I interferon responses earlier than IFI16 [40]. MRE11, functioning as a DNA damage sensor usually in the form of the MRN complex (MRE11, RAD50, and Nijmegen breakage syndrome 1 (NBS1)), binds with RAD50 and exogenous DNA to induce IFN-Is by promoting STING translocation from the endoplasmic reticulum to the Golgi apparatus. The DDX41 and MRE11 expression increased both in IT and IA patients, suggesting they were involved in chronic HBV infection. Although there is still no report concerning the role of DDX41 and MRE11 playing in HBV infection, other reports have shown that they are involved in control of virus infections [42–44]. Thus, further investigation of their roles in HBV infection is warranted. Although activation of the cGAS-STING pathway was demonstrated to be able to inhibit HBV replication in cell culture and mice [18–19][45–46], HBV could also actively suppress cGAS expression and function in vitro and humanized liver chimeric mice [46]. Our results showed cGAS expression level was not altered in chronic HBV patients, suggesting HBV may evade from cGAS sensing. In parallel with the increased expression of IFI16, DDX41, and MRE11, the adaptor STING expression was also enhanced in chronic HBV patients. But the induction of effector IFN-β was not observed in chronic HBV patients, implying the DNA sensor-STING pathway is suppressed. This may be explained by Liu’s report showing HBV polymerase blocked DNA-sensing pathways by disrupting k63-linked ubiquitination of STING [21]. Taken together, these data suggest HBV can be sensed by DNA sensors in PBMCs in chronic HBV-infected patients and support the notion that HBV plays an active role in evading and inhibiting innate immune responses [6].
Different from the expression profile of DNA sensors in chronic HBV infection, IFI16 was found to be the only upregulated DNA sensor in AHB patients, in accordance with a previous report which showed induced expression of the IFI16 gene in an acute HBV infection model of chimpanzees [33]. However, the other DNA sensors were significantly downregulated as compared with HCs. This is reminiscent of the report by Dunn in which he demonstrated that with abundant production of IL-10 in AHB patients, the responses of NK cells along with CD4+ and CD8+ HBV-specific T-cells from PBMCs were reduced [26]. Interestingly, the varicella patients and AHB patients displayed a similar expression pattern of DNA sensors, except IFI16. Despite with severe liver injury and a rapid short-term drop in HBV DNA levels in these AHB patients, the STING and IFN-β expression was not induced by acute HBV infection. The absence of a robust IFN-I response in AHB is in accordance with other studies [26][33]. Of note, the IFN-β expression in varicella patients was significantly suppressed as compared with HCs, which may be explained by the evidence that the IFN-β expression is inhibited by VZV ORF47 kinase via preventing the IRF3 activation in VZV infected cells [47]. Taken together, these data suggest that HBV can be sensed by IFI16 and utilizes an immunosuppressive strategy to actively prevent innate immune responses in acute HBV infection.
Next, we determined the cellular source of IFI16. Early reports showed IFI16 was constitutively expressed in all CD34+ myeloid progenitor cells from human bone marrow and later exclusively on the CD14+ monocytoid lineage [27]. IFI16 is present in peripheral blood monocytes and lymphocytes, but not in granulocytes. In lymphocytes, it is not regulated by IFN-α, IFN-γ, and IL-6 [29]. In 2010, IFI16 was first described as a STING-dependent dsDNA sensor in monocytes and macrophages by Bowie’s group [31]. Recently, Liang’s group revealed that HBV was not sensed by the infected hepatocytes due to lack of DNA sensing machinery but could induce human peripheral macrophage activation [30]. Based on these data, we divided PBMCs into monocytes and non-monocytes by magnetic separation. Quantitative RT-PCR analyses revealed that IFI16, whether in HCs, the CHB group or the AHB group, is mainly expressed in monocytes. Furthermore, IFI16 expression in monocytes from both AHB and CHB patients was significantly upregulated as compared with that from HCs. These findings along with the data from the aforementioned studies indicate that HBV can be sensed by monocytes and its infection results in enhanced expression of IFI16 in monocytes from AHB and CHB patients.
Lastly, we investigated the response of the IFI16-STING pathway in monocytes to VACV ds 70mer, a ligand for IFI16, to further confirm the impact of HBV infection on IFN-β expression. In concordance with the results from patients, the response of the IFI16-STING pathway to VACV ds 70mer was significantly attenuated in monocytes from CHB patients in comparison with HCs, suggesting this DNA sensing pathway is impaired in CHB patients. These data further support the view that HBV is not always invisible to innate immunity but has evolved strategies to actively counteract recognition by innate immunity [6].
Our study has some limitations. First, the expression of DNA sensors in PBMCs may not be representative of the expression profile in liver. So studies of the liver tissues should be included. Second, more cases of AHB patients are needed to verify the expression characteristics of these DNA sensors in AHB. Third, the response of the IFI16-STING pathway to VACV ds 70mer in monocytes from AHB patients was not assayed because of the difficulty of obtaining adequate specimens from AHB patients. So additional studies are warranted. Last, the detailed mechanism of suppression of the IFI16-STING pathway by HBV is needed to identify.