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

Abstract

OBJECTIVES:CD4+ regulatory T cells (Tregs) seem to have a key role in persistence of hepatitis B virus (HBV) infection. Notch and transforming growth factor (TGF-β) signaling independently help in the differentiation and regulation of CD4+T cells, including T-helper (TH) 1, TH2, and Tregs. Whether, the two pathways have modulatory role on different stages of HBV infection and severity of liver disease is not clear. We investigated Notch and TGF-β families' gene expression in peripheral blood and intrahepatic lymphocytes in patients with different stages of chronic HBV (CHB) infection.METHODS:Peripheral blood mononuclear cells (PBMCs), CD4+, and CD8+ T cells were isolated from patients with acute HBV (AVH-B, n=15), CHB (n=16), and controls (HC, n=10). In addition to PBMCs, intrahepatic lymphocytes were obtained from liver biopsies from CHB (n=12), cirrhosis (n=12), hepatocellular carcinoma (HCC, n=5), and healthy livers (n=5). Notch family (Notch1–4, Hes1, Jag1, and NF-kβ) and TGF-β family gene expressions were studied by real-time PCR, flow cytometry, and immunohistochemistry.RESULTS:Relative expression of Notch signaling target genes, Hes1 and NF-kβ, was higher in the total PBMCs of AVH-B and CHB patients than that in HC patients (Log relative quantification (RQ); 1.1 AVH-B vs. 0.3 HC, 1.3 CHB vs. 0.3 HC; P=0.02). CD8+ T cells showed upregulated expression of Hes1 and Notch1 (P=0.02 and 0.01, respectively) in AVH-B than in CHB patients. Also, in AVH-B patients, HBV-specific CD8+ T-cell proliferation (5.74% vs. 2.7%) and TGF-β signaling activity were higher. All Notch receptors and ligands were upregulated in the PBMCs in CHB infection (CHB vs. cirrhosis, P=0.001; CHB vs. HCC, P=0.023; and cirrhosis vs. HCC, P=NS). Intrahepatic expression of Notch1 and FoxP3 were significantly higher in cirrhotics and HCCs, and further blockage of Notch signaling reduced the FoxP3 expression. Array data of TGF-β family showed increased TGF-β3, TGF-α, SMAD3, SMAD4, SMAD6, and GDF9 expression on intrahepatic lymphocytes in cirrhotic and HCC patients compared with CHB.CONCLUSIONS:Our findings suggest that there is a complementary association between Notch1 and Hes1 in CD8+T cells during AVH-B infection. On development of CHB infection, repression of the Notch receptors mediates the regulation of immune response in patients, who progress to cirrhosis and HCC. Finally, HBV infection drives increased Notch1, TGF-β, and FoxP3 expression on intrahepatic T cells in cirrhosis, resulting in fibrogenesis and disease progression.

Notch in fibrosis and as a target of anti-fibrotic therapy

“…Notch3 and Notch4 are not expressed in normal liver [50, 112] but Notch4 is expressed by hepatocytes at the edge of regenerative nodules and in cell planes adjacent to fibrous septa [112], While Notch3 is significantly up-regulated in fibrotic liver tissues from patients with chronic active hepatitis [50]. Expression of Notch1 and Hes1 is reduced in activated hepatic stellate cells (HSCs) [9] but hepatitis B virus (HBV) infection drives increased Notch1 expression in intrahepatic T cells in cirrhosis [113]. Notch 2, Notch 3, Hey2 and HeyL expression increases significantly during activation of quiescent HSCs to myofibroblastic HSCs [49].…”

From Cirrhosis to Hepatocellular Carcinoma: New Molecular Insights on Inflammation and Cellular Senescence

“…Reports of chronic inflammation in autoimmune disorders such as rheumatoid arthritis suggest that increased infiltration of Foxp3+Tregs in damaged organs and at the sites of inflammation has a suppressive role in combating injurious inflammation [26]. In nonautoimmune diseases such as chronic viral hepatitis and primary biliary cirrhosis, studies have suggested that Tregs are involved in the maintenance of chronic inflammation [27,28]. In support of the latter finding, recent work from our group has indicated a role for Tregs coupled to activation of Notch and TGF-β in perpetuation of the inflammatory response during liver disease progression in cirrhosis and HCC in chronically hepatitis B virus (HBV)-infected patients [27] (fig.…”

“…4 Graphical representation showing Notch/TGF-β signaling and T cell activation during liver disease progression through phases of acute and chronic HBV infection with respect to the later stages of end-stage liver disease, i.e., cirrhosis and HCC. Activation is seen during the acute and end stages of liver disease, whereas attenuation is noted during the chronic stage of infection [28]. …”

“…Activation is seen during the acute and end stages of liver disease, whereas attenuation is noted during the chronic stage of infection [28]. …”

Function and Regulation of MicroRNAs and Their Potential as Biomarkers in Paediatric Liver Disease

“…This suggests that a panel of miRNAs could be used to identify the immunological status of patients. From the above identified miRNA target genes the majority have been described as regulators or suppressors of carcinogenesis or HBV replication [174,175,176,177,178]. This, once more, highlights the role of microRNA during the course of infection and liver damage; however, further studies are required to clarify the mechanisms involved.…”