Endothelial Cell-Derived TGF-β Promotes Epithelial-Mesenchymal Transition via CD133 in HBx-Infected Hepatoma Cells

Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies.Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls.Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGFβR1, VIM, and CD133. ELISAs revealed increased levels of TGF-β in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-β stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-β significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-β induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells.Conclusion: The study indicates that secretory factors like TGF-β from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC

Soluble factors and suppressive monocytes can predict early development of sepsis in acute‐on‐chronic liver failure

Abstract Patients with acute‐on‐chronic liver failure (ACLF) have a high probability of developing systemic inflammation and sepsis due to immune dysregulation. Fifty‐nine patients with ACLF (12 without and 19 with systemic inflammation, and 28 with sepsis) were serially monitored for clinical and immunological changes at baseline, 6 hours, 24 hours, day 3, and day 7 following hospitalization. Ten healthy controls were also included. At all time points, soluble plasma factors and monocyte functions were studied. Patients with ACLF and systemic inflammation showed higher interleukin (IL)–6, vascular endothelial growth factor‐a, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1β than patients with no systemic inflammation. Patients with ACLF with sepsis had raised (p < 0.001) levels of IL‐1Ra, IL‐18, and triggering receptor expressed on myeloid cells 1 (TREM1) compared to patients with ACLF‐systemic inflammation. Five of the 19 (26.3%) patients with systemic inflammation developed sepsis within 48–72 hours with a rapid rise in plasma levels of IL‐1Ra (1203–35,000 pg/ml), IL‐18 (48–114 pg/ml), and TREM1 (1273–4865 pg/ml). Monocytes of patients with ACLF with systemic inflammation and sepsis showed reduced human leukocyte antigen–DR but increased programmed death ligand 1 (PD‐L1) and T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM3) (p < 0.04) expression with increased ETosis by monocytes at baseline and until day 7. Conclusion: High and rising levels of plasma IL‐1Ra, IL‐18, TREM1 soluble factors, and increased suppressive monocytes (PDL1+ve, TIM3+ve) at baseline can stratify patients with ACLF at high risk of developing sepsis within 48–72 hours of hospitalization