Xinmin Si1, Ji Chen2, Lei Huang3. 1. Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, No.300 of Guangzhou Road, Nanjing, 210029, China. 2. Department of General Surgery, Children's Hospital of Nanjing Medical University, No.72 of Guangzhou Road, Nanjing, 210008, China. 3. Department of General Surgery, Children's Hospital of Nanjing Medical University, No.72 of Guangzhou Road, Nanjing, 210008, China. surgeonhuang@126.com.
Abstract
BACKGROUND: Persistent inflammation induced by viral infection may contribute to the pathogenesis of biliary atresia (BA). Moreover, CD4+ helper cells and CD8+ killer cells are the main effector cells involved in BA and intrahepatic bile duct injuries. OBJECTIVE: Thus, we aimed to explore the dynamics of inflammatory cell infiltration and inflammation-regulated pathways in liver-specific inflammatory responses. METHODS: Neonatal Balb/C mice were intraperitoneally infected with 1 × 106 PFU rhesus rotavirus (RRV; BA + group), 1 × 105 PFU RRV (BA- group), or DMEM (control group). Mice were sacrificed 7 or 14 days post-infection and their bile ducts, livers, and spleen-derived tissues were examined via H & E staining. The number of CD4+T lymphocytes helper cells (CD4+Th), CD8+T lymphocytes killer cells (CD8+Tc), natural killer (NK) cells, and macrophages (Mac) in the liver and spleen were quantified by flow cytometry. The expression of inflammatory genes was analyzed via a PCR-array. Western blotting was conducted to quantify the protein expression of Notch receptor active fragments (NICD). Finally, some mice were injected with DAPT (a γ-secretase inhibitor) 12 h post-infection followed by analysis of liver and bile duct tissues after 14 days. RESULTS: The numbers of CD4+Th cells were increased in the livers of BA- mice after 14 days (P < 0.05). After RRV infection, the number of CD8+Tc, CD4+Th, NK, and Mac were increased in the livers of BA + mice after 7 and 14 days. Notably, NK cell numbers remained elevated in the BA + group, but the number of Mac first increased and then decreased in both the treatment groups. PCR-array analyses indicated that the expression of many genes related to T cell proliferation and differentiation significantly increased in the livers of BA. The most upregulated gene was Jagged2 (20.34-fold). Increased NICD (Notch receptor active fragments) protein expression was found in the BA + group. Finally, DAPT injection could reduce inflammation, CD8+Tc infiltration, NICD expression, and bile duct damage after RRV infection. We found that CD8+Tc played the most important role in damaging bile ducts and promoting BA. CONCLUSION: The DAPT-based intervention could reduce expression of CD8+Tc and bile duct damage in BA mouse livers post-RRV infection. We believe that the Notch signaling pathway regulates CD8+Tc functions and inflammatory dynamics in BA mouse livers.
BACKGROUND: Persistent inflammation induced by viral infection may contribute to the pathogenesis of biliary atresia (BA). Moreover, CD4+ helper cells and CD8+ killer cells are the main effector cells involved in BA and intrahepatic bile duct injuries. OBJECTIVE: Thus, we aimed to explore the dynamics of inflammatory cell infiltration and inflammation-regulated pathways in liver-specific inflammatory responses. METHODS: Neonatal Balb/C mice were intraperitoneally infected with 1 × 106 PFU rhesus rotavirus (RRV; BA + group), 1 × 105 PFU RRV (BA- group), or DMEM (control group). Mice were sacrificed 7 or 14 days post-infection and their bile ducts, livers, and spleen-derived tissues were examined via H & E staining. The number of CD4+T lymphocytes helper cells (CD4+Th), CD8+T lymphocytes killer cells (CD8+Tc), natural killer (NK) cells, and macrophages (Mac) in the liver and spleen were quantified by flow cytometry. The expression of inflammatory genes was analyzed via a PCR-array. Western blotting was conducted to quantify the protein expression of Notch receptor active fragments (NICD). Finally, some mice were injected with DAPT (a γ-secretase inhibitor) 12 h post-infection followed by analysis of liver and bile duct tissues after 14 days. RESULTS: The numbers of CD4+Th cells were increased in the livers of BA- mice after 14 days (P < 0.05). After RRV infection, the number of CD8+Tc, CD4+Th, NK, and Mac were increased in the livers of BA + mice after 7 and 14 days. Notably, NK cell numbers remained elevated in the BA + group, but the number of Mac first increased and then decreased in both the treatment groups. PCR-array analyses indicated that the expression of many genes related to T cell proliferation and differentiation significantly increased in the livers of BA. The most upregulated gene was Jagged2 (20.34-fold). Increased NICD (Notch receptor active fragments) protein expression was found in the BA + group. Finally, DAPT injection could reduce inflammation, CD8+Tc infiltration, NICD expression, and bile duct damage after RRV infection. We found that CD8+Tc played the most important role in damaging bile ducts and promoting BA. CONCLUSION: The DAPT-based intervention could reduce expression of CD8+Tc and bile duct damage in BA mouse livers post-RRV infection. We believe that the Notch signaling pathway regulates CD8+Tc functions and inflammatory dynamics in BA mouse livers.
Authors: M Riepenhoff-Talty; K Schaekel; H F Clark; W Mueller; I Uhnoo; T Rossi; J Fisher; P L Ogra Journal: Pediatr Res Date: 1993-04 Impact factor: 3.756