Background & Aims

Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells.

Methods

We performed studies with Mdr2^-/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ⁺ cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR⁺ cells.

Results

Mdr2^–/– mice had collagen deposition around hepatic bile ducts and periportal–bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2^–/– mice had increased numbers of IL17A⁺ γδTCR⁺ cells—particularly of IL17A⁺ Vγ6Jγ1 γδ TCR⁺ cells. Fecal samples from Mdr2^–/– mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2^–/– mice also had increased intestinal permeability. The γδ TCR⁺ cells isolated from Mdr2^-/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice with L gasseri led to increased serum levels of IL17 and liver infiltration by inflammatory cells; injection of these mice with anti-γδ TCR reduced serum level of IL17. Intravenous injections of Mdr2^-/- mice with anti-γδ TCR reduced fibrosis; liver levels of IL17, and inflammatory cells; and serum levels of IL17. γδTCR⁺ cells isolated from livers of patients with primary sclerosing cholangitis, but not hepatitis C virus infection, produced IL17.

Conclusions

In Mdr2^-/- mice, we found development of liver fibrosis and inflammation to require hepatic activation of γδ TCR⁺ cells and production of IL17 mediated by exposure to L gasseri. This pathway appears to contribute to development of cholestatic liver disease in patients.

中文翻译：

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肠道微生物群的改变导致肝内 γδ T 细胞受体阳性细胞产生白细胞介素 17 以及胆汁淤积性肝病的发病机制

 背景与目标

ABCB4或MDR2基因座（编码胆汁转运蛋白）的变异与一系列胆汁淤积性肝病相关。肝脏疾病的恶化与肝脏白介素 (IL) 17 水平升高有关，但这种升高的机制尚不清楚。我们研究了Mdr2被破坏的小鼠，以确定肝脏缺陷和微生物群的改变如何影响肝内 γδ T 细胞产生 IL17。

 方法

我们对Mdr2 ^-/-和同窝 FVB/NJ（对照）小鼠进行了研究。通过酶联免疫吸附测定测定血清样品中的 IL17。给小鼠注射针对 γδ T 细胞受体（TCR；抗γδ TCR）或小鼠 IL17A（抗 IL17A）的中和抗体。收集肝脏并通过培养程序鉴定匀浆中的细菌；通过流式细胞术分离TCRγδ ⁺细胞。从小鼠身上收集粪便样本并通过 16S 核糖体 DNA 测序进行分析。用抗体或细菌刺激细胞，并测量细胞因子的产生。我们从 10 名因原发性硬化性胆管炎或慢性丙型肝炎病毒感染而接受肝移植的患者身上获取了组织。分析组织中 γδ TCR ⁺细胞产生的细胞因子。

 结果

与对照小鼠相比， Mdr2 ^–/–小鼠肝胆管周围有胶原沉积，门静脉桥接纤维化，伴有炎症细胞流入，血清 IL17 水平升高。施用抗IL17A可减少肝纤维化。 Mdr2 ^–/–小鼠的肝脏中 IL17A ⁺ γδTCR ⁺细胞的数量增加，尤其是 IL17A ⁺ Vγ6Jγ1 γδ TCR ⁺细胞的数量。与对照小鼠相比， Mdr2 ^–/–小鼠的粪便样本中富含乳杆菌，肝组织中也富含格氏乳杆菌。 Mdr2 ^–/–小鼠的肠道通透性也增加。从Mdr2 ^-/-肝脏中分离出的 γδ TCR ⁺细胞响应热灭活的加氏乳杆菌而产生 IL17。对照小鼠腹腔注射Lgasseri导致血清 IL17 水平升高，炎症细胞浸润肝脏；给这些小鼠注射抗 γδ TCR 降低了 IL17 的血清水平。静脉注射Mdr2 ^-/-小鼠抗γδ TCR可减少纤维化；肝脏 IL17 和炎症细胞水平；和血清 IL17 水平。从原发性硬化性胆管炎患者的肝脏中分离出的 γδTCR ⁺细胞（而非丙型肝炎病毒感染者）产生了 IL17。

 结论

在Mdr2 ^-/-小鼠中，我们发现肝纤维化和炎症的发展需要肝脏激活 γδ TCR ⁺细胞并产生 IL17，这是由暴露于加氏乳杆菌介导的。该途径似乎有助于患者发生胆汁淤积性肝病。