HBV, HCV, and HEV are considered the principal etiologic agents for viral hepatitis and FHF, and extremely high mortality caused mainly by HEV infection occurs in Indian pregnant women compared with their nonpregnant counterparts (1
). This high mortality was evident in the present study. Out of 20 pregnant FHF patients, 18 (90%) of which 14 (77.8%) were infected with HEV died within 24 h of their admission in the hospital, whereas none of five non-pregnant women with FHF died. We performed electrophoretic mobility super-shift assays and immunoblotting using antibodies against p50 and p65 with the nuclear extracts prepared from blood samples as well as liver tissues of dying FHF patients along with controls. We observed a very high DNA binding activity of NF-kB in pregnant FHF patients compared with that of healthy controls, but further dissection of components in band-supershift assays revealed that in spite of high binding of NF-kB, the p65 always remained absent in NF-kB complex formation (). The complete absence of p65 expression is also confirmed by immunoblotting (). Thus the major component showing high DNA binding activity and expression was p50, which was found to be forming a homodimer in absence of its canonical dimerization partner, p65. Interestingly, the two pregnant FHF patients who received treatment and returned to normal health showed reappearance of p65, although in a low amount, as a heterodimerization partner both in gel shift assay () as well as in immunoblotting (, lane c). Most interestingly, an inverse correlation between p65 expression and viral load of HEV has been observed in pregnant FHF patients who showed an extremely high titer of viral load compared with that of pregnant women with AVH or nonpregnant women with FHF (Jilani et al, unpublished data).
In an ingenious experiment by Baltimore and his group [Beg et al. (14
)], it has been demonstrated that mice lacking the p65 component of NF-kB show widespread hepatic apoptosis and die at 15–16 d of embryonic development. Also, experiments done with fibroblasts from 13-day-old embryos revealed that p65 deficiency interferes with inducible but not basal levels of NF-kB activity, and p65 has been shown to be essential for liver development, enhanced cell proliferation, and liver regeneration. Therefore, NF-kB is mostly activated after partial hepatectomy. Because the principal factors responsible for the poor clinical outcome and high mortality of FHF patients are severe liver damage, lack of liver regeneration, and impaired immunity, our observation of selective suppression of p65 and an increased homodimerization of p50 subunits leading to disruption of normal NF-kB complex and its function seems to play a crucial role during viral hepatitis and FHF. This hypothesis gains credence from our observation of partially upregulated expression of p65 in two pregnant FHF women who recovered after treatment. It is also important to note that although there is apparently no significant difference in increased NF-kB binding activity between pregnant and nonpregnant FHF patients, nonpregnant patients did show similarly moderate to low levels of p65 expression, as observed in patients who recovered. Also, an almost similar pattern of high binding activity of NF-kB but a low expression of p65 was observed in pregnant AVH patients who served as an additional disease control group (see and , lane d and e). Thus our results establish that the presence of p65 is most essential, and its absence is responsible for severe liver damage and high mortality in pregnant FHF patients. This conclusion gains further support from the recent observation that decreased expression of p65 causes liver fibrosis and liver damage in patients with HCV-induced chronic liver disease (22
The expression of transcription factor NF-kB, which is generally downregulated during pregnancy, was recently demonstrated to play a pivotal role in regulating the maternal immune responses throughout gestation (21
). NF-kB expression also regulates apoptosis of certain specific cell types through transcriptional control of cell-cycle regulatory and other protective genes (23
), as is evident from the massive degeneration of liver by apoptosis leading to embryonic lethality in p65 knockout mice. Recently, McCracken et al (25
) have showed that NF-kB is downregulated in T-cells during pregnancy, which in turn increases the susceptibility of T-cells to apoptosis. We found very high NF-kB activity in pregnant FHF patients, which may be indicative of highly increased T-cell response leading to a severe imbalance in maternal immune responses. It is well established that T-cell-mediated immunity is highly decreased during pregnancy owing to marked reduction in T-cells and increased B cell counts (26
). Recently, low CD4 and high CD8 T-cell counts and a decreased CD4/CD8 ratio leading to a low or loss of immunity in pregnant women was demonstrated (29
). This finding indicates that HEV infection induces high production of CD8 cells, which appear to be recognized by specific viral proteins that are expressed specifically in liver cells (). As is the case in HCV (30
) and HBV (32
) infection, the expression of specific HEV protein can also enhance NF-kB activation, which modulates immunoregulatory molecules and leads to production of serum and intrahepatic inflammatory cytokine including TNF-α (31
). CD8 cells that kill hepatocytes may cause degeneration of liver and release of cytokines such as TNF-α, IL-1, and IL-6, which in turn activate NF-kB in PBMCs, leading to further release of the above cytokines (see ). It is also known that viral hepatitis and FHF can induce local production of proinflammatory cytokines such as TNF-α, IL-1, and IL-6 (33
), which not only activate NF-kB but also exert effects on liver regeneration and hepatocyte apoptosis and liver necrosis (35
). In addition, the decreased CD4/ CD8 ratio causes severe immune deficiency in pregnant FHF women. Because the fetus is also infected through maternal circulation, similar phenomena occur in fetal liver, which is the site for development of the immune system. Therefore both mother and fetus are equally affected. The growing fetus is highly susceptible and affected first, and may die in utero or be aborted, or both the fetus and mother may die together. A schematic model showing the possible signaling pathways involved during hepatitis virus-induced FHF leading to death in advanced stage pregnant women is presented in .
Diagrammatic representation of the possible signaling pathways involved in HEV-induced pathogenesis during pregnancy.
In contrast, immune function in the nonpregnant FHF patients was found to be much better than in their pregnant counterparts. A further deterioration in immune response during advanced pregnancy, when mortality due to FHF is significantly higher, may be contributed by selective suppression of p65 and/or disruption of normal NF-kB complexes.
The immunosuppressive functions of steroid hormones in lymphocytes are well documented (36
) and are mediated through interactions between steroid receptors and NF-kB, leading to inhibition of NF-kB DNA binding activity. It is paradoxical, however, to find a high NF-kB binding activity but a low cellular immunity in pregnant FHF patients. The formation of NF-kB p50 homodimers, which also act as repressors of NF-kB–dependent transcription, and absence of p65 might explain this finding. Recently, a similar pattern of homodimerization of p50 subunits leading to functional inhibition of NF-kB was reported in laryngeal papilloma (37
) and cervical (38
) and oral carcinoma (39
The level of sex steroid hormones, particularly progesterone and estrogens that are increased during later half of pregnancy, are also known to directly influence viral replication and viral gene expression through their effects on viral regulatory elements (40
). Therefore pregnancy appears to be a potential risk factor for enhanced viral replication/ expression and along with this extremely low immunity in Indian/Asian pregnant women, a vast majority of whom suffer from malnutrition and folate deficiency (43
), which also contribute to liver injury, low immunity, and disease severity, leading to death (see ). Highly reduced immunocompetence is known to be associated with folate deficiency (43
), which increases the multiple viral infection and/or increased viral load (43
), including multifactorial disorders, in the Asian population (46
). A selective high susceptibility of Asian pregnant women to viral hepatitis leading to liver failure could also be due to ethnicity-associated host genetic susceptibility, particularly associated with the genes of the major histocompatibility complex (MHC), which has a strong effect on immune response to viral antigens (48
). Thus it is possible that a specific HLA allele(s) or haplotype(s) prevalent in Indian women might be associated with severe immune deficiency that influences the persistence HEV infection.
In conclusion, we suggest that the NF-kB signaling pathway is differentially regulated at the transcriptional level through upregulation and homodimerization of p50 subunits but selective suppression of its canonical dimerization partner p65. This suppression correlates with severe liver damage and complete breakdown of the immune system, which leads to multiple organ failure and death of both the mother and the fetus.