- Research Article
- Open Access
Selective Suppression of NF-kBp65 in Hepatitis Virus-Infected Pregnant Women Manifesting Severe Liver Damage and High Mortality
© Feinstein Institute for Medical Research 2007
- Received: 12 May 2007
- Accepted: 9 July 2007
- Published: 1 September 2007
Fulminant hepatitis in Asian pregnant women is generally caused by hepatitis E virus infection, and extremely high mortality is most common in them. Decreased cell-mediated immunity is considered a major cause of death in these cases, but what exactly influences decreased immunity and high mortality specifically during pregnancy is not known. We used electrophoretic mobility shift assays, immunoblotting, and immunohistochemical analysis to study the expression and DNA binding activity of NF-kB p50 and NF-kB p65 in pregnant fulminant hepatic failure (FHF) patients and compared them with their nonpregnant counterparts. In both PBMC and postmortem liver biopsy specimens the DNA-binding activity of NF-kB was very high in samples from pregnant FHF patients compared with those from nonpregnant women as well as pregnant women with acute viral hepatitis (AVH) without FHF. Further dissection of the NF-kB complex in supershift assays demonstrated complete absence of p65 in the NF-kB complex, which is formed by homodimerization of the p50 component in pregnant FHF patients. Western blotting and immunohistochemical analysis of the expression of p50 and p65 proteins both showed higher levels of p50 expression and a complete absence or a minimal expression of p65, indicating its nonparticipation in NF-kB-dependent transactivation in pregnant FHF patients. We suggest that the exclusion of p65 from the NF-kB transactivation complex seems to be a crucial step that may cause deregulated immunity and severe liver damage, leading to the death of the patient. Our findings provide a molecular basis, for developing novel therapeutic approaches.
Viral hepatitis constitutes a major public health problem in developing countries, including India. In addition to parenterally transmitted hepatitis B and C viruses, which cause majority of hepatitis, enterically transmitted hepatitis E virus (HEV) infection is mainly responsible for sporadic as well as large waterborne hepatitis epidemics related to poor hygiene and sanitation. HEV-induced viral hepatitis is the most common cause of death in Indian pregnant women (1). Studies carried out in Iran, Africa, the Middle East, and other Asian countries have also found a high mortality due to fulminant hepatic failure (FHF) during pregnancy in women with HEV infection (2–5). In contrast, reports from the United States and Europe have failed to find any significant correlation between death during pregnancy and viral hepatitis (6). The mortality rate in pregnant women with FHF has been found to be specifically higher during second and third trimesters of pregnancy (1,2,7–9), which are associated with an altered status of hormones and immunity, but what exactly influences high mortality during pregnancy is not known.
NF-kB, a eukaryotic dimeric transcription factor formed by hetero- or homodimerization of proteins of the Rel family, is involved in a wide range of cellular effects, including immune and inflammatory responses, proliferation, cell survival, and apoptotic stimuli (10). Different members of the Rel family, such as p50, p52, p65, cRel, and RelB possess a rel homology domain that confers DNA binding and protein dimerization properties (11). NF-kB remains in an inactive form in the cytoplasm by binding to the labile cytoplasmic inhibitor IκB (12,13), which masks the RelA nuclear localization signal. The release of IκB in response to intracellular signals leads to nuclear translocation of the p50 and p65 subunits and subsequent activation of a whole set of NF-kB responsive effector genes. Disruption of the RelA locus in mice lacking the p65 subunit of NF-kB has been demonstrated to lead to embryonic lethality at 15–16 d of gestation due to massive degeneration of the fetal liver by programmed cell death (14). Studies done with p65 knock-out mice also indicated that p65 is indispensable for liver development and causes enhanced cell proliferation during embryonic development (9). This finding prompted us to investigate the probable role of NF-kB during the death of hepatitis virus infected pregnant women with FHF. We report that the suppression of p65 expression appears to be associated with the breakdown of immunity and with severe liver degeneration leading to death of the patient.
. p50 and p65 Expression of NF-kB Components and the Type of Hepatitis Virus Infection in Pregnant and Nonpregnant Fulminant Hepatic Failure Patients and Controls.
Patient type and number
Type of Hepatitis
Pregnant women 15
+ + +
Pregnant Women 3
+ + +
Pregnant Women 2
+ + +
Pregnant women (disease control) 5
+ + +/+ +
Nonpregnant women 5
+ + +
Healthy pregnant women 10
Pregnant FHF patients recovered 2
All women recruited had undergone clinical assessment, a urine test for human chorionic gonadotropin, and pelvic ultrasound examination for diagnosis of pregnancy and duration of gestation. In pregnant women, a careful clinical assessment, hematological and biochemical investigations, and liver imaging were performed to further define whether liver disease unique to pregnancy could be the cause of acute liver failure (ALF). Fulminant hepatitis was considered in patients with no history of preexisting liver disease who suffered typical acute-onset hepatitis with severe liver injury and then became deeply jaundiced and went into hepatic encephalopathy within 8 wk of the onset of the disease with no past history of liver disease (15). AVH was considered in patients who had suffered acute self-limiting disease and a serum aminotransferases (ALT and AST) elevation of at least fivefold or jaundice, or both. We also obtained five normal liver biopsy specimens from patients who had undergone abdominal surgeries at Lok Nayak Hospital for conditions other than liver problems and had normal viral markers for hepatitis viruses and normal liver function tests. We obtained informed consent from these patients.
Serology and RT-PCR
The sera from approximately 10 mL of patient blood was separated and stored at −70 °C for subsequent viral assays. All sera collected during the acute phase of illness were tested for markers of HAV (IgM anti-HAV), HBV (HBsAg and IgM anti-HBc), and HCV (anti-HCV s generation) with commercially available enzyme-linked immunosorbent assay (ELISA) kits, strictly following manufacturer instructions. All sera were also tested for IgM and IgG antibodies to HEV with an ELISA kit that uses two recombinant HEV antigens corresponding to structural region of the HEV (Dignostic Biotechnology, Singapore) (16,17). DNA and RNA were extracted from the serum samples of all the patients and employed for detection of HEV and HCV RNA by RT-PCR and HBV DNA by PCR using standard procedures routinely followed in our laboratory (18). On the basis of the above viral markers, AVH was classified as hepatitis A (presence of IgM anti-HAV), or acute hepatitis B (presence of HBsAg and IgM anti-HBc). Hepatitis E was diagnosed by the presence of IgM anti-HEV (n = 11) and/or HEV RNA (n = 4) in acute phase sera or seroconversion to IgG anti-HEV alone in convalescent sera (16,17). Presence of anti-HCV (n = 1) and HCV RNA (n = 2) was taken as evidence of hepatitis C infection.
Preparation of Nuclear Extract and Western Blot Analysis
Frozen liver biopsies were minced finely and immediately processed for preparation of nuclear extract using previously described methods (19). The blood samples collected were also immediately processed for nuclear extract preparation using the same protocol as for tissue biopsies but with the exception that peripheral blood mononuclear cells (PBMCs) were first separated from whole blood using Histopaq solution (Sigma, St. Louis, MO, USA) and then processed for protein extraction. Western blot analysis was carried out as described earlier (19,20). The bands were visualized with an anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) conjugated with horseradish peroxidase in a dilution of 1:10000, using the Santa Cruz Luminol detection kit. To confirm equal protein loading, Ponceau red staining was used and in addition, the filters were reincu-bated, after stripping, with a polyclonal actin antibody raised against a peptide mapping at the carboxy terminus of a normal actin gene of human origin in a dilution of 1:1000 (Santa Cruz Biotechnology, Cat No. sc-1615). The bands were visualized and quantitated using the BioRad Gel Doc 2000TM gel documentation system with Quantity One Quantitation Software (BioRad Laboratories, Hercules, CA, USA). The densitometric analysis of fold increase in the level of expression was calculated in comparison to that of normal controls. The densitometric value of actin was used to normalize the signal. The blots were stripped of and reincubated with different antibodies when required. Stripping of antibody was done by the standard method of incubating the membranes in a buffer containing 2% SDS, 62.5 mM TrisCl, pH 6.8, and 0.1 M β-mercaptoethanol for 30 min.
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay (EMSA) was carried out using previously described techniques (19). For EMSA, the following oligonucleotides were used: an NF-kB consensus sequence 5′-AGTTGAGGGGACTTTCCCAGGC-3 ′ and an Oct-1 consensus oligonucleotide primer 5′-TGTCGAATGCAAATCACTAGAA-3′. The antibodies used were: NF-kB p50 Ab, an epitope corresponding to the nuclear localization signal region of NF-kB p50 of human origin (Cat No. sc-114) and NF-kB p65 Ab, an epitope corresponding to the amino terminus of NF-kB p65 of human origin (Cat No. sc-7151, Santa Cruz Biotechnology). The dried gels were visualized and bands were quantitated using a BioRad Gel Doc 2000TM gel documentation system with Quantity One Quantitation Software (BioRad).
Immunohistochemistry was also carried out using the previously described protocol (21). Briefly, after deparaffnization and rehydration, the tissue sections were blocked in 3% bovine serum albumin for 30 min and incubated overnight at 37°C with the primary antibody. Immunoreactivity was visualized using an ABC Staining System Kit from Santa Cruz Biotechnology following the manufacturer’s protocol. The slides were then counterstained regressively in Mayer’s hematoxylin, dipped in methanol for a few seconds, cleared in xylene, and mounted in Permount. To assess the specificity of staining, sections were processed without primary or without secondary antibodies as controls. The same antibodies were used for the immunohistochemical study.
Of the 20 pregnant women with FHF, 15 were infected with HEV, three with HBV, and two with HCV, as revealed by both ELISA and PCR tests. The age of the patients ranged from 20–35 years. Postmortem liver biopsies were collected from 18 patients who died during the study. Two patients recovered following treatment. Ten healthy pregnant women constituted the control group, and an additional disease control group consisted of five pregnant women with AVH. The details of hepatitis viral infection, disease, and NF-kB p50 and p65 status are presented in Table 1.
Constitutive Activation and High Binding Activity of NF-kB in Pregnant Women with FHF
Absence of p65 Component in NF-kB Complex Formation in Pregnant FHF Patients
Partial Participation of p65 in FHF Patients after Recovery
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 (1,3). 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 (Figure 3B, C). The complete absence of p65 expression is also confirmed by immunoblotting (Figure 5). 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 (Figure 3D, E) as well as in immunoblotting (Figure 7, 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 Table 1 and Figure 7, 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).
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 (40–42). 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 Figure 8). Highly reduced immunocompetence is known to be associated with folate deficiency (43–45), which increases the multiple viral infection and/or increased viral load (43), including multifactorial disorders, in the Asian population (46,47). 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 (48,49). 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.
The study was funded by Indian Council of Medical Research and Department of Biotechnology, Government of India, New Delhi. BKP received SRF funding from the Council of Scientific and Industrial Research, Government of India. Authors thank Dr. Alok C. Bharti for critical discussion.
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