Our results show that HCV-exposed neonates had a relative suppression of T cell activation and pro-inflammatory markers compared with controls, which was offset by a higher production capacity for IFN-γ. This indicates a balance between pro- and anti-inflammatory responses in the HCV-exposed neonate. To our knowledge, this is the first description of the immunologic characteristics of UCB from HCV-exposed neonates.
Taken together, these results suggest that HCV contacts the fetal immune system in utero, likely via transplacental passage. There are several potential sites in the placenta for the passage of free or cell-associated virus [23
], but it is also possible that HCV may directly infect the placenta. Many of the putative HCV receptors and attachment factors have been detected in the placenta, including claudin-1, occludin, SR-B1, LDLr, and DC-SIGN [24
]. Alternatively, it is possible that HCV proteins cross the placenta as soluble antigens rather than as whole virions. In particular, the HCV core protein can be secreted in soluble form and has known immunomodulatory effects including the suppression of T cell proliferation and activation [27
]. An attractive hypothesis is that the HCV core protein may be playing a role in the alteration of fetal immunity in utero.
While it seems likely that HCV crosses into the fetal circulation and contacts the fetal immune system, antigen-specific immune responses were not detected in HCV-exposed neonates. One possible explanation is that neonatal responses were below the assay limit of detection despite the excellent sensitivity of these assays for adult samples. Second, it is possible that anti-HCV responses were being actively suppressed or resulted in Th2 cytokine production, and experiments to test these hypotheses are ongoing. Third, it is possible that HCV might not contact the fetal immune system directly. For example, the increased IFN-γ production seen in HCV-exposed neonates could be a result of maternal pro-inflammatory cytokines [29
] or anti-idiotype antibodies that mimic exposure to HCV antigens [30
]. These possibilities seem less likely, but we were unable to exclude them entirely because we did not have paired maternal blood samples. It is also possible that maternal chronic liver disease itself, irrespective of HCV, might have indirectly affected the immune system of the HCV-exposed neonates, perhaps as a result of maternal cytokine dysregulation [32
]. We did not have any women in the control group with chronic liver disease in order to test this hypothesis because liver disorders in pregnancy are rare [33
], but no woman in either group had cirrhosis and there were no differences in maternal ALT values between groups. Last, it is possible that the innate immune system or unconventional T cell responses might play a larger role than classic adaptive immune responses in protecting the fetus from in utero infection [34
What mechanism underlies the altered immune balance seen in HCV-exposed neonates? The levels of Tregs, IL-10, and IDO activity were not different between groups. HCV-exposed neonates did have lower levels of HLA-DR+
Tregs, a subset of Tregs that are reported to be more highly suppressive [16
]. However, because the level of HLA-DR+
Tregs was highly correlated with the level of CD4+
T cell activation, the presence of HLA-DR+
Tregs might in fact be a surrogate marker of immune activation in this setting. Another possible mechanism for the altered immune balance in HCV-exposed neonates is the immunomodulatory HCV core protein, as discussed above. Finally, it is possible, although unlikely, that the frequency of unmeasured maternal coinfections was higher in the control group, thereby resulting in greater in utero antigen exposure and T cell activation.
Although most of the pro-inflammatory markers and immune activation indices were lower in HCV-exposed neonates, IFN-γ production capacity was higher. This balance between pro- and anti-inflammatory mechanisms in response to infectious disease exposure in utero has been described elsewhere [39
]. On the one hand, low levels of T cell activation may be beneficial in promoting T cell function for the clearance of HCV [42
]. On the other hand, IFN-γ produced in response to in utero exposure might play a role in protecting infants from infection, as in the case of HIV [29
]. Therefore, the balance of pro- and anti-inflammatory mechanisms might serve to protect against in utero HCV infection. Indeed, 5 of 6 neonates tested did not show evidence of in utero infection. The mechanism behind the increased IFN-γ production in HCV-exposed neonates remains to be determined, but one possibility is via an epigenetic process that reverses the neonatal hypermethylation of the IFN-γ promoter [43
]. Given our finding that HCV-exposed neonates had a higher proportion of TEMRA
cells among IFN-γ+
T cells, it is also possible that HCV exposure alters the maturation pathway of memory T cells.
The clinical effect of this altered fetal immune balance in HCV-exposed neonates remains to be determined. For example, will the increased IFN-γ production decrease the incidence of allergy or augment the clinical response to vaccination and infection? The maintenance of a Th2 skew and poor IFN-γ production after birth have been associated with the development of asthma and weaker responses to vaccination [44
]. Conversely, the presence of a Th1-biased immune response in the setting of congenital Trypanosoma cruzi
infection has been shown to augment the IFN-γ response to neonatal vaccination [46
]. Longitudinal studies that follow the clinical outcomes of HCV-exposed infants are needed.
This study has several limitations. Our small sample size may have limited our ability to find subtle differences in immune parameters with considerable biological variation, such as Tregs. However, our results showed a consistent pattern across multiple assays and for both absolute numbers and percentages, thereby lending increased credibility to these findings. That said, our conclusions are still preliminary and should be confirmed in a larger study of HCV-exposed neonates. Another potential limitation is that we could not compare maternal immune responses between groups, which could have provided insight into differences seen in the neonates. This is an important component to include in future studies of HCV-exposed neonates. Finally, it is possible that the inclusion of one neonate born to an HIV/HCV-co-infected mother could have influenced the results of our HCV-exposed group. However, in the assays in which this neonate's results were not near the group median, exclusion from the analysis did not alter our results.
Our findings indicate that neonates exposed to HCV in utero have an altered immune reactivity manifest as a relative suppression of immune activation and pro-inflammatory markers that was counterbalanced by an increased production capacity for IFN-γ. These results suggest that HCV encounters the fetal immune system in utero and alters the balance between suppressive and pro-inflammatory responses. More research on the mechanisms of HCV transmission across the placenta and its effects on the fetal immune system are needed. These results have important implications for our understanding of the fetal immune response to antigens encountered in utero and may contribute to the development of interventions aimed at interrupting the MTCT of infectious agents.