The purpose of the present study was to investigate the relationship between trophoblast TLR3 signaling pathway and the HB vaccine response of the infants who were born to HBsAg-positive mothers. Considering the high morbidity and mortality of the HBV infection sequelae, it is imperative to clarify the possible cause and mechanism of non- or hypo-response in this population who is suffer from the high risk of HBV infection and considerably inadequate response rate to the HB vaccine. Our results showed that the TLR3 signaling pathway proteins, TLR3 and NF-κB, were significantly decreased in the non- or hypo-responders. Moreover, by comprehensively analyzing information from HBsAg-positive mothers, trophoblast cells TLR3 signaling pathway protein expression and the babies, we found that HBsAg-positive mother’s HBV infection status impaired trophoblast cells TLR3 signaling pathway and the impaired TLR3 pathway eventually affected infant immune response to the HB vaccine. The findings suggested that, as the intermediary, trophoblast cells TLR3 signaling pathway may involve in the vaccine non- or hypo-response.
It has been demonstrated that HBV can affect TLR3 signaling [29]. In the case of long-term HBV infection, intrahepatic TLR3 expression was lower and TLR3 elevation was slower compared with normal controls [30]. Besides, the mRNA of TLR3 was significantly down-regulated in peripheral blood mononuclear cell of patients with chronic HBV infection [31]. Consistent with these researches, our study found that trophoblast cells TLR3 expression was significantly decrease in the non- or hypo-response. The placental function can be modified by the maternal environment [32, 33]. By analyzing the maternal HBV infection, we found that the HBsAg-positive mothers with both HBV DNA- and HBeAg-positive had a significant reduction of TLR3 expression. Furtherly, the Bayesian network model indicated that the maternal HBeAg status was directly contacted to TLR3. These findings indicated that the affected TLR3 involved in the non- or hypo-response and the reduced TLR3 expression may attribute to the HBsAg-positive mother’s HBV infection status.
The impairment of trophoblast cells’ TLR3 signaling pathway was multifaceted in the non- or hypo-responsiveness to the HB vaccine. In this study, the expressions of the TLR3 downstream protein NF-κB were significantly decreased among inadequate responders. This phenomenon may be related to the diminished expression of TLR3, because TLR3 expression directly positively correlated to NF-κB expression in our investigation. However, it has also recently been proven that the HBV polymerase (Pol) inhibited the activation of NF-κB by suppressing the phosphorylation of IκB kinase, blocking degradation of IκBα and by restraining translocation of NF-κB to the nucleus [34]. In Bayesian network model, the direct contact between NF-κB and non- or hypo-response suggested a complicated underlying mechanism of decreased trophoblast cell NF-κB expression, which requires further investigation.
In our study, the expressions of TRIF and IRF3 were lowered in the inadequate response cases, even though the difference was not statistically significant. The Bayesian model indicates that IRF3 directly contacted to the non- or hypo-response condition. It has been proven that the HBX protein encoded by the HBV genome reduces the TRIF protein expression in human hepatoma cell lines and liver tissue samples by affecting protein ubiquitination [35, 36]. In addition, the HBX protein could suppress IRF3 activation by inhibiting IRF3 phosphorylation, dimerization and nuclear translocation [35]. However, the increased expression of HBX is mainly in HBV-related hepatocellular carcinoma [37]. Therefore, it is possible that HBX expression might be low in HBsAg-positive mothers, which results in insignificant decreases in TRIF and IRF3. However, better understanding of this mechanism needs to be supported with additional confirmations.
As an intermediary, placental transfers maternal pathogenic microbiota exposure to baby and affects the development of the offspring’s immune system [38]. In our study, significantly decreased trophoblast cells TLR3 expression was observed in HBeAg positive neonate. The result indicated that the suppressed expression of trophoblast cells TLR3 signaling pathway proteins might relate to neonate HBV infection status. Besides, the trophoblast cells NF-κB expression was positively correlated with infant IL-6, IL-12 and TNF-α, that is, the decrease NF-κB affected these cytokines expression. Studies have shown that decreased levels of cytokines such as IL-6, TNF and IL-12are involved in inadequate response to the HB vaccine, and even lead to the vaccination failure [11, 39–41]. Not only that, the Bayesian model presented in this study, suggested that trophoblast cells TLR3 signaling pathway influenced the offspring immune response profoundly, and IL-6 might be a crucial cytokine because IL-6 levels were not only contacted to the non- or hypo-response status but also to IL-12, TNF-α, IFN-α and IFN-γ. All these indicated that by affecting baby cytokines, mainly IL-6, the impaired trophoblast cells TLR3 signaling pathway involved in the HB vaccine non- or hypo-responsiveness. Although TLR signaling pathway can be triggered via vaccination [20], how the activation of TLR3 signaling pathway followed HB vaccine need to be illuminated furtherly.
In summary, the findings presented in this study indicated that impaired TLR3 signaling pathway of trophoblast cells involved in the HB vaccine non- or hypo-response. The vaccines containing TLR ligands as adjuvants can enhance innate immunity and activate adaptive immunity [42]. Therefore, TLR3 ligands should be considered as an alternative adjuvant of HB vaccine. Our study was limited by its sample size, and by the ethical constraints of performing invasive diagnostics and sampling techniques on the newborn, which resulted in a small neonatal blood sample volume and an inability to perform more extensive analysis on the neonatal blood. Therefore, further studies are needed to focus on inadequate immune response resulting from inhibition of the TLR3 pathway and other TLR pathways in trophoblast as well as in other cell types of the placenta.