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Hepatitis B virus (HBV) is a serious public health problem around the world and major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Approximately 2 billion people have serological evidence of past or present HBV infection and more than 360 million people are with chronic HBV (CHB) infection.1 It is reported that 15–40% of HBV-infected patients would develop cirrhosis, liver failure, or HCC.2 Around 78% of chronic hepatitis B patients live in Asia and the Western Pacific countries. Out of 6,50,000 people who die from HCC each year, two-thirds are from Asia.3 HCC is one of the most common malignant tumors in Asia and also in India.4 Notch is an evolutionally conserved molecule and controls cell-fate decision in a variety of cells. Notch signaling regulates its activities through the expression of several target genes, including Hes, HEY, transcription nuclear factor kB (NF-kB),T-bet, the pro-inflammatory cytokine interferon-γ (IFN-γ), interleukin-4, and enhancer CNS2.5 Involvement of Notch signaling is being reported in different cancers,5, 6, 7 including aberrant Notch3 and 4 expression in HCC,8 in early stages of T-cell development, peripheral T-cell activation, differentiation of various CD4+ T-helper subsets (including T-helper (TH) cells TH1, TH2, and regulatory T cells (Tregs)), and the generation of T-cell tumors.5, 9, 10 Overexpressed Notch1 and Hes1 were reported in skewed expression of CD4/CD8 T cells.9 Transforming growth factor beta 1 (TGF-β1) is a pleiotropic anti-inflammatory cytokine that also has been implicated in regulatory T-cell differentiation. Human and mouse CD4+CD25+ Tregs express the transcription factor FoxP3, and naive T cells can be converted into Tregs with stimulation and in the presence of TGF-β1.11 Although the in vivo relevance of peripheral regulatory T-cell generation is not completely clear, it is thought that TGF-β1 is required for the maintenance of the peripheral Treg pool,12 and the cross-talk between the Notch and TGF-β signaling pathways through intracellular mediators, Smad3, is evident.13, 14 The spectrum of HBV infection varies from acute hepatitis to chronic hepatitis B, cirrhosis, and HCC. Because Notch signaling relates to the organ of inflammation and injury, we undertook to study the Notch signaling pathways at different stages of HBV-related hepatic injury and also assessed the involvement of Notch in regulating the FoxP3-expressing Tregs in the peripheral blood mononuclear cells (PBMCs) and liver. Here we have demonstrated that the target gene Hes1 of Notch signaling is upregulated in CD8T cells in acute HBV (AVH-B) infection, making them to proliferate and transform into IFN-γ-producing effector T cells. Although there was downregulation of Notch signaling molecules during CHB infection, the expression was again increased with the development of cirrhosis and HCC. This study shows that Notch1 induces FoxP3 expression in the intrahepatic lymphocytes in HBV-infected cirrhosis. A close correlation of TGF-β1 expression with Notch signaling from the stage of CHB to cirrhosis and HCC could explain the association between the two pathways in disease progression with CHB infection. The tissues were collected and stored in liquid nitrogen. Paraffin-embedded biopsy samples were used for immunohistochemistry. Biopsy and tissue samples were also used to make protein extract for western blotting. From the selected patients and healthy controls, peripheral blood was collected in an EDTA-coated tube. Plasma samples were stored at −20 °C. The biochemical assessment was done according to the study protocol. Quantitative real-time PCR for Notch signaling molecules and FoxP3 was performed in triplicate in a 7900 ABI Prism Sequence Detection system using the Syber Green kit and specific primers (Table 1) for Notch1, Notch2, Notch3, Notch4, Hes1, Jag1, NF-kβ, and FoxP3, with Primer Express 1.5 software (Applied Biosciences, Carlsbad, CA, USA). Amplification of β-actin and 18S was used as the control for normalization. For TGF-β signaling, we have used a 48-format custom-designed array of TGF-β signaling from ABI, where we have included all the genes (Table 2). To normalize results within each individual group, total RNA was extracted from pooled PBMCs or LILs (n=6) per group using the Qiagen RNA extraction easy kit (Valencia, CA, USA) and cDNA was prepared. Relative quantification of each gene was analyzed by calculating the Log RQ of each sample Ct value. Anti-Notch1 FACs antibody was procured from eBiosciences (clone mN1A), and mN1A antibody reacts with the intracellular domain of human Notch1. The mN1A antibody has a low affinity for the full-length (unprocessed or heterodimeric cell surface) forms of Notch1. Therefore, Notch1 expression was considered intracellular not surface expression. After staining, the cells were acquired for flow-cytometric analyses using FACS Calibur and the results were analyzed using the Flow-Jo software. Grading on Notch1 and Notch3 expression was given as: strong, moderate, weak, and no staining. Cellular localization of the respective protein expression was also carefully observed. In total PBMCs, Notch1 and Jag1 expression were lower in AVH-B compared with HC and CHB subjects (Figure 1a), whereas Hes1 and NF-kβ expression were increased in both HBV-infected groups (Figure 1a, P=0.02, P=0.001). In CD4+ T cells, Hes1 expression was lower among those with AVH-B, whereas their expression in CHB-infected subjects was similar to HC (Figure 1b, P=0.02). In contrast, Notch1, Jag1, and Hes1 expression were upregulated in CD8+ T cells of AVH-B subjects compared with the other groups (Figure 1c, P=0.01). The expression of Notch1 in CD8 T cells during AVH-B infection was higher (Figure 1d). Significantly, higher percentage of proliferative CD8+ T cells from AVH-B subjects responded to HBV pooled peptides by secreting IFN-γ (Figure 1e) than those from CHB and healthy controls (P=0.02). Quantitative PCR results of ABI custom-designed TGF-β signaling array (Table 2) also showed increased expression of TGF-β1, TGF-β2, SMAD1, SMAD4, MAP kinases, BMP6, and PPP2CB mRNA expression in AVH-B than that in CHB patients (Figure 1f). In liver, TGF-α, TGF-β2, TGF-β3, SMAD3, SMAD4, SMAD6, and interleukin-6 were more expressed in cirrhosis patients than those in HCC patients (Figure 4a). GDF9, PPP2CB, and RASGRP3 were upregulated both in the PBMCs and liver of HCC patients. Western analysis showed the TGF-β1 expression in cirrhotic tissues, but faint expression of phospho-Smad3C (Ser423/425) in one of cirrhotic tissue. However, in HCC tissue, we did not observe expression of phospho-Smad3C (Ser423/425) and TGF-β1 (Figure 4b). Our data demonstrate that, in AVH-B infection, there is an increased expression of Notch1 in CD8 T cells (Figure 1), which may favor proliferation of CD8+ T cells and its activity, which in turn is vital for development of protective immunity and resolution of acute infection. However, during early chronic infection, Notch signaling is not activated but in subsequent stages of cirrhosis and HCC, there is an enhanced expression of Notch family genes in the liver and associated increased hepatic TGF-β signaling and emergence of CD4+ FoxP3-expressing T cells probably contributing to fibrogenesis (Figure 2, 3, 4). Several mammalian Notch receptors are oncogenic when constitutively active, including Notch1, though Notch does not obviously cause unregulated cell proliferation or genetic alterations associated with tumor progression.16 It can alter the developmental state of a cell and consequently maintain cells in a proliferative or undifferentiated state.9 We found, that Hes1 expression was significantly higher in the PBMCs and CD8+T cells but was attenuated in CD4+T cells of AVH-B patients (Figure 1). Further, a significantly higher percentage of proliferative CD8+ T cells from AVH-B respond to HBV pooled peptides by secreting IFN-γ than those from CHB patients. Therefore, a complementary association between Notch1 and Hes1 expression in CD8 T cells is likely in AVH-B than in CHB patients. These data suggest that skewed expression of Hes1 in CD4+ T cells may facilitate cell fate towards CD8+ T cells in acute stage of HBV infection (AVH-B) and strengthen the role of Notch signaling to maintain TH1 (CD8+ T cells) than TH2 cell pool in AVH-B. Multiple Notch family members act in a redundant fashion during thymic development of CD4 or CD8 T cell.9, 17 Notch1 gene activation results in decreased CD4 single positive thymocytes and a corresponding increase in CD8 single positive thymocytes.9 Altered or truncated Notch functionality is also documented to prevent differentiation of cells and predispose the undifferentiated cells to malignant transformation.6, 7, 18, 19, 20, 21, 22, 23 Onset of chronicity is thought to involve an imbalance of T-helper (Th) 1/Th2 cells. Although, there is no sequential progression from chronic hepatitis B to cirrhosis and HCC, CHB infection offers a unique opportunity to study development of carcinogenesis as it often involves nearly all stages from necroinflammation of chronic hepatitis to fibrosis and cirrhosis. In this study, in CHB, repression of Notch receptors was observed leading to immune dysfunction. Of course, it cannot be ascertained whether this alteration could contribute to ongoing fibrosis, cirrhosis, and HCC, but repression of notch receptors in CHB stage is suggestive of repression in immune regulation, i.e., no differentiation, no proliferation of effector cells, leading to further pathogenesis of disease. Peripheral and hepatic lymphocytes showed significant increased expression of all Notch receptors (Notch1, Notch2, Notch3, Notch 4 (P=0.001), Jag1, and NF-kβ in cirrhosis patients as compared with CHB patients (Figure 2b). Increased TGF-β signaling molecules and FoxP3 was also observed in cirrhosis and HCC. Therefore, enhanced Notch, TGF-β, and FoxP3 expression was found to be associated with and possibly resulting in fibrogenesis. Studies show that Tregs with FoxP3 expression have an important role in modulating the required T-cell functions15 and in the presence of TGF-β1, naive T cells can be differentiated into Tregs and maintain peripheral Tregs pool.24, 25, 26, 27, 28 TGF-β1 also mounts tumor-suppressive functions at early stages of liver damage. Whereas during cancer progression TGF-β signaling in hepatocytes shifts from tumor-suppressive pSmad3C to oncogenic pSmad3L,29, 30, 31, 32 in our study, we did not observe pSmad3C in liver tissue of HCC patients (Figure 4b). Present study showed increased TGF-β expression and enhanced SMAD1 and SMAD4; SMAD6 in intrahepatic lymphocytes in cirrhosis. In HCC patients, TGF- β and these molecules showed increased expression in PBMCs not in intrahepatic lymphocytes. This data may be suggestive of increased fibrosis in cirrhosis liver due to TGF- β, but in HCC disease is at end stage and oncogenic. In the present study, we were able to link the expression of Notch signaling with dual expression of FoxP3 and enhanced TGF-β signaling on the intrahepatic T cells (Figures 3). Flow-cytometric analysis also showed that Notch1 and FoxP3 dual expression was much higher in liver lymphocytes than peripheral lymphocytes of cirrhosis and HCC patients. Blocking the Notch signaling in LIL and PBMCs with DAPT has significantly lowered the FoxP3 expression, which strongly suggests that Notch signaling influences FoxP3 expression. In the same pool of PBMCS and LIL’s, expression of TGF-β signaling molecules was also high. This indicates that these changes may be associated with changes in TGF-β signaling expression, leading to progressive fibrosis/cirrhosis and HCC. Larger sample pool of patients with AVH-B infection would have enabled us to study the dual expression in this group of patients also.  Guarantor of the article: Nirupama Trehanpati, PhD. Specific author contributions: Planning and conducting the study, interpretation and manuscript writing, and editing and corresponding: Nirupama Trehanpati; conducting RT-PCR: Shikha Shrivastav, Sukriti, Binayak Kumar, and Bhavana Shivakumar; Notch inhibitor assay: Ritu Khosla; Western blots: Suvercha Bhardwaj and Jaya Chaturvedi; collection of samples: Sujoy Bose, Dinesh Mani Tripathi, Binayak Kumar, and Subhash Gupta; immunohistochemistry: Puja Sakhuja, Archna Rastogi, and Chagan Bihari; editing the manuscript: Shiv Kumar Sarin and Shyam Kottilil. Financial support: This study was in part supported by grant no. BT/PR12759/MED/29/135/2009 received from the Department of Biotechnology, Government of India, New Delhi, India. Potential competing interests: None.

Analysis of Notch and TGF-β Signaling Expression in Different Stages of Disease Progression During Hepatitis B Virus Infection