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.

中文翻译：

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RRV 诱导的新生小鼠胆道闭锁涉及 CD8 + T 淋巴细胞杀伤细胞和 Notch 信号通路

背景

由病毒感染引起的持续炎症可能有助于胆道闭锁 (BA) 的发病机制。此外，CD4 ⁺辅助细胞和CD8 ⁺杀伤细胞是参与BA和肝内胆管损伤的主要效应细胞。

客观的

因此，我们旨在探索炎症细胞浸润的动力学和肝脏特异性炎症反应中炎症调节的途径。

方法

^{用1×10 6} PFU 恒河猴轮状病毒（RRV；BA+组）、1×10 ⁵ PFU RRV（BA-组）或DMEM（对照组）对新生Balb/C小鼠进行腹膜内感染。在感染后 7 或 14 天处死小鼠，并通过 H & E 染色检查它们的胆管、肝脏和脾源组织。CD4 ⁺ T淋巴细胞辅助细胞（CD4 ⁺ Th）、CD8 ⁺ T淋巴细胞杀伤细胞（CD8 ⁺通过流式细胞术对肝脏和脾脏中的 Tc)、自然杀伤 (NK) 细胞和巨噬细胞 (Mac) 进行量化。通过 PCR 阵列分析炎症基因的表达。进行蛋白质印迹以量化 Notch 受体活性片段 (NICD) 的蛋白质表达。最后，一些小鼠在感染后 12 小时注射 DAPT（一种 γ-分泌酶抑制剂），然后在 14 天后分析肝脏和胆管组织。

结果

14天后，BA-小鼠肝脏中CD4 ^{+ Th细胞数量增加（P < 0.05）。}RRV感染后7天和14天后，BA+小鼠肝脏中CD8 ⁺ Tc、CD4 ^{+ Th、NK和Mac的数量增加。}值得注意的是，BA + 组的 NK 细胞数量仍然升高，但两个治疗组的 Mac 数量先增加后减少。PCR-array 分析表明，许多与 T 细胞增殖和分化相关的基因在 BA 肝脏中的表达显着增加。上调最多的基因是 Jagged2（20.34 倍）。在 BA + 组中发现增加的 NICD（Notch 受体活性片段）蛋白表达。最后，DAPT 注射可以减少炎症、CD8 ⁺RRV 感染后 Tc 浸润、NICD 表达和胆管损伤。我们发现CD8 ⁺ Tc在破坏胆管和促进BA方面发挥了最重要的作用。

结论

基于 DAPT 的干预可以减少 RRV 感染后 BA 小鼠肝脏中 CD8 ⁺ Tc 的表达和胆管损伤。我们认为 Notch 信号通路调节 BA 小鼠肝脏中的 CD8 ⁺ Tc 功能和炎症动力学。