Inhibiting TIM-4 function in Kupffer cells induces iTreg cell proliferation and thus immune tolerance in mice after liver transplantation

Hao Wu; Xuesong Xu; Yakun Wu; Yiming Liu; Jinzheng Li; Jianping Gong

doi:10.16016/j.1000-5404.201607147

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Inhibiting TIM-4 function in Kupffer cells induces iTreg cell proliferation and thus immune tolerance in mice after liver transplantation

Basic Medicin | 更新时间：2020-08-05

- Inhibiting TIM-4 function in Kupffer cells induces iTreg cell proliferation and thus immune tolerance in mice after liver transplantation
- J Third Mil Med Univ Vol. 39, Issue 2, Pages: 143-150(2017)
- 作者机构：
  
  400010 重庆, 重庆医科大学附属第二医院肝胆外科
- 作者简介：
- 基金信息：
  
  Supported by the General Program of National Natural Science Foundation of China(81370580);and the National Natural Science Foundation for Young Scholars of China(81300364)
- DOI：10.16016/j.1000-5404.201607147
  CLC： R392.2;R392.4;R657.3
- Published：30 January 2017，
  
  Received：22 July 2016，
  
  Accepted：11 October 2016
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Hao Wu, Xuesong Xu, Yakun Wu, et al. Inhibiting TIM-4 function in Kupffer cells induces iTreg cell proliferation and thus immune tolerance in mice after liver transplantation. [J]. J Third Mil Med Univ 39(2):143-150(2017)
DOI：

Hao Wu, Xuesong Xu, Yakun Wu, et al. Inhibiting TIM-4 function in Kupffer cells induces iTreg cell proliferation and thus immune tolerance in mice after liver transplantation. [J]. J Third Mil Med Univ 39(2):143-150(2017) DOI： 10.16016/j.1000-5404.201607147.

摘要

目的

探讨阻断Kupffer细胞(Kupffer cells， KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4， TIM-4)诱导小鼠原位肝移植术后iTreg细胞的产生及其相关机制。

方法

建立小鼠原位肝移植急性排斥反应模型，分为sham组、control mAb组、TIM-4 mAb组。流式细胞仪检测KCs活化，Western blot检测肝脏TIM-4表达，TUNEL检测肝细胞凋亡指数(apoptotic index，AI)。提取KCs，激光共聚焦检测KCs TIM-4表达，将KCs和提取的初始CD4

T细胞进行共培养，激光共聚焦检测Kupffer细胞TIM-4表达，分为control组、control mAb组以及TIM-4 mAb组；CFSE检测CD4

T细胞增殖，流式细胞仪检测CD4

CD25

Foxp3

T细胞，ELISA检测IL-4、IL-6、IL-13水平，Western blot检测STAT6表达。建立小鼠移植模型，分为3组：iTreg组移植模型注入iTreg细胞(预先用TIM-4 mAb处理的TIM-4

KCs与初始CD4

T细胞共培养所诱导)，sham组和单纯肝移植组(LT)注入相应的PBS作为对照，检测各组AST、ALT、TBIL水平，HE染色评价肝组织排斥活动指数(rejective activity index，RAI)，观察小鼠生存率。

结果

肝移植术后24、48、72 h KCs的活化分别为15.9%、21.6%、28.3%，术后1、3、7 d TIM-4蛋白表达水平明显高于sham组(

＜0.05)；control mAb组AI值(29.23±2.56)明显高于sham组和TIM-4 mAb组。KCs与CD4

T细胞共培养后， control、control mAb以及TIM-4 mAb组CD4

T细胞的增殖率分别为32.5%、31.6%、 17.2%，CD4

CD25

Foxp3

T细胞分化分别为12.4%、13.9%、28.1%，共培养上清液TIM-4 mAb组IL-4、IL-6、IL-13 分泌水平明显低于control mAb(

＜0.05)，且加入TIM-4 mAb明显降低了与TIM-4

KCs共培养的CD4

T细胞p-STAT6蛋白表达(

＜0.05)，外源性加入IL-4可明显增高CD4

T细胞p-STAT6蛋白的表达(

＜0.05)。移植模型注入iTreg细胞，iTregz组肝功指标明显好转，与LT组比较差异有统计学意义(

＜0.05)。肝组织病理学检查，iTreg组RAI平均得分为(3.97±0.67)，明显低于LT组，且iTreg组小鼠平均存活时间延长。

结论

阻断KCs TIM-4能够抑制初始CD4

T细胞IL-4/STAT6信号通路的激活，从而诱导更多iTreg细胞的生成，致使宿主对移植物产生免疫耐受。

Abstract

Objective

To determine the effect of inhibiting T-cell immunoglobulin and mucin-domain-containing molecule 4 (TIM-4) function in Kupffer cells (KCs) on the proliferation in iTreg cells after liver transplantation in mice and the relevant mechanism.

Methods

The mouse models of orthotopic liver transplantation were established

and the mice were divided randomly into 3 groups: sham group

control mAb group

and TIM-4 mAb group. The number of activated KCs was assayed by flow cytometry. The expression of TIM-4 in the liver tissues was assayed by Western blotting

and the apoptotic index (AI) was analyzed by TUNEL assay. KCs were extracted from the liver. The expression of TIM-4 in the KCs was observed by laser confocal microscopy. The KCs and naive CD4

T cells were co-cultured

and then divided into 3 groups: control group

control mAb group

and TIM-4 mAb group. The proliferation of naive CD4

T cells was assayed with CellTrace CFSE cell proliferation kit. Furthermore

the number of CD4

CD25

Foxp3

T cells was assayed by flow cytometry. The levels of IL-4

IL-6 and IL-13 were assayed by ELISA

and the protein level of p-STAT6 in T cells was assayed by Western blotting. Then

the mice in the iTreg group were injected with iTreg cells

which were induced with co-culture system of KCs treated with TIM-4mAb and naive CD4

T cells through portal vein. The sham group and liver transplantation (LT) group were treated with commensurable PBS. In 7 d after operation

the serum levels of AST

ALT and TBIL were tested in each group. The liver tissues in each group were stained with HE to observe the rejective activity index (RAI) under light microscope. The survival time of the mice was observed.

Results

The ratios of activated KCs after liver transplantation at 24

48 and 72 h were 15.9%

21.6% and 28.3%

respectively

and the protein level of TIM-4 in liver tissues was significantly higher than that in the sham group in 1

3 and 7 d after operation (

＜0.05). The AI (29.23±2.56) of the control mAb group was significantly higher than that (0.40±0.49) in sham group

and that (11.04±2.28) of TIM-4 mAb group (

＜0.05). Then

after KCs and naive CD4

T cells were co-cultured

the growth ratios of CD4

T cells were 32.5%

31.6% and 17.2%

respectively in the control

control mAb and TIM-4 mAb groups

and the differentiation ratios of CD4

CD25

Foxp3

T cells were 12.4%

13.9%

and 28.1%

respectively. The IL-4

IL-6 and IL-13 levels in TIM-4 mAb group were significantly lower than those in control mAb group (

＜0.05). However

the addition of TIM-4 antibody blocked the increased expression of p-STAT6 in CD4

T cells co-cultured with TIM-4

KCs but not in the cells co-cultured with TIM-4- KCs (

＜0.05). Furthermore

exogenous IL-4 was added to the co-culture model and overwhelmed the inhibition of p-STAT6 expression by TIM-4 mAb (

＜0.05). After injection of iTreg cells through the portal vein

the index of liver function in the iTreg group was improved compared with the LT group (

＜0.05). And the mean RAI score in the iTreg group was 3.97±0.67

which was significant lower than 8.47±0.90 in the LT group (

＜0.05). The survival time of the iTreg group was markedly prolonged.

Conclusion

The blockade of TIM-4 function in Kupffer cells can inhibit the activation of IL-4/STAT6 signaling and induce proliferation of iTreg cells

and as a result

better immune tolerance is induced after liver transplantation.

关键词

T细胞免疫球蛋白黏蛋白4Kupffer细胞Treg细胞白介素4肝移植

Keywords

T-cell immunoglobulin and mucin-domain-containing molecule 4Kupffer cellsTreg cellsinterleukin-4liver transplantation

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